Elicitation, Analysis, and Biological Activity of Secondary Metabolites of Ziziphus spina-christi (L.) Desf. In Vitro Cultures

Abstract

Ziziphus spina-christi (L.) Desf. (Rhamnaceae) or “Christ's Thorn”, "Sider" in Arabic is an evergreen tree widely distributed in the Mediterranean region, North and East Africa. It has been used in traditional medicine to treat several medical conditions including dermatitis, eye inflammation, toothache, stomachache, rheumatoid arthritis and diabetes. This study investigated chemical properties and biological activity of the secondary metabolites extracted from the in vitro-grown cotyledonary-callus. Yield percentage of the extract was measured in callus grown on MS media in which chloroform:ethanol 3:1 gave 0.852%, 90% alcohol gave 1.648% and aqueous extract gave 0.120%. The antioxidant power of the extract was measured by ferric reducing antioxidant power (FRAP) assay after culturing the callus on three different growth media; B5, QL and MS. FRAP assay results (absorbance) were 0.189, 0.327 and 0.38 respectively indicating that growth on B5 media gave the highest antioxidant power. The effect of alcoholic extract was examined using disc diffusion assay (DDA) on four different bacterial isolates; E. coli, methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) and Pseudomonas auregenosa. Addition of 2000 µl of extract on assay discs showed inhibition zone only on MSSA. Minimum inhibitory concentration (MIC) showed positive results on S. aureus with MIC of 3.0mg/100µl. Other bacterial species that gave negative results on DDA were not tested by MIC. The UV active fractions was separated from the TLC plates and was applied on two cancer cell lines; A549, a human lung carcinoma, and U205 an osteosarcoma. Cell toxicity on osteosarcoma was observed at EC50 = 395 µg/ml. Mass spectrometry (MS) was carried out in the negative mode which gave a single peak at 485, therefore the compound is corresponded to triterpenoid acid. Salicylic acid (SA) was used as an elicitor in the cell suspension culture at three different concentrations; 25, 50 or 75 mM, HPLC analysis revealed that adding 50.0 mM SA resulted in the highest area under the curve (8575 mAU) for the total crude extract compared to control treatment (7440mAU), 25.0Mm (8143mAU) or 75mM (7483mAU).