Phenolic Profile and In Vitro Assessment of Cytotoxicity and Antibacterial Activity of Ziziphus spina-christi Leaf Extracts

Background: Ziziphus spina-christi is a tropical evergreen tree of Sudanese origin. The plant is commonly used in ethno-medicine for the treatment of many diseases such as malaria, digestive disorders, weakness, hepatic diseases, obesity, urinary problems, diabetes, skin infections, fever, diarrhea and insomnia. However, the pharmaceutical properties and bioactive compounds present in Z. spina-christi are still unknown. Therefore, the aim of the study was to significantly improve and deepen our knowledge about the phenolic composition of Z. spina-christi leaf extracts as well as their antibacterial and potential cytotoxic effects. Methods: The phytochemical profile of the known compounds in the methanol/water (7:3 v/v) extract of Z. spinachristi leaves were tentatively assigned on the basis of reported analytical data from three to four pieces of independent methods including retention time (tR), UV/Vis spectroscopy, high resolution mass spectrometry (HR-MS) and tandem mass spectrometry (MSn). Four bioactive compounds were purified via preparative-HPLC and identified by means of HPLC-ESI-TOF, HPLC-ESI-MSn, 1H-NMR, 13C-NMR, literature data and authentic reference standards. Antibacterial activities of aqueous and methanol leaf extracts were carried out with six bacterial strains (Bacillus subtilis, Bacillus aquimaris, Clavibacter michiganensis, Escherichia coli, Erwinia amylovora, and Pseudomonas syringae) using an agar diffusion assay. Cytotoxic effects towards the human epidermal keratinocyte cell line HaCaT and rat intestine epithelial IEC-6 cells was assess by cytoskeletal and plasma membrane integrity or analyses of mitochondrial and proliferative activities. Results: We tentatively assigned fifty-seven phenolic compounds (fifty-two of them are known) to their regioisomeric level in the methanol/water extract of Z. spina-christi leaves. To our knowledge 45 of them were not reported previously in Z. spina-christi and five for the first time in nature. Highly glycosylated flavonoids, proanthocyanidins, and chlorogenic acids were identified as the major components. Preparative-HPLC let to the isolation of four known phytochemicals including quercetin 3-O-(6-O-rhamnosyl-glucoside) (rutin) 26, quercetin 3-O-(2-O-rhamnosyl-arabinoside) 27, phloretin 3',5' di-C-glucoside 39 and quercetin 3-O-(2,6-di-O-rhamnosyl-glucoside) 50. Regarding the antimicrobial assays, the methanol extracts of the plant were found to show higher activity than the aqueous extracts indicating hydrophobic substances of Ziziphus leaves exerting antibacterial activity. The extracts revealed no cytotoxic effects towards the HaCaT or IEC-6 cells. Conclusions: Several previously known and unknown compounds were indentified in Z. spina-christi leaf extracts. The plant possesses strong antimicrobial activity and no toxicity. Our results therefore suggest that Z. spina-christi extracts might be potentially suitable for topical applications and support the use of the plant in traditional medicine to treat infectious diseases.