Screening selected medicinal plants for potential anxiolytic activity using an in vivo zabrafish model.
Maphanga, Veronica Busisiwe
Maphanga, Veronica Busisiwe
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Abstract
Anxiety is a feeling characterized by an unpleasant state of distress which, can often lead to excessive nervousness, fear and worry. When an individual often feels anxious even under normal circumstances, this might result in an anxiety disorder. Anxiety disorders are still problematic to the health care system and co-morbid with a range of medical conditions. Moreover, anxiety disorders can lead to a reduction in the quality of life and current therapeutic options are perceived as insufficient. The search for new drugs that demonstrate central nervous system (CNS) activity has shifted towards medicinal plants. Traditionally prepared medicines are used in most African rural communities; in some cases, can be the only method of treatment available. In this study, several medicinal plants were selected based on literature reports of their ethnopharmacological use, for anxiolytic activity study using an in vivo zebrafish model. Crude extracts were prepared with solvents of different polarities and the maximum tolerated concentration (MTC) of these crude extracts established. The anxiolytic activity of the crude extracts was determined using the 5-days post fertilisation (dpf) zebrafish larvae model. General locomotor activity and reverse-thigmotaxis behaviour (indicative of anxiolytic activity) were analysed under continuous illumination and alternating light-dark challenges, which induced anxiety in the zebrafish larvae. Of the 28 extracts tested, 13 were toxic according to the MTC values obtained. Larvae were subsequently treated with the 15 non-toxic extracts, at a dose determined in the MTC assay or with 1% DMSO as control, in 24 well plates, and exposed to stress-inducing light-dark transitions. The anxiolytic activity (reverse-thigmotaxis) was demonstrated by an increase in the percentage time spent by the larvae in the central arena of the well. Of the 15 non-toxic extracts, the Sceletium tortuosum water extract exhibited the highest anxiolytic activity. Sceletium tortuosum (L.) N.E. Br. (Mesembryanthemaceae) is an indigenous plant in South Africa, which has demonstrated possible beneficial effects to alleviate anxiety, stress and depression. Alkaloids from Sceletium tortuosum suspected to be responsible for anxiolytic activity were further examined using validated chromatographic techniques. UPLC-MS was used to determine the chemical profile of
the plant. The chromatographic method was validated by determining the linearity, recovery, limit of detection (LOD) and quantification (LOQ). Prep-HPLC was used to target the identified alkaloids; eluents were fractionated and collected into 220 drops per tube. Various fractions of targeted alkaloids were collected, combined and concentrated to give residues, which were analyzed by UPLC-MS. The isolated alkaloids were mesembrenone, mesembrenol, mesembrine and mesembranol. Zebrafish larvae at 5-dpf were further exposed to the alkaloids to study their anxiety-related behaviours under continuous illumination and alternating light-dark transitions. Significant increases in the percentage time spent in the central arena during the dark phase were observed when larvae were treated with different concentration of single alkaloids (mesembrenone, mesembrenol, mesembrine and mesembranol) as compared to the control and diazepam. Mesembrenone and mesembranol demonstrated more anxiolytic activity than other alkaloids when compared with both the control and diazepam. This indicated that Sceletium tortuosum alkaloids demonstrated an anxiolytic effect on the zebrafish larvae model. This signified that the zebrafish larvae model was effective in studying anxiety-related behaviour. Thus, the study confirmed that Sceletium tortuosum and related alkaloids mitigates anxiety in zebrafish larvae, a step towards the full in vivo validation of the traditional use of the plant.
Description
Submitted in partial fulfilment of the requirements for the degree Magister Technologiae Pharmaceutical Sciences in the Department of Pharmaceutical Sciences, Faculty of Science at the Tshwane University of Technology.
Date
2019-06-01
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Tshwane University of Technology
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Keywords
Screening, Medicinal plants, Anxiolytic activity, In vivo zebrafish model