Isolation and In Vitro Pharmacological Evaluation of Phytochemicals from Medicinal Plants Traditionally Used for Respiratory Infections in Limpopo Province

<b>Background/Objectives</b>: Tuberculosis (TB) remains one of the most pressing global health challenges, ranking among the leading infectious causes of mortality worldwide. Medicinal plants possess antimycobacterial potential, warranting the isolation and characterization of their bioactive compounds to address bacterial infections. The study aimed to determine five selected traditional medicinal plants' in vitro antioxidant and antibacterial activities and the isolation of active phytoconstituents. <b>Methods</b>: Powdered leaf material was extracted using <i>n</i>-hexane, dichloromethane, acetone, methanol, and water. The quantity of phytochemicals and antioxidants was determined using colorimetric assay, The antimycobacterial activity and combination effects were determined using microbroth dilution assay. Cell viability was determined using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT reduction assay. Bioassay-guided fractionation was used to isolate bioactive compounds. <b>Results</b>: Polar solvents had high extraction yields, and all extracts had varying phytoconstituents. Active extracts were selected for fractionation and isolation of pure compounds using gradient elution column chromatography. <i>Rhoicissus tridentata</i> water extracts had the highest total phenolic (335.20 ± 8.26 mg GAE/g) and tannin (103.48 ± 7.36 mg GAE/g) content, while <i>Rosmarinus officinalis</i> (45.90 ± 11.04 mg QE/g) methanol extract had the highest total flavonoid. <i>Ximenia caffra</i> had promising antioxidant activity. <i>R. officinalis</i> had prominent antimycobacterial. <i>Rhoicissus tridentata</i> had the highest percentage cell viability. Two compounds were isolated, and they were active against <i>Mycobacterium smegmatis</i> with minimum inhibitory concentration values ranging from 0.125 to 0.25 mg/mL. <b>Conclusions</b>: The selected medicinal plants contain phytochemicals with antioxidant and antimycobacterial activities, supporting their pharmacokinetic studies and evaluation against <i>Mycobacterium tuberculosis</i> H37Rv.