LC‐DAD‐MSⁿ and HR‐LC‐QTOF Analysis of <i>Ziziphus budhensis</i> Leaves and Evaluation of Their In Vitro and In Vivo Biological Activities

Ziziphus budhensis Bhattarai & M.L. Pathak is a specie described in 2015 in Nepal poorly studied for its phytochemical composition and possible bioactivities. In this study, six extracts were obtained from Z. budhensis leaves using ultrasound assisted extraction with solvents in increasing polarity namely hexane, dichloromethane, chloroform, ethyl acetate, acetone, and methanol. The phytochemical contents, along with the antioxidant, antibacterial, and antifungal capabilities, were evaluated for all the extracts to screen possible bioactivities. Chemical composition was studied combining liquid chromatography coupled with diode array and multiple stage mass spectrometry (LC-DAD-MSⁿ) and liquid chromatography coupled with quadrupole time of flight high resolution mass spectrometry (HR-MS-QTOF). Forty-six secondary metabolites belonging to the classes of phenolics, benzyl-isoquinolinic alkaloids, cyclopeptide alkaloids, triterpene aglycone and saponins were identified. Among them 23 different derivatives were subjected to quantitative analysis and quercetin 3-O-β-neohesperidoside (179.63 mg/g), quercetin-3-O-rutinoside (123.20 mg/g), and quercetin-3-O-rhamnoside (116.62 mg/g) were the most abundant in the methanol extract while quercetin 3-O-β-neohesperidoside (236.06 mg/g), medicagenic acid (192.80 mg/g) were the most abundant in acetone extract and oleanolic acid (163.14 mg/g) was the most abundant in dichloromethane extract. To screen possible bioactivities extracts were tested against Gram-positive (Bacillus subtilis and Staphylococcus aureus), Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae), and Candida albicans and the ethyl acetate and acetone extracts presented significant activities. In vivo data were obtained using brine shrimp lethality test to evaluate the toxicity levels of the Z. budhensis leaves extracts. Acetone (LC₅₀ = 145.04 µg/mL), methanol (50.22 µg/mL), and ethyl acetate (124.86 µg/mL) extracts exhibit toxic effects on nauplii, highlighting the importance of understanding extract-specific toxicity profiles in ecological assessments. In addition, in vivo acute oral toxicity test on mice was performed on all six extracts and showed no toxic effects, suggesting their safety at tested doses during oral administration. The findings of this study advocate for further in-depth research into the use of Z. budhensis leaves for medicinal purposes.