Targeted 1H qNMR Metabolomic Analysis of Betulinic Acid Variability in the Stem-Bark of Curtisia dentata (Burm.f.) C.A. Sm. Trees: Implications for Traditional Medicines

Curtisia dentata is traditionally used across southern Africa to treat gastrointestinal issues, sexually transmitted infections, malaria, tuberculosis, as an emetic, purgative, blood purifier, and aphrodisiac. Betulinic acid, a key triterpenoid isolated from the stem-bark of C. dentata , is linked to several of these traditional therapeutic applications and demonstrates broad pharmacological activity. However, natural variation and its impact on the doses delivered in C. dentata traditional preparations remain poorly characterized, constraining our ability to assess their therapeutic consistency. This study quantified inter-individual and seasonal variation in betulinic acid in stem-bark collected from ten C. dentata trees in Nkandla Forest Reserve, KwaZulu-Natal, South Africa, between January and September 2019, and evaluated the implications for the consistency and biological activity of ethnomedicinal doses. Quantitative proton nuclear magnetic resonance spectroscopy ( 1 H qNMR) targeting the isopropenyl proton signals at H δ 4.61 and 4.74 ppm revealed concentrations ranging from 0.046 to 0.291 mg/g of dry stem-bark. Significant variability occurred among individual trees and across seasons, with the highest levels in mid-summer and early autumn and the lowest in mid-winter. These fluctuations indicate that traditional decoctions where small quantities of stem-bark are boiled in large water volumes may yield markedly different betulinic acid concentrations, in some cases below thresholds required for pharmacological activity. The results highlight the need for improved standardization and dosage guidance to enhance the therapeutic reliability of C. dentata -based traditional medicines.