Quantitative Analysis of Betulinic Acid in Different Lamiaceae Herbs using GC-MS and Studies on its Fragmentation Pattern

Betulinic acid (3 beta-hydroxy-lup20(29)-en-28-oic acid) is a pentacyclic lupane-type triterpenoid that is widely distributed throughout the whole plant kingdom. Recently the compound has gained considerable interest owing to a variety of biological and pharmacological activities that have been ascribed to this compound including anti-inflammatory, antibacterial, antiviral, anti-malarial, anti-HIV and antitumor effects. Therefore betulinic acid is still under development as a therapeutic agent for treatment of metastatic malignant melanoma. Hundreds of published reports have described the occurrence of betulinic acid across numerous different plant sources, but quantitative investigations are generally extended to only a few species, mainly from Betulaceae, Rhamnaceae, Platanaceae and Ebenaceae family. Unfortunately, insufficient literature data is available concerning its distribution in the Lamiaceae family. In the present study, betulinic acid was successfully separated, identified and determined in five different species of Lamiaceae family (rosemary-Rosmarinus officinalis L., sage-Salvia officinalis L., winter savory-Satureja montana L., clary sage-Salvia sclarea L. and sticky sage-Salvia glutinosa L.) using GC-MS technique. The sample preparation procedure involves solvent ultrasonic extraction, clean-up by solid phase extraction followed by size exclusion chromatography and derivatization. Also, the studies were performed on fragmentation pattern of TMS derivative of betulinic acid and characteristic fragments were proposed. Results showed that the content of betulinic acid in air dried leaves of different Lamiaceae species varied from traces up to 0.6% per dry weight. Described GC-MS procedure is efficient and sensitive as it offers high-resolution separation and simultaneous determination of pentacyclic triterpenoids in complex plant matrices even if they are present in minor quantities.