DEVELOPMENT OF THE COMBINED METHOD OF EXTRACTION, PURIFICATION AND ANALYTICAL HPLC PROCEDURES FOR OBTAINING THREE MAJOR TRITERPENE ACIDS FROM ALMOND HULLS

Almond hulls represent a low-cost and rich source of fruit-derived bioactive compounds with the potential of being processed for its multifunctional clinical, nutritional, and pharmaceutical value. The pentacyclic triterpenoids - ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are particularly abundant in almond hulls and have attracted a lot of attention due to their unique and strong pharmacological activities. The aim of this study was to develop an alternative, selective, reproducible, low-cost, robust and high-yield extraction-based method by ultrasound-assisted technique combined with a rapid, effective and specific analytical HPLC procedure for obtaining three major triterpene acids – ursolic, oleanolic and betulinic acids from almond hulls as a waste material in agriculture and their quantitative determination in the obtained extracted product and almond hulls as well. The three-stage sequential ultrasound-assisted extraction was carried out under ultrasonication at 40 kHz. A dried powdered extracted product was obtained after the stepwise extraction process with a high yield 0.2-0.3%. The HPLC analysis was performed using a column - C18 4.6×250 mm, 5 μm with an isocratic elution of mobile phase and detection at 205 nm to quantitatively determine the assay of each analyte in the obtained extracted product and the waste material. The proposed method was validated with respect to the following method performance characteristics: system suitability test, specificity, precision, linearity-range, sensitivity, limits of quantification and detection, accuracy and robustness. The average recovery of the combined method is >93%. The average content of ursolic acid, oleanolic acid and betulinic acid equals to 0.891 mg, 0.697 mg and 1.014 mg per 1 g of the almond hulls, respectively. The purity of the dried extracted product is not less than 92%