## 3 Results and Discussion  
### Flash Chromatography and HPTLC  
Olive leaves were pre-treated using the freeze-thaw method to maximize extraction. The freeze-thaw method causes disintegration of plant tissue and cell membrane, and induces the maximum degree of cell membrane permeabilization [24]. Prepared extracts in different solvents were separated on HPTLC plates, with n-hexane-ethyl acetate-acetic acid (60:36:4, \(v/v/v\)) as the mobile phase, and then observed under white light, 254 nm and 366 nm, and after derivatization with anisaldehyde/sulfuric acid reagent. HPTLC fingerprint profiles of extracts and standards of oleanolic, ursolic and maslinic acids are shown in Figure 1. Derivatization of separated components on HPTLC plates, with anisaldehyde/sulfuric acid reagent was necessary, in order to visualize and detect the presence oleanolic, unsolic, and mashinic acids. These acids are non-chromophoric and non-UV active, and therefore require derivatization to make them visible. Although scraping the zones of the analyte of interest from developed HPTLC plates can be used to extract the bioactive compounds into a solvent, it does not yield significant amounts of pure compounds. Another important consideration is the detection of oleanolic and mashinic acids on the plate. Identifying non-chromophoric compounds such as oleanolic and mashinic acid from complex mixtures is difficult. Additionally, compounds with very similar retention factor (\(R_{\text{F}}\)) values are hard to differentiate and separate by scraping the respective bands from the HPTLC plate. In this work, the extracts were fractionated with flash chromatography, using n-hexane and ethyl acetate as mobile phases A and B, respectively. Conventional flash chromatography, used for isolating natural products, cannot detect compounds that are non-chromophoric. However, the use of evaporative light scattering detection (ELSD), in addition to UV detection, allows for detection and isolation of both chromophoric and non-chromophoric compounds (deficient in a UV-absorbing chromophore) in a single run. Collected fractions were then analyzed via HPTLC in order to detect fractions with high amounts of oleanolic and mashinic acids. The pres  
Figure 1: HPTLC fingerprints of olive leaf extracts and triterpenoid acids under (**a**) white light (b) 254 nm (**c**) 366 nm (**d**) after derivatization with anisaldehyde/sulfuric acid under white light. Track 1, methanol extract; track 2, ethanol extract; track 3, ethyl acetate extracts; track 4, ethyl acetate phase from the water: ethyl acetate extract; track 5 oleanolic acid; track 6, unsolic acid; track 7, mashinic acid. ence of oleanolic and maslinic acids in these fractions was confirmed using NMR and ATR-FTIR spectroscopy. Separation efficiency of flash chromatography from the combined methanol and ethanol extracts was not sufficient to allow for the full resolution of oleanolic and maslinic acids from the extracts (Figure 2). Both acids started to elute when the mobile phase contained 21% of ethyl acetate (79% n-hexane). Therefore, maslinic acid could not be isolated in a pure form, since all fractions with maslinic acid also contained either oleanolic acid, and/or more polar compounds with lower \(R_{\text{F}}\) values, as impurities. Due to the higher solubility of maslinic acid in ethyl acetate, larger amounts of maslinic acid were expected to be isolated from the ethyl acetate extract. In a longer chromatographic run from ethyl acetate extracts, oleanolic acid started eluting at 21% of ethyl acetate, while maslinic acid started eluting at 60% of ethyl acetate (Figure 3). While collected fractions 13-19 contained oleanolic acid of high purity, maslinic acid rich fractions 27-30 also contained oleanolic acid and more polar compounds with lower \(R_{\text{F}}\) values (the lower part of the HPTLC chromatogram). Figure 2: (a) Separation of combined methanol and ethanol extracts and (b) HPTLC assessment of collected fractions (A–oleanolic acid and B–maslinic acid).