Bioassay-guided detection, identification and assessment of antibacterial and anti-inflammatory compounds from olive tree flower extracts by high-performance thin-layer chromatography linked to spectroscopy.

Olive trees are one of the most widely cultivated fruit trees in the world. The chemical compositions and biological activities of olive tree fruit and leaves have been extensively researched for their nutritional and health-promoting properties. In contrast, limited data have been reported on olive flowers. The present study aimed to analyse bioactive compounds in olive flower extracts and the effect of fermentation-assisted extraction on phenolic content and antioxidant activity. High-performance thin-layer chromatography (HPTLC) hyphenated with the bioassay-guided detection and spectroscopic identification of bioactive compounds was used for the analysis. Enzymatic and bacterial in situ bioassays were used to detect COX-1 enzyme inhibition and antibacterial activity. Multiple zones of antibacterial activity and one zone of COX-1 inhibition were detected in both, non-fermented and fermented, extracts. A newly developed HPTLC-based experimental protocol was used to measure the high-maximal inhibitory concentrations (IC50) for the assessment of the relative potency of the extracts in inhibiting COX-1 enzyme and antibacterial activity. Strong antibacterial activities detected in zones 4 and 7 were significantly higher in comparison to ampicillin, as confirmed by low IC50 values (IC50 = 57-58 µg in zone 4 and IC50 = 157-167 µg in zone 7) compared to the ampicillin IC50 value (IC50 = 495 µg). The COX-1 inhibition by the extract (IC50 = 76-98 µg) was also strong compared to that of salicylic acid (IC50 = 557 µg). By comparing the locations of the bands to coeluted standards, compounds from detected bioactive bands were tentatively identified. The eluates from bioactive HPTLC zones were further analysed by FTIR NMR, and LC-MS spectroscopy. Multiple zones of antibacterial activity were associated with the presence of triterpenoid acids, while COX-1 inhibition was related to the presence of long-chain fatty acids.

Evaluation of bioactive compounds from Ficus carica L. leaf extracts via high-performance thin-layer chromatography combined with effect-directed analysis.

This study compares different solvent systems with the use of spontaneous fermentation on the phytochemical composition of leaf extracts from a locally grown white variety of common fig (Ficus carica Linn.). The aim was to detect and identify bioactive compounds that are responsible for acetylcholinesterase (AChE), α-amylase and cyclooxygenase-1 (COX-1) enzyme inhibition, and compounds that exhibit antimicrobial activity. Bioactive zones in chromatograms were detected by combining High-performance thin-layer chromatography (HPTLC) with enzymatic and biological assays. A new experimental protocol for measuring the relative half-maximum inhibitory concentration (IC50) was designed to evaluate the potency of the extracts compared to the potency of known inhibitors. Although the IC50 of the fig leaf extract for α-amylase and AChE inhibition were significantly higher when compared to IC50 for acarbose and donepezil, the COX-1 inhibition by the extract (IC50 = 627 µg) was comparable to that of salicylic acid (IC50 = 557 µg), and antimicrobial activity of the extract (IC50 = 375-511 µg) was similar to ampicillin (IC50 = 495 µg). Four chromatographic zones exhibited bioactivity. Compounds from detected bioactive bands were provisionally identified by comparing the band positions to coeluted standards, and by Fourier transform infrared (FTIR) spectra from eluted zones. Flash chromatography was used to separate selected extract into fractions and isolate fractions that are rich in bioactive compounds for further characterisation with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) analysis. The main constituents identified were umbelliferon (zone 1), furocoumarins psoralen and bergapten (zone 2), different fatty acids (zone 3 and 4), and pentacyclic triterpenoids (calotropenyl acetate or lupeol) and stigmasterol (zone 4).

Effect directed analysis of bioactive compounds in leaf extracts from two Salvia species by High-performance thin-layer chromatography.

Extracts of two Salvia species, Salvia apiana (white sage) and Salvia officinalis (common sage) were screened for phytoconstituents with the ability to act as antidiabetic, cognitive enhancing, or antimicrobial agents, by hyphenation of high-performance thin-layer chromatography with enzymatic and microbial effect directed assays. Two bioactive zones with α-amylase inhibition (zone 1 and zone 2), 3 zones for acetylcholinesterase inhibition (zones 3, 4 and 5), and two zones for antimicrobial activity (zones 4 and 5) were detected. The compounds from the five bioactive zones were initially identified by coelution with standards and comparing the RF values of standards to the bioautograms. Identity was confirmed with ATR-FTIR spectra of the isolated compounds from the bioactive zones. A significantly higher α-amylase and acetylcholinesterase inhibition of S. apiana leaf extract was associated with a higher flavonoid and diterpenoid content. Fermented S. officinalis extract exhibited a significantly higher ability to inhibit α-amylase compared to other non-fermented extracts from this species, due to increased extraction of flavonoids. The ATR-FTIR spectra of 2 zones with α-amylase inhibition, indicated that flavonoids and phenolic acids were responsible for α-amylase inhibition. Multiple zones of acetylcholinesterase inhibition were related to the presence of phenolic abietane diterpenoids and triterpenoid acids. The presence of abietane diterpenoids and triterpenoid acids was also found responsible for the mild antimicrobial activity. Flash chromatography was used to isolate sufficient amounts of bioactive compounds for further characterisation via NMR and MS spectroscopy. Five compounds were assigned to the zones where bioactivity was observed: cirsimaritin (zone 1), a caffeic acid polymer (zone 2), 16-hydroxyrosmanol (zone 3), 16-hydroxycarnosic acid (zone 4), oleanolic and ursolic acids (zone 5).