Polyamide

All reagents and solvents for extraction, as well as UPLC and HPLC-MS analyses (methanol, acetonitrile, formic acid, dimethylsulfoxide, polyamide and chloroform), were from Sigma-Aldrich (Poznań, Poland), and ultrapure water was obtained from a Millipore Direct Q3 device. Standards of flavonoids (apigenin, apigenin-7-O-glucoside, apigenin-6-C-glucoside, apigenin-8-C-glucoside, isorhamnetin, luteolin, luteolin 7-O-glucoside, luteolin 4′-O-glucoside, luteolin-8-C-glucoside, luteolin-6-C-glucoside, quercetin 3-O-galactoside, quercetin, rutin, isorhamnetin, isorhamnetin 7-O-glucoside) were purchased from Extrasynthese (Genay, France).
Extracts were prepared according to [21 (link)]. Weighted samples of frozen leaves (about 100 mg) were placed in Eppendorf tubes with 1.4 mL of 80% methanol and the internal standard, homogenized using a ball mill (MM 400, Retsch, Haan, Germany) and subsequently placed in an ultrasonic bath for 30 min and centrifuged (11,000× g) for 10 min. apigenin (5 µL of 1 mg/mL solution in dimethylsulfoxide) was used as the internal standard in extracts prepared for the UPLC analyses. Samples of the secondary metabolite extract were directly subjected to HPLC-MS analysis and, prior to the UPLC analysis, 200 µL were diluted with 80% methanol (800 µL).

Low-pressure column chromatography was performed on a BioLogic chromatograph (BioRad, Hercules, CA, USA), and concentration to dryness of the supernatant (n-heptane phase) containing terpene lactones was performed on a silica gel 60 (Macherey-Nagel, Duren, Germany) fraction of 0.2–0.5 mm. A mixture of water–methanol–tetrahydrofuran was used as the mobile phase in the corresponding ratios of 75:20:10, and a column was used with a size of 30 × 300 mm.
Chromatography of flavon glycosides was performed on polyamide (Sigma-Aldrich, Germany) packed in a 5.3 × 250 mm chromatographic column on a BioLogic low-pressure chromatograph (BioRad, Hercules, CA, USA) using gradient eluting mixtures, as follows: chloroform–methanol (100:0 → 60:40), then water–ethanol (100:0 → 0:100).
For complete separation of the components and their purification, silica gel rechromatography on a silica gel 60 (Macherey-Nagel, Duren, Germany) fraction of 0.2–0.5 mm was used, using the following eluent mixture: chloroform–petroleum ether in ratios of 30:70, followed by recrystallization of the substances [48 (link),54 (link),55 (link),56 (link)].

Air-dried, powdered pericarp of L. australis (1.75 kg) was extracted with 70% ethanol (5 L) by cold maceration for 72 h, and then filtered through filter paper. The marc was re-extracted again until exhaustion, and the collected extracts was dried under vacuum at 45 °C to afford the crude extract (340 g). A part of the dried extract (200 g) was fractionated using n-hexane, dichloromethane and ethyl acetate to give 22, 14 and 6 g, respectively. The n-hexane fraction (6 g) was chromatographed on a silica gel (E-Merck, Germany) column using hexane with 10% ethyl acetate increments to afford one major compound 1 (500 mg). The ethyl acetate fraction (5 g) was chromatographed on a polyamide (E-Merck, Germany) column starting with 100% water as the eluent, then with decreasing polarity by 20% MeOH to afford five sub-fractions. The sub-fraction eluted with 20% water (500 mg) was re-chromatographed on a Sephadex LH-20 column using methanol to afford compounds 2 and 3 (25, 10 mg, respectively). The sub-fraction eluted with 60% water (100 mg) was re-chromatographed on a Sephadex LH-20 column (Sigma-Aldrich, USA) using methanol as an eluent to afford three compounds 4–6 (15, 12, 20 mg, respectively).

NMR analysis was conducted on a Bruker High-Performance Avance III FTNMR spectrometer (¹H-NMR: 400 MHz & ¹³C-NMR: 100 MHz) using TMS as the internal standard. Analytical TLC was performed on Merck TLC plates with KGF Silica gel 60 and KGF RP-18 Silica gel 60, Kenilworth, NJ, USA and the spots were visualized under UV light (254 & 365 nm) after spraying with aluminum chloride. Column chromatography was carried out on flash silica gel 60 (Merck, Kenilworth, NJ, USA, particle size 230, 400 mesh), RP-C18 (silica gel, 40 × 10⁶³ mm; Merck, Kenilworth, NJ, USA), and polyamide (Merck, Kenilworth, NJ, USA).

Mass spectrometry analysis was performed with Agilent G6550A LC-QTOF-MS (Agilent Technologies, Inc., CA, United States). RP-HPLC analysis was performed with Agilent 1260 Infinity equipped with a diode array detector (DAD). VersaMax (VersaMax Molecular Devices, Sunnyvale, CA, United States) ELISA Microplate Reader and Spectramax i3x (Spectramax i3x Molecular Devices, San Jose, CA, United States) were used for the measurement of absorbance to determine the enzyme inhibitions. Polyamide (Merck KGaA, Darmstadt, Germany for column chromatography, 6), LiChroprep^® RP-18 (Merck KGaA, Darmstadt, Germany, 25–40 µm) were used for column chromatography. Silica gel 60 (Merck, Darmstadt, Germany, F₂₅₄ 0.2 mm) plates were used for thin layer chromatography studies (TLC). After this process the plates were dried and spots were detected on TLC under UV₂₅₄ and UV₃₆₆ or by heating after spraying the plates with sulfuric acid (5%). Solvents, chemicals used in HPLC and LC-QTOF-MS analysis, and all enzymes and substrates were purchased from Sigma-Aldrich (St. Louis, MO, United States) and Carlo Erba (Milan, Italy) companies. In addition, Acarbose, orlistat, and simvastatin were purchased from Bayer Group (Istanbul-Turkey), Roche (Basel, Switzerland), and Nobel (Düzce, Turkey), respectively.