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Isolation of amylase regulators from the leaves of Ixeridium dentatum

Two new compounds, one sesquiterpene lactone ( 1 ) and one phenylethanoid tautomer ( 2 ), together with eleven known compounds ( 3 – 13 ) were isolated from the leaves of Ixeridium dentatum . Their structures were determined by extensive spectroscopic methods, including 1D-, 2D-NMR, and mass spectrometry. All compounds were evaluated for their amylase secretion activity in human salivary gland cells after treatment in 40 mM of high glucose. All compounds showed increased amylase secretion activity. Moreover, previously undescribed compounds ( 1 – 2) , luteolin 7- O - β - D glucopyranoside ( 10 ), quercimeritrin ( 11 ), and quercetin 3- O - β - D -xylopyranoside ( 13 ) exhibited significant amylase activity comparable to the positive control.


Extraction and isolation
The dried leaves of I. dentatum (5.0 kg) were extracted with MeOH (3 × 10 L, 50℃) under sonication for 4 h to yield 290.0 g extract after solvent evaporation. This extract was suspended in H 2 O and successively partitioned with n-hexane and EtOAc to obtain the n-hexane (ID1, 91.0 g), EtOAc (ID2, 15.1 g) and H 2 O (ID3, 178.0 g) extracts after removal of the solvents in vacuo.

Amylase assay
HSG cells were obtained from the American Type Culture Collection and cultured in RPMI 1640 media (Sigma R1363, 2 mM L-glutamine, without glucose & NaHCO 3 ) supplemented with 10% fetal bovine serum (Gibco), 5 mM glucose (Sigma, G-8270), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), 100 μg/mL gentamycin (Gibco), 10 mM HEPES (Sigma, H3375), and sodium bicarbonate (Sigma, S6014) (pH 7.4). After 70% confluency, cells were sub-cultured in RPMI 1640 media containing high glucose (40 mM) for 7 days. Cells were washed, fed with fresh media and treated the isolated compounds (20 μg/mL) every day. At 7 th day, media was collected from the cultured plates and placed in an ultrafiltration (Vivaspin 15R, Sartorius, United Kingdom) for collecting the high concentration of proteins from the cell media. They were then spun at a speed of 3000 g for 20 min at 4℃. Protein concentration was measured using the Bradford method (Bradford Assay, BioRad, Hercules, CA, USA). 100 μg Proteins were loaded in the 96 well plates to measure amylase activity.
Amylase activity was assessed using a commercial kit (K711-100, BioVision Inc., Milpitas) and used ethylidene-pNP-G7 as a substrate. Absorbance, which is directly related to a-amylase activity, was measured at 405 nm at 25℃ by using an auto-analyser (Alcyon 300® analyser, Molecular Devices Corp., Sunnyvale, CA, USA).

Protein quantification assay
Protein quantification was determined using a Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Cambridge, MA). The methodology from the Bio-Rad instruction manual was followed.
Briefly, 20 μL of the concentrated media was added to 980 μL of 20% Bio-Rad Protein Assay Dye Reagent then mixed it properly. To detect protein concentration in a sample, a cuvette method using an Eppendorf BioPhotometer (Eppendorf AG, Hamburg, Germany) at a wavelength of 595 nm.