Inhibitory Potential of Constituents from Osmanthus fragrans and Structural Analogues Against Advanced Glycation End Products, α-Amylase, α-Glucosidase, and Oxidative Stress

Inhibition of α-amylase and α-glucosidase, advanced glycation end products (AGEs) formation, and oxidative stress by isolated active constituents of Osmanthus fragrans flowers (9,12-octadecadienoic acid and 4-(2,6,6-trimethyl-1-cyclohexenyl)-3-buten-2-one) and their structural analogues were evaluated. 9,12-Octadecadienoic acid was 10.02 and 22.21 times more active against α-amylase and α-glucosidase, respectively, than acarbose and ascorbic acid, followed by 9,12,15-octadecatrienoic acid, 9-octadecenoic acid, 4-(2,6,6-trimethyl-1-cyclohexenyl)-3-buten-2-one, 4-(2,6,6-trimethyl-2-cyclohexenyl)-3-buten-2-one, 1-heptadecanecarboxylic acid, and 1-pentadecanecarboxylic acid. Concerning the inhibition of AGEs formation, similar with data for 2,2’-diphenyl-1-picrylhydrazl radical scavenging activities, 9,12-octadecadienoic acid was 3.54 times more active than aminoguanidine, followed by 9,12,15-octadecatrienoic acid, and 9-octadecenoic acid. These results indicate that 4-(2,6,6-trimethyl-1-cyclohexenyl)-3-buten-2-one, 9,12-octadecadienoic acid and their analogues inhibit α-amylase and α-glucosidase, AGEs formation, and oxidative stress have potential value in alleviating diabetic pathological conditions.

Osmanthus fragrans (Oleaceae family) has been domesticated as a local herb in East Asia and is the source of medicinal compounds 9 . O. fragrans flowers are also used as additives in foods and beverages 9 , and are considered natural essences and are commonly used in expensive cosmetics and perfumes 9 . O. fragrans flowers are used to alleviate pain and coughing, have antioxidant activity, and can provide neuroprotection 10 . Various compounds isolated from O. fragrans flowers, including tyrosyl acetate, phillygenin, ligustroside, rutin, and verbascoside findings, indicate that O. fragrans flowers may have important pharmacological properties 11 .
Little is known of the potential inhibitory effects of the active constituents isolated from O. fragrans flowers on α -amylase and α -glucosidase activities, AGEs formation, and oxidative stress. In this study, the active constituents of O. fragrans flowers were identified, and their inhibitory activities were evaluated.

Results and Discussion
Inhibition of α -amylase and α -glucosidase by the hexane, chloroform, ethyl acetate, butanol, and distilled water fractions partitioned from the methanol extract of O. fragrans flowers were evaluated ( Table 1). The IC 50 values for α -amylase and α -glucosidase inhibition were 275.6 and 134.5 μ g/mL, respectively. Among the five fractions, the respective IC 50 value of the chloroform fraction against α -amylase and α -glucosidase was 134.5 and 60.5 μ g/mL. The IC 50 values of the hexane fraction were 250.2 and 120.4 μ g/mL, respectively. The inhibitory effect of the chloroform fraction against α -amylase and α -glucosidase was 1.18 and 1.25 times higher than that of the acarbose positive control (IC 50 , 158.4 and 75.5 μ g/mL), respectively. A prior study reported strong inhibitory activity (IC 50 12.5 μ g/mL) of O. fragrans extract against α -glucosidase compared with acarbose (IC 50 1,081.27 μ g/mL) 12 . Treatment with O. fragrans extract can decrease PBGL and fasting blood glucose 12 . In the same study, treatment with O. fragrans extract (500 mg/kg) significantly decreased the content of serum malondialdehyde and increased the level of superoxide dismutase in diabetic rats, and oral administration of 160 mg/kg of the extract significantly decreased the level of serum triglyceride and serum cholesterol in diabetic rats, and significantly increased liver glycogen content 12 . The present findings bolster the idea that the chloroform fraction derived from O. fragrans flowers could efficiently inhibit α -amylase and α -glucosidase, and could possibly play a role in treatment of hypoglycemia through oxidative mechanisms.
The initial velocity 'v' of the hydrolysis reactions catalyzed by α -amylase and α -glucosidase was measured using starch or p-nitrophenyl-α -D-glucopyranoside (PNPG) as the substrate in the presence and absence of the chloroform fraction (0.2-1.6 mg/mL) are presented in Fig. 1. The regression and extrapolation lines consist of a series of lines crossing on the horizontal and vertical axes. The intercept of the vertical axis (1/Vm) increased as the concentration of the chloroform fraction increased. However, the intercept of the horizontal axis (-1/Km) remained the same. The reaction velocity catalyzed by α -amylase and α -glucosidase slowed and were correlated with an increase in the concentration of the chloroform fraction. The Km values of α -amylase and α -glucosidase were not affected by the concentration of the chloroform fraction, typical of non-competitive inhibition. The results indicate that the chloroform fraction and the substrate did not bind to α -amylase and α -glucosidase at the same site. The data are similar to a prior description of the non-competitive inhibition of α -glucosidase and porcine pancreatic amylase by Rhus chinensis extract 13 .

Isolation and identification of bioactive constituents.
To isolate the active constituents from the chloroform subfraction of the methanol extract of O. fragrans flowers, column chromatography (4 cm i.d.× 60 cm L.) was conducted with silica gel. Ethyl acetate and methanol were the elution solvents. The flow rate was 4.9 ml/min. The chromatographic analysis yielded five fractions (OF1-OF5) were obtained. OF1 and OF4 were subjected to chromatography using a Sephadex LH-20 column (GE Healthcare, Schenectady, NY, USA). From OF1, five fractions (OF11-OF15) were obtained. OF12 was isolated by prep HPLC as a single peak. From OF4, six fractions (OF41-OF46) were obtained. OF45 was isolated using a Jaigel-W253 column (2 cm i.d.× 50 cm L., JAI Co., Tokyo, Japan) with 100% methanol at a flow rate of 2.9 mL/min, resulting in the five fractions (OF451-OF455). OF453 was examined further.
Mass spectra (m/z) were obtained by mass spectrometry using a QP-2010 quadrupole device (Shimadzu Co., Kyoto, Japan) equipped with an electrospray source, operating in electron ionization (EI) mode at 70 Ev 16 . Mass spectrometry (MS) parameters were as follows: negative ionization mode, capillary, 2.89 kV; column temperature, 51 °C and gradually increased to 209 °C; source temperature, 250 °C; and mass range, 10-300 eV. The chemical structures of the isolated active compounds were analyzed in CDCl 3 on a JNM EX-600 spectrometer (JEOL Co., Tokyo, Japan). 1 H (600 MHz), 13 C (150 MHz), and DEPT (100 MHz) NMR were measured. 2D NMR ( 1 H-1 H COSY and HMQC) was performed to study the relationships between protons and carbons. Tetramethylsilane was used as a standard. The chemical shift was described as δ (ppm).
α-Glucosidase inhibition. The inhibitory activity of each sample against α -glucosidase was measured as previously described 24 . Sample (25 μ L) and phosphate buffer (25 μ L; PB, 100 mM and pH 6.8) containing α -glucosidase (0.2 U/mL) was preincubated in a 96 well plate at 37 °C for 10 min. After 10 min, 5 mM PNPG (50 μ L) in 100 mM PB was added and incubated at 37 °C for 15 min. The reaction was stopped by adding 150 μ L of 200 mM NaCO 3 . Absorbance at 405 nm was recorded with a SpectraMax ® microplate reader (Molecular Devices, Sunnyvale, CA, USA) and compared with the control contained 25 μ L of 100 mM PB in place of the sample.
α-Amylase inhibition. The inhibitory activity of each sample against α -amylase was measured as described previously 8 . Sample (40 μ L) dissolved in sodium phosphate buffer (SPB, 20 mM and pH 6.9) with 6 mM NaCl was added to 1.0 U/ml α -amylase (200 μ L) in SPB and incubated at 30 °C for 10 min. After 10 min, 400 μ L of 0.3% starch solution in SPB was added to each tube. This reaction was carried out at 37 °C for 10 min and stopped with the treatment of reagent (100 μ L) consisting of 1% 3,5-dinitrosalicylic acid, 12% sodium potassium tartrate, and 400 mM NaOH. Each tube was incubated in boiling water for 20 min and cooled to 27 °C. The reaction was diluted by adding 10 ml distilled water and absorbance at 540 nm was measured using a UV-Vis spectrophotometer. The control contained 20 mM SPB (pH 6.9, 200 μ L) instead of α -amylase.
Inhibitory kinetics against α-glucosidase and α-amylase. The inhibition mode of the chloroform subfraction from the methanol extract of O. fragrans flowers against α -glucosidase and α -amylase was evaluated with increasing concentrations of PNPG or starch as the substrate in the absence or presence of the chloroform fraction at a concentration of 0.2, 0.4, or 1.6 mg. The inhibition type was determined by Lineweaver-Burk plot analysis of the data, calculated from the results using Michaelis-Menten kinetics. DPPH radical scavenging activity. DPPH radical scavenging activity of each sample for the inhibition of diabetic complications was evaluated as described previously 25,26 . Methanol solution (100 μ L) containing some concentrations of the sample or ascorbic acid (0.2 mg/mL) as a positive control was mixed with 0.2 mM DPPH (100 μ L) in wells of a 96-well microplate. Each reaction volume was mixed and allowed to stand in the dark at 28 °C for 30 min. After the reaction time, the absorbance change of the resulting solution was measured at 517 nm with the aforementioned SpectraMax ® microplate reader. The DPPH solution was freshly prepared daily, and was covered and kept in the dark at 4 °C until the measurements were made. Measurements were performed at least in triplicate.
Inhibition of AGEs formation. The inhibitory activity against AGEs formation was measured as described previously 27 . To prepare the AGEs reaction solution, bovine serum albumin (10 mg/mL) dissolved in 50 mM SPB (pH 7.4) was added to 200 mM fructose, 200 mM glucose, and 0.02% NaN 3 to prevent the growth of bacteria. The reaction solution (950 μ L) was mixed with various concentrations of the samples (50 μ L) in 10% (CH 3 ) 2 SO. After incubation at 37 °C for 7 days, the fluorescence intensity of the reaction solution was analyzed using a spectrofluorometric detector (Bio-Tek Inc., Winooski, VT, USA) with an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Aminoguanidine hydrochloride served as a positive control.