SUPPLEMENTARY MATERIAL

Triterpenes as uncompetitive inhibitors of -glucosidase from flowers of Punicagranatum. L

Riham Salah El Dine,aQiongMa,bZeinabA.Kandilaand Ali M. El-Halawany,a,c*

aDepartment of Pharmacognosy, Faculty of Pharmacy, Cairo University,11562,Cairo, Egypt. bSchool of Life Sciences, Inner Mongolia University, Huhhot,China,.cFaculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia.

  1. Experimental
  2. General.

Nuclear magnetic resonance (NMR) spectra, were recorded on a JHA-LAA 400 WB-FT (1H, 400 MHz; 13C, 100 MHz; Jeol Co., Tokyo, Japan) spectrometer, the chemical shifts being represented as ppm with tetramethylsilane as an internal standard. TLC was carried out on pre-coated silica gel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm, Merck Co., Darmstadt, Germany). Column chromatography (CC) was carried out on BW-820MH silica gel, Wakosil C-300 silica gel (40-63 μm) (Wako Chem. Co., Osaka, Japan).

1.2.Plant material

The flowers ofP. granatumLinn were purchased in 2009 from the herbal store Harraz, Cairo, Egypt. The identity of the plant was authenticated by Dr. Rim Hamdy, Plant Taxonomy Department, Faculty of Science, Cairo University. A voucher specimen(No. P.24-03-2014) was kept in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

1.3.Extraction, isolation and identification

The air dried powdered flowers of P. granatumL(2 kg) were extracted with methanol (4-5 L) at room temperature, till exhaustion.The combined extracts were filtered and concentrated to give a brown residue of 45.6 g. Part of the residue (30 g) was suspended in distilled water (500 ml) and partitioned successively with n-hexane, chloroform (4 x500 ml), to yield 8.5 g. and 12 g, respectively. The CHCl3fraction was chromatographed over silica gel 60 H for column chromatography (6x 50cm) with n-hexane, n-hexane–chloroform mixtures, chloroform-ethyl acetate mixtures with a gradual increase in polarity up to 100% ethyl acetate. Fractions (100 ml each) were collected and monitored by TLC. The spots were visualized after spraying with p-anisaldehyde followed by heating. Similar fractions were pooled to give four main fractions (A–D). Fraction A (20-80% chlororform-n-hexane, 1.5 g) was subjected to silica gel column (20 x 2 cm) usingn- hexane-chloroform mixtures yielding Compound 1 (10 mg). Fraction B (40–50% chloroform–n-hexane, 3.5 g) was rechromatographed over silica gel column (25 cmx 2 cm, 50 g), using n-hexane: ethyl acetate (8:2 and 7:3 v/v) mixtures as eluent to afford compound 2(20 mg) and 3(28 mg). Fraction D (60-80% chloroform–n-hexane, 2.9 g) was rechromatographed over silica gel 60 column (25 cmx2 cm), using CHCl3: MeOH(9.5:0.5 v/v) as an eluent to give two subfractions. The two subfractions were purified over sephadex LH-20 column using CHCl3–MeOH (1:1v/v) as an eluent to yield twocompounds 4(23 mg),and 5(31 mg).

1.4.Determination of inhibitory activity against -glucosidase

The inhibitory activities on -glucosidase were determined using reported method (Meng and Ma 2013)on 96-well plates. Ten microlitres of sample solution (in DMSO) and 80 μL of substrate solution (2 mM of 4-nitrophenyl -D-glucopyranoside in 100 mM potassium phosphate buffer, pH 7.0) were added to each well. After 10 μL of enzyme solution (0.40 U/ml of Bacillus Stearothermophilus, Sigma, Lot# 090M1360V) in buffer was added per well, the plates were incubated at 37 C for 20 min. In the control wells, sample solution was replaced with DMSO. The absorbance at 405 nm was measured on a plate reader before and after incubation. The increase in absorbance (ΔA) was used to calculate the inhibition:

Inhibition% = Acontrol- Asample/Acontrol

Each sample was measured at 4 concentrations in triplicate (final concentrations of 100, 10, l and 0.1μg/mL).

IC50 values(the concentration at which the compound inhibits 50% of enzymeactivity) (Table S1) were calculated from the inhibition%-versus concentrationcurves.

1.5.Determination of inhibitory activity against maltase

The intestinal disaccharidases were prepared by using the reported method(Matsuo et al. 1992, Meng and Ma 2013). The small intestines from 3 mice were slit opened and washed with phosphate buffer (pH 7). The mucosa was scraped off and pulled to a centrifuge tube containing 2 mL of phosphate buffer. The mixture was vortexed for 1 min followed by centrifuge for 1 min (relative centrifugal force: 1073 g). Then the supernatant was filtered with a 0.22 lm microfilter. The filtrate (disaccharidases) was diluted 10 times to measure the maltase activity and 5 times to measure the sucrase activity. The assay was carried out on a 96-well plate. The diluted disaccharidase (7 μL) was incubated with 3 μL compound solution and 20 μL sucrose or maltose solution (2 mg/mL) at 37 C for 20 min. After the incubation, 10μL of DMSO was added to each well and the released glucose was measured by glucose oxidase method using the assay kit from Nanjing Jiancheng Bio Company (Nan Jing, China). IC50 values (Table S1) were calculated using the same method as for -glucosidase. The average logPvalues (the ratio of concentrations of solute between octanol and water) of these compounds were calculated using ALOGPS 2.1 from vcclab.org (VCCLAB, 2005).

3.6. Kinetics of inhibition against α-glucosidase.

-glucosidase inhibition was determined by monitoring spectrometric changes (405 nm) associated with cleavage of the substrate used by inhibitor in the presence or absence of the enzyme over time.In order to evaluate the inhibition type of the triterpene acids against α-glucosidase activities, increasing concentrations of 4-nitrophenyl α-D-glucopyranoside were used as a substrate in the absence or presence of ursolic acid at different concentrations.

3.7. Lineweaver-Burk Plots (double reciprocal plots).

The Inhibition type was determined by Lineweaver-Burk plot analysis of the data, which were calculated from the result according to Michaelis-Menten kinetics.

The enzyme concentration was kept constant at 0.4 u/mL in a phosphate buffer (pH 7.0), while substrate concentrations varied from 0.2 to 2 mM(2, 0.5, 0.286, 0.2 nM). Absorbance at 405 nmwas measured every 3 min, to get the initial velocity (v). Plots of 1/v versus 1/S were constructed for DMSO, ursolic acid (35μM) and (72μM), and a known α-glucosidase inhibitor, acarbose (0.9 μM).

References

Matsuo T, Odaka H, Ikeda H. 1992. Effect of an intestinal disaccharidase inhibitor (AO-128) on obesity and diabetes. Am J Clin Nutr.55:314S-317S.

Meng HC, Ma CM. 2013. Flavan-3-ol-cysteine and acetylcysteine conjugates from edible reagents and the stems of Cynomorium songaricum as potent antioxidants. Food Chem.141:2691-2696.

Table S1: Inhibitory effects of the different extracts and isolated compounds of p.granatumLon -glucosidase and maltase enzymes.

Sample / -glucosidase IC50 (μg/mL)(µM) / maltase
IC50 (μg/mL)
PGFMe / 4.05 / 49.36
PGFH / 47.04 / 100
PGFC / 5.21 / 86.52
PGFW / 0.83 / 46.44
-sitosterol / 100 / 100
Ursolic acid / 17.74 (39) / 100
Oleanolic acid / 15.52 (35) / 100
p-Coumaric acid / 100 / 100
Apigenin / 100 / 100
Acarbose / 0.6 (0.93) / 0.005 (7.7 nM)