Two new compounds from leaves of Bruguiera cylindrica (L.) Blume with the in vitro a -glucosidase inhibitory activity

Introduction: Bruguiera cylindrica is one of the mangrove plants belonging to Bruguiera genus. This genus is characterized by the presence of a large number of compounds, but the research on bioactivities has not been investigated so far. In the present research, the a -glucosidase inhibitory activity, as well as chemical constituents of the ethyl acetate extract of this plant, were studied. Methods : The chemical structures of two new compounds were elucidated by spectroscopic and computational methods. Results : Two new compounds, benzobrugierol ( 1 ) and bruguierine ( 2 ), were isolated from leaves of Bruguiera cylindrica (L.) Blume, together with nine known ones, including lupeol ( 3 ), betulin ( 4 ), chrysoeriol ( 5 ), glut-5-ene-3-ol ( 6 ), cholesta-4-ene-3-one( 7 ), 3 a -( Z )-coumaroyllupeol( 8 ), 3 a -( E )-coumaroyllupeol( 9 ), 3 b -hydroxycholesta-5-ene-7-one ( 10 ) and b -sitosterol 3- O - b -D-glucopyranoside ( 11 ). Extracts and some isolated compounds were evaluated for a - glucosidase inhibitory activities. Conclusion : The results showed that most of the extracts and tested compounds exhibited activities better than the positive control acarbose, es-pecially two new compounds 1 and 2 with their IC 50 values of 17.9 (cid:6) 0.4 and 34.6 (cid:6) 0.7 (mg/mL), respectively.


INTRODUCTION
Bruguiera cylindrica (L.) Blume grows widely at Can Gio mangrove forest, Vietnam. Three others of this genus are also found in Vietnam as Bruguiera gymnorrhiza, Bruguiera parviflora, and Bruguiera sexangula. The genus Bruguiera is characterized by the presence of a large number of compounds, many of which show a broad range of biological activities. These include insect antifeedant, antioxidant, antifungal, cytotoxic, antimalarial, and antibacterial activities 1,2 . Two sulfur-containing compounds, gymnorrhizol, and bruguiesulfurol, from Bruguiera gymnorrhiza, showed antidiabetic activities with IC 50 values of 14.9 and 17.5 µM, respectively 3 . Bruguiera cylindrica has traditionally been used for treating diarrheoa, hepatitis, blood pressure, ulcers, infections, anti-inflammatory agent, and diabetes 4 . Following up with our interest in mangrove plants, the chemical constituent of Bruguiera cylindrica was also carried out.

General experimental procedures
The NMR spectra were recorded on a Bruker Avance III, Institute of Chemistry (Vietnam Academy of Science and Technology, Hanoi, Vietnam). HR-ESI-MS spectra were obtained on a Shimadzu +IDA TOF MS. TLC was performed on precoated silica gel 60 F 254 (Merck, Darmstadt, Germany). Gravity column chromatography was performed on silica gel 60 (0.040-0.063 mm, Merck) and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden). α-Glucosidase (EC 3.

Extraction and isolation
The dried powder (8 kg) of leaves was macerated with ethanol (20 L) at room temperature for two days. After filtration, the ethanol solution was evaporated to dryness under reduced pressure to yield a crude ethanol residue (900 g). This crude ethanol residue was fractionated according to the solid phase extraction method and eluted consecutively with -hexane, ethyl acetate, and finally with ethanol to yield -hexane (100 g), ethyl acetate (300 g), and ethanol (380 g).

α -glucosidase inhibitory assay
The α-glucosidase inhibitory activity was evaluated on some isolated compounds according to the method of Apostolidis et al. 5 . Acarbose was used as a positive control. All experiments were carried out in triplicate.

Computational Details
All DFT calculations were performed with Gaussian 09 package 6 . The geometric optimization of predicted structures was done at B3LYP/6-311++G(2d,p) in both the gas phase and in the methanol solvent. The frequency calculations were also taken at the same level to ensure these structures are minimum on the potential energy surface. The relative energies with ZPE correction for optimized structures were evaluated based on the difference between the total energy of each configuration. Theoretical 1 H and 13 C NMR chemical shifts were deduced from the isotropic magnetic shielding tensors by using Gauge-Independent Atomic Orbital (GIAO) methodology at B3LYP/6-311+G(d,p) 7-9 . The modified DP4+ probability was performed to assign the exact conformer using online implementation available from http://www-jmg.ch.ca m.ac.uk/tools/nmr/DP4/ 10 .

RESULTS
The crude extract of Bruguiera cylindrica leaves was fractionated and eluted with n-hexane, ethyl acetate, and ethanol, to yield the corresponding residues:hexane, ethyl acetate, and ethanol fractions. These fractions were evaluated on the α-glucosidase inhibitory activity. The result indicated that except for the ethanol fraction, the other fractions were potent inhibitors. There was a dose-dependent increase in the percentage inhibitory activity against the αglucosidase enzyme. The ethyl acetate fraction was the most efficient one with the IC 50 value of 61.8 ± 0.3 mg/mL (Table 1). Then, it was chromatographed on silica gel and Sephadex LH-20 to give two new compounds, benzobrugierol (1) and bruguierine (2), and nine known ones 3-11 ( Figure 1). The known compounds were identified from spectroscopic analysis and comparison with literature data, including lupeol (3) 11 , betulin (4) 11 , chrysoeriol (5) 12 , glut-5-ene-3-ol (6) 13 , cholesta-4-ene-3-one (7) 14 , 3α-(Z)-coumaroyllupeol (8) 15 , 3α-(E)-coumaroyllupeol (9) 16 , 3β -hydroxycholesta-5-ene-7-one (10) 17 and β -sitosterol 3-O-β -Dglucopyranoside (11) 18,19 . Except for two compounds 8 and 9, all of them were isolated from leaves of B. cylindrica for the first time. The α-glucosidase inhibitory activity was evaluated on two new compounds, 1 and 2, and some of the known ones, 3, 4, 5, and 11 (the other compounds were not tested because the samples did not well dissolve in the tested media). The results showed that all of the test compounds exhibited better activities than the positive control acarbose. Among them, two new compounds, benzobrugierol (1) and bruguierine (2) were the most potent inhibitors with IC 50 values of 17.9 ± 0.4 and 34.6 ± 0.7 µg/mL, respectively (Table 2). These spectral data resembled those of indol 3carboxylic acid 20 . However, its molecular formula (C 9 H 7 O 2 N+H, 162.0555 amu) did not fit the experimental HR-MS spectrum of 1. The combination of 2D-NMR and HR-ESI-MS data suggested that 1 could be composed of the 1,2-disubstituted benzene ring (counted for a structural formula of C 6 H 4 ) fused with a certain x-membered ring whose partial structure formula of C 2 H 2 O 2 S. This structure was also confirmed via the HMBC correlations of the H-4 proton at δ H 8.15 to three carbons at δ C 123.3 (C-5,C-6) and 137.8 (C-7a), of the H-7 proton at δ H 7.51 to two carbons at δ C 122 (C-4) and 127.5 (C-3a), of H-bons at δ C 112.8 (C-7), 122.0 (C-4), 127.5 (C-3a) and 137.8 (C-7a). Additionally, the HMBC correlations of a singlet at δ H 8.03 (1H, s) to three carbons at δ C 127.5 (C-3a), 137.8 (C-7a) and 166.3 (an oxygenated olefin carbon of the C 2 H 2 O 2 S moiety) demonstrated that the hydroxy group located at C-2 or C-3 of the five-membered ring bearing a S=O group. Up to this point, there were two structures 1x and 1y, that could satisfy all the NMR and HR-MS data (Figure 4). In order to assign the correct structure of the isolated compound (1x or 1y), their stable geometries were optimized as given in Figure 3, and the DP4 probability was performed based on their parameters of NMR chemical shift to determine the true configuration. Following the relative energy, the results in the Table 1 showed that the isomer 1y was estimated to be more stable than the remaining one by 1.9 kcal.mol −1 in gas phase and by 4.35 kcal.mol −1 in methanol solvent. The DP4 calculation resulted in the prediction of 1y with 99.85% probability. Accordingly, 3-hydroxybenzo[b]thiophene-1-dioxide (1y) was assigned for 1 and was named benzobrugierol. Five-membered ring compounds containing a thiolane oxide group, e.g. brugierol, isobrugierol, had been reported in some species of this genus such as in Bruguiera conjugata 21  ) revealed the presence of three symmetrical 1,4-disubstituted benzene rings. Furthermore, the singlet proton signal at δ H 9.76 (2H, s) as well as the carbon signal at δ C 192.8 in the 13 C-NMR spectrum of 2 suggested the presence of an aldehyde group in the molecule. In addition, five oxygenated aromatic carbon signals were observed at δ C 165.2 (C-1' ,C-1"'), 158.8 (C-4'), and 116.9 (C-4,C-5) along with twelve aromatic methine carbon signals at δ C 133.4 (C-3",C-5",C-3"' ,C-5"'), 129.0 (C-2' ,C-6'), 116.9 (C-2",C-6",C-2"' ,C-6"') and 115.8 (C-3' ,C-5'), one methine carbon-bearing two heteroatom at δ C 104.9 (C-2) and three quaternary aromatic carbon signals at δ C 130.5 (C-1') and 130.3 (C-4",C-4"'). The combination of NMR and HR-ESI-MS data suggested that 2 could be composed of three 1,4-disubstituted benzene rings (A, B, C rings), one imidazole, one hydroxy, and two aldehyde groups. The HSQC and HMBC correlations of two aldehyde proton signals with C-2",C-3",C-4",C-5",C-6" and C-2"' , C-3"' ,C-4"' ,C-5"' ,C-6"' indicated the attachment of the first aldehyde group at C-4" of the aromatic ring and of the second aldehyde one at C-4"' of the C ring ( Figure 5). The HMBC cross-peak of H-2 with C-3' ,C-5' ,C-2' ,C-6' and C-4' indicated the attachment of the C-1' of the aromatic A ring with carbon C-2 of the imidazole ring. Based on these analyses, there were two structures 2x and 2y (Figure 7), which could fit all the experimental HR-MS and NMR data. It was obvious that the geometric structure 2x possessed an imidazole ring while 2y having a dioxole one. The calculated results in Figure 6 showed that 2x was the more energetic-favorable structure as compared to 2y by the relative energy of 16.01 kcal.mol −1 in the gas phase and 18.06 kcal.mol −1 in methanol. The PD4 analysis gave a great probability of 100% for 2x. Thus, 2 x was predicted to be the structure of compound 2, named 4,5-di(4-formylphenoxy)-2-(4-hydroxyphenyl)-2,3-dihydro-1H-imidazole, or ruguierine.

CONCLUSION
From the ethyl acetate fraction of leaves of Bruguiera cylindrica (L.) Blume, two new compounds namely benzobrugierol (1) and bruguierine (2), together with nine known ones, were isolated and the chemical structure elucidated. Extracts and some isolated compounds were evaluated for α-glucosidase inhibitory activities. Among them, benzobrugierol (1) and bruguierine (2) were the potent inhibitors with IC 50 values of 17.9 ± 0.4 and 34.6 ± 0.7 (mg/mL), respectively.

CONFLICTS OF INTEREST
The authors declare no competing financial interest