In vitro α-glucosidase inhibitory activity of compounds isolated frommangrove Lumnitzera littorea leaves

Introduction: Lumnitzera littorea grown at CanGio Mangrove Forest has been investigated. The present study reports the isolation, characterization and evaluation of the α-glucosidase inhibitory activity of isolated compounds from Lumnitzera littorea leaves. Methods: Their structures were elucidated by spectroscopic methods (including MS, 1D and 2D–NMR) and comparison with values from the literature. From the n-hexane extract, nine compounds including lupeol (1), betulin (2), betulinic acid (3), oleanolic acid (4), corosolic acid (5), β-sitosterol (6), β-sitosterol 3-Oβ-D-glucopyranoside (7), stigmast-5-ene-3β-O-(6-O-hexadecanoyl-β-D-glucopyranoside) (8), and stigmast-4-ene-3-one (9) were isolated and identified. Results: The results of the α-glucosidase inhibitory activity showed thatcorosolic acid (5) and oleanolic acid (4)were the most potent, with IC50 values of 17.86 ± 0.42 and 18.82 ± 0.59 μg/mL, respectively. Five of the other seven compounds exhibited inhibitory activity with IC50 values below 100 μg/mL, and higher than the positive control acarbose (127.64± 0.64 μg/mL).


INTRODUCTION
Diabetes is a chronic disease associated with unusually high levels of glucose in the blood. The goal of diabete therapy is the maintenance of normal blood glucose levels after a meal. Postprandial hyperglycemia plays an important role in the development of type 2 diabetes and its complications. One of the therapeutic approaches for decreasing blood glucose rise after a meal is to slow down the absorption of glucose by inhibition of carbohydrate hydrolyzing enzymes, such as α-glucosidase. α-Glucosidase is an intestinal enzyme that breaks down α-1,4 linked polysaccharides to α-glucose, which leads to the high blood sugar levels. The development of an α-glucosidase inhibitor derived from natural products is an important contribution for the treatment of diabetes. Lumnitzera littorea, a woody tree of the Combretaceae family, grows at the Can Gio Mangrove Forest in Vietnam. The antimicrobial activities of n-hexane, ethyl acetate and methanol extracts of leaves of this species were evaluated against six human pathogenic microbes and the former extract was the most active 1 . Our published research showed that the αglucosidase inhibitory activity on all extracts and isolated flavonoids from the leaves of Lumnitzera littorea were very strong 2 . The aim of this study was to isolate phytoconstituents and evaluate the inhibition of αglucosidase activity of the compounds isolated from the n-hexane extract of L. littorealeaves. Merck (Darmstadt, Germany). Other chemicals were of the highest grade available.

In vitro α-glucosidase inhibitory assay
The α-glucosidase inhibitory activity was evaluated on all compounds according to the method of Apostolidis et al. 3 . A reaction mixture containing 60 µL of 100 mM phosphate buffer (pH 6.8), 20 µL of sample (at the different concentrations), and 100 µL of 200 µM p-nitrophenyl-α-D-glucopyranoside solution (in 100 mM phosphate buffer) was incubated in 96-well plates at 37 o C for 10 min. Then, 20 µL of 0.3 U/mL α-glucosidase in the phosphate buffer was added to the mixture. The reaction mixtures were incubated at 37 o C for 10 min. Then, the reaction was stopped by adding 20 µL of 50 mM NaOH. Absorbances were recorded at 405 nm by a microplate reader and compared to a control which had 20 µL of buffer solution in place of the sample. Acarbose was used as a positive control. The α-glucosidase inhibitory activity was expressed as % inhibition and was calculated as follows: The inhibitory concentration (IC50) for each sample was calculated using a regression analysis from the graph plotting scavenging activity against concentration. All experiments were carried out in triplicate and the results were expressed as the mean ± SD of three determinations.

Statistical analysis
All assays were conducted in triplicate. Statistical analyses were performed with Statgraphics Plus Professional 16.0.03 for an analysis of variance (ANOVA), followed by Duncan's test. Differences at P<0.05 were considered significant.

Structural elucidation
The phytochemical study of Lumnitzera littorea led to the isolation and identification of nine compounds whose structures are shown in Figure 2. The spectral properties of these known compounds, including 1 H-NMR and 13 C-NMR data, were identical to those previously described in the literature. Lupeol

In vitro α-glucosidase inhibitory assay
The α-glucosidase inhibitory effects of the isolated compounds (1-9) were evaluated. The inhibition % and IC50 values of all compounds are shown in Table 1.

DISCUSSION
The 1 H NMR spectrum of compounds 1-5 showed the presence of several singlet signals in the high shielded region at d 0.71-1.69, that was characteristic of methyl protons. The 13 C NMR spectrum of compounds 1-5 revealed 30 carbon signals, including seven methyl carbons, nine methylene carbons, seven methine carbons, and seven non-hydrogenated carbons. The result showed characteristic of a pentacyclic triterpenoid. On the other hand, the skeleton of 1 was recognized to be lupane triterpenoid by the NMR spectra, with the typical olefinic proton signals at d 4.56 (s, H-29b) and 4.68 (brs, H-29a) in the 1 H NMR spectrum and two olefinic carbons of the exocyclic double bond at d 109.5 (C-29) and 151.1 (C-20) in the 13 C NMR spectrum. Moreover, the assignment of the hydroxyl group at C-3 was performed by the presence of one secondary hydroxyl proton signal at d 3.18 (dd, 11.5, 5.0 Hz, H-3), correlating with a carbon signal at d 79.1 (C-3). Thus, 1 was determined as lupeol that was consistent with the reported values in the literature 4 . The NMR spectra of 2 were similar to those of 1, including the proton and carbon signals for the terpenoid of lupane skeleton. The 1 H NMR spectrum of bon signal at d 60.7 (C-28), thus confirming that there was a second hydroxyl group at C-28 in the structure of 2. Comparison of the spectroscopic data of 2 with those in the literature suggested 2 was betulin 4 . Similar to the NMR spectra of 2, the 1 H NMR and 13 C NMR spectra of 3 also possessed the signals of a lupane skeleton. However, the 1 H NMR spectrum of 3 differed from that of 2 in the absence of a pair of proton signals at d 3.30-3.80 of H-28 position. It corresponded to the presence of a carboxyl carbon signal at d 180.5, instead of an oxygenated methylene carbon signal at d 60.7 (C-28) as in 2. Thus, compound 3 was betulinic acid whose NMR data were in good compatibility with those in the literature 5 . The 1 H NMR spectrum of compound 4 displayed one olefinic proton signal at d 5.29 (t, 3.5 Hz, H-12), together with a signal at d 2.83 (dd, 13.5, 4.0 Hz, H-18) which indicated the oleanan-12-ene skeleton. One methine proton signal at d 3.22 (dd, 11.5, 4.0 Hz, H-3) showed that 4 had one hydroxyl group. The 13 C NMR spectral data exhibited signals at d 122.9 and 143.7, corresponding to the carbons C-12 and C-13, respectively. The signal at d 177.8 was assigned to the carboxyl group at C-28. This data allowed the identification of compound 4 as oleanolic acid which is isolated for the first time from Lumnitzera littorea. Hz, H-6) appeared to be characteristic of the sterols. Furthermore, the proton signal connected to the C-3 hydroxyl group appeared as a multiplet at d 3.52 (m, H-3). The 13 C NMR spectrum exhibited 29 carbon signals, including two carbon signals at d 121.9 (C-6) and 140.9 (C-5), characteristic of a double bond and an oxymethine carbon signal at d 72.0 (C-3). Thus, the structure of 6 was assigned as β-sitosterol and was consistent with values reported in the literature 7 . Detailed analysis of NMR spectra of 7 indicated that it also possessed the proton and carbon signals of a βsitosterol skeleton. Additionally, the 1 H NMR spectrum of 7 confirmed the presence of one β-glucose unit through a doublet signal at d 4.21 (d, 8.0 Hz, H-1'), assigned for anomeric proton, and multiplet signals from d 2.89 to 3.15, assigned for the carbinol protons of the sugar part. In the 13 C NMR spectrum which displayed an anomeric carbon signal at d 100.  4 . The 1 H NMR spectrum of 9 closely resembled that of 6. In addition, the 1 H NMR spectrum confirmed the presence of one olefinic proton signal at d 5.72 (s, H-4) and the absence of a multiplet proton signal at d 3.10-3.60 of H-3 position. The 13 C NMR spectrum showed the carbonyl carbon signal at d 199.8 (C-3) and two olefinic carbon signals at d 171.9 (C-5) and 123.9 (C-4). Based on the spectral data obtained and comparison with literature data, the structure of 9 was confirmed as stigmast-4-ene-3-one 9 . Although 1-5, 8,and 9 are known compounds, this is the first time their presence in leaves of Lumnitzera littorea has been reported. α-Glucosidases are a series of enzymes located on the human intestine. The most important carbohydrates in food are hydrolyzed to monosaccharide by α-glucosidase, then absorbed into the blood to increase blood glucose level. This is the reason for development of diabetes. The α-Glucosidase inhibitors may have the potential to delay or prevent the rise of blood glucose level. However, the mechanism of the inhibitions against αglucosidase has not yet clear. In our experiments, five compounds of triterpenoids and four compounds of steroids from Lumnitzera littorea showed different activity against α-glucosidase ( Table 1). From the structures of compounds 1-3, we can infer that the α-glucosidase inhibitory acitvity is strengthened when the methylene group at C-28 is altered to an oxygenated methylene or a carboxylic group. As the result, the IC50 values of lupeol (1), betulin (2) and betulinic acid (3) were 97.95 ± 0.85, 38.74 ± 0.63 and 28.82 ± 0.37 µg/mL, respectively. Furthermore, a carboxylic acid group or a CH2-OH group at C-17 is important for the action of compounds 2-5.
Comparison of the chemical structures and the αglucosidase inhibitory activity indicates that the presence of a hydroxyl group at C-3 plays an important role in the α-glucosidase inhibitory activity. Thus, the data from this study also demonstrated that the IC50 values of compounds 2-6 were lower than those of compounds 7 and 8-9.Of note, it is interesting that for 9 it is not an -OH group but an =O group. However, an oxygen is not enough at 7 and 8 have low activity. This could be ascribed to the more bulky structure of the inhibitor. Thus, the presence of one β-glucose unit at C-3 of βsitosterol 3-O-β-D-glucopyranoside (7) or the attachment of the palmitoyl moiety at C-6' of the glucose unit of stigmast-5-ene-3β-O-(6-O-hexadecanoyl-β-D-glucopyranoside) (8) decreased the α-glucosidase inhibitory activity. This demonstrated that the IC50 values of compounds 6-8 had increased to 34.45 ± 0.34, 114.19 ± 0.61 and 174.51 ± 0.58 µg/mL, respectively. When the methylene group at C-2 was altered to a hydroxyl group, the α-glucosidase inhibitory activity increased. This also indicated that IC50 values of corosolic acid (5), as the most effective compound, displayed a significantly inhibitory activity against αglucosidase with IC50 values of 17.86 ± 0.42 µg/mL.