Home | About PM | Editorial board | Search | Ahead of print | Current Issue | Archives | Instructions | Subscribe | Advertise | Contact us |  Login 
Pharmacognosy Magazine
Search Article 
  
Advanced search 
 


 
  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 68  |  Page : 161-167  

A new stilbene derivative and isolated compounds from Bauhinia pottsii var. pottsii with their anti-alpha-glucosidase activity


1 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand
2 Department of Biochemical and Chemical Engineering, Technical University of Dortmund, Dortmund, Germany

Date of Submission30-Sep-2019
Date of Decision13-Nov-2019
Date of Web Publication31-Mar-2020

Correspondence Address:
Sukanya Dej-Adisai
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112
Thailand
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_433_19

Rights and Permissions
   Abstract 


Background: Natural products are becoming important alternative medicines for diabetes treatment. Many Bauhinia plants in Thailand have been used in Thai traditional medicines for diabetic patients. Bauhinia plants have been reported on anti-alpha-glucosidase activity. However, Bauhinia pottsii var. pottsii G. Don which is a local plant in Southern Thailand has not been reported on antidiabetic activity and phytochemistry. Hence, it was selected for further study. Objectives: Isolation of chemical constituents from B. pottsii var. pottsii and determination of their biological activity on alpha-glucosidase inhibition. Materials and Methods: Chromatographic techniques, enzymatic assay, and molecular docking were done in this study. Results: A new stilbene derivative, 1,3-benzenediol-5-(-2-phenylethyl)-6-(2',3'-butanediol)-3', methyl (1) and five known compounds such as 3,4-dihydroxybenzoic acid (2), quercetin (3), 3-O-methylquercetin (4), a mixture of beta-sitosterol and stigmasterol (5), and 3,4,5-trimethoxyphenyl-beta-D-glucopyranoside (6) were isolated from B. pottsii var. pottsii. The plant extracts and isolated compounds were determined for anti-alpha-glucosidase activity. The results showed that the compounds 2, 3, and 4 were inhibited alpha-glucosidase enzyme activity with inhibition concentration at 50% (IC50) values of 3.637 mM, 0.486 mM, and 0.292 mM, respectively. While positive control, acarbose exhibited IC50value as 0.166 mM. The mechanism of action was proved using computer molecular docking. Compound 2 was predicted as mixed-type inhibitor, whereas compounds 3 and 4 were predicted as competitive inhibitors. Conclusion: This study will be the first report of phytochemical and biological study from B. pottsii var. pottsii including the first report of new compound.

Keywords: Anti-alpha-glucosidase activity, Bauhinia pottsii, molecular docking, phytochemistry, stilbene derivative


How to cite this article:
Phoopha S, Wattanapiromsakul C, Pitakbut T, Dej-Adisai S. A new stilbene derivative and isolated compounds from Bauhinia pottsii var. pottsii with their anti-alpha-glucosidase activity. Phcog Mag 2020;16, Suppl S1:161-7

How to cite this URL:
Phoopha S, Wattanapiromsakul C, Pitakbut T, Dej-Adisai S. A new stilbene derivative and isolated compounds from Bauhinia pottsii var. pottsii with their anti-alpha-glucosidase activity. Phcog Mag [serial online] 2020 [cited 2020 Jun 1];16, Suppl S1:161-7. Available from: http://www.phcog.com/text.asp?2020/16/68/161/281709



SUMMARY

  • Chemical constituents from B. pottsii var. pottsii and determination of their biological activity on alpha-glucosidase inhibition were reported as the first time
  • A new stilbene derivative; 1,3-benzenediol-5-(-2-phenylethyl)-6- (2',3'-butanediol)-3', methyl was isolated from B. pottsii var. pottsii
  • Three isolated compounds as protocatechuic acid and two flavonoids, quercetin and 3-O-methylquercetin, exhibited alpha-glucosidase inhibition
  • The mechanism of action of these compounds was predicted using in silico study which showed mixed-type inhibition and competitive inhibition of protocatechuic acid and two flavonoids, respectively.




Abbreviations used: A°: Angstrom; IC50: Inhibition concentration at 50%; ESI: Electrospray ionization; m/z: Mass-to-charge ratio; MHz: Megahertz; NMR: Nuclear magnetic resonance, 1H NMR: proton NMR, 13C NMR: Carbon-13 NMR; ppm: Parts-per million.


   Introduction Top


Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both.[1] Type 2 diabetes is referred to non-insulin-dependent diabetic mellitus that pancreatic beta-cells lost some ability to produce and secrete insulin, including insulin-resistant is also one of the causes of type 2 diabetes.[2]

Type 2 diabetes is a metabolic disease that can be prevented through lifestyle modification, diet control, and control of overweight and obesity.[3] The antidiabetic drugs which are used for Type 2 diabetes were treated through various mechanisms, increase insulin secretion, increase insulin sensitivity, increase glucose uptake to muscle, and decrease glucose absorption. In this research, we studied how to decrease glucose absorption using an anti-alpha-glucosidase activity model. Alpha-glucosidase or maltase is the most important enzyme which is located in the small intestine. The enzyme is used for digesting polysaccharide or sugar into monosaccharide, especially glucose molecules. Hence, the inhibition of this enzyme activity will decrease glucose absorption in intestine and bloodstream.

At present, natural products are becoming important alternative medicines for diabetes treatment. Over 170 different natural medicines are used for combating diabetes. However, only a fraction of these products has reliable clinical evidences of effectiveness. Herbal formulae containing multiple herbs may have synergistic effects.[4] Thus, searching for antidiabetic drugs from nature will find out new sources of lead compounds which can be used in drug discovery and diabetic treatment.

Many Bauhinia plants in Thailand have been used in Thai traditional medicines for diabetic patients. From our previous report,[5] the plants in genus Bauhinia have been reported on anti-alpha-glucosidase activity. However, Bauhinia pottsii var. pottsii G. Don which is a local plant in southern Thailand has not been reported on antidiabetic activity and phytochemistry. Hence, this will be the first report concerning anti-alpha-glucosidase and its isolated compounds of this plant.


   Materials and Methods Top


General experimental procedures

One-dimensional and two-dimensional NMR spectrometry were observed by Fourier Transform NMR Spectrometer (1 H-NMR 500 MHz and 13 C-NMR 125 MHz), model UNITY INNOVA, Varian. LC-MS/MS was analyzed on a Finnigan LCQ-Duo ion trap mass spectrometer with an ESI source (ThermoQuest) coupled to a Finnigan Surveyor HPLC system with an EC 150/3 Nucleodur 100-3 C18ec column (Macherey-Nagel). A gradient of water and acetonitrile with 0.1% formic acid each was applied from 2% to 70% acetonitrile in 60 min at 30°C. The flow rate was 0.5 ml/min. The injection volume was about 20 μL. NMR and LC-MS/MS experiments were observed at the Institute of Pharmacy and Molecular Biotechnology, Department of Pharmaceutical Biology, Heidelberg University, Germany. High-resolution mass spectrometry was observed using positive mode of TOF/Q-TOF mass spectrometer combined with Dual AJS ESI detector (Agilent Technologies ®, USA) at Scientific Equipment Centre, Prince of Songkla University, Thailand.

Phytochemical investigation was done using classical column chromatography. The normal stationary phase was performed using Silica Flash ® P60 (Ultrapure Silica Gel, particle size 40–63 μm, SiliCycle ®, Canada). Sephadex ® LH-20 (bead size 25–100 μm, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was used for column chromatography to purify the compounds. Silica gel 60 F254 (Merck, Germany) was used for thin-layer chromatography.

Plant preparation

The wood and leaf of B. pottsii var. pottsii G. Don were collected from the swamp forest and identified by the botanist of The Pikun Thong Royal Development Study Centre, Narathiwat, Thailand. The plant specimen was deposited at the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand, with herbarium number as SKP 072 02 16 01.

The dried samples were macerated with various solvents for 72 h. The filtrate was evaporated with a rotary evaporator under reduced pressure at 40°C. The crude extracts were kept at 4°C. The marc was repeated for 3 times of extraction with the same solvent. After that, the marc was macerated with the other solvents, ethyl acetate and ethanol, respectively. Finally, the marc was boiled with water at 70°C for 6 h. The filtrate was also evaporated with a rotary evaporator under reduced pressure at 40°C. The crude extracts were kept at 4°C until examination.

Phytochemical study techniques

Chromatographic techniques such as column chromatography, thin-layer chromatography, size-exclusion chromatography and reversed phase RP-18 chromatography were used for isolation and purification of chemical constituents of this plant. After that, the isolated compounds were interpreted using NMR and LC-MS/MS techniques.

Assay on anti-alpha-glucosidase activity

The assay on anti-alpha-glucosidase activity was followed by previously reported.[5] The enzyme activity was measured using the colorimetric method. The yellow color of the product was observed using UV-spectrometry at 405 nm. The color of reaction produced from hydrolysis reaction between the substrate (p-nitrophenyl-alpha-D-glucopyranoside) and alpha-glucosidase enzyme.

Briefly, the tested samples and positive standard (acarbose) were dissolved in 20% of dimethyl sulfoxide in water. Alpha-glucosidase enzyme from S. cerevisiae (EC 3.2.1.20) and p-nitrophenyl-alpha-D-glucopyranoside were dissolved in 0.1 M phosphate buffer pH 7, which was supplemented with bovine serum albumin and NaN3. 50 μL of phosphate buffer, enzyme, and sample were added to the 96-well plate and incubated for 2 min. Then, 50 μL of the substrate was added and the enzyme activity was measured following the kinetic parameter at 405 nm (interval time 30 min and 20 cycles).

The inhibition of enzyme activity was measured by calculating the velocity of the reaction. The UV absorption was plotted using linear regression between the difference of absorbance and time as following Eq. (1). The highest velocity (V) from the initial reaction of each sample was collected and calculated the percentage of inhibition by Eq. (2) as follows:





Computer molecular docking of alpha-glucosidase enzyme

The crystal structure of alpha-glucosidase from S. cerevisiae, PDB: 3a4a,[6],[7] was downloaded from the RCSD Protein Data Bank (http://www.rcsb.org) and this targeted protein was prepared for docking experiment by AutoDockTools version 1.5.6.[8] Whereas, the 3D structures of the interesting compounds were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and these structures were optimized by the geometry and GAFF optimizations from Avogadro version 1.2.0[9] before subjection to AutoDockTools for the docking preparation process. Glucose as a native ligand was used to indicate the active site, which was described as a grid box in the 3D structure as the center of X-axis = 21.1, the center of Y-axis =-7.4, and center of Z-axis = 24.2, respectively. Sizes of this grid box were presented as 16 Š ×16 Š ×16 Š. Moreover, the native ligand was extracted and redocked for the docking validation method.

AutoDock Vina version 1.1.2[10] was selected to proceed the docking experiment in this study. All parameters in this experiment were set up as default values except the exhaustiveness was adjusted as 20. Eventually, ViewDock package from Chimera version 1.11.2[11] was performed to analyze all results from this experiment. The best confirmation of the interesting compounds was selected from the outcomes based on the combination of the lowest binding affinity and the alignment of those structures. For the validation protocol, RMSD was measured between the native ligand and redocked ligand to confirm the reliability of this experiment.[12]


   Results and Discussion Top


Phytochemical investigation of Bauhinia pottsii var. pottsii

Six compounds were isolated from B. pottsii var. pottsii. Compound 1 is a new compound, a stilbene derivative named as 1,3-benzenediol-5-(-2-phenylethyl)-6-(2',3'-butanediol)-3', methyl. This is the first time to report its structure and bioactivity of this compound. Five known compounds were interpreted as 3,4-dihydroxybenzoic acid (protocatechuic acid) (2), quercetin (3), 3-O-methylquercetin (4), a mixture of beta-sitosterol and stigmasterol (5), and 3, 4, 5-trimethoxyphenyl-beta-D-glucopyranoside (6) [Figure 1].
Figure 1: The structure of isolated compounds from Bauhinia pottsii var. pottsii. 1 = 1,3-benzenediol-5-(-2-phenylethyl)-6-(2',3'-butanediol)-3', methyl, 2 = 3,4-dihydroxybenzoic acid; 3 = quercetin, 4 = 3-O-methylquercetin, 5 = a mixture of beta-sitosterol and stigmasterol, 6 = 3,4,5-trimethoxyphenyl-beta-D-glucopyranoside

Click here to view


Compound 1 (A new stilbene derivative)

Compound 1 was obtained as a yellow amorphous and dissolved in methanol. Chemical formula: C19H24O4 and ultraviolet (UV) λmax: 217, 236, and 279 nm. The 1 H nuclear magnetic resonance (NMR) spectrum (500 MHz in MeOH-d 4) showed the chemical shifts of aromatic protons at 6.19 ppm (d, J = 2.51 Hz, H-2), 6.22 ppm (d, J = 2.45 Hz, H-4), 7.19 ppm (dd, J = 8.30, 1.5 Hz, H-2''), 7.24 ppm (ddd, J = 7.54, 7.06, 1.61 Hz, H-3''), 7.15 ppm (tt, J = 6.68, 1.33 Hz, H-4''), 7.24 ppm (ddd, J = 7.54, 7.06, 1.61 Hz, H-5''), and 7.19 ppm (dd, J = 8.30, 1.50 Hz, H-6''). The chemical shifts of -CH2-CH2- that linked between two aromatic rings showed at 2.81 ppm (m, H-7) and 2.82 ppm (m, H-8). The substitution which identified as butanediol showed the signals at 2.85 ppm (dd, J = 14.60, 10.8 Hz, H-1'), 2.57 ppm (dd, J = 14.50, 10.50 Hz, H-1'), 3.45 ppm (d, J = 10.50, 1.80 Hz, H-2'), 1.23 ppm (d, J = 2.90 Hz, H-4'), and 1.23 ppm (d, J = 2.90 Hz, H-5').

The 13 C spectrum (125 MHz in MeOH-d 4) showed the chemical shifts of aromatic carbons at 156.43 (C-1), 100.88 (C-2), 155.82 (C-3), 107.91 (C-4), 142.18 (C-5), 116.23 (C-6), 141.87 (C-1''), 128.06 (C-2''), 127.95 (C-3''), 125.44 (C-4''), 127.95 (C-5''), and 128.06 (C-6''). The chemical shifts of -CH2-CH2- that linked between two aromatic rings showed at 35.54 (C-7) and 35.37 (C-8). The chemical shifts of butanediol showed at 27.18 (C-1'), 80.13 (C-2'), 72.44 (C-3'), 23.43 (C-4'), and 24.32 (C-5').

The positive ESI/HR/MS spectrum of compound 1 showed an [M + Na]+ ion peak at m/z 339.1581 [M = 316.1687] supporting the molecular formula ([C19H24O4]+Na)+ (calcd. for C19H24O4, 316.1675, diff −3.83 ppm).1 H-1 H and 1 H-13 C from COSY and HMBC correlations at the position C-7, C-8, C-1', C-2', and C-3' were the important correlations.

Due to the Karplus equation,[13] the coupling constant at the chiral proton showed the correlation at 3.45 ppm (d, J = 10.50, 1.80 Hz, H-2') which coupled with methylene proton at 2.85 and 2.57 ppm that could be used to explain the configuration of this structure. The chiral proton was coupled with methylene protons at 2.85 ppm (Hβ) with θ nearly to 180° which showed the J = 10.50 Hz and coupled with 2.57 ppm (Hα) with θ nearly to 60° which showed the J = 1.80 Hz [Figure 2].
Figure 2: COSY and HMBC correlation of compound 1

Click here to view


The result of ESI-positive mass spectroscopy of compound 1 showed the molecular ion peak at 317.06 m/z ([M + H]+) [Figure 3]. There were several main fragments from ESI-mass which showed at 299.12, 281.16, and 227.19 m/z. These fragments were broken down from the main structure by losing of water molecules and substitution. The benzenediol derivative fragment showed at 135.09 m/z. The spectrum of compound 1 was interpreted as a new stilbene derivative which was substituted with butanediol. The compound was named as 1,3-benzenediol-5-(-2-phenylethyl)-6-(2',3'-butanediol)-3', methyl [Figure 2].
Figure 3: Mass fragmentation of a new stilbene derivative from Bauhinia pottsii var. pottsii

Click here to view


Compound 2 (Protocatechuic acid)

Compound 2 was obtained as brown needle crystals and dissolved in methanol. Chemical formula: C7H6O4. ESI mass: 155.06 m/z ([M + H]+). UV λmax: 250 nm. The 1 H NMR spectrum (500 MHz in MeOH-d 4) showed the chemical shifts of aromatic protons at 7.44 ppm (d, J = 2.00 Hz, H-2), 6.81 ppm (d, J = 8.10 Hz, H-5), and 7.42 ppm (dd, J = 8.10, 2.00 Hz, H-6). The 13 C spectrum (125 MHz in MeOH-d 4) showed the chemical shifts of aromatic carbons at 121.77 (C-1), 116.38 (C-2), 144.61 (C-3), 150.06 (C-6), 114.44 (C-5), and 122.62 (C-6). The carbonyl of carboxylic acid showed at 168.98 (C-7). The chemical shifts of compound 2 were compared to a previous report as 3,4-dihydroxybenzoic acid.[14]

Compound 3 (Quercetin)

Compound 3 was obtained as yellow powder and dissolved in methanol. Chemical formula: C15H10O7. ESI mass: 303.27 m/z ([M + H]+). UV λmax: 250 and 346 nm. The 1 H NMR spectrum (500 MHz in MeOH-d 4) showed the chemical shifts of quercetin structure at 6.17 ppm (d, J = 1.50 Hz, H-6), 6.38 ppm (d, J = 1.60 Hz, H-8), 7.74 ppm (d, J = 2.00, H-2'), 6.88 ppm (d, J = 8.50 Hz, H-5'), and 7.64 ppm (dd, J = 8.50, 2.10 Hz, H-6'). The 13 C spectrum (125 MHz in MeOH-d 4) showed the chemical shifts of quercetin at 146.54 (C-2), 137.39 (C-3), 179.44 (C-4), 161.11 (C-5), 98.27 (C-6), 164.58 (C-7), 92.99 (C-8), 156.55 (C-9), 103.11 (C-10), 120.18 (C-1'), 114.71 (C-2'), 144.77 (C-3'), 149.23 (C-4'), 114.76 (C-5'), and 120.26 (C-6'). The chemical shifts of compound 3 were compared to previous report as quercetin.[15]

Compound 4 (3-O-methoxyquercetin)

Compound 4 was obtained as yellow needle crystals and dissolved in methanol. Chemical formula: C16H12O7. ESI mass: 317.20 m/z ([M + H]+). UV λmax: 251 nm. The 1 H NMR spectrum (500 MHz in MeOH-d 4) showed the chemical shifts of quercetin structure at 6.38 ppm (d, J = 2.00 Hz, H-6), 6.19 ppm (d, J = 2.00 Hz, H-8), 7.61 ppm (d, J = 2.20 Hz, H-2'), 6.89 ppm (d, J = 8.50 Hz, H-5'), and 7.52 ppm (d, J = 8.50, 2.20 Hz, H-6'). The substitution at ring C which identified as methoxy showed the signal at 3.77 ppm (s, 3-OCH3). The 13 C spectra (125 MHz in MeOH-d 4) showed the chemical shifts of quercetin at 156.99 (C-2), 138.10 (C-3), 178.56 (C-4), 161.65 (C-5), 93.28 (C-6), 164.58 (C-7), 98.35 (C-8), 156.55 (C-9), 104.40 (C-10), 121.49 (C-1'), 115.03 (C-2'), 145.03 (C-3'), 148.52 (C-4'), 114.98 (C-5'), and 120.87 (C-6'). The substitution at ring C which identified as methoxy showed the signal at 59.08 (C-3-OCH3). The chemical shifts of compound 4 were compared to a previous report as 3-O-methylquercetin.[16]

Compound 5 (a mixture of beta-sitosterol and stigasterol)

Compound 5 was obtained as white needles. It was soluble in chloroform. Molecular formula: C29H50O (beta-sitosterol) and C29H48O (stigmasterol)*. The 1 H NMR spectrum (500 MHz in CHCl3-d) showed the chemical shifts of olefinic protons of beta-sitosterol and stigmasterol* at 5.35 (d, J = 5.20 Hz, H-6), 5.01* (dd, J = 15.10, 8.6 Hz, H-22), and 5.15* (dd, J = 15.21, 8.6, H-23). The methyl protons of beta-sitosterol and stigmasterol were assigned at 0.69 and 0.70* (H-18), 1.01 (H-19), 0.92 (H-21), 0.81-0.82 (H-26 and 27), and 0.83 (H-29), respectively. The 13 C spectrum (125 MHz in CHCl3-d) showed the chemical shifts of olefin carbons at 140.73 (C-5) and 121.73 (C-6), 129.25* (C-22), and 138.29* (C-23). The methyl carbons were assigned at 11.85 (C-18), 19.38 (C-19), 18.69 (C-21), 19.80 (C-26), 19.20 (C-27), and 11.97 (C-29). The spectra of compound 5 were compared to a previous report of β-sitosterol and stigmasterol.[17] The proportion of the mixture between beta-sitosterol and stigmasterol approximately calculated the ratio at 4:1 using integration of 1 H-NMR at H-18 of methyl protons.

Compound 6 (3, 4, 5-trimethoxyphenyl-beta-D-glucopyranoside)

Compound 6 was obtained as brown needle crystals and dissolved in methanol. Chemical formula: C15H22O9. ESI mass: 346.88 m/z m/z ([M + H]+). UV λmax: 217, 236 and 270 nm. The 1 H NMR spectra (500 MHz in MeOH-d 4) showed the chemical shifts of aromatic protons at 6.48 ppm (s, H-2,6). The sugar substitution which was identified as glucopyranoside showed the chemical shifts at 4.80 ppm (d, J = 7.43 Hz, H-1'), 3.43 ppm (m, H-2'), 3.30-3.43 (m, H-3',4',5'), 3.65 ppm (dd, 12.01, 2.27 Hz, H-6'), and 3.91 ppm (dd, 12.06, 6.60 Hz, H-6'). Beta form of sugar was considered from coupling constant value at anomeric proton at H-1'. The 13 C spectra (125 MHz in MeOH-d 4) showed the chemical shifts of aromatic carbons at 154.61 (C-1), 94.68 (C-2,6), 153.35 (C-3,5), and 133.00 (C-4). The anomeric proton of glucopyranoside showed the correlation to C-1 of aromatic carbon which was confirmed by HMBC. The sugar substitution which was identified as glucopyranoside showed the chemical shifts at 101.76 (C-1'), 73.60 (C-2'), 76.99 (C-3' or 5'), 70.27 (C-4'), 76.65 (C-3' or 5'), and 61.29 (C-6'). The chemical shifts of compound 6 were compared to a previous report as 3, 4, 5-trimethoxyphenyl-beta-D-glucopyranoside.[14]

Bioactivity on alpha-glucosidase inhibition of plant extracts

Wood and leaf of B. pottsii var. pottsii were extracted with various solvents as hexane, ethyl acetate, ethanol, and water, respectively to provide eight crude extracts which were continued to determine the bioassay. They were used at the concentration of 2 mg/mL for preliminary screening of anti-alpha-glucosidase activity. Afterward, the extracts which exhibited enzyme inhibitory activity would be figured out the inhibition concentration at 50% (IC50) values. The result of plant extracts is shown in [Table 1].
Table 1: Anti-alpha-glucosidase activity of Bauhinia pottsii var. pottsii extracts

Click here to view


The results of plant extracts were suggested that the ethanol and water extracts from both leaf and wood showed the potential enzyme inhibitory effect. However, the highest potency of alpha-glucosidase inhibition was water extract of the leaf which showed the IC50 value as 520 μg/mL.

Bioactivity on alpha-glucosidase inhibition of isolated compounds

Six isolated compounds were determined on anti-alpha-glucosidase activity to find out the active compound using IC50 values. The result of their activity is shown in [Table 2].
Table 2: Anti-alpha-glucosidase activity of isolated compounds

Click here to view


According to the result in [Table 2], three compounds which were interpreted as 3,4-dihydroxybenzoic acid (2), quercetin (3), and 3-O-methylquercetin (4) exhibited enzyme inhibitory activity with IC50 values as 3.637, 0.486, and 0.292 mM, respectively. The highest active compound for alpha-glucosidase inhibition was 3-O-methylquercetin (4).

The anti-alpha-glucosidase activity of flavonoid compounds was proved by explaining of structure–activity relationship of their structures. The substitution of hydroxyl on ring A and B at C-5, C-7, C-3', and C-4' is significantly enhancing the activity and the methylation at C-3 on ring C is the slightly decreased activity of flavonoid when compared with the other position of the structure.[18]

Computer molecular docking of active compounds

The inhibition of enzyme activity has proved using docking experiment. The crystal structure of alpha-glucosidase from Saccharomyces cerevisiae, PDB: 3a4a [6],[7] was downloaded from the RCSD Protein Data Bank (http://www.rcsb.org) and this targeted protein was prepared for docking experiment by AutoDockTools version 1.5.6.[8] Whereas, the three-dimensional (3D) structures of the interesting compounds were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and these structures were optimized by the geometry and General AMBER Force Field (GAFF) optimizations from Avogadro version 1.2.0[9] before subjection to AutoDockTools for the docking preparation process. Glucose as a native ligand was used to indicate the active site, which was described as a grid box in the 3D structure as the center of X-axis = 21.1, the center of Y-axis = −7.4, and center of Z-axis = 24.2, respectively. Sizes of this grid box were presented as 16 Š × 16 Š × 16 Š. Moreover, the native ligand was extracted and redocked for the docking validation method.

AutoDock Vina version 1.1.2 (The Scripps Research Institute, San Diego, California, USA)[10] was selected to proceed the docking experiment in this study. All parameters in this experiment were set up as default values except the exhaustiveness was adjusted as 20. Eventually, ViewDock package from Chimera version 1.11.2[11] was performed to analyze all results from this experiment. The best confirmation of the interesting compounds was selected from the outcomes based on the combination of the lowest binding affinity and the alignment of those structures. For the validation protocol, root-mean-square deviation (RMSD) was measured between the native ligand and redocked ligand to confirm the reliability of this experiment.[12]

The result from the redocking experiment of the extracted native ligand, as a glucose, indicated that this docking method is liable based on the RMSD values which was lower than 3.50 Š (RMSD = 0.92 Š).[12]

All the best conformations, as well as native ligand from the experiment, are shown in [Figure 4]a. The catalytic domains (GLU 277 and ASP 352) and the stabilizer domain (ARG 442)[6] were involved in the inhibitory mechanism between quercetin, 3-O-methoxyquercetin, 3,4-dihydroxybenzoic acid, and alpha-glucosidase enzyme. The hydrogen bonds were found between the hydroxy groups on ring A and B from quercetin and GLU 277 and ARG 442 in the active site of the enzyme [Figure 4]b. Moreover, the hydroxy groups on ring A from 3-O-methylquercetin and on the aromatic ring from 3,4-dihydroxybenzoic acid could also bind with ASP 352 in the same pocket [Figure 4]c and [Figure 4]d. Quercetin and 3-O-methylquercetin showed the same affinity energy at −7.5 Kcal/mol, whereas 3,4-dihydroxybenzoic acid had a lower energy, −5.6 Kcal/mol.
Figure 4: The positions of the best conformation in the active site of alpha-glucosidase (Amber yellow) and the interaction of quercetin (Pink), 3-O-methylquercetin (Light blue), 3,4-dihydroxybenzoic acid (Green) and glucose as a native ligand (Red) from docking experiments. (a) An overlays positions of quercetin, 3-O-methylquercetin, 2,3-dihydroxy-benzoic acid and glucose in the active site of alpha-glucosidase. (b) H bonds (orange line) between quercetin and alpha-glucosidase. (c) H bond (orange line) between 3-O-methylquercetin and alpha-glucosidase. (d) H bonds (orange line) between 3,4-dihydroxybenzoic acid and alpha-glucosidase

Click here to view


Molecular docking experiment was wildly and internationally used by researchers to study the molecular interaction between the interested ligands and the targeted protein, which provides the better understanding. However, the validation of the protocol is required to ensure that the obtained data are reliable. The result [as shown in [Figure 4] from the redocking experiment in this study was accepted due to RMSD ≤3.5 Š.[18] Even though the affinity energy of flavonoids (quercetin and 3-O-methoxyquercetin) was identical and this result was a contradiction with the actual anti-alpha-glucosidase test, it could be differentiated the activity between flavonoids and benzoic acid, because they have the different pharmacophore. The position and the interaction between quercetin, 3-O-methylquercetin, and glucosidase enzyme indicated the competitive inhibition behavior. This finding strongly agreed with the previously reported.[19] In addition, benzoic acid derivatives were showed the mixed type inhibition,[20] which was also correlated with docking result from 2,3-dihydroxybenzoic acid, which interacted with one of the catalytic domains.


   Conclusion Top


Six compounds were isolated from B. pottsii var. pottsii including a new stilbene derivative and five five known compounds, protocatechuic acid, quercetin, 3-O-methoxyquercetin, a mixture of beta-sitosterol and stigmasterol, and 3, 4, 5-trimethoxyphenyl-beta-D-glucopyranoside. The crude extracts and isolated compounds were studied on antidiabetic activity using in vitro and in silico models, anti-alpha-glucosidase activity and computer molecular docking, respectively. The ethanolic and water extracts of leaf and wood showed the potential effect on anti-alpha-glucosidase. However, only three isolated compounds as protocatechuic acid and two flavonoids, quercetin and 3-O-methylquercetin, exhibited alpha-glucosidase inhibition. The mechanism of action of these compounds was predicted by using in silico study which showed mixed-type inhibition and competitive inhibition of protocatechuic acid and two flavonoids, respectively.

Acknowledgements

The authors would like to thank the Department of Pharmacognosy and Pharmaceutical Botany and the Pharmaceutical Laboratory Service Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, for the lab space and equipment.

Financial support and sponsorship

The authors would like to give many thanks to all supportive grants for Ph.D. study of Prince of Songkla University, Thailand; The Scholarship Awards for Ph.D. Studies Thailand's Education Hub for Southern Region of ASEAN Countries, Graduate School Dissertation Funding for Thesis and Overseas Thesis Research Scholarship, Graduate School, Prince of Songkla University, Thailand.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2010;33 Suppl 1:S62-9.  Back to cited text no. 1
    
2.
Wilson CO. Hormone-Relate Disoder: Nonsteroidal Therapies Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry. Ch. 20. 12th ed. Pennsylvania, USA: Wolters Kluwer Health/Lippincott Williams and Wilkins; 2011.  Back to cited text no. 2
    
3.
Olokoba AB, Obateru OA, Olokoba LB. Type 2 diabetes mellitus: A review of current trends. Oman Med J 2012;27:269-73.  Back to cited text no. 3
    
4.
Prabhakar PK, Doble M. Mechanism of action of natural products used in the treatment of diabetes mellitus. Chin J Integr Med 2011;17:563-74.  Back to cited text no. 4
    
5.
Dej-Adisai S, Pitakbut T. Determination of a-glucosidase inhibitory activity from selected Fabaceae plants. Pak J Pharm Sci 2015;28:1679-83.  Back to cited text no. 5
    
6.
Yamamoto K, Miyake H, Kusunoki M, Osaki S. Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltose. FEBS J 2010;277:4205-14.  Back to cited text no. 6
    
7.
Rivera-Chávez J, González-Andrade M, González Mdel C, Glenn AE, Mata R. Thielavins A, J and K: α-Glucosidase inhibitors from MEXU 27095, an endophytic fungus from Hintonia latiflora. Phytochemistry 2013;94:198-205.  Back to cited text no. 7
    
8.
Chetty S, Soliman ME. Possible allosteric binding site on gyrase B, a key target for novel anti-TB drugs: Homology modelling and binding site identification using molecular dynamics simulation and binding free energy calculations. Med Chem Res 2015;24:2055-74.  Back to cited text no. 8
    
9.
Hanwell MD, Curtis DE, Lonie DC, van dermeersch T, Zurek E, Hutchison GR. Avogadro: An advanced semantic chemical editor, visualization, and analysis platform. J Cheminform 2012;4:17.  Back to cited text no. 9
    
10.
Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010;31:455-61.  Back to cited text no. 10
    
11.
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera – A visualization system for exploratory research and analysis. J Comput Chem 2004;25:1605-12.  Back to cited text no. 11
    
12.
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 2009;30:2785-91.  Back to cited text no. 12
    
13.
Matsumori N, Kaneno D, Murata M, Nakamura H, Tachibana K. Stereochemical determination of acyclic structures based on carbon-proton spin-coupling constants. A method of configuration analysis for natural products. J Org Chem 1999;64:866-76.  Back to cited text no. 13
    
14.
Nguyen DM, Seo DJ, Kim KY, Park RD, Kim DH, Han YS, et al. Nematicidal activity of 3,4-dihydroxybenzoic acid purified from Terminalia nigrovenulosa bark against Meloidogyne incognita. Microb Pathog 2013;59-60:52-9.  Back to cited text no. 14
    
15.
Joshi H, Joshi AB, Gururaj MP, Sati H. Flavonoids of Memecylon umbellatum (Burm.). Asian J Chem 2009;21:1639-41.  Back to cited text no. 15
    
16.
Wang J, Gao H, Zhao J, Wang Q, Zhou L, Han J, et al. Preparative separation of phenolic compounds from Halimodendron halodendron by high-speed counter-current chromatography. Molecules 2010;15:5998-6007.  Back to cited text no. 16
    
17.
Elgendy EM, Al-Ghamdy H. Thermal and photooxidation reactions of the steroids: Beta-sitosterol, stigmasterol and diosgenin. Taiwan Pharm J 2007;59:113-2.  Back to cited text no. 17
    
18.
Cao H, Chen X. Structures required of flavonoids for inhibiting digestive enzymes. Anticancer Agents Med Chem 2012;12:929-39.  Back to cited text no. 18
    
19.
Proença C, Freitas M, Ribeiro D, Oliveira EFT, Sousa JLC, Tomé SM, et al. α-Glucosidase inhibition by flavonoids: An in vitro and in silico structure-activity relationship study. J Enzyme Inhib Med Chem 2017;32:1216-28.  Back to cited text no. 19
    
20.
Jeong SY, Nguyen PH, Zhao BT, Ali MY, Choi JS, Min BS, et al. Chemical Constituents of Euonymus alatus (Thunb.) Sieb. and their PTP1B and α-glucosidase inhibitory activities. Phytother Res 2015;29:1540-8.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
   
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
    Results and Disc...
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed150    
    Printed0    
    Emailed0    
    PDF Downloaded47    
    Comments [Add]    

Recommend this journal