α-Glucosidase Activity of Oleanolic Acid and Its Oxidative Metabolites: DFT and Docking Studies

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α-Glucosidase Activity of Oleanolic Acid and Its Oxidative Metabolites: DFT and Docking Studies

El Hassane Anouar1,2,*, Nur Shahidatul Shida Zakaria2, Ali Alsalme1 and Syed Adnan Ali Shah2,3,*

1Chemistry Department, College of Sciences and Humanities, Prince Sattam bin Abdulaziz University, P.O. Box 83, Al-Kharij 11942, Saudi Arabia; 2Atta-ur-Rahman Institute For Natural Product Discovery (AuRIns), Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D.E, Malaysia; 3Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D.E, Malaysia

Abstract: A natural pentacyclic triterpenoid, oleanolic acid 1 and its biotransformed metabolites 2 and 3 are potential -glucosidase inhibitors. To elucidate the inhibitory mechanism of compounds 1-3 against -glucosidase, (i) their electronic and optical

properties were calculated using DFT and TD-DFT methods at the B3LYP/6-31G(d) level of theory in gas and IEF-PCM solvent; and (ii) their binding energies to -glucosidase were determined via a docking study. DFT results showed that the -glucosidase inhibtion is mainly dependent on the polarity parameters of the studied compounds. Docking results revealed that the activity is increased with binding energies (i.e. the stability of ligand-receptor complex). The NMR spectroscopic data revealed that 13C and 1H chemical shifts of 1 and its hydroxylated metabolites 2 and 3 are well predicted with R2 of 99% and 90%, respectively. The UV/vis spectra of substrate 1 and its transformed products 2 and 3 are well reproduced. The detailed assignments of 1H and 13C chemical shifts, and bathochromic shift of λMAX absorption bands have been presented.

Keywords: Chemical shifts, DFT, Docking, α-Glucosidase, NMR, TD-DFT, Oleanolic acid, UV/visible.

1. INTRODUCTION

Oleanolic acid (1: 3β-hydroxyolean-12-en-28-oic acid), Fig. 1 is a pentacyclic triterpenoid with a widespread occurrence throughout the plant kingdom [1-3]. It has been isolated from more than 1600 plant species, including many food and medicinal plants. In nature, oleanolic acid 1 exists either as a free acid or as an aglycone of triterpenoid saponins, in which it can be linked to one or more sugar moieties. Oleanolic acid 1 is especially prevalent in plants belonging to the Oleaceae family, among which are olives (Olea europaea), the plant species after which the compound was named, and that still serve as the main source of commercial oleanolic acid. Oleanolic acid 1 and its derivatives possess several promising pharmacological activities, such as anti-inflammatory [4], inhibitory activity of tumor growth and therapeutic utility of protecting bone marrow from the damages of radiation [5-7], hepatoprotective [8-10], anti-HIV [11-13], skin protective [14-16], antioxidant [17], and

*Address correspondence to these authors at the Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA Campus Puncak Alam, 42300 Bandar Puncak Alam, Selangor D.E, Malaysia; Tel: +603 3258 4771; Fax: +603 3258 4770; E-mail: [email protected]; and Faculty of Pharmacy, Universiti Teknologi MARA, (UiTM) Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E, Malaysia; Tel: +603-3258 4616/4837; Fax: +603 3258 4770; E-mails: [email protected]; [email protected]

cytotoxic activity toward many cancer cell lines in culture [4]. In higher plants, the various glycoconjugated oleanolic acids may function as defensive compounds against herbivores or pathogens, or as allelopathic agents. Our research group has been investigated on the fungal transformation of oleanolic acid (1) with filamentous fungus, Furarium lini, which afforded two oxidative metabolites, 2α,3β-dihydroxyolean-12-en-28-oic acid (2, Fig. 1), and 2α,3β,11β-trihydroxyolean-12-en-28-oic acid (3). Oleanolic acid 1 and its biotransformed products 2 and 3 were reported first time as potent -glucosidase inhibitors [18, 19]. α-Glucosidase (EC 3.2.1.20) is a typical exo-type glycosidase that catalyze the releases of α-glucosides from the nonreducing end of the substrates. Diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose levels. Among the pharmacotherapy to prevent high blood glucose levels, the α-glucosidase inhibitors offer an effective strategy to lower the glucose absorption. Oxidative metabolites 2 and 3 showed more potent α-glucosidase inhibitory activities than a clinically used drug, acarbose [19]. The docking studies performed by Ming Liu et al. showed that hydrophobic forces and hydrogen bonds are the main interecations in favour of driven the inhibitory mechanisms of Bis(2,3-dibromo-4,5-dihydroxybenzyl) Ether (BDDE) against α-glucoside enzyme [20]. Recently, Saqib et al. reported a 3D-QSAR studies of -glucosidase inhibtion by xanthone derivatives; and they concluded that the biological activity of

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α-Glucosidase Activity of Oleanolic Acid and Its Oxidative Metabolites Mini-Reviews in Medicinal Chemistry, 2015, Vol. 15, No. 14 1149

ligands depend on the steric and electrostaic interactions of structural features of ligand-receptor bindings [21].

Nowadays, the computational chemistry offers a better understanding of correlation of experimental data with the predicted spectroscopic data and rationalize the structure-activity relationships (SAR). To predict ECD and UV/Vis spectra of molecular systems, time-dependent density functional theory (TD-DFT) has rapidly emerged as an important tool to evaluate excited states of diversified organic compounds especially natural products with complex structures like triterpenoids [22]. In order to predict the 1H and 13C chemical shifts of molecular systems, GIAO theory has mostly used in several studies in literature. Borkowski et al showed a better correlation between the experimental and predicted chemical shift of a series of pentacyclic triterpenoids using GIAO theory at the B3LYP/6-31G(d) level of theory [23].

Herein, we particularly focus on interactions between the active sites of -glucosidase and oleanolic acid (1) and its two biotransformed products 2 and 3 by (i) calculating their electronic properties by means of density functional theory (DFT) and (ii) their molecular docking interactions with -glucosidase. The 1H and 13C NMR, and UV/vis spectra of compounds 1-3 have been predicted using DFT and TD-DFT methods in order to validate the reported experimental data.

2. THEORETICAL METHODS

2.1. DFT Calculations

The geometry optimization of the ground states (GS), cationic and anionic forms of oleanolic acid (1) and its two oxidative metabolites 2α,3β-dihydroxyolean-12-en-28-oic acid (2) and 2α,3β,11β-trihydroxyolean-12-en-28-oic acid (3)

have been performed by density functional theory (DFT) using B3LYP hybrid functional combined with 6-31G(d) basis set. The frequency analyses were conducted at the same level of theory (B3LYP/6-31G(d)). The absence of imaginary frequencies has confirmed the ground state minima. The electronic properties such as electronic affinity (EA), ionization potential (IP), electronegativity (χ), chemical hardness (η), electrophilicity (ω), gap energy (Egap), dipole moment (µ) and the isotropic polarizability (α) were calculated as the same level of theory. These electronic properties can also be calculated by different approaches [24-29]. In the first approach, they are determined based on the use of the classical finite difference approximation, in which the change of one electron is usually involved ΔN=±1 [24]. In this approach, EA=E0 –E-1 and IP = E+1-E0 where E0, E-1 and E+1 are the electronic energies of a neutral molecule, when adding and removing of an electron to the neutral molecule. In the second approach, the electronic properties are calculated based on Koopman’s theorem, in which IP=-EHOMO and EA=-ELUMO. In the third approach named internally resolved hardness tensor (IRHT), the electronic properties are also calculated based on orbital energies [25, 26]. This approach deals with fractional occupation numbers based on the Janak’s extension of DFT [27]. Three approaches were tested by De Luca et al. to study the solvent effects on the hardness values of a series of neutral and charged molecules, and they concluded that three approaches lead to similar results in the presence of solvent [28]. In the present study, the electronic descriptors were calculated in solvent (IEF-PCM) using the first and second approaches. The electronegativity. (χ), hardness (η), softness (S), electrophilicity (ω), dipole moment (µ’) and polarizability(α) were calculated using the following formula:

Fig. (1). Bioconversion of oleanolic acid 1 by F. lini.

H3C

CH3H3C

HOH

H

CH3

H3CCH3

O

OH

H

CH3

1

2

34

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1213

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1516

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3029

CH3

CH3H3C

HOH

H CH3

H3CCH3

O

OHHCH31

2

34

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67

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1213

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3029

CH3

CH3H3C

HOH

H CH3

H3CCH3

O

OHHCH31

2

34

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67

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1112

13

14

1516

1718

1920

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3029

Oleanolic Acid (1)

Metabolite (2)

Metabolite (3)

HO

HO

HO

A

BC

DE

1150 Mini-Reviews in Medicinal Chemistry, 2015, Vol. 15, No. 14 Anouar et al.

� ��

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In the hardness formula, N is the number of electrons, υ the external potential, and E the electronic energy [30, 31]. Excited singlet state (ES) energies were calculated from the optimized geometries, using TD-DFT formalism at the same level of theory (B3LYP/6-31G(d)) [32]. The allowed vertical electronic excitation energies were thus obtained, which consequently give the absorption energies in the UV/vis range with their CI (configuration interaction) description as well as their oscillator strength (f>0). 1H and 13C NMR magnetic isotropic shielding tensors (σ) were calculated within the standard GIAO (Gauge-Independent Atomic Orbital) approach at the same level of theory (B3LYP/6-31G(d)) [33]. The isotropic shielding values were used to calculate the isotropic chemical shifts δ with respect to tetramethylsilane (TMS). δiso(X) = σTMS(X) – σiso(X), where δiso is isotropic chemical shift and σiso isotropic shielding constant. The predicted chemical shifts were obtained using the equation δexp=aδcal+b, where δcal= δiso. The DFT and TD-DFT calculations were performed in gas and in solvent. The solvent effects were taken into account implicitly using the polarizable continuum model (IEF-PCM) [34]. In PCM, the substrate is embedded into a shape-adapted cavity surrounded by solvent, which is implicitly described as a dielectric continuum characterized by its dielectric constant (εCH3OH=32.61). Explicit molecule solvent have been investigated [35]. The PCM model generally gives a relatively good description compared to the explicit model. Kozlowski et al. were tested hybrid models (i.e., one or two molecules in the surrounding of the OH groups + PCM) to calculate electronic properties of quercetin [36]. They observed a slight difference between hybrid and a pure PCM models, while computational time was dramatically increased using hybrid model [36]. For the present study, we are limited to IEF-PCM model using methanol as solvent. All the theoretical calculations for neutral and anionic systems were performed with Gaussian09 [37]. The linear correlations were obtained using the DataLab package (http://www.lohninger.com/datalab/en_home.html), while the visualization of molecular orbitals was done using Molden software (http://www.cmbi.ru.nl/molden/).

2.2. Molecular Docking

The 3D crystal structure of -glucosidase was downloaded from the RCSB data bank with PDB ID code 2ZQO, in which -glucosidase bound with Cadmium,

imidazole, N-acetyl-d-galactosamine and phosphate ion [38]. Heteroatoms were removed, polar hydrogen atoms and Kollman were added to the receptor structure using the automated tool in AutoDoc 4.2 [39]. The active site residues were identified based on co-crystallized ligands structures in

-glucosidase complex structure [38]. Firstly, oleanolic acid 1 and its oxidative metabolites 2 and 3 were validated by docking of co-crystallized ligands structures into the receptor active sites (RMSD of the docked ligand into binding pocket must be less than 2 Å). The PDB of 1 and its oxidative metabolites 2 and 3 were obtained from their corresponding B3LYP/6-31G(d) optimized geometries. Nonpolar hydrogens were merged and rotatable bonds were defined for ligands. Docking studies were performed by Lamarckian genetic algorithm, with a total of 100 runs for each binding site. In each respective run, a population of 150 individuals with 27000 generations and 250000 energy evaluations were employed. Operator weights for crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively. Based on the active site, binding site was defined using a grid of 30 × 30 × 30 points each with a grid space of 0.375 Å centered at coordinates x = 30.809, y = 17.205, and z = 2.282. The docking studies were performed using an Intel (R) Core (TM) i7-3770 CPU @ 3.40 GHz workstation. Cluster analyses were performed on docked results using a root mean- square deviation (RMSD) tolerance of 2.0 Å. The protein−ligand complexes were visualized and analyzed using AutoDockTools and Discovery Studio.

3. RESULTS AND DISCUSSION

3.1. Electronic Properties Relationship to α -glucosidase Inhibitory Activity

The inhibitory activity of the oleanolic acid (1), and its two oxidative metabolites 2 and 3 are presented in Tables 1 and 2. The oleanolic acid (1) bearing one hydroxyl group at C-3 position exhibited more inhibitory activity than metabolite 2 `with two hydroxyl groups at C-2 and C-3 positions and metabolite 3 with three hydroxyl groups at C-2, C-3 and C-11 positions. On the basis of in vitro inhibitory results, it has been concluded that the inhibitory activity of metabolites decreased as the number of hydroxyl groups increased, thus their polarity also enhanced. For better understanding of α-glucosidase inhibition, electronic descriptors were calculated for the three pentacyclic triterpenoids 1-3 at the B3LYP/6-31G(d) in gas and PCM (Table 1 and 2). These electronic properties were calculated with the help of two different approaches as defined previously. To rationalize the inhibitory activity, we focused on the results obtained with energy consideration in PCM as presented in Table 2. The comparison of electronic descriptors of oleanolic acid (1) and oxidative metabolite 2 revealed similar values regarding EA, IP, hardness, electronegativity, electrophilicity, where the difference was around 0.01 eV, while the variation of permanent dipole moment (µ) and polarizability (α) were significant as presented in Table 1. Dipole moments are calculated as 1.66, 2.67 and 5 Debye for oleanolic acid (1), metabolite 2 and metabolite 3, respectively. The inhibtion activity increased with decreasing the polarity of the studies compounds (i.e., The oleanolic acid with the lowest dipole


Table 1. Electronic properties of compounds 1-3. calculated at B3LYP/6-31G(d) level of theory (Energy consideration).

EA (eV) IP (eV) η (eV) χ (eV) ω (eV) � (D) α (Å) IC50 (µM)±SEM

Gas phase

Oleanolic acid 1 -1.40 7.17 4.29 2.89 2.14 1.05 307.18 12.8±0.00

Metabolite 2 -1.36 7.22 4.29 2.93 2.14 2.11 310.36 444.0±8

Metabolite 3 -0.97 7.43 4.20 3.20 2.10 4.43 313.97 666.0±20

PCM solvent

Oleanolic acid 1 0.51 5.76 2.62 3.14 1.31 1.66 408.24 12.8±0.00

Metabolite 2 0.52 5.78 2.63 3.15 1.31 2.67 411.7 444.0±8

Metabolite 3 0.62 5.97 2.68 3.30 1.34 5.00 416.26 666.0±20

Table 2. Electronic properties of compounds 1-3.calculated at B3LYP/6-31G(d) level of theory (Orbital consideration).

EA (eV) IP(eV) Egap (eV) η (eV) χ (eV) ω (eV) � (D) α (Å) IC50 (µM)±SEM

Gas

Oleanolic acid 1 -0.30 5.98 170.95 3.14 2.84 1.29 1.05 307.18 12.8±0.00

Metabolite 2 -0.26 6.04 171.34 3.15 2.89 1.33 2.11 310.36 444.0±8

Metabolite 3 0.15 6.25 165.91 3.05 3.20 1.68 4.43 313.97 666.0±20

PCM solvent

Oleanolic acid 1 -0.19 6.04 169.42 3.11 2.93 1.37 1.66 408.24 12.8±0.00

Metabolite 2 -0.18 6.06 169.82 3.12 2.94 1.38 2.67 411.7 444.0±8

Metabolite 3 6.29 0.00 -171.07 -3.14 3.14 -1.57 5.00 416.26 666.0±20

moment and polarizability is the most active). The energy gaps for the pentacyclic triterpenoids were very similar (Table 2). The use of a polar solvent (using IEF-PCM) induced an increase of the dipole moment, which was probably due do the solvent-solute interactions, which increases the polarity of the compounds.

3.2. Docking Analysis of Oleanolic Acid and its Metabolites with α-glucosidase

Docking analyses were performed to investigate the interaction of the potent inhibitors with the active site residues of -glucosidase. The accuracy of the results is based on the free binding energies and the hydrogen bond interactions involved in the binding mode for potential inhibitors in -glucosidase active binding sites. For compound 1 and its metabolites 2 and 3, the number of multi-member conformational clusters was found to be 1, out of 100 runs. The estimated free binding energies values of 1-3 with -glucosidase enzyme were calculated as -7.45, -6.28 and -5.99 kcal/mol, respectively (Fig. 2). The variation of binding energies are in good agreement with the observed activities. Indeed, the most active inhibitor 1 possesses the lowest energy, and the less active metabolite 3 possesses the highest energy. It is worth highlighting that no hydrogen

bindings were found between the amino acid residues of -glucosidase and oleanolic acid 1, and its biotransfromed products 2 and 3. Therefore, it can be concluded that these interactions are purely electrostatics.

3.3. UV/Vis Spectra

The experimental UV/vis spectra of oleanolic acid 1 and its oxidative metabolites 2 and 3 were recorded on advance ultraviolet spectrophotometer, using methanol as solvent. The experimental λ MAX for the oleanolic acid 1, and its metabolites 2 and 3 are 204, 204 and 208 nm, respectively (Table 3). The predicted UV/vis spectra for the three pentacyclic triterpenoids 1-3 were obtained at the B3LYP/6-31G(d) in gas and PCM (methanol) (Table 3). The UV/vis spectra are presented in Fig. 3. The main absorption bands at 185, 185 and 196 nm are corresponding to HOMO→LUMO+1 electronic transition with high oscillator strength. It can be predicted in HOMO and LUMO+1 molecular orbital shapes, HOMO→LUMO+1 electronic transition is related to a π→π* electronic transition (Fig. 4). The bathochromic shift of λMAX in metabolite 3 was probably due to the presence of OH group at C-11 position, which increased the delocalization of LUMO+1 orbital. The metabolite 2 bearing OH group at C-2 position had no


Fig. (2). Docked pose oleanolic acid (1) and its metabolites 2 and 3 inside the active binding site of -glucosidase enzyme.

Fig. (3). Calculated UV-visible spectra of compounds 1-3. Table 3. λλ max (nm), Emax (eV), f and MO→ MO’ for compounds 1-3 calculated at B3LYP/6-31G(d) level of theory.

Gas phase PCM-Model Experimental

λmax Emax f MO→ MO’ λmax Emax f MO→ MO’ λmax Emax

Oleanolic acid 1 227 5.46 0.01 H→L (67%) 229 5.40 0.01 H→L (69%) 204 6.10

183 6.77 0.20 H→L +1(55%) 185 6.70 0.20 H→L +1(55%)

Metabolite 2 226 5.48 0.01 H→L (67%) 228 5.42 0.01 H→L (69%) 218 5.70

184 6.75 0.17 H→L+1 (53%) 185 6.70 0.26 H→L +1(55%)

Metabolite 3 234 5.29 0.01 H→L (64%) 225 5.50 0.01 H→L (60%) 208 5.98

190 6.54 0.10 H-1→L+1 (42%) 196 6.30 0.13 H→L +1(55%)

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Oleanolic Acid

Metabolite M2 (M2)

Metabolite M3 (M3)

HOMO LUMO

HOMO LUMO+1


Fig. (4). HOMO, LUMO, and LUMO+1 molecular orbitals of compounds 1-3. significant effect on π→π* transition, which explains the similar λ MAX of 1 and 2. IEF-PCM solvent induced a bathochromic shift of the maximum wavelength λ MAX by 2 nm as in oleanolic acid (1). The red shift can be rationalized by the the stabilization of HOMO and LUMO molecular orbitals by 0.05 and 0.07 eV, respectively. Theoretically, oleanolic acid (1) and oxidative metabolite 2 showed a shoulder at 227 and 226 nm respectively, which corresponds to HOMO→LUMO electronic transition. Mentioned electronic transition induced a charge transfer from one part to another part of a molecule (Fig. 4). The low intensity of the shoulder (f ≈ 0.01) provided information that indicated absorption band was corresponding to n→π* electronic transition of the carbonyl group.

3.4. NMR Spectra

The structure elucidation of 1 and its two oxidative metabolites 2 and 3 was accomplished by mean of 1D and 2D NMR, IR, UV/vis spectroscopic data and HREIMS techniques. The 1H and 13C NMR spectra of the compound 1-3 were recorded using TMS as an internal standard and deuterated chloroform (CDCl3) as solvent. The experimental and theoretical 1H and 13C NMR chemical shifts values (with respect to TMS) of the oleanolic acid (1) and its oxidative metabolites 2 and 3 are presented in Tables 3 and 4. Linear regressions (δexp=aδexp+b, where a is the slope of the linear curve and b the intercept) were obtained by plotting the experimental 1H and 13C NMR chemical shifts (δexp) vs (δcal)

Oleanolic Acid 1

Metabolite 2

Metabolite 3

HOMO HOMO+1 LUMO

HOMO HOMO+1 LUMO

HOMO HOMO+1 LUMO


Table 4. Experimental and predicted 13C NMR chemical shifts of compounds 1-3.

Oleanolic acid 1 Metabolite 2 Metabolite 3

B3LYP B3LYP B3LYP

Gas Solvent Exp Gas Solvent Exp Gas Solvent Exp

C1 37.6 37.6 40.9 44.2 44.1 48.1 45.5 45.4 48.7

C2 29.2 29.2 28.2 68.1 68.2 69.5 70.6 70.8 69.7

C3 78.1 78.1 78.0 77.9 78.5 84.4 80.9 81.6 84.0

C4 40.4 40.4 39.4 38.3 38.7 39.3 40.2 40.6 39.5

C5 56.4 56.4 55.8 53.5 53.5 55.6 56.3 56.2 54.6

C6 18.9 18.9 18.8 17.3 17.7 19.5 19.0 19.2 19.9

C7 33.0 33.0 33.3 30.9 31.3 34.2 32.9 33.3 35.2

C8 42.4 42.4 39.8 40.2 40.7 40.8 42.1 42.7 38.8

C9 49.8 49.8 48.1 47.4 47.7 54.5 53.3 52.9 58.6

C10 39.4 39.4 37.4 51.5 39.0 39.2 42.2 42.7 39.1

C11 23.7 23.7 23.8 22.0 22.5 24.4 66.3 67.7 65.0

C12 126.2 126.2 122.5 121.5 120.7 123.1 128.8 127.8 125.0

C13 141.6 141.6 144.8 137.2 138.4 145.7 144.9 147.6 145.0

C14 43.8 43.8 42.0 41.6 42.3 40.5 44.4 44.9 38.4

C15 29.0 29.0 28.3 27.0 27.3 28.5 28.3 28.9 28.5

C16 28.7 28.7 23.8 27.0 27.5 24.2 28.9 29.3 24.5

C17 48.3 48.3 46.7 45.8 46.7 43.3 46.4 47.8 43.6

C18 45.3 45.3 42.0 42.9 43.6 42.9 45.2 45.6 43.9

C19 42.7 42.7 46.7 40.5 40.7 47.2 41.7 42.0 47.6

C20 30.8 30.8 31.0 28.9 29.5 31.6 30.2 30.9 31.8

C21 34.0 34.0 34.3 32.0 32.1 35.0 33.4 33.6 35.2

C22 35.2 35.2 33.3 33.2 34.0 33.9 33.4 34.9 33.7

C23 14.9 14.9 28.7 14.6 15.2 24.1 16.0 16.3 27.1

C24 27.0 27.0 16.5 25.5 26.0 17.2 27.2 27.7 17.5

C25 15.6 15.6 15.5 15.3 15.6 17.5 19.0 19.1 16.5

C26 16.5 16.5 17.5 15.0 15.5 17.9 18.4 19.2 17.0

C27 27.0 27.0 26.2 25.3 25.7 26.4 26.7 26.0 25.4

C28 177.5 177.5 180.2 171.9 173.5 180.0 178.0 179.3 181.3

C29 21.4 21.4 30.3 19.7 20.1 29.3 21.3 21.3 29.2

C30 32.1 32.1 32.8 30.1 30.2 33.6 31.6 31.6 28.8

in CDCl3 for 1-3. These linear equations were used to calculate the predicted NMR chemical shifts of 1H and 13C for the compounds 1-3. The correlation between experimental and the theoretical chemical shifts were better for 13C chemical shifts than 1H NMR shifts. Indeed, for carbons, the correlation coefficient R2 is higher than 98%,

whereas for the 1H NMR shifts is less than 90% as shown in (Figs. 5 and 6). The PCM solvent had no significant effect on the 13C NMR chemical shift, which can probably be explained by the low abundance of the carbon isotope (1.1%), whereas the 1H NMR chemical shifts were downfield shifted, probably due to the higher abundance of


1H isotope (99.9%) and solute-solvent interactions that is due to hydrogen bonding. The bioconversion of oleanolic acid (1) to: (i) metabolite 2, with an OH group at C-2 position induced a downfield chemical shift of 13C value of C-2 at 70 ppm and 1H NMR chemical shift resonated at 3.60 ppm (Tables 4 and 5), which was probably due to the electronegativity effect of hydroxyl group; (ii) metabolite 3, bearing two OH groups at C-2 and C-11 positions, both 13C and 1H NMR chemical shifts of C-2, C-11, H-2 and H-11 were resonated at downfield region (Tables 4 and 5). The downfield shift of the theoretical chemical shifts of C-2, C-11, H-2 and H-11 were in good agreement with experimental results.

4. CONCLUSIONS

In summary, we have highlighted the role of dipole moment and the polarizability descriptors on the -glucosidase inhibitory activity of the oleanolic acid 1 and its two oxidative metabolites 2 and 3. The biological activity decreased with the increase of the polarity of triterpenoids (i.e. increase of the dipole moment and polarity values by polar hydroxyl groups). The binding energies of 1 and its oxidative metabolites 2 and 3 with -glucosidase were well correlated with the biological activity, and also confirmed using DFT results. B3LYP is a reliable hybrid functional to

Fig. (5). Correlation curve between experimental and theoretical 13C NMR chemical shifts of compounds 1-3.

Fig. (6). Correlation curve between experimental and theoretical 1H NMR chemical shifts of compounds 1-3.

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Table 5. Experimental and predicted 1H NMR chemical shifts of compounds 1-3.

Oleanolic acid 1 Metabolite 2 Metabolite 3

B3LYP B3LYP B3LYP

Gas Solvent Exp Gas Solvent Exp Gas Solvent Exp

H1(a) 1.54 1.54 1.54 1.58 1.74 1.94 2.14 2.18 2.03

H1(b) 1.12 1.16 1.20 1.09 1.04 1.21 1.32 1.31 1.92

H2(a) 1.71 1.66 1.93 3.35 3.43 3.60 3.38 3.49 3.62

H2(b) 1.16 1.31 1.25 - - - - - -

H3 3.04 3.10 2.95 2.95 2.93 2.90 2.90 2.92 2.91

H5 1.09 1.14 1.51 1.15 1.18 1.52 1.20 1.24 2.51

H6(a) 1.51 1.50 2.05 1.52 1.50 2.05 1.58 1.56 2.03

H6(b) 1.59 1.61 2.13 1.58 1.59 2.11 1.69 1.69 2.12

H7(a) 1.52 1.54 1.49 1.52 1.54 1.49 1.52 1.56 1.24

H7(b) 1.47 1.49 1.24 1.49 1.50 1.24 1.55 1.58 1.46

H9 1.61 1.66 1.43 1.67 1.70 1.41 1.39 1.49 2.25

H11(a) 1.75 1.75 1.81 1.75 1.77 1.81 3.74 3.77 4.04

H11(b) 1.78 1.81 2.03 1.79 1.83 2.04 - - -

H12 4.80 4.73 5.22 4.91 4.87 5.23 4.74 4.78 5.28

H15(a) 1.77 1.80 1.51 1.78 1.81 1.51 1.82 1.84 1.56

H15(b) 1.55 1.61 1.31 1.56 1.61 1.38 1.60 1.58 1.34

H16(a) 2.41 2.35 1.61 2.44 1.71 1.36 2.24 2.30 1.61

H16(b) 1.63 1.71 1.33 1.64 2.39 1.61 1.81 1.82 1.35

H18 2.95 2.85 2.85 3.00 2.92 2.86 2.78 2.85 2.86

H19(a) 1.73 1.79 1.44 1.74 1.80 1.44 1.77 1.85 1.49

H19(b) 1.46 1.44 1.28 1.46 1.43 1.29 1.55 1.53 1.20

H21(a) 1.46 1.52 1.57 1.46 1.51 1.55 1.57 1.61 1.65

H21(b) 1.33 1.37 1.23 1.33 1.36 1.23 1.44 1.46 1.21

H22(a) 1.73 1.71 1.75 1.74 1.71 1.75 1.74 1.77 1.72

H22(b) 1.52 1.54 1.47 1.53 1.54 1.47 1.69 1.66 1.45

H23 1.04 1.03 0.98 0.88 1.10 0.99 1.43 1.19 0.80

H24 1.10 0.63 0.94 1.06 0.64 0.95 0.74 0.76 0.92

H25 0.94 1.03 0.9 1.09 1.08 0.89 2.46 2.19 0.99

H26 0.78 0.98 0.8 0.76 0.95 0.79 0.98 0.86 0.78

H27 1.11 1.06 1.13 1.12 1.03 1.15 1.09 1.04 1.18

H29 1.09 0.81 1.05 0.84 0.77 1.00 0.93 0.91 1.00

H30 0.85 0.93 1.23 1.11 0.90 1.10 1.02 1.02 1.30

predict 1H and 13C NMR chemical shifts, and reproduce entire UV-visible absorption bands of 1 and its oxidative

metabolites 2 and 3. There was better 13C NMR chemical shifts correlations obtained for compounds 1-3 as compared


to 1H NMR chemical shifts correlations. This could be explained by the overlapping of protons signals and make the assignment a significant challenge for experimentalists.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

This research was supported by King Saud University, deanship of Scientific Research, College of Science Research center. Authors thank Prof. Dr. Sharifuddin Bin Md Zain and Mr. Mohamad Safwan bin Jusof for help to access to supercomputer (PTMLXSMP, Universiti Malaya, Kuala Lumpor, Malaysia).

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s web site along with the published article.

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Received: July 26, 2014 Revised: March 24, 2015 Accepted: March 30, 2015

Documents

α-Glucosidase Activity of Oleanolic Acid and Its Oxidative Metabolites: DFT and Docking Studies