Sesquiterpenoids and 2-(2-phenylethyl)chromones respectively acting as α-glucosidase and tyrosinase inhibitors from agarwood of an Aquilaria plant

Abstract The ethyl ether extract of agarwood from an Aquilaria plant afforded six new sesquiterpenoids, Agarozizanol A − F (1−6), together with four known sesquiterpenoids and six known 2-(2-phenylethyl)chromones. Their structures were elucidated via detailed spectroscopic analysis, X-ray diffraction, and comparisons with the published data. All the isolates were evaluated for the α-glucosidase and tyrosinase inhibitory activities in vitro. Compounds 5, 7, 8, and 10 showed significant inhibition of α-glucosidase with IC50 values ranging between 112.3 ± 4.5 and 524.5 ± 2.7 µM (acarbose, 743. 4 ± 3.3 µM). Compounds 13 and 14 exhibited tyrosinase inhibitory effect with IC50 values of 89.0 ± 1.7 and 51.5 ± 0.6 µM, respectively (kojic acid, 46.1 ± 1.3). In the kinetic studies, compounds 5 and 14 were found to be uncompetitive inhibitors for α-glucosidase and mixed type inhibitors for tyrosinase, respectively. Furthermore, molecular docking simulations revealed the binding sites and interactions of the most active compounds with α-glucosidase and tyrosinase.


Introduction
Type 2 diabetes mellitus (T2DM), a chronic metabolic disorder disease, can cause life-threatening long-term complications including heart disease, strokes, blindness, kidney failure, and poor blood flow in the limbs 1 . In 2015, an estimated 1.6 million deaths directly resulted from diabetes, which is among the top 10 causes of death worldwide 2 . a-Glucosidase (EC 3.2.1.20) is an exo-acting enzyme, which implicates in the glycoprotein processing and carbohydrate metabolism. Additionally, a-glucosidase catalyzes the cleavage of a-1,4 glycosidic bonds from the nonreducing ends of oligosaccharides and provides high amount of intestine absorbable glucose, which results in the postprandial hyperglycaemia and complications associated with T2DM 3,4 . Therefore, a-glucosidase inhibition is a very effective therapy to delay glucose absorption and lower blood glucose level after food intake, which can potentially postpone the progression of T2DM 5 .
As a precious traditional Chinese medicine, agarwood has been used for the treatment of joint pain, inflammatory related dailments, and diarrhoea, as well as a stimulant, sedative, and cardioprotective agent [6][7][8] . It is also popular as an incense for cultural and religious ceremonies and its essential oil is further processed into perfume in cosmetics. Previous chemical studies revealed that sesquiterpenoids and 2-(2-phenylethyl)chromone derivatives are the two principal and aroma components in agarwood. To date, over 130 sesquiterpenoids and 120 2-(2-phenylethyl)chromone derivatives have been isolated from agarwood 9 and many of them exhibited various pharmacological properties including antibacterial 10 , anti-inflammatory 11 , a-glucosidase inhibitory 12 , acetylcholinesterase inhibitory 13 , cytotoxic 12 , neuroprotective 14 , and antidepressant activities 15 . As part of our long-term project to chemically and biologically characterize chemical constituents from agarwood, the ethyl ether extract of agarwood from an Aquilaria plant was found to potently inhibit a-glucosidase. Then, a phytochemical investigation on the agarwood of an Aquilaria plant was performed and 10 a-glucosidase inhibitory sesquiterpenoids including 6 new sesquiterpenoids, Agarozizanol A À F (1À6), together with 4 known sesquiterpenoids ( Figure 1) were isolated and identified. Additionally, 6 known 2-(2-phenylethyl)chromones were also obtained. 2-(2-Phenylethyl)chromones are structurally similar to the flavones except for presence of additional ethyl group and many flavones show intriguing tyrosinase inhibitory activity [16][17][18] . Tyrosinase, a rate-limiting enzyme for the melanin biosynthesis, involves in pigmentation of skin 19 , browning in fresh vegetables and fruits 20 , cuticle formation in insects 21,22 , and neurodegeneration associated with Parkinson's disease 23 . Hence, tyrosinase inhibitors have broad application in medicines, food preservatives, bio-insecticides, and cosmetic products. Inspired by this, All the 2-(2-phenylethyl)chromones were assayed for their tyrosinase inhibitory activity. Moreover, the inhibition mechanism were investigated. Herein, we report the isolation, structure elucidation, a-glucosidase and tyrosinase inhibitory activities, as well as kinetic and molecular docking studies of these compounds.

Plant material
The agarwood was purchased from Bangkok, Thailand in August 2014 and its original plant was identified as a species of the genus Aquilaria by gene sequence analysis of the ITS region. A voucher specimen (201408SLLK) was retained at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

a-Glucosidase inhibitory assay
The a-glucosidase (G5003, Sigma-Aldrich) was obtained from Saccharomyces cerevisiae and the enzyme inhibitory assay was performed using formerly described method 24 with some modifications. The samples were dissolved in DMSO and 2-fold diluted to afford a serial concentrations (the highest final concentration was set at 750 mM because of low solubility of 1-10 in buffer). 10 mL sample was incubated with 100 mL a-glucosidase solution (0.2 U/mL in 100 mM phosphate buffer (pH: 6.8)) at 37 C for 15 min. Then, 40 mL of 2.5 mM p-nitrophenyl-a-D-glucopyranoside (p-NPG) were added and further incubated at 37 C for 15 min. DMSO instead of compound was used as control and the blank wells contained buffer in place of substrate. The OD values were measured at 405 nm with microplate reader. Acarbose was used as reference compound. The percentage inhibition was calculated using the following equation: Tyrosinase inhibitory assay All the isolated compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were tested for the inhibitory activity against tyrosinase (T3824, Sigma-Aldrich) from mushroom according to the previously reported method 16 with slight modifications. Briefly, 130 mL of tyrosinase (50 U/mL) solvated in 50 mM phosphate buffer (pH: 6.8) were mixed with 20 mL of 2-fold serial dilutions (from 1 mM to 0.03125 mM) of compounds in DMSO, and transferred into a 96-well plate. After 5 min pre-incubation of the mixture at 37 C, 50 mL of 2 mM L-tyrosine in buffer were added and incubated for additional 20 min at 37 C. The control wells contained DMSO instead of compound, and the blank wells were added with buffer in place of L-tyrosine. Kojic acid was used as a positive control. The absorbance was measured at 495 nm using microplate reader. The percentage inhibition was calculated using the formula as below: Kinetic analysis according to the inhibition assay described above. The data were expressed as double-reciprocal Lineweaver À Burk plot and the inhibition constants (K I and K IS ) were obtained from secondary plots of slope and y intercept of lines from Lineweaver-Burk plot against inhibitor 25 .

Molecular docking study
The X-ray structures of mushroom tyrosinase (PDB: 2Y9X) at a resolution of 2.78 Å and a-glucosidase (PDB: 3A4A) co-crystalized with glucose at a resolution of 1.6 Å were achieved from RCSB Protein Data Bank (www.rcsb.org). All hydrogen atoms and gasteiger charges in the protein structure were added by AutoDockTools and molecular docking simulations were performed with Autodock 4.2 software using the Lamarckian Genetic Algorithm 26,27 . Compounds 5 and 10 were docked into the catalytic site of a-glucosidase with a grid box of 50 Å Â 50 Å Â 50 Å at 0.375 Å space, centered at coordinates of x ¼ 21.275, y ¼ À0.741, and z ¼ 18.635. Genetic Algorithm parameters were set at Runs 50 and the maximum number of evals was set as medium (25000000). For the compounds 13 and 14, mixed type tyrosinase inhibitors, a blind molecular docking simulation was performed (Runs 20 and the maximum number of evals was set as short (250000)). The size of grid box was set at 100 Å Â 100 Å Â 100 Å in the x, y, and z dimensions at 0.375 Å space. This computational docking identified the site that bound tightly to the ligand. Then, a refined docking simulation was conducted with smaller grid box of 30 Å Â 44 Å Â 30 Å at 0.375 Å space, centered at the above identified binding site (x ¼ À16.345, y ¼ À36.541, z ¼ À22.916).
The Genetic Algorithm parameters were set at Runs 50 and the maximum number of evals was set as medium (25000000). The predicted geometries were ranked by the binding energies and clustered at RMSD value of 2 Å, and the best pose was selected for further analysis.

Structure elucidation
Compound 1 was obtained as colorless crystals and its molecular formula was assigned to be C 15 (Table 2) of 1 showed fifteen carbon signals which were categorized by HSQC spectrum as two methyl groups, six methylenes (one oxygenated), four methines, and three quaternary carbons. Apart from the double bond (d C 162.6, 108.2), the remaining three degrees of unsaturation combined with above mentioned spectroscopic data implied that 1 was a tricyclic sesquiterpenoid. In the 1 H-1 H COSY spectrum (Figure 2), two spin systems were observed: H 2 -12-H-2-H 2 -3-H 2 -4 and H-11-H-8-H 2 -9-H 2 -10. The two spin systems in association with key HMBC correlations from H 2 -12 to C-1, from H 2 -4 to C-1, C-5, and C-6, from H-5 to C-6, C-7, C-1, C-10, and C-11, from H 2 -13 to C-6, C-7, and C-8, and from H 3 -14 and H 3 -15 to C-5, C-6, and C-7 constructed the planar structure of 1, possessing a prezizaane skeleton, as shown in Figure 1. The relative configuration of 1 was clarified unambiguously from analysis of the ROESY spectrum. The ROE cross peak of H 2 -12 with H-10a revealed that H 2 -12 and H-10a located at the same side and assumed that they were a oriented. H-11 showed ROESY correlations with H-5 and H 3 -14, indicating they were cofacial and assigned to be b orientation. Thus, the structure of 1 was determined to be as shown in Figure 1, named Agarozizanol A.
Compound 2 was isolated as colorless crystals and had a molecular formula C 15 (Table 2) and HSQC spectra of 1 showed the resonances for three methyl groups, six methylenes (one oxygenated), three methines, and three quaternary carbons (one oxygenated). The aforementioned spectroscopic data were similar to those of 8, a co-occurring known prezizaane sesquiterpenoids Jinkohol II 28 . The only difference was the presence of additional oxidized quaternary carbon signal (d C 83.6) and the absence of a methine signal in the NMR spectra of 2. The HMBC correlations (Figure 2) of H 2 -13, H 2 -11, and H 2 -10 with this carbon suggested that the oxygenated carbon was C-8. H 3 -12 showed ROESY correlations with H-10a, suggesting they were cofacial. Other ROESY interactions of H-2 with H-5, H-11b and H 3 -14 with H-5 and H-7 were found, revealing that these protons were resided on the same face. Therefore, the structure of 2 was deduced as Agarozizanol B shown in Figure. 1 Table 1 and 2) of 3 exhibited the characteristic signals for prezizane sesquiterpenoid including four methyls, four methylenes, four methines, and three quaternary carbons. The NMR spectroscopic data of 3 closely resemble that of 7, Jinkohol I 29 , with exception of deshieled C-11 at d C 83.5. This chemical shift indicated the attachment of a hydroxyl to C-11, which was confirmed by HMBC correlation from H-11 to C-9/C-10/ C-5/C-7 and 1 H-1 H COSY correlations of H-11-H-8-H-9-H 2 -10 ( Figure 2). The ROESY correlations of H-2/H-11/H-5 suggested that these protons were cofacial and b oriented. Additional ROESY cross peaks of H 3 -12/H-10a and H 3 -13/H-9b permitted assignment of a orientation to H 3 -12 and H 3 -13. Summarizing these data, the structure of 3 was determined as shown in Figure 1 and named Agarozizanol C.
Obtained as white amorphous powder, compound 4 showed an ion peak [M þ Na] þ at m/z 261.1826 (calcd for C 15 H 26 NaO 2 , 261.1825) in the HRESIMS, consistent with a molecular formula of C 15 H 26 O 2 . The structure of 4 was similar to that of known compound 7, except for the presence of additional hydroxyl at C-13 in 4, which was verified by the deshielded chemical shift of C-13 at d C 62.7 and the molecular formula of 4 with one more oxygen atom than that of 7. The ROESY interactions of H 3 -12 with H 2 -10, H 2 -9 with H 2 -13 and H 3 -15 (Figure 2), indicated these protons located at the same side of the molecule and were assigned to be a oriented. While H-5, H 3 -14, H-2, and H-11 a were oriented on the other side by their ROESY correlations. Hence, the structure of 4 was established as a new epimer of synthetic (þ)-prezizaene diol 30 , named Agarozizanol D.
Compound 5 was obtained as white amorphous powder and was shown to have a molecular formula C 15 (Table 2) and HSQC spectra of 5 showed the presence of three methyl groups, six methylenes (one oxygenated), two methines, and four quaternary carbons including two olefinic carbons. These spectroscopic data of 5 were similar to those of compound 10 31 , a known zizaene sesquiterpenoid. The major difference was that the hydroxyl, which located at C-12 in the 10, was attached to the C-13 in the compound 5. This assignment was confirmed by the deshielded chemical shifts of C-13 at d C 66.7 and the HMBC correlations between H 2 -13 and C-6/C-14/C-7/ C-8 ( Figure 2). The ROESY correlations of H 3 -12/H-10a, H 2 -13/H-9a, and H 3 -14/H-11b were observed, allowing the assignment of a orientation for H 3 -12 and H-9a, and b orientation for H 3 -14 and H-11b. Based on all the above evidence, the structure of 5 was identified as shown in Figure 1 and named as Agarozizanol E.
Compound 6 was obtained as white amorphous powder and displayed an ion peak [M þ Na] þ at m/z 259.1662 (calcd for C 15 H 24 NaO 2 , 259.1669) in its HRESIMS, corresponding to a molecular formula C 15 H 24 O 2 with one more oxygen atom than that of 10. The 1 H and 13 C NMR data of 6 were closely similar to those of 10 31 with only difference being the deshieled chemical shift of CH-4 (d H 4.29, d C 81.3), which indicated the presence of hydroxyl at C-4. This deduction was further verified by HMBC correlations of H-4 with C-1/C-5/C-6 ( Figure 2). The a orientation of CH 2 OH-12, OH-4, and CH 2 -10 was assigned from the ROESY correlations of H 2 -12/H-10b and H-2/H-4. Accordingly, the structure of 6 was characterized as shown in Figure.1, named Agarozizanol F.
On the basis of their structural similarity, compounds 1À6 should be generated from a common precursor in their biosynthetic pathway (Supplementary material, Figure S1), and possessed the same stereochemistry at C-1 and C-2. Fortunately, single-crystals of 2 were obtained and subjected to X-ray diffraction experiment with Cu Ka radiation ( Figure 3). However, the imperfect Flack parameter [0. 3(8)] only allowed confirmation of the above deduced planar structure and relative configuration of 2. Therefore, further effort was needed to determine the absolute configuration of 126.
By comparing their experimental spectroscopic data with reported data in the literature, the known compounds were identified as Jinkohol I (7) 29 , Jinkohol II (8) 28 , Jinkoholic acid (9) 32 , isokhusenol (10)  Bioactivity assays, kinetic analysis, and molecular docking simulations a-Glucosidase inhibitory activity assay, kinetic analysis, and molecular docking study All the isolated compounds were assessed for the inhibition against a-glucosidase. The results were listed in the Table 3. Compounds 5, 7, 8, and 10 showed inhibitory effect on a-glucosidase (IC 50 values ranging from 112.3 ± 4.5 to 524.5 ± 2.7 mM) superior to acarbose (IC 50 , 743. 4 ± 3.3 mM). Compounds 2, 4, and 9 also possessed inhibitory activity comparable to the positive control.
To elucidate the type of inhibition of isolated sesquiterpenoids with a-glucosidase, compound 5 with most potency was selected for the kinetic analysis. The Lineweaver-Burk plot (Figure 4(A)) of 5 was established and revealed that K m and V max decreased in a parallel fashion, indicating that 5 acted as an uncompetitive a-glucosidase inhibitor by binding only with the enzyme-substrate complex 37 . The inhibition constant was calculated using secondary plot of y intercept versus concentration of inhibitor, yielding K I value of 168.0 mM (Figure 4(B)).
In order to rationalize the uncompetitive inhibition mechanism indicated by kinetic study, molecular modeling studies were performed. The most active compounds 5 and 10 were well docked into the catalytic site of the a-glucosidase (Figure. 5) with binding energies of À7.2 and À7.1 kcal/mol, respectively. The docking results revealed that compounds 5 and 10 bound to the entrance part of the active site cavity of a-glucosidase, therefore blocking the release of product (glucose) after the enzyme catalyzed reaction was completed 37 . This may explain why compound 5 behaved as an uncompetitive inhibitor of a-glucosidase. Specifically, compound 5 bound to the active site through hydrogen bonds with residues Asp 352 and Arg 442, and hydrophobic interactions with Tyr 158, Phe 303, and Arg 315 ( Figure 5(B)). Compound 10 took hydrogen bonding with Asn 415 and hydrophobic interactions with Lys 156, Phe 314, and Arg 315 ( Figure  5(C)). These interactions may provide hints for designing of a new class of a-glucosidase inhibitor.
As shown in the Lineweaver-Burk plot of the most active compound 14 (Figure 6(A)), increasing inhibitor concentration resulted in a decrease in V max and an increase in K m , which indicated a  mixed type inhibition for 14 against tyrosinase 25 . Thus, compound 14 inhibited the tyrosinase not only by binding with the free enzyme but also with the enzyme À substrate complex.
The inhibition constants of 14 binding with the free enzyme (K I ) and with enzyme-substrate complex (K IS ) were determined by the slope and the y intercept versus the inhibitor concentration, respectively. From Figure 6(B,C), the inhibition constants K I and K IS of 14 were calculated to be 39.6 mM and 72.8 mM, respectively, by secondary re-plots, indicating that compound 14 effectively bound to free enzyme as compared to enzyme-substrate complex.
To further gain insight into the binding mechanism of 2-(2phenylethyl)chromones with tyrosinase, molecular docking simulations were performed. As presented in Figure 7, compounds 13 and 14 were able to anchor the same binding pocket of tyrosinase with the binding energy of À7.49 and À7.75 kcal/mol, respectively. Compound 14 formed hydrogen bonds with residues Gln   41, Lys 180, and hydrophobic interactions with residues His 178 and Gln 44. However, compound 13 bound to the binding site with a different mode and interacted via hydrogen bonds with residues Gln 41, Lys 180, Asn 174, and Glu173, and hydrophobic interactions with residues Gln 44 and Ala 45. The differences may stem from the substitution of ring A and B of 2-(2-phenylethyl)chromones. These results unveiled a possible allosteric site of tyrosinase by 2-(2-phenylethyl)chromones and will be helpful for understanding the binding interactions between 2-(2-phenylethyl)chromones and tyrosinase.

Conclusions
The ethyl ether extract of agarwood from an Aquilaria plant afforded 6 previously undescribed sesquiterpenoids, 4 known sesquiterpenoids, as well as 6 known 2-(2-phenylethyl)chromones. Among them, compounds 2, 4, 5, and 7-10 showed intriguing inhibitory activity against a-glucosidase. The combined enzyme kinetic and molecular docking studies were performed and revealed that the most effective compound 5, a new zizaane-type sesquiterpenoid, behaved as an uncompetitive a-glucosidase inhibitor and blocked catalytic reaction by interacting with the entrance of active site of a-glucosidase. Uncompetitive inhibitors are considered to be superior to competitive and noncompetitive inhibitors in drug development and are expected to display better in vivo efficacy 37,38 . Therefore, compound 5 could serve as a new promising lead compound for synthesis of more potent derivatives as a-glucosidase inhibitors. Additionally, compounds 13 and 14 manifested remarkable tyrosinase inhibitory effect. Compound 14 with the most potency was revealed to have affinity for the  allosteric site of tyrosinase as a mixed type inhibitor. Agarwood formation is due to a self-defense mechanism initiated by the tree to fight off pests and pathogens invading through holes and wounds on the tree surface 6 . 2-(2-Phenylethyl)chromone is one of the two main components of agarwood and our study revealed the 2-(2-phenylethyl)chromones exert inhibitory activity on tyrosinase, which may provide the chemical evidence for the selfdefense mechanism of the agar-producing tree against pests.