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.

Keywords:

• Agarwood
• aquilaria plant
• sesquiterpenoid
• 2-(2-phenylethyl)chromone
• α-glucosidase inhibition
• tyrosinase inhibition

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 limbs¹. In 2015, an estimated 1.6 million deaths directly resulted from diabetes, which is among the top 10 causes of death worldwide². α-Glucosidase (EC 3.2.1.20) is an exo-acting enzyme, which implicates in the glycoprotein processing and carbohydrate metabolism. Additionally, α-glucosidase catalyzes the cleavage of α-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, α-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 T2DM⁵.

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^6–8. 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 agarwood⁹ and many of them exhibited various pharmacological properties including antibacterial¹⁰, anti-inflammatory¹¹, α-glucosidase inhibitory¹², acetylcholinesterase inhibitory¹³, cytotoxic¹², neuroprotective¹⁴, and antidepressant activities¹⁵. 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 α-glucosidase. Then, a phytochemical investigation on the agarwood of an Aquilaria plant was performed and 10 α-glucosidase inhibitory sesquiterpenoids including 6 new sesquiterpenoids, Agarozizanol A − F (1−6), 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^16–18. Tyrosinase, a rate-limiting enzyme for the melanin biosynthesis, involves in pigmentation of skin¹⁹, browning in fresh vegetables and fruits²⁰, cuticle formation in insects^21,22, and neurodegeneration associated with Parkinson’s disease²³. 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, α-glucosidase and tyrosinase inhibitory activities, as well as kinetic and molecular docking studies of these compounds.

Figure 1. Structures of 1–16.

Materials and methods

General experimental procedures

Optical rotations were measured on an Anton Paar MCP 5100 polarimeter. IR spectra were obtained using a Nicolet 380 FT-IR instrument (Thermo, USA) using KBr pellets. HRESIMS were determined by a Waters Autospec Premier (Waters, USA) mass spectrometer. The NMR spectra were recorded on Bruker Avance 500 NMR spectrometers (Bruker, Germany) with TMS as an internal standard. The high performance liquid chromatography (HPLC) was performed with an analytic reversed-phased column (YMC–packed C₁₈, 250 mm × 10 mm, 5 µm) (YMC, Japan) using a G1311C 1260 Quat Pump VL and detected with a G1315D 1260 DAD VL detector (190–500 nm) (Agilent Technologies 1260 infinity, USA). For column chromatography, silica gel (60–80, 200–300 mesh, Qingdao Haiyang Chemical Co., Ltd, China), ODS gel (20–45 µm, Fuji Silysia Chemical Co., Ltd, USA), and Sephadex LH-20 (Merck, Germany) were used. TLC analysis was performed on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd, China), and spots were visualized by spraying with 5% H₂SO₄ in EtOH followed by heating. Thermo fisher scientific plate reader was used for enzyme inhibition assays.

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.

Extraction and isolation

Air-dried agarwood (384.0 g) was ground and extracted with ethyl ether (1.5 L) for three times. The obtained extract was tested for the α-glucosidase inhibitory activity and showed inhibition rate of 44.13 ± 1.92% at concentration of 50 µg/mL (acarbose, 62.06 ± 4.77%). Then, the ethyl ether extract (20.4 g) was subjected to ODS column chromatography (CC) eluting with MeOH/H₂O (v/v, 2:3, 1:1, 3:2, 7:3, 4:1, 9:1, 1:0) to obtain 16 fractions (Fr.1–Fr.16). Fr.4 (0.88 g) was separated by silica gel CC (petroleum ether/EtOAc, 50:1–5:2) to afford 12 subfrctions (Fr.4.1–Fr.4.12), Fr.4.5 was purified by silica gel CC (petroleum ether/EtOAc, 50:4) to yield compound 6 (3.5 mg). Purification of subfraction Fr.4.6 using silica gel CC (petroleum ether/EtOAc, 50:4) furnished compound 10 (3.3 mg). Fr.5 (5.3 g) was applied to silica gel CC eluted by petroleum ether/EtOAc (1:0–5:1), yielding 15 subfrctions (Fr.5.1–Fr.5.15). Separation of Fr.5.2 by silica gel CC (petroleum ether/CHCl₃/EtOAc, 50:10:1) afforded compounds 1 (2.9 mg) and 5 (3.2 mg). Compounds 2 (2.9 mg) and 3 (2.8 mg) were obtained by separation of subfraction Fr.5.3 with silica gel CC (petroleum ether/EtOAc/isopropyl alcohol, 100:10:1). Subfraction Fr.5.4 was separated by silica gel CC (petroleum ether/CHCl₃/isopropyl alcohol, 100:10:1) and preparative TLC, yielding compounds 9 (2.2 mg) and 4 (1.2 mg). Subfraction Fr.5.7 was chromatographed by silica gel CC (petroleum ether/CHCl₃/isopropyl alcohol, 100:15:1) to obtain compounds 7 (1.2 mg) and 8 (2.3 mg). Fr.6 (0.6 g) was fractioned by silica gel CC (petroleum ether/CHCl₃/CH₃OH, 2:1:1), leading to isolation of 7 subfractions (Fr.6.1–Fr.6.7). Isolation of subfraction Fr.6.6 using silica gel CC and semi-preparative HPLC obtained compounds 15 (18.0 mg) and 16 (5.4 mg). Fr.7 (1.3 g) was divided into 13 subfractions (Fr.7.1–Fr.7.13) by silica gel CC using petroleum ether/EtOAc (500:1–0:1) as eluent. Subfraction Fr.7.11 was isolated via silica gel CC and semi-preparative HPLC to yield compounds 12 (2.2 mg) and 13 (3.0 mg). Fr.8 (0.8 g) was purified by silica gel CC (petroleum ether/EtOAc, 200:1-0:1), affording compounds 11 (0.8 mg) and 14 (3.5 mg).

Agarozizanol A (1)

Colorless crystals; [α]25_D -3 (c 0.28, MeOH); IR (KBr) ν_max cm⁻¹: 3252, 2956, 2870, 1629, 1462, 1090, and 1044; ¹H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 259.1667 [M + Na]⁺ (calcd for C₁₅H₂₄NaO₂, 259.1669); 1 D and 2 D NMR, IR spectra, and HRESIMS, see Figure S2–S9 in Supplementary material.

Table 1. ¹H NMR (500 MHz) data for compounds 1−6.

position	1^a	2^a	3^a	4^b	5^a	6^a
2	2.20, m	1.76, m	2.12, m	1.73, m	1.77, m	1.94, m
3a	1.84, m	1.85, m	1.88, m	1.76, m	1.80, m	2.38, dd (12.5,7.1)
3b	1.22, m	1.14, m	1.14, m	1.04, m	1.25, m	1.36, m
4a	1.70, m	1.53, m	1.59, m	1.42, m	2.24, dd (17.2, 9.1)	4.29, t (7.3)
4b			1.50, m		2.11, m
5	1.66, m	1.54, m	1.74, m	1.64, dd (11.7, 8.5)
7		1.80, m
8	2.72, d (7.0)		1.87, m	2.18, dd (6.6, 4.7)	2.13, dd (7.5, 5.6)	1.83, m
9a	2.18, m	2.00, m	1.92, m	1.53, m	1.81, m	1.83, m
9b	1.65, m	1.56, m	1.60, m		1.63, m	1.66, m
10a	1.74, m	1.55, m	1.68, m	1.15, m	1.42, m	1.67, m
10b	1.53, m	1.26, m	1.32, m		1.18, m	1.51, m
11a	3.34, m	1.89, d (10.1)	4.01, brs	1.54, d (10.4)	1.57, dd (10.5, 5.5)	1.49, t (7.7)
11b		1.09, dt (10.1, 2.2)		1.25, dd (10.4, 4.8)	1.32, m	1.30, m
12a	3.53, t (10.7)	0.87, d (6.7)	1.01, d (6.9)	0.81, d (6.1)	0.95, d (6.5)	3.70, dd (10.6, 6.1)
12b	3.42, dd (10.7, 4.5)					3.55 dd (10.6,7.7)
13a	4.81, d (1.5)	3.89, dd (11.1, 9.4)	1.19, s	3.40, d (4.5)	3.60, d (10.9)	1.06, s
13b	4.79, d (1.5)	3.77, dd (11.1, 2.7)			3.54 d (10.9)
14	1.13, s	1.06, s	0.87, s	0.85, s	1.07, s	1.03, s
15	1.10, s	0.79, s	0.95, s	0.80, s	1.41, s	1.60, s

^aRecorded in CD₃OD, ^bRecorded in DMSO-d₆.

Table 2. ¹³C NMR (125 MHz) data for compounds 1−6.

position	1^a	2^a	3a	4^b	5^a	6^a
1	58.4, C	53.0, C	57.0, C	51.9, C	54.0, C	52.3, C
2	47.5, CH	40.2, CH	39.3, CH	38.7, CH	41.1, CH	44.7, CH
3	25.8, CH2	31.5, CH2	33.0, CH2	30.5, CH2	33.5, CH2	35.9, CH2
4	22.3, CH2	21.9, CH2	23.3, CH2	20.7, CH2	28.4, CH2	81.3, CH
5	59.1, CH	60.4, CH	56.7, CH	55.6, CH	146.7, C	143.1, C
6	37.4, C	35.8, C	40.2, C	37.4, C	126.1, C	137.0, C
7	162.6, C	58.1, CH	80.3, C	76.1, C	46.5, C	41.9, C
8	55.4, CH	83.6, C	58.4, CH	43.0, CH	43.0, CH	48.0, CH
9	27.9, CH2	21.8, CH2	24.4, CH2	25.5, CH2	25.1, CH2	25.3, CH2
10	20.2, CH2	32.2, CH2	20.4, CH2	21.0, CH2	29.7, CH2	39.5, CH2
11	84.3, CH	52.2, CH2	83.5, CH	37.4, CH	37.8, CH2	30.9, CH2
12	62.6, CH2	14.4, CH3	18.4, CH3	19.1, CH3	14.4, CH3	64.3, CH2
13	108.2, CH2	62.4, CH2	23.9, CH3	62.7, CH2	66.7, CH2	28.6, CH3
14	27.9, CH3	33.9, CH3	27.6, CH3,	27.8, CH3	23.3, CH3	25.4, CH3
15	32.1, CH3	17.8, CH3	21.8, CH3	14.5, CH3	13.3, CH3	12.8, CH3

^aRecorded in CD₃OD, ^bRecorded in DMSO-d₆.

Agarozizanol B (2)

Colorless crystals; [α]25_D +15 (c 0.28, MeOH); IR (KBr) ν_max cm⁻¹: 3413, 2956, 2870, 1450, 1381, 1261, and 1059; ¹H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 261.1820 [M + Na]⁺ (calcd for C₁₅H₂₆NaO₂, 261.1825). 1 D and 2 D NMR, IR spectra, and HRESIMS, see Figures S10–S17 in Supplementary material.

Agarozizanol C (3)

White amorphous powder; [α]25_D -11 (c 0.25, MeOH); IR (KBr) ν_max cm⁻¹: 3444, 2956, 2868, 1456, 1376, 1250, 1091, and 1027; ¹H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 261.1817 [M + Na]⁺ (calcd for C₁₅H₂₆NaO₂, 261.1825). 1 D and 2 D NMR, IR spectra, and HRESIMS, see Figure S18-Figure S25 in Supplementary material.

Agarozizanol D (4)

White amorphous powder; [α]25_D +14 (c 0.10, MeOH); IR (KBr) ν_max cm⁻¹: 3439, 2950, 2867, 1457, 1374, and 1067; ¹H and 13C NMR data, see Table 1 and 2; HRESIMS m/z 261.1826 [M + Na]⁺ (calcd for C₁₅H₂₆NaO₂, 261.1825). 1 D and 2 D NMR, IR spectra, and HRESIMS, see Figure S26–S33 in Supplementary material.

Agarozizanol E (5)

White amorphous powder; [α]25_D -7 (c 0.33, MeOH); IR (KBr) ν_max cm⁻¹: 3447, 2938, 2863, 1627, 1456, 1377, 1260, 1094, and 1034; ¹H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 243.1707 [M + Na]⁺ (calcd for C₁₅H₂₄NaO, 243.1719). 1 D and 2 D NMR, IR spectra, and HRESIMS, see Figure S34–S41 in Supplementary material.

Agarozizanol F (6)

White amorphous powder; [α]25_D +2 (c 0.36, MeOH); IR (KBr) ν_max cm⁻¹: 3446, 2928, 2867, 1617, 1454, 1382, and 1084; ¹H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 259.1662 [M + Na]⁺ (calcd for C₁₅H₂₄NaO₂, 259.1669). 1 D and 2 D NMR, IR spectra, and HRESIMS, see Figure S42–S49 in Supplementary material.

X-ray crystallographic analysis of compound 2

A suitable crystal of 2 obtained from MeOH was selected and determined on a Bruker D8 QUEST diffractometer at 296.15 K. Crystal data for compound 2: C₁₅H₂₆O₂ (M = 238.36 g/mol), orthorhombic, space group P2₁2₁2₁, a = 6.7545(7) Å, b = 8.8069(8) Å, c = 23.886(2) Å, V = 1420.9 (2) ų, Z = 4, T = 296(2) K, µ (Cu Kα)=0.556 mm⁻¹, Dcalc = 1.114 g/cm³, 9320 reflections collected (7.402° ≤ 2θ ≤ 136.888°), 2598 unique (Rint = 0.1110, Rsigma = 0.0937). The final R1 was 0.0559 (I > 2σ(I)) and wR2 was 0.1624 (all data), Flack parameter = 0.3(8). The crystallographic data for 2 have been deposited at the Cambridge Crystallographic Data Centre under the reference number CCDC 1873716. This data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or from CCDC, 12 Union Road, Cambridge, CB21EZ, UK; (fax: [phone] or e-mail: [email protected]).

α-Glucosidase inhibitory assay

The α-glucosidase (G5003, Sigma-Aldrich) was obtained from Saccharomyces cerevisiae and the enzyme inhibitory assay was performed using formerly described method²⁴ 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 µM because of low solubility of 1–10 in buffer). 10 µL sample was incubated with 100 µL α-glucosidase solution (0.2 U/mL in 100 mM phosphate buffer (pH: 6.8)) at 37 °C for 15 min. Then, 40 µL of 2.5 mM p-nitrophenyl-α-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: $$\% \text{inhibition}=\left[1–\left({\text{OD}}_{\text{compound}}–{\text{OD}}_{\text{blank}}\right)/\left({\text{OD}}_{\text{control}}–{\text{OD}}_{\text{blank}}\right)\right]\times 100$$

Tyrosinase inhibitory assay

All the isolated compounds (1–16) were tested for the inhibitory activity against tyrosinase (T3824, Sigma-Aldrich) from mushroom according to the previously reported method¹⁶ with slight modifications. Briefly, 130 µL of tyrosinase (50 U/mL) solvated in 50 mM phosphate buffer (pH: 6.8) were mixed with 20 µL 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 µL 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: $$\% \text{inhibition}=\left[1–\left({\text{OD}}_{\text{compound}}–{\text{OD}}_{\text{blank}}\right)/\left({\text{OD}}_{\text{control}}–{\text{OD}}_{\text{blank}}\right)\right]\times 100$$

Kinetic analysis

To determine the mechanisms by which the most effective compound 5 inhibited α-glucosidase and compound 14 inhibited tyrosinase, the enzyme kinetic analysis was investigated at various concentrations of substrate (2.5, 1.25, 0.625, and 0.3125 mM p-NPG for α-glucosidase; 4.0, 2.0, 1.0, and 0.5 mM tyrosine for tyrosinase) in the absence or presence of different test inhibitor concentrations (0, 187.5, and 750 µM of 5; 0, 25, and 100 µM of 14) according to the inhibition assay described above. The data were expressed as double-reciprocal Lineweaver − 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 inhibitor²⁵.

Molecular docking study

The X-ray structures of mushroom tyrosinase (PDB: 2Y9X) at a resolution of 2.78 Å and α-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 α-glucosidase with a grid box of 50 Å × 50 Å × 50 Å at 0.375 Å space, centered at coordinates of x = 21.275, y = −0.741, and z = 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 × 100 Å × 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 × 44 Å × 30 Å at 0.375 Å space, centered at the above identified binding site (x = −16.345, y = −36.541, z = −22.916). 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.

Results and discussion

Structure elucidation

Compound 1 was obtained as colorless crystals and its molecular formula was assigned to be C₁₅H₂₄O₂ based on the HRESIMS ion peak [M + Na]⁺ at m/z 259.1667 (calcd for C₁₅H₂₄NaO₂, 259.1669), requiring 4 degrees of unsaturation. The ¹H NMR data (Table 1) of 1 showed resonances for two methyl groups at δ_H 1.10 (3H, s) and 1.13 (3H, s), two exocyclic olefinic protons at δ_H 4.81 (1H, d, J = 1.5 Hz) and 4.79 (1H, d, J = 1.5 Hz), and one oxygenated methylene at δ_H 3.53 (1H, t, J = 10.7 Hz) and 3.42 (1H, dd, J = 10.7, 4.5 Hz). The ¹³C NMR data (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 (δ_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 ¹H-¹H COSY spectrum (Figure 2), two spin systems were observed: H₂-12–H-2–H₂-3–H₂-4 and H-11–H-8–H₂-9–H₂-10. The two spin systems in association with key HMBC correlations from H₂-12 to C-1, from H₂-4 to C-1, C-5, and C-6, from H-5 to C-6, C-7, C-1, C-10, and C-11, from H₂-13 to C-6, C-7, and C-8, and from H₃-14 and H₃-15 to C-5, C-6, and C-7 constructed the planar structure of 1, possessing a prezizaane skeleton, as shown in Figure 1.

Figure 2. Key ¹H-¹H COSY (bold lines), HMBC (arrows), and ROESY (dashed double arrows) correlations of compounds 1−6.

The relative configuration of 1 was clarified unambiguously from analysis of the ROESY spectrum. The ROE cross peak of H₂-12 with H-10a revealed that H₂-12 and H-10a located at the same side and assumed that they were α oriented. H-11 showed ROESY correlations with H-5 and H₃-14, indicating they were cofacial and assigned to be β 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 C₁₅H₂₆O₂ on the basis of its HRESIMS and 13C NMR spectroscopic data. The ¹H NMR data (Table 1) of 2 revealed the presence of three methyl group at δ_H 0.87 (3H, d, J = 6.7 Hz), 1.06 (3H, s), and 0.79 (3H, s) and one oxygenated methylene at δ_H 3.89 (1H, dd, J = 11.1, 9.4 Hz) and 3.77 (1H, d, J = 11.1, 2.7 Hz). The ¹³C NMR (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 II²⁸. The only difference was the presence of additional oxidized quaternary carbon signal (δ_C 83.6) and the absence of a methine signal in the NMR spectra of 2. The HMBC correlations (Figure 2) of H₂-13, H₂-11, and H₂-10 with this carbon suggested that the oxygenated carbon was C-8. H₃-12 showed ROESY correlations with H-10a, suggesting they were cofacial. Other ROESY interactions of H-2 with H-5, H-11b and H₃-14 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.

Compound 3, a white amorphous powder, was found to have a molecular formula of C₁₅H₂₆O₂ from the ion peak [M + Na]⁺ at m/z 261.1817 (calcd for C₁₅H₂₆NaO₂, 261.1825) in the HRESIMS. The ¹H and ¹³C NMR data (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 I²⁹, with exception of deshieled C-11 at δ_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 ¹H-¹H COSY correlations of H-11–H-8–H-9–H₂-10 (Figure 2). The ROESY correlations of H-2/H-11/H-5 suggested that these protons were cofacial and β oriented. Additional ROESY cross peaks of H₃-12/H-10a and H₃-13/H-9b permitted assignment of α orientation to H₃-12 and H₃-13. 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 + Na]⁺ at m/z 261.1826 (calcd for C₁₅H₂₆NaO₂, 261.1825) in the HRESIMS, consistent with a molecular formula of C₁₅H₂₆O₂. 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 δ_C 62.7 and the molecular formula of 4 with one more oxygen atom than that of 7. The ROESY interactions of H₃-12 with H₂-10, H₂-9 with H₂-13 and H₃-15 (Figure 2), indicated these protons located at the same side of the molecule and were assigned to be α oriented. While H-5, H₃-14, 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 (+)-prezizaene diol³⁰, named Agarozizanol D.

Compound 5 was obtained as white amorphous powder and was shown to have a molecular formula C₁₅H₂₄O based on its ion peak [M + Na]⁺ at m/z 243.1707 (calcd for C₁₅H₂₄NaO, 243.1719) in HRESIMS. Resonances of three methyls at δ_H 0.95 (3H, d, J = 6.5 Hz), 1.07 (3H, s), and 1.41 (3H, s), and one oxygenated methylene at δ_H 3.60 (1H, d, J = 10.9 Hz) and 3.54 (1H, d, J = 10.9 Hz) were observed in the ¹H NMR spectrum of compound 5. The ¹³C NMR (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 10³¹, 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 δ_C 66.7 and the HMBC correlations between H₂-13 and C-6/C-14/C-7/C-8 (Figure 2). The ROESY correlations of H₃-12/H-10a, H₂-13/H-9a, and H₃-14/H-11b were observed, allowing the assignment of α orientation for H₃-12 and H-9a, and β orientation for H₃-14 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 + Na]⁺ at m/z 259.1662 (calcd for C₁₅H₂₄NaO₂, 259.1669) in its HRESIMS, corresponding to a molecular formula C₁₅H₂₄O₂ with one more oxygen atom than that of 10. The ¹H and ¹³C NMR data of 6 were closely similar to those of 10³¹ with only difference being the deshieled chemical shift of CH-4 (δ_H 4.29, δ_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 α orientation of CH₂OH-12, OH-4, and CH₂-10 was assigned from the ROESY correlations of H₂-12/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 1−6 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 Kα 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 1−6.

Figure 3. X-ray crystallographic structure of 2.

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)³¹, 6-hydroxy-2-[2-(3-methoxy-4-hydroxyphenyl)ethyl]chromone (11)³³, 6-methoxy-7-hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone (12)³⁴, 6-hydroxy-7-methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone (13)¹⁴, 6,8-dihydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone (14)¹², 8-chloro-2–(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydrochromone (15)³⁵, 8-chloro-2–(2-(4-methoxyphenyl)ethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydrochromone (16)³⁶.

Bioactivity assays, kinetic analysis, and molecular docking simulations

α-Glucosidase inhibitory activity assay, kinetic analysis, and molecular docking study

All the isolated compounds were assessed for the inhibition against α-glucosidase. The results were listed in the Table 3. Compounds 5, 7, 8, and 10 showed inhibitory effect on α-glucosidase (IC₅₀ values ranging from 112.3 ± 4.5 to 524.5 ± 2.7 µM) superior to acarbose (IC₅₀, 743. 4 ± 3.3 µM). Compounds 2, 4, and 9 also possessed inhibitory activity comparable to the positive control.

Table 3. α-Glucosidase inhibitory activities of compounds 1−16^a.

Compound	α-glucosidase inhibitory activity^b
	750 μM (%)	IC50 ± SD (μM)
1	37.4 ± 1.4	N.T.
2	42.0 ± 2.7	N.T.
4	49.9 ± 1.8	N.T.
5	93.4 ± 3.1	112.3 ± 4.5
7	86.9 ± 0.4	407.0 ± 3.3
8	77.3 ± 1.0	524.5 ± 2.7
9	48.9 ± 1.1	N.T.
10	92.1 ± 3.4	218.8 ± 5.6
acarbose^c	50.7 ± 1.2	743. 4 ± 3.3

^aCompounds 3, 6, and 11−16 were inactive toward α-glucosidase at 750 μM (inhibition rate <30%). ^bValues represent means ± SD based on three individual experiments. ^cPositive control. N.T.: not tested.

To elucidate the type of inhibition of isolated sesquiterpenoids with α-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 α-glucosidase inhibitor by binding only with the enzyme-substrate complex³⁷. The inhibition constant was calculated using secondary plot of y intercept versus concentration of inhibitor, yielding K_I value of 168.0 µM (Figure 4(B)).

Figure 4. Kinetics of α-glucosidase inhibition by 5. Lineweaver-Burk plot of 5 against α-glucosidase (A). Secondary plot of y intercept vs. inhibitor for determination of inhibition constant (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 α-glucosidase (Figure. 5) with binding energies of −7.2 and −7.1 kcal/mol, respectively. The docking results revealed that compounds 5 and 10 bound to the entrance part of the active site cavity of α-glucosidase, therefore blocking the release of product (glucose) after the enzyme catalyzed reaction was completed³⁷. This may explain why compound 5 behaved as an uncompetitive inhibitor of α-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 α-glucosidase inhibitor.

Figure 5. Docking analysis of 5 (yellow sticks), 10 (green sticks), and acarbose (red sticks) with α-glucosidase co-crystalized with glucose (cyan sticks). Binding mode of 5, 10, acarbose, and glucose in the active site of α-glucosidase (A). 3D cartoon diagram of the interactions of 5 (B), 10 (C), and acarbose (D) with α-glucosidase.

Tyrosinase inhibitory actvity assay, kinetic analysis, and molecular docking study

2-(2-Phenylethyl)chromones 11−16 as well as sesquiterpenoids 1−10 were assayed for the inhibition toward the tyrosinase. As illustrated in Table 4, Compounds 14 and 13 displayed potent inhibitory activity against tyrosinase with IC₅₀ values of 51.5 ± 0.6 and 89.0 ± 1.7 µM, respectively (kojic acid, 46.1 ± 1.3 µM). Compound 15 also exhibited weak inhibition effect with inhibition rate of 34.8 ± 1.4% at 100 µM. Whereas, compounds 1−12 and 16 were inactive to the tyrosinase up to concentration of 100 µM (inhibition rate <30%).

Table 4. Tyrosinase inhibitory activities of compounds 1−16^a.

compound	tyrosinase inhibitory activity^b
	100 μM (%)	IC50 ± SD (μM)
13	53.4 ± 0.7	89.0 ± 1.7
14	65.0 ± 1.8	51.5 ± 0.6
15	34.8 ± 1.4	N.T.
kojic acid^c	72.5 ± 0.5	46.1 ± 1.3

^aCompounds 1–12, and 16 were inactive against tyrosinase at 100 μM (inhibition rate < 30%). ^bValues represent means ± SD based on three individual experiments. ^cPositive control. N.T.: not tested.

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 tyrosinase²⁵. Thus, compound 14 inhibited the tyrosinase not only by binding with the free enzyme but also with the enzyme − 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 µM and 72.8 µM, respectively, by secondary re-plots, indicating that compound 14 effectively bound to free enzyme as compared to enzyme-substrate complex.

Figure 6. Kinetics of tyrosinase inhibition by 14. Lineweaver-Burk plot of 14 against tyrosinase (A). Secondary re-plot of slope vs. inhibitor for determination of inhibition constant KI (B). Secondary re-plot of y intercept vs. inhibitor for determination of inhibition constant KIS (C).

To further gain insight into the binding mechanism of 2-(2-phenylethyl)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 −7.49 and −7.75 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.

Figure 7. Docking analysis of 13 (green sticks), 14 (yellow sticks), and kojic acid (red sticks) with tyrosinase. Binding mode of 13, 14, and kojic acid with tyrosinase (A). 3D cartoon diagram of the interactions of 14 (B), 13 (C), kojic acid (D) with 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 α-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 α-glucosidase inhibitor and blocked catalytic reaction by interacting with the entrance of active site of α-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 α-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 surface⁶. 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 self-defense mechanism of the agar-producing tree against pests.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This work was supported by the Innovative Research Team Grant of the Natural Science Foundation of Hainan Province (No.2017CXTD020), Major Technology Project of Hainan Province (ZDKJ2016004-03), Central Public-interest Scientific Institution Basal Research Fund for Innovative Research Team Program of CATAS (17CXTD-15), and China Agriculture Research System (CARS-21).