A New Aurone and Other Constituents From the Seeds of Psoralea corylifolia With Their Diacylglycerol Acyltransferase Inhibitory Activity

Abstract

A new aurone (1), together with 5 known compounds (2-6), were isolated from EtOAc-soluble extract of the seeds of Psoralea corylifolia. Their structures were elucidated on the basis of spectroscopic methods. All compounds were evaluated for in vitro inhibitory activity against diacylglycerol acyltransferase (DGAT). Among them, compound 1 showed selective inhibitory activity on DGAT1 with IC₅₀ value of 54.9 ± 1.4 µM.

Diacylglycerol acyltransferase (DGAT) is a key enzyme in triacylglycerol (TG) synthesis, which catalyzes the final step of the TG synthesis pathway. ¹ There are 2 isoforms of DGAT including DGAT1 and DGAT2, and lots of studies showed that DGAT1 is responsible for the synthesis of TG. ² Excessive accumulation of TG can lead to obesity. Therefore, inhibiting the activity of DGAT1 is a promising therapy to treat obesity.

Psoralea corylifolia L. (Leguminosae), an important Chinese folk medicine, is distributed widely in Southeast Asia. The seeds of P. corylifolia are traditionally used to treat bacterial infections, ³ osteoporosis, ³ asthma, ⁴ spermatorrhea, ³ and gynecological bleeding. ⁵ Published studies have shown that P. corylifolia has a large number of biological constituents including flavonoids, ⁶ meroterpenes, ⁷ and coumarins ⁸ with reported biological activities such as antioxidant, ⁹ anti-inflammatory, ¹⁰ and antibacterial ¹¹ properties.

During the course of our search for DGAT inhibitors from P. corylifolia, a new aurone was isolated from the EtOAc extract of the seeds of P. corylifolia, together with 5 known compounds (2-6). Herein, we report the isolation and structural elucidation of the new aurone and the evaluation of DGAT inhibitory activity.

Compound 1 was obtained as a yellow amorphous powder, and the molecular formula was established as C₂₁H₂₀O₅ based on HREIMS at m/z 352.1307 for the [M]⁺. The IR (KBr) spectrum showed absorptions attributable to hydroxyl (3415 cm⁻¹), carbonyl (1742 cm⁻¹) groups, and olefinic bond (1658 cm⁻¹). The ¹H nuclear magnetic resonance (NMR) spectrum (Table 1, Supplementary Material 1) of 1 showed a set of ABX spin system at δ _H 7.60 (1H, d, J = 2.0 Hz, H-2′), 7.40 (1H, dd, J = 8.0, 2.0 Hz, H-6′), 6.94 (1H, d, J = 8.0 Hz, H-5′); a set of AX spin system at δ _H 7.44 (1H, d, J = 8.5 Hz, H-4), 6.81 (1H, d, J = 8.5 Hz, H-5); an olefinic proton at δ _H 6.62 (1H, s, H-10); a prenyl group at δ _H 3.55 (2H, d, J = 7.5 Hz, H-1″), 5.38 to 5.42 (1H, m, H-2″), 1.87 (3H, s, H-4″), and 1.68 (3H, s, H-5″); and a methoxy proton at δ _H 3.81 (3H, s). The ¹³C NMR spectrum of 1 revealed the presence of 21 carbons including 2 sets of aromatic rings and a prenyl group. The above NMR data indicated that 1 is most likely a prenylated aurone. Compared with the NMR data of licoagroaurone, it could also be further confirmed. ¹² In addition, the position of prenyl group could be deduced by the key HMBC corrections of H-1″ (δ _H 3.55) with C-2″ (δ _C 122.3), C-3″ (δ _C 132.9), and C-7 (δ _C 113.2). The HMBC spectrum also showed key correlations of δ _H 3.81 (H-1″) to 166.8 (C-6), which revealed that the methoxy was linked at C-6 (Figure 1). The Z-stereochemistry of the double bond at C-10 in 1 was determined by the chemical shift of C-10 (δ _C 112.1). On the basis of the above data, the structure of 1 was elucidated as (2Z)-2-[(3′,4′-dihydroxyphenyl)methylene]-6-methoxy-7-prenyl-3(2H)-benzofurane.

Table 1

NMR Data of Compound 1 in Acetone-D ₆ (¹H: 500 MHz, ¹³C: 125 MHz).

Position	δ C	δ H (multiplicity)
2	147.6
3	183.1
4	125.8	7.44 (1H, d, 8.5)
5	113.0	6.81 (1H, d, 8.5)
6	166.8
7	113.2
8	163.9
9	115.1
10	112.1	6.62 (1H, s)
1′	123.5
2′	118.9	7.60 (1H, d, 2.0)
3′	146.3
4′	148.3
5′	116.6	6.94 (1H, d, 8.0)
6′	125.6	7.40 (1H, dd, 8.0, 2.0)
1″	22.7	3.55 (2H, d, 7.5)
2″	122.3	5.38-5.42 (1H, m)
3″	132.9
4″	18.2	1.87 (3H, s)
5″	18.2	1.68 (3H, s)
OCH3	56.3	3.81 (3H, s)

Figure 1

The key HMBC correlations of compound 1.

Moreover, 5 known compounds (2-6) were obtained from P. corylifolia and identified as bavachromene, ^2,13 isobavachalcone, ^3,14 (2S)-5,7-dihydroxy-4′-methoxy-8-prenylflavanone, ^4,15 corylifol D, ^5,14 and corylifol E ^6,14 by comparing their NMR data with those in literatures (Figure 2).

Figure 2

Structures of compounds 1 to 6.

All the isolates were evaluated for their inhibitory activity against DGAT1 and DGAT2, and the known DGAT inhibitor, kuraridine, was used as the positive control. ¹⁶ The result showed 6 compounds inhibited DGAT1 activity with IC₅₀ values ranging from 54.9 ± 1.4 to 124.3 ± 1.1 µM (Table 2). Among these isolates, compound 1 showed the highest inhibitory activity on DGAT1 with IC₅₀ value of 54.9 ± 1.4 µM. Furthermore, 6 (IC₅₀ = 70.9 ± 1.2 µM) displayed high inhibitory activity on DGAT1 compared with 5 (IC₅₀ = 93.1 ± 1.0 µM), which indicated that the DGAT1 inhibitory activity might be associated with the hydroxyl group substituted at C-1″. Moreover, 6 also showed moderate inhibitory activity on DGAT2 with IC₅₀ value of 124.1 ± 1.1 µM, suggesting that C-1″-OH might be playing a role in inhibiting DGAT2.

Table 2

Inhibitory Effects of Compounds 1 to 6 (MM) on DGAT Activity (Expressed as IC₅₀ Values).

Compound	IC50 a
	DGAT1	DGAT2
1	54.9 ± 1.4	>200
2	124.3 ± 1.1	151.7 ± 1.2
3	63.1 ± 1.2	>200
4	85.5 ± 0.9	>200
5	93.1 ± 1.0	>200
6	70.9 ± 1.2	124.1 ± 1.1
Kuraridine b	10.4 ± 0.8	19.3 ± 0.9

^aIC₅₀ values were determined by regression analyses and expressed as means ± SD of 3 replicates.

^bPositive control.

Experimental

General Experimental Procedures

Optical rotations were determined on a Horiba SEPA-300 polarimeter (Horiba Scientific, Kyoto, Japan). UV spectra were recorded in MeOH using Shimadzu UV-2401PC spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra were recorded with a JASCO FT-IR 620 spectrophotometer. NMR spectra were obtained from Bruker Avance-III 600, DRX-500, and AM-400 MHz spectrometers (Bruker BioSpin GmBH, Rheinstetten, Germany) using TMS as the internal standard. Mass spectra were obtained on a QTOF2 high-resolution mass spectrometer (Micromass, Wythenshawe, UK). Column chromatography was conducted using silica gel 60 (200 μm particle size, Yantai Xinde Chemical Co., Ltd, Yantai, China) and RP-18 (20, 45 μm particle size, Fuji Silysia Chemical Ltd., Kasugai, Japan). For thin-layer chromatography (TLC), precoated TLC silica gel 60 F₂₅₄ plates from Merck were used. HPLC was carried out using a Waters 2535Q HPLC system fitted with a 2998 photodiode array detector (Waters, USA).

Plant Material

The seeds of P. corylifolia were collected in Guangyuan, Sichuan province, Republic of China, and authenticated by Professor Lei Zhang (College of Pharmacy, Sichuan University). A voucher specimen of the plant (No. 20160425) was deposited at Chengdu University of Traditional Chinese Medicine, Sichuan, China.

Extraction and Isolation

The dried seeds of P. corylifolia (5.0 kg) were extracted with 95% EtOH aqueous at room temperature for 3 days and then concentrated under vacuum at 45°C to obtain a residue (812.3 g). This extract was suspended in H₂O, partitioned successively with petroleum ether, EtOAc, and n-BuOH to afford petroleum ether-, EtOAc-, and n-BuOH-soluble fractions, respectively. Part of the EtOAc-soluble fraction (100.0 g) was chromatographed over a silica gel column using a gradient of CH₂Cl₂/MeOH (from 0:1, 200:1, 100:1, 50:1, 25:1, 10:1, 5:1, 3:1 to 0:1) and was separated into 18 fractions (Fr.1-Fr.18). Fr.6 (12.1 g) was subjected to silica gel column eluted with CH₂Cl₂-MeOH (from 0:1, 100:1, 50:1, 10:1, 5:1 to 0:1) to get 8 fractions (Fr.6.1-Fr.6.8). Fr.6.5 (3.5 g) was subjected to RP-18 column (40.0 × 3.5 cm) and eluted with MeOH/H₂O (3:7 to 10:0) to afford 10 fractions. Further purification of Fr.6.5.3 (851.2 mg) was subjected to RP-18 column, eluted with a stepwise gradient of MeOH/H₂O (4:6 to 6:4) to afford 12 subfractions (Fr.6.5.3.1-Fr.6.5.3.12). After that, Fr.6.5.3.5 (120.1 mg) was separated by HPLC, using a gradient solvent system 50% to 70% MeOH in H₂O over 80 minutes, yielded compounds 5 (21.4 mg, t _R = 53.7 minutes) and 2 (17.5 mg, t _R = 71.2 minutes). Further purification of Fr.6.5.3.6 (103.4 mg) via semipreparative HPLC using a gradient solvent system of 50% to 55% MeOH in H₂O over 60 minutes yielded compounds 1 (10.9 mg, t _R = 42.2 minutes) and 3 (8.4 mg, t _R = 55.3 minutes). Fr.5.6.3.9 (82.7 mg) was purified via preparative HPLC using an isocratic solvent system of 50% MeOH in H₂O over 90 minutes yielded compounds 6 (14.8 mg, t _R = 48.2 minutes) and 4 (15.2 mg, t _R = 75.2 minutes).

(2Z)-2-[(3′,4′-Dihydroxyphenyl)methylene]-6-Methoxy-7-Prenyl-3(2H)-Benzofurane (1)

Yellow amorphous powder.

IR (KBr) ν _max: 3415, 1752, 1658, 1604 cm⁻¹.

UV (MeOH) λ _max (nm) (log ε): 255 (4.01), 397 (4.38).

¹H (500 MHz) and ¹³C NMR (125 MHz) in acetone-d ₆: Table 1.

HREIMS m/z: 352.1307 [M]⁺ (calcd for C₂₁H₂₀O₅, 352.1311).

DGAT1 and DGAT2 Assays

DGAT activity assays were carried out as described previously. ¹⁷ Briefly, DGAT activity in total membranes prepared from DGAT2- or DGAT1-overexpressing Sf-9 and HEK293 Tet-on cells was determined by measuring the formation of [¹⁴C]-TG from [¹⁴C]-oleoyl CoA. The reaction mixture for the DGAT1 assay contained 175 mM Tris (pH 7.5), 100 mM MgCl₂ (5 mM MgCl₂ for DGAT2 assay), 200 µM sn-1,2-diacylglycerol, 20 µM [1-¹⁴C]-oleoyl CoA (5.5 µCi), 2 mg/mL BSA, and 32 µg of the membrane protein. The mixture was incubated for 20 minutes at 37°C and then the reaction was stopped by the addition of 1.5 mL of stop solution [2-propanol-heptane-water (80:20:2, v/v/v)] and vortexed with 1.0 mL of heptane and 0.5 mL of water. The top heptane phase was collected and washed with 2.0 mL alkaline ethanol solution [ethanol-0.5 N NaOH-water (50:10:40, v/v/v)]. The radioactivity of the top phase was determined by liquid scintillation counting (ALOKA LSC-8000, Aloka, Inc., Tokyo, Japan).

Supplemental Material

Supporting Information - Supplemental material for A New Aurone and Other Constituents From the Seeds of Psoralea corylifolia With Their Diacylglycerol Acyltransferase Inhibitory Activity

Supplemental material, Supporting Information, for A New Aurone and Other Constituents From the Seeds of Psoralea corylifolia With Their Diacylglycerol Acyltransferase Inhibitory Activity by Bo Ren, Jia-Yue Dong, and Ying Liu in Natural Product Communications