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Abstract

Objectives: To investigate the phytochemical constituents of Vietnamese Dalbergia oliveri and their inhibition of NO production. Methods: The ethyl acetate soluble fraction was subjected to column chromatography using silica gel and Sephadex LH-20 to isolate compounds. The chemical structures of isolated compounds were identified by nuclear magnetic resonance data and comparison with previously reported literature. The anti-inflammation effects of the isolated compounds on the lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells were measured using the Griess reaction. Results: Nine secondary metabolites (1−9) were isolated successfully from the heartwood of D. oliveri. The chemical structures of these compounds were identified as daidzein (1), formononetin (2), 3,7-dihydroxy-4^′-methoxyflavone (3), liquiritigenin (4), 3^′-methoxydaidzein (5), dalbergin (6), butin (7), sativanone (8), and isoliquiritigenin (9). This is the first time that compounds 1, 3, 5−8 have been isolated from D. oliveri. In the NO production inhibition, compounds 7 and 9 exhibited the most potent inhibitory activity, with IC₅₀ values of 7.6 and 11.2 μM, respectively, followed by 3−6, with IC₅₀ values from 19.6 to 28.7 μM. Conclusion: The results suggested that D. oliveri and its natural products might exert anti-inflammatory effects due to their NO-inhibiting actions.

Keywords

flavonoid inflammation NO production cytotoxic RAW264.7 cells

Introduction

Inflammation is a normal protective reaction that occurs following trauma, infection, or tissue injury.¹ In the inflammation process, nitric oxide (NO) is produced and activated inflammatory cells are increased dramatically.² NO is produced by inducible nitric oxide synthases (iNOS) in macrophages, hepatocytes, and renal cells, under the stimulation of LPS, tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), or interferon-gamma (IFN-γ).³ The overproduction of NO by iNOS has been implicated in the pathology of several inflammatory disorders, including septic shock, tissue damage after inflammation, and rheumatoid arthritis.^4–6 Therefore, NO production induced by LPS through iNOS can reflect the degree of inflammation, and a change in the NO level by inhibiting iNOS enzyme activity could be a proxy with which to assess the anti-inflammatory effects of plant extracts.

During the screening program, we found that the ethyl acetate (EtOAc) fraction from the heartwood of Vietnamese Dalbergia oliveri Gamble ex Prain (Fabaceae) exhibited appreciable inhibitory activity in LPS-induced NO production in RAW264.7 cells in vitro. The Dalbergia genus consists of 274 accepted species and is widely distributed in tropical and sub-tropical regions.⁷ Numerous Dalbergia species are used for the treatment of several diseases in the traditional medicine system.^7–9 D. oliveri is a woody climber, and is widely distributed in southeast Asian countries, including in Myanmar, Thailand, Laos, Cambodia, and Vietnam.^10,11 D. oliveri has been used to treat chronic ulcers,¹⁰ and has been reported to possess various pharmacological activities, such as anti-tumor promoting,¹² cytotoxic and anti-fungal,¹³ larvicidal,^14,15 and cell proliferation activity.¹⁶ Previous studies on chemical constituents of D. oliveri showed the presence of isoflavones, isoflavanones, isoflavans, neoflavones, coumestones, coumarins, flavanones, chalcones, and pterocarpans.^12–17 Despite the number of studies, there has been no investigation of chemical constituents and inhibitory activity of NO production of Vietnamese D. oliveri. For that reason, this paper describes the isolation and structural elucidation of the isolated compounds as well as evaluating their inhibitory action on NO production.

Experimental

General experimental procedure

¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were measured on a Bruker Avance 500 MHz spectrometer (Bruker Daltonics, Ettlingen, Germany). Chromatography column (CC) was carried out on silica gel Si 60 F₂₅₄, 40–63 mesh (Merck, St Louis, MO, USA), YMCGEL ODS-A, 12 nm S-150 μm (YMC Co Ltd, Kyoto, Japan), and Sephadex LH-20 (Sigma–Aldrich, MO, USA). Compounds were visualized under UV radiation (254, 365 nm) and by spraying plates with 10% H₂SO₄ followed by heating with a heat gun.

Plant material

The heartwood of D. oliveri was collected at Dak Lak Province, a central province of Vietnam. Botanical identification was performed by Nguyen Quoc Binh. PhD, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology (VAST). A voucher specimen (Cam Lai-CL-Lõi) is deposited with the Natural Product Chemistry Lab, Tay Nguyen University, Vietnam.

Extraction and isolation

The heartwood of D. oliveri (4.0 kg) was reflux extracted with 96% ethanol (EtOH) for 1 h. The extract was then filtered before being evaporated under reduced pressure to give a crude EtOH extract. The EtOH extract (150 g) was then suspended in hot water and partitioned with ethyl acetate (EtOAc) successively. The resulting fraction was concentrated under decreased pressure to give EtOAc (120 g) and water (H₂O) fractions. Following activity-guided fractionation, the EtOAc soluble fraction (120 g) was chromatographic on a silica gel (Kieselgel 60, 230–400 mesh) CC (15 × 50 cm) using a stepwise gradient of CH₂Cl₂-MeOH (100:1 to 0:1, each 3.0 L), to yield 14 fractions (E1–E14) based on their TLC profiles. Fraction E4 (8.2 g) was applied to a silica gel (Kieselgel 60, 230–400 mesh) CC (6 × 50 cm) and eluted with a gradient of n-hexane-EtOAc (5:1 to 1:1) to obtain four fractions (E4.1–4.4). Further purification of sub-fraction E4.3 (800 mg) by Sephadex LH-20 (25 μm particle size) CC (3 × 90 cm), eluting with a gradient of MeOH:H₂O (1:1), yielded compound 1 (15 mg). Fraction E5 (4.5 g) was subjected to silica gel (Kieselgel 60, 230–400 mesh) CC (6 × 40 cm), using n-hexane-acetone (8:1 to 1:1) as the eluent to obtain five sub-fractions (E5.1–5.5). Further purification of E5.3 (500 mg) using an ODS silica gel (5 μm particle size) CC (2 × 40 cm), eluting with a gradient of MeOH:H₂O (1:2 to 2:1), yielded 2 (18 mg), 5 (16 mg), and 8 (23 mg). Fraction E6 (5.1 g) was subjected to silica gel (Kieselgel 60, 230–400 mesh) CC (6 × 40 cm), using CH₂Cl₂-EtOAc (4:1 to 1:1) as the eluent to obtain four sub-fractions (E6.1–6.4). Further purification of E6.2 (820 mg) using an ODS silica gel (5 μm particle size) CC (2 × 40 cm), eluting with a gradient of acetonitrile:H₂O (1:2 to 2:1), yielded 4 (11 mg) and 6 (12 mg). Fraction E7 (7.6 g) was subjected to silica gel (Kieselgel 60, 230–400 mesh) CC (6 × 40 cm), using CH₂Cl₂-acetone (5:1 to 1:1) as the eluent to obtain four sub-fractions (E7.1–7.4). Further purification of E7.3 (950 mg) using an ODS silica gel (5 μm particle size) CC (2 × 40 cm), eluting with a gradient of MeOH:H₂O (1:3 to 1:1) yielded 7 (20 mg) and 9 (14 mg). Fraction E8 (2.5 g) was applied to a silica gel (Kieselgel 60, 230–400 mesh) CC (4 × 40 cm) and eluted with a gradient of CH₂Cl₂-acetone (3:1 to 0:1) to obtain four fractions (E8.1–8.4). Further purification of sub-fraction E8.3 (250 mg) by Sephadex LH-20 CC, eluting with a gradient of MeOH:H₂O (1:1), yielded 3 (10.5 mg).

Daidzein (1) - Pale yellow prisms; ¹H-NMR (500 MHz, DMSO-d₆) δ_H (ppm): 10.76 (1H, br s, 7-OH), 9.50 (1H, br s, 4^′-OH), 8.28 (1H, s, H-2), 7.96 (1H, d, J = 8.5 Hz, H-5), 6.93 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.85 (1H, d, J = 2.5 Hz, H-8), 7.37 (2H, d, J = 8.5 Hz, H-2^′/H-6^′), 6.81 (2H, d, J = 8.5 Hz, H-3^′/H-5^′); ¹³C-NMR (125 MHz, DMSO-d₆) δ_C (ppm): 174.6 (C-4), 162.4 (C-7), 157.1 (C-4^′), 157.4 (C-9), 152.7 (C-2), 130.0 (C-2^′/C-6^′), 127.2 (C-5), 123.4 (C-3), 122.5 (C-1^′), 116.6 (C-10), 114.9 (C-6), 115.1 (C-3^′/C-5^′), 102.1 (C-8).

Formononetin (2) - Colorless needles; ¹H-NMR (500 MHz, DMSO-d₆) δ_H (ppm): 8.33 (1H, s, H-2), 7.97 (1H, d, J = 9.0 Hz, H-5), 6.94 (1H, dd, J = 9.0, 2.0 Hz, H-6), 6.87 (1H, d, J = 2.0 Hz, H-8), 7.49 (2H, d, J = 8.5 Hz, H-2^′/H-6^′), 7.01 (2H, d, J = 8.5 Hz, H-3^′/H-5^′), 3.79 (3H, s, 4^′-OCH₃); ¹³C-NMR (125 MHz, DMSO-d₆) δ_C (ppm): 174.6 (C-4), 162.6 (C-7), 158.9 (C-4^′), 157.4 (C-9), 153.1 (C-2), 130.0 (C-2^′/C-6^′), 127.2 (C-5), 124.2 (C-1^′), 123.1 (C-3), 116.5 (C-10), 115.2 (C-6), 113.6 (C-3^′/C-5^′), 102.8 (C-8), 55.1 (4^′-OCH₃).

3,7-dihydroxy-4^′-methoxyflavone (3) - Yellow powder; ¹H-NMR (500 MHz, DMSO-d₆) δ_H (ppm): 7.97 (1H, d, J = 9.0 Hz, H-5), 6.94 (1H, dd, J = 9.0, 2.0 Hz, H-6), 6.86 (1H, d, J = 2.0 Hz, H-8), 7.50 (2H, d, J = 8.5 Hz, H-2^′/H-6^′), 6.98 (2H, d, J = 8.5 Hz, H-3^′/H-5^′), 3.78 (3H, s, 4^′-OCH₃); ¹³C-NMR (125 MHz, DMSO-d₆) δ_C (ppm): 175.1 (C-4), 163.0 (C-7), 159.4 (C-4^′), 157.9 (C-9), 153.6 (C-2), 130.5 (C-2^′/C-6^′), 127.8 (C-5), 124.7 (C-3), 123.6 (C-1^′), 117.1 (C-10), 115.7 (C-6), 114.1 (C-3^′/C-5^′), 102.6 (C-8), 55.6 (4^′-OCH₃).

Liquiritigenin (4) - Yellow amorphous powder; ¹H-NMR (500 MHz, DMSO-d₆) δ_H (ppm): 7.64 (1H, d, J = 8.5 Hz, H-5), 7.32 (2H, d, J = 8.5 Hz, H-2^′/H-6^′), 6.79 (2H, d, J = 8.5 Hz, H-3^′/H-5^′), 6.50 (1H, dd, J = 2.0, 8.5 Hz, H-6), 6.33 (1H, d, J = 2.0 Hz, H-8), 5.43 (1H, dd, J = 3.0, 13.0 Hz, H-2), 3.20 (1H, dd, J = 13.0, 17.0 Hz, H-3_ax), 2.62 (1H, dd, J = 3.0, 17.0 Hz, H-3_eq); ¹³C-NMR (125 MHz, DMSO-d₆) δ_C (ppm): 190.0 (C-4), 164.6 (C-7), 163.2 (C-9), 157.6 (C-4^′), 129.3 (C-1^′), 128.4 (C-5), 128.2 (C-2^′/C-6^′), 115.1 (C-3^′/C-5^′), 114.1 (C-10), 110.5 (C-6), 102.5 (C-8), 78.9 (C-2), 43.1 (C-3).

3^′-methoxydaidzein (5) - Pale yellow plates; ¹H-NMR (500 MHz, DMSO-d₆) δ_H (ppm): 8.27 (1H, s, H-2), 7.96 (1H, d, J = 8.5 Hz, H-5), 6.93 (1H, dd, J = 8.5, 2.0 Hz, H-6), 6.85 (1H, d, J = 2.0 Hz, H-8), 7.49 (1H, d, J = 2.0 Hz, H-2^′), 6.92–6.94 (2H, overlap, H-5^′/H-6^′), 3.79 (3H, s, 3^′-OCH₃), 8.99 (1H, s, 4^′-OH); ¹³C-NMR (125 MHz, DMSO-d₆) δ_C (ppm): 174.5 (C-4), 162.5 (C-7), 157.3 (C-9), 157.4 (C-9), 152.9 (C-2), 147.5 (C-3^′), 146.0 (C-4^′), 127.2 (C-5), 124.7 (C-3), 123.3 (C-1^′), 119.6 (C-6^′), 116.6 (C-10), 116.4 (C-5^′), 115.1 (C-6), 111.9 (C-2^′), 102.0 (C-8), 55.6 (3^′-OCH₃).

Dalbergin (6) - Yellow prisms; ¹H-NMR (500 MHz, Methanol-d₄) δ_H (ppm): 7.57 (3H, m, H-3^′/H-4^′/H-5^′), 7.51 (2H, m, H-2^′/H-6^′), 7.06 (1H, s, H-8), 6.88 (1H, s, H-5), 6.21 (1H, s, H-3), 3.99 (3H, s, 7-OCH₃); ¹³C-NMR (125 MHz, Methanol-d₄) δ_C (ppm): 163.8 (C-2), 158.2 (C-4), 153.6 (C-7), 150.4 (C-9), 145.2 (C-6), 137.1 (C-1^′), 130.8 (C-4^′), 129.9 (C-3^′/C-5^′), 129.5 (C-2^′/C-6^′), 112.3 (C-3), 111.9 (C-5), 113.1 (C-10), 101.1 (C-8), 56.8 (7-OCH₃).

Butin (7) - Yellow amorphous powder; ¹H-NMR (500 MHz, DMSO-d₆) δ_H (ppm): 7.63 (1H, d, J = 9.0 Hz, H-5), 6.88 (1H, s, H-2^′), 6.74 (2H, d, J = 8.5 Hz, H-5^′/H-6^′), 6.49 (1H, dd, J = 2.0, 9.0 Hz, H-6), 6.32 (1H, d, J = 2.0 Hz, H-8), 5.37 (1H, dd, J = 3.0, 12.5 Hz, H-2), 3.02 (1H, dd, J = 12.5, 17.0 Hz, H-3_ax), 2.62 (1H, dd, J = 3.0, 17.0 Hz, H-3_eq); ¹³C-NMR (125 MHz, DMSO-d₆) δ_C (ppm): 193.5 (C-4), 164.5 (C-9), 163.0 (C-7), 145.5 (C-4^′), 145.1 (C-3^′), 129.8 (C-1^′), 128.3 (C-5), 117.8 (C-6^′), 115.3 (C-5^′), 114.2 (C-2^′), 113.5 (C-10), 110.4 (C-6), 102.5 (C-8), 78.9 (C-2), 43.2 (C-3).

Sativanone (8) - White amorphous powder; ¹H-NMR (500 MHz, Acetone-d₆) δ_H (ppm): 7.77 (1H, d, J = 9.0 Hz, H-5), 7.01 (1H, d, J = 8.5 Hz, H-6^′), 6.58 (2H, overlap, H-3^′/H-5^′), 6.48 (1H, dd, J = 9.0, 2.5 Hz, H-6), 6.40 (1H, d, J = 2.0 Hz, H-8), 4.56 (1H, t, J =11.0 Hz, H-2_ax), 4.45 (1H, dd, J = 11.0, 5.5 Hz, H-2_eq), 4.17 (1H, dd, J = 11.0, 5.5 Hz, H-3), 3.79 (3H, s, 2^′-OCH₃), 3.78 (3H, s, 4^′-OCH₃); ¹³C-NMR (125 MHz, Acetone-d₆) δ_C (ppm): 190.7 (C-4), 164.5 (C-7), 164.3 (C-9), 161.1 (C-4^′), 159.1 (C-2^′), 131.2 (C-5), 129.7 (C-6^′), 117.0 (C-1^′), 115.6 (C-10), 110.8 (C-3^′), 105.3 (C-5^′), 103.1 (C-6), 99.2 (C-8), 71.4 (C-2), 55.6 (4^′-OCH₃), 55.2 (2^′-OCH₃), 47.6 (C-3).

Isoliquiritigenin (9) - Yellow amorphous powder; ¹H-NMR (500 MHz, DMSO-d₆) δ_H (ppm): 8.14 (1H, d, J = 8.5 Hz, H-6^′), 7.74 (4H, ovelap, H-7/H-8/H-2/H-6/), 6.83 (2H, d, J = 8.5 Hz, H-3/H-5), 6.39 (1H, dd, J = 2.0, 8.5 Hz, H-5^′), 6.26 (1H, d, J = 2.0 Hz, H-3^′); ¹³C-NMR (125 MHz, DMSO-d₆) δ_C (ppm): 191.4 (C-9), 165.8 (C-4^′), 165.2 (C-2^′), 160.3 (C-4), 144.2 (C-7), 132.8 (C-6^′), 131.2 (C-2/C-6), 125.7 (C-1), 117.4 (C-8), 115.8 (C-3/C-5), 112.9 (C-1^′), 108.2 (C-5^′), 102.6 (C-3^′).

Biological assay

Cell culture, cell viability assay and determination of NO production were performed according to the methods previously described.^18,19

Statistical analysis

The NO production inhibitory activity assay was performed in triplicate. The results are presented as the means ± standard error of the mean.

Results

Chemical structure identification

The ethanol extract was separated with EtOAc to obtain EtOAc soluble fraction, which was then subjected to column chromatography using silica gel and Sephadex LH-20 to obtain nine secondary metabolites (1–9) (Figure 1 and Supplemental data).

Figure 1.

Structure of compounds 1−9 from D. oliveri.

The ¹H-nuclear magnetic resonance (NMR) spectrum of compounds 1, 2, and 5 exhibited typical signals due to aromatic protons of 2 benzene rings, together with those of hydroxyl (C-7 and C-4^′) and methoxy groups, while their ¹³C-NMR spectrum revealed the signals of oxygen-substituted carbons, and 3 quaternary carbons (C-3, C-10, and C-1^′) (Figure 1 and Supplemental data). The presence of an olefinic group [δ_H 8.27–8.33 (H-2)/δ_C 152.7–153.1 (C-2), and δ_C 123.1–124.7 (C-3)] and a ketone carbon δ_C 174.5–174.6 (C-4) in the ¹H- and ¹³C-NMR spectra indicated 1, 2, and 5 to be isoflavones.^16,20 Comprehensive analysis of the ¹H- and ¹³C-NMR spectra of 1 showed that 1 possessed 7 aromatic proton signals and four oxygenated carbons (C-2, C-7, C-9, and C-4^′). Similar to 1, compound 2 also possessed four oxygenated carbons but showed six aromatic protons and a methoxy carbon at C-4^′. Compound 5 also possessed six aromatic protons but showed five oxygenated carbons (C-2, C-7, C-9, C-3^′, and C-4^′), and a methoxy carbon at C-3^′ (Figure 1 and Supplemental data). Comparison of the ¹H- and ¹³C-NMR data of these compounds with those published in the literature led to the structures of 1, 2, and 5 being identified as daidzein,²¹ formononetin,²⁰ and 3-methoxydaidzein,²² respectively. This is the first report of daidzein (1) and 3-methoxydaidzein (5) from Vietnamese D. oliveri.

Compound 3 was obtained as a yellow powder. The ¹H-NMR spectrum of 3 showed signals of a trisubstituted benzene ring [δ_H 7.97 (H-5), 6.94 (H-6), and 6.86 (H-8)], an A₂B₂ spin system [δ_H 7.50 (H-2^′/H-6^′) and 6.98 (H-3^′/H-5^′)], and a methoxy group [δ_H 3.78 (4^′-OCH₃)] (Figure 1). The ¹³C-NMR spectrum of 3 exhibited 16 carbon signals attributable to twelve sp² carbons of two benzene rings, and a methoxy carbon [δ_C 55.6 (4^′-OCH₃)]. A ketone at δ_C 175.1 (C-4), and two oxymethine carbons [δ_C 153.6 (C-2), and δ_C 124.7 (C-3)] in the ¹³C-NMR spectrum indicated 3 to be a flavonol.²³ (Figure 1). Based on this evidence, and in comparison with the published data, 3 was identified as 3,7-dihydroxy-4^′-methoxyflavone.²³ This is the first time that 3,7-dihydroxy-4^′-methoxyflavone (3) has been recognized in D. oliveri.

Both compounds 4 and 7 were obtained as yellow amorphous powder. Their ¹H-NMR spectra displayed characteristic signals of aromatic protons of two benzene rings, an oxymethine (H-2) and methylene (2H-3), while their ¹³C-NMR spectra revealed the signals of a ketone (C-4), an oxymethine carbon (C-2), a methylene carbon (C-3), and twelve sp² carbons of two benzene rings (Figure 1). The above observation indicated that these compounds were flavanone derivatives.^24,25 Detailed analysis of the ¹H- and ¹³C-NMR spectra revealed that 4 possessed seven protons of two benzene rings, an oxymethine [δ_H 5.43 (H-2)/δ_C 78.9 (C-2)], a methylene [δ_H 3.20 and 2.62 (H-3)/δ_C 43.1 (C-3)], and a ketone carbon at δ_C 190.0 (C-4). Compound 7 also possessed one ketone carbon (C-4), an oxymethine (C-2), and methylene (C-3), but showed only six aromatic protons and one additional hydroxy group (C-3^′) (Figure 1 and Supplemental data). Following comparison of the ¹H- and ¹³C-NMR data of these compounds with the published literature, 4 and 7 were identified to be liquiritigenin²⁴ and butin,²⁵ respectively. This is the first time that butin (7) has been identified from D. oliveri.

Compound 6 was obtained as yellow prisms. The presence of an olefinic group [δ_H 6.21 (H-3)/δ_C 112.3 (C-3), and δ_C 158.2 (C-4)] and a carboxyl carbon at δ_C 163.8 (C-2) in the ¹H- and ¹³C-NMR spectra indicated 6 to be 4-aryl-coumarin.²⁶ Detailed analysis of the ¹H- and ¹³C-NMR spectra of compound 6 revealed that 6 possessed a methoxy group [δ_H 3.99 (7-OCH₃)/δ_C 56.8 (7-OCH₃)], seven aromatic protons, and twelve sp² carbons of two benzene rings (Figure 1). Thus, compound 6 was identified as dalbergin.²⁶

Compound 8 was isolated as a white amorphous powder. The ¹H-NMR spectrum of 8 showed signals of two trisubstituted benzene rings [δ_H 7.77 (H-5), 7.01 (H-6^′), 6.58 (H-3^′/H-5^′), 6.48 (H-6), and 6.40 (H-8)], and two methoxy groups [δ_H 3.79 (2^′-OCH₃), and 3.78 (4^′-OCH₃)]. The ¹³C-NMR spectrum showed 17 carbon signals due to twelve sp² carbons of two benzene rings, and two methoxy carbons [δ_C 55.6 (4^′-OCH₃), and 55.2 (2^′-OCH₃)]. The presence of an oxygenated methylene [δ_H 4.45 and 4.56 (H-2)/δ_C 71.4 (C-2)], a methine [δ_H 4.17 (H-3)/δ_C 47.6 (C-3)], and a carbonyl carbon at δ_C 190.7 (C-4) in the ¹H- and ¹³C-NMR spectra indicated 8 to be an isoflavanone (Figure 1 and Supplemental data).²⁰ Based on the above evidence, 8 was identified as sativanone.²⁰ It is worthwhile to note that sativanone (8) was identified for the first time from D. oliveri.

Compound 9 was obtained as a yellow amorphous powder. The presence of a trans-olefinic group [δ_H 7.74 (H-7/H-8)/δ_C 144.2 (C-7), and δ_C 117.4 (C-8)] and a carbonyl carbon at δ_C 191.4 (C-9) in the ¹H- and ¹³C-NMR spectra indicated 9 to be a chalcone.^27,28 Detailed analysis of the ¹H- and ¹³C-NMR spectra of 9 revealed a 1,2,4-trisubstituted benzene ring characterized by an ABX system and a 1,4-disubstituted benzene ring characterized by an A₂B₂ system, seven aromatic protons, and twelve sp² carbons of two benzene rings.^27,28 Thus, 9 was identified as isoliquiritigenin by comparison of the NMR data with published literature.^27,28

Anti-inflammation activity

In the preliminary test, a cytotoxic assay was performed to define the safe and non-toxic concentration of isolated compounds (1−9) in RAW 264.7 cells using MTT assay. Nontoxicity of the isolated compounds was indicated by over 90% of cell viability¹⁸; however, at a concentration of 100 μM, most of these compounds were toxic to RAW 264.7 cells (Figure 2). Consequently, a concentration range from 1 to 30 μM was chosen for further study (Figure 2).

Figure 2.

Effect of compounds 1−9 on cell viability by LPS stimulation. Cell viability was determined by MTS assay and expressed as a percentage of the control without the addition of indicated 1−9.

To check for NO production inhibitory activity, the RAW 264.7 cells were treated with isolated compounds (1–30 μM), and the level of NO production was quantified using the Griess reaction.¹⁹ As the results in Table 1 demonstrate, butin (7) showed the strongest inhibitory effect on NO production among the isolates, with an IC₅₀ value of 7.6 ± 0.3 μM, followed by isoliquiritigenin (9), which exhibited inhibitory effects with an IC₅₀ value of 11.2 ± 0.4 μM. Compounds 3−6 showed moderate inhibition of NO production, with IC₅₀ values of 24.8 ± 1.9, 19.6 ± 1.6, 27.2 ± 0.4, and 28.7 ± 0.6 μM, respectively, while 1, 2, and 8 were inactive (IC₅₀ > 30 μM). Celastrol, a positive inhibitor, significantly inhibited LPS-induced NO production, with an IC₅₀ value of 1.0 ± 0.1 μM.¹⁹

Table 1.

NO production inhibition in RAW264.7 cells of isolated compounds.

Compounds	IC₅₀ value (μM)^a
1	>30
2	>30
3	24.8 ± 1.9
4	19.6 ± 1.6
5	27.2 ± 0.4
6	28.7 ± 0.6
7	7.6 ± 0.3
8	>30
9	11.2 ± 0.4
Celastrol^b	1.0 ± 0.1

^aThe inhibitory effects are represented as the molar concentration (μM) giving 50% inhibition (IC₅₀) relative to the vehicle control. Values are mean ± SD (n = 3).

^bPositive control.

Neither LPS nor the samples were added to the control group. Thus, the inhibitory effects of these compounds on NO production were not attributable to any cytotoxic effect. After LPS (1 μg/mL) stimulation, NO production increased approximately 12-fold after 24 h in the control. Compounds 7 and 9 reduced NO production 24 h after LPS stimulation in a dose-dependent manner (Figure 3).

Figure 3.

Inhibitory effect of 7 and 9 on the LPS-induced NO production in RAW 264.7 cells. RAW 264.7 cells were pretreated with 7 and 9 (1–30 μM) for 1 h, then with LPS (1 μg/mL), and incubated for 24 h. Control values were obtained in the absence of LPS and compound. The data are expressed as the mean ± SD (n = 3).

Discussion

Lipopolysaccharide inflammation stimuli, known as lipoglycans or endotoxins, stimulate macrophages to induce the expression of iNOS protein to produce NO. NO has been found to be an inflammatory mediator by acting as an activator of macrophages to kill microorganisms through signal transduction.²⁹ On the other hand, the overproduction of NO in the immune system might cause immune hypersensitivity reactions that are subsequent to tissue or cell injury.¹⁹ Thus, the inhibition of iNOS activity or downregulation of iNOS expression could be beneficial to inhibit inflammatory responses. From our results, the flavanone butin (7) was the strongest inhibitor of NO production (IC₅₀ = 7.6 ± 0.3 μM) among the isolated compounds, presumably because of the presence of the 3,4-hydroxylation(s) in the benzene ring (Figure 1), and this result was similar to those previously reported.¹⁹ Meanwhile, the inhibitory activities on NO production of 3−6 were significantly reduced (IC₅₀ values of 24.8, 19.6, 27.2, and 28.7 μM, respectively) and 1, 2, and 8 were inactive, presumably because of the lack of 3-hydroxylation (1 and 4) of the benzene ring or the presence of the methoxy groups (2, 3, 5, 6, and 8) in the benzene ring.^19–21 Cuong et al. also tested the effects of the chalcone derivatives from Caesalpinia sappan on NO production. Their in vitro results indicated that chalcone derivatives as 3-deoxysappanchalcone inhibited NO production from LPS-activated RAW 264.7 cells with an IC₅₀ value of 8.1 ± 0.9 μM.¹⁹ This compound revealed the presence of the conjugated double bond of the olefinic group with carbonyl carbon in the structures. In our experiment, isoliquiritigenin (9), a derivative of chalcone, also exhibited NO production, with an IC₅₀ value of 11.2 ± 0.4 μM, presumably because of the presence of the conjugated double bond of the olefinic group with carbonyl carbon in 9 (Figure 1). Our obtained data suggested that phenolic compounds, particularly the flavanones bearing the 3,4-hydroxylation(s) of the benzene ring or chalcones bearing the double bond conjugated with carbonyl carbon, could be considered as new lead compounds for the development of agents against NO production.

Formononetin (2), an isoflavone, has been found to regulate inflammatory responses and lipid metabolism by reducing the expression of enzymes such as COX-2,^30,31 inhibit neuroinflammation, improve cerebral vascular angiogenesis in human umbilical vein endothelial cells and increase the expression of estrogen receptor beta protein in BV2 microglia,³¹ and restore the function of the epithelial barrier, resulting in the inhibition of cytokines derived from E-cadherin and the production of thymic stromal lymphopoietin.³⁰ Liquiritigenin (4) and isoliquiritigenin (9) have previously been isolated from D. oliveri¹⁶ and were found to suppress the LPS (1 μg/mL)-induced iNOS expression by inhibiting NF-κB/IκBa activation in RAW264.7 macrophages in a dose-dependent manner.³² In the same study, these compounds suppressed Ca-stimulated synthesis of Th2 cytokine and levels of IL-4 and IL-5 in D10 cell culture media supernatants in a concentration-dependent manner without affecting cell viability, and significantly blocked NO levels in the presence of IL-1β without cytotoxicity.³² Furthermore, liquiritigenin (4) and isoliquiritigenin (9) have been found to reduce both iNOS mRNA and protein levels in primary rat hepatocytes and to downregulate TNF-α and IL-6 mRNA levels.³² However, the anti-inflammatory activities against the LPS-induced NO production of isolated compounds 1−3 and 5−8 have not been investigated to date. Our study showed that compounds 3−7 and 9 potentially inhibited NO production activity, with IC₅₀ values ranging from 7.6 to 28.7 μM, respectively.

In this study, we obtained some positive results, such as compounds 1, 3, 5–8, which were isolated from Vietnamese D. oliveri for the first time. The anti-inflammatory activity of all isolated compounds was evaluated. However, only the inhibitory activity of NO production was assessed, while other inflammatory agents such as prostaglandin E2 (PGE2) or proinflammatory agents such as IL (s), TNF-α, and IFN-γ were not evaluated. Moreover, this study did not carry out the inhibitory property on a molecular level, such as via western blotting experiments to evaluate the ability to inhibit iNOS and cyclooxygenase-2 (COX-2), from which the anti-inflammatory mechanism can be proposed. The main reason for some of these limitations is a lack of experimental facilities and equipment. However, we will try to overcome the above limitations to carry out further in-depth studies, and publish the obtained results in the near future.

Conclusion

In conclusion, based on a bioassay-guided extraction and isolation, nine natural compounds including daidzein (1), formononetin (2), 3,7-dihydroxy-4^′-methoxyflavone (3), liquiritigenin (4), 3^′-methoxydaidzein (5), dalbergin (6), butin (7), sativanone (8), and isoliquiritigenin (9) were isolated from the heartwood of Vietnamese D. oliveri. This is the first time that compounds 1, 3, and 5−8 have been identified from D. oliveri. Butin A (7) and isoliquiritigenin (9) showed the most potent inhibitory activities against the LPS-induced NO production. These results suggest that the active constituents from D. oliveri can be useful for the research and development of inflammatory agents.

Supplemental Material

Supplemental Material - Anti-inflammatory activity of ingredients from the heartwood of Vietnamese Dalbergia oliveri Gamble ex Prain

Supplemental Material for Anti-inflammatory activity of ingredients from the heartwood of Vietnamese Dalbergia oliveri Gamble ex Prain by Ngu Truong Nhan, Phi Hung Nguyen, Dam Thi Bich Hanh, Dang Thi Thuy My, Nguyen Phuong Dai Nguyen, Truong Ba Phong, Nguyen Thi Mai Huong, Manh Hung Tran, Nguyen Thị Thu Tram and Dao Cuong To in European Journal of Inflammation

Footnotes

Author’s contributions

PHN, NPDN, NTTT designed and planned the experiments. NTN, DTBH, DTTM, TBP, NTMH implemented the experiments. MHT, DCT investigated the data and wrote the manuscript. All the authors agreed the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Dao Cuong To

Supplemental Material

Supplemental material for this article is available online.

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