Anti-inflammatory Flavanones and Flavanols from the Roots of Pongamia pinnata

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Computational methods
• Cell culture, viability assay, and measurement of nitric oxide production
• References

Abstract

A phytochemical study of the roots of Pongamia pinnata afforded 29 flavanones and flavanols, including 7 previously undescribed compounds. The structures of the isolated compounds were determined by 1D and 2D NMR and mass spectroscopy data. The absolute configurations of the compounds were assigned via analysis of the specific rotations and electronic circular dichroism spectra, application of Mosherʼs method, and by comparing the calculated and experimental electronic circular dichroism spectra. The isolates were evaluated for their inhibitory effects on nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells. All of the isolated compounds exhibited inhibitory effects against nitric oxide production, and most of them showed obvious anti-inflammatory activities (IC₅₀ < 20 µM), among which 26 was the most active compound with an IC₅₀ of 9.6 µM.

Introduction

Pongamia pinnata (L.) Pierre (Leguminosae) is a semi-mangrove tree that is distributed along the Pacific coast from Southeast Asia to Northern Australia. Different parts of this plant have been widely used as a traditional medicine to treat a broad spectrum of diseases and wounds [1]. The root of P. pinnata is used for the treatment of gonorrhea, urethritis, and skin diseases [2]. Phytochemical studies on this plant resulted in the discovery of various types of compounds, including flavanones and flavanols. In particular, several furanoflavanones, pyranoflavanones, and prenylated flavanones have been isolated from P. pinnata [3]. Flavanones exhibit antioxidant, anti-inflammatory, antiproliferative, and vasorelaxant activities [4], [5], [6], [7], while flavans show aromatase inhibitory, antioxidant, and anti-inflammatory effects [8], [9], [10]. Moreover, flavanones have exhibited impressive anti-inflammatory properties in various animal models [11], [12], [13]. To search for new flavanones and flavanols from P. pinnata, a 95% aqueous ethanol extract of the roots was investigated and afforded 29 flavanones and flavanols, including 7 previously undescribed compounds. Herein, we describe the isolation and structural characterization of compounds 1–7 and the inhibitory effects of these isolates on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 microglial cells.

Results and Discussion

The 95% aqueous EtOH extract of the roots of P. pinnata was suspended in H₂O and partitioned with CH₂Cl₂. The CH₂Cl₂-soluble portion was subjected to silica gel and Sephadex LH-20 column chromatography (CC) followed by semipreparative RP-HPLC to afford 7 previously undescribed (1–7) and 22 known (8–29) flavanones and flavanols ([Fig. 1]). By comparing their NMR data with that reported in the literature, the known compounds were identified as ovalichromene B (8) [14], ponganone III (9) [14], isoglabrachromene (10) [15], pongachin (11) [16], 6-methoxy-6″,6″-dimethylchromeno-[2″,3″:7,8]-flavanone (12) [14], isolonchocarpin (13) [17], maxima flavanone A (14) [18], 6-(γ,γ-dimethylallyl)-3′,4′-dimethoxy-6″,6″-dimethylpyran[2″,3″:7,8]flavanone (15) [19], pongamone C (16) [20], 3′,4′,7-trimethoxyflavanone (17) [21], pinostrobin (18) [22], liquiritigenin (19) [23], 7-hydroxy-8-prenylflavanone (20) [24], ovaliflavanone C (21) [25], 7-hydroxy-6,8-diprenylflavanone (22) [26], isoderricine A (23) [27], ovaliflavanone D (24) [25], derrivanone (25) [28], dehydroisoderricin (26) [29], griffinone C (27) [30], griffinone A (28) [30], and pongaflavanol (29) [31]. The absolute configuration of compound 29 was determined as (2S, 4S) by Mosherʼs method ([Fig. 2]). The absolute configuration of 25 was assigned as (2S, 3″R, 4″R) by comparing the calculated electronic circular dichroism (ECD) spectra of (2S, 3″R, 4″R)- and (2S, 3″S, 4″S)-diastereomers with the experimental spectrum ([Fig. 3]). Compounds 14, 15, 17–19, 20, 21, 23, 24, and 26–28 are described for the first time in P. pinnata.

Compound 1 was obtained as a yellow oil. Its molecular formula was determined to be C₂₇H₃₂O₅ by HRESIMS (m/z 435.2163 [M – H]⁻, calcd. for C₂₇H₃₁O₅, 435.2171). The ¹H NMR spectrum of 1 showed proton signals at δ _H 2.83 (1H, dd, J = 2.9, 16.8 Hz), 3.01 (1H, dd, J = 13.0, 16.8 Hz), and 5.39 (1H, dd, J = 2.9, 13.0 Hz) in combination with a carbonyl carbon (δ _C 191.5), a methylene carbon (δ _C 44.3), and an oxygenated methine carbon (δ _C 79.5) in the ¹³C NMR spectrum, which were characteristic of a flavanone. The ¹H NMR data and ¹H-¹H COSY correlations revealed the presence of two isopentenyl groups [δ _H 3.32 (2H, d, J = 7.1 Hz, H-1″), 5.30 (1H, t, J = 7.1 Hz, H-2″), 1.77 (3H, s, H-4″), 1.79 (3H, s, H-5″), and 3.42 (2H, d, J = 7.1 Hz, H-1‴), 5.26 (1H, t, J = 7.1 Hz, H-2‴), 1.75 (3H, s, H-4‴), 1.74 (3H, s, H-5‴)], a 1,2,4-trisubstituted phenyl unit [δ _H 7.02 (1H, d, J = 2.0 Hz, H-2′), 7.01 (1H, m, H-6′), 6.90 (1H, d, J = 8.1 Hz, H-5′)], and an aromatic singlet [δ _H 7.62 (1H, s, H-5)]. The NMR data ([Table 1]) showed that 1 contained similar features to those of ovaliflavanone D (24) [25], except for the replacement of a methylenedioxy group in the B-ring by two methoxy singlets, which were deduced to be located at C-3′ and C-4′ by the HMBC correlations. The ECD spectrum of 1 showed positive Cotton effects (CEs) at 219 and 336 nm and a negative CE at 307 nm, which is in accordance with the 2S-configuration [32], [33]. On the basis of the above data, the structure of 1 was identified as (2S)-7-hydroxy-6,8-di-(3-methylbut-2-enyl)-3′,4′-dimethoxyflavanone.

Compound 2 was obtained as a yellow oil with a molecular formula of C₂₇H₃₂O₅, determined by HRESIMS (m/z 435.2162 [M – H]⁻, calcd. for C₂₇H₃₁O₅, 435.2171). Through comparison of the NMR data of 2 and 1, these two compounds were found to possess similar carbon and proton resonances, with the major differences located at the A-ring. The ¹H NMR data and ¹H-¹H COSY correlations of 2 showed signals for two ortho-coupled aromatic protons [δ _H 7.67 (1H, d, J = 8.9 Hz), 6.83 (1H, d, J = 8.9 Hz)], a terminal double bond [δ _H 6.17 (1H, dd, J = 10.9, 17.6 Hz), 5.20 (1H, d, J = 10.9 Hz), 5.25 (1H, d, J = 17.6 Hz)], an isopentenyl group [δ _H 3.37 (2H, t, J = 6.0 Hz, H-1‴), 5.25 (1H, m, H-2‴), 1.67 (6H, s, H-4‴, 5‴)], and two other methyl groups [δ _H 1.55 (6H, s)] ([Table 1]). In the HMBC spectrum, the correlations from H-1‴ to C-7/C-8/C-8a ([Fig. 4]) suggested that the isopentenyl group was connected to C-8. HMBC correlations of the proton signals of the terminal double bond and the two methyl groups to the carbon resonance at δ _C 80.7 revealed the presence of a 2-methylbut-3-enyl unit. The chemical shifts of δ _C 80.7 and δ _C-7 161.0 indicated that an oxygen atom was located between these two carbon atoms [30]. The ECD spectrum of 2 showed positive CEs at 218 and 337 nm and a negative CE at 306 nm, which was in accordance with the 2S-configuration. Thus, the structure of 2 was identified as (2S)-7-(2-methylbut-3-enyloxy)-8-(3-methylbut-2-enyl)-3′,4′-dimethoxyflavanone.

Compound 3 was obtained as a brown oil. HRESIMS gave a protonated molecular ion at m/z 421.2016 [M + H]⁺ (calcd. 421.2015), suggesting a molecular formula of C₂₆H₂₈O₅. Its 1D and 2D NMR data showed many similarities to those of 2, except for the replacement of two methoxy singlets in the B-ring by a methylenedioxy group. The absolute configuration at C-2 was determined to be 2S from its positive CEs at 220 and 337 nm and a negative CE at 302 nm in the ECD spectrum. Therefore, the structure of 3 was deduced as (2S)-7-(2-methylbut-3-enyloxy)-8-(3-methylbut-2-enyl)-3′,4′-methylenedioxyflavanone.

Compound 4, a red brown oil, gave a molecular ion [M + H]⁺ at m/z 453.2280 in the HRESIMS, indicating a molecular formula of C₂₇H₃₂O₆ (calcd. 453.2277). Analysis of the HSQC and ¹H-NMR spectra revealed signals ascribable to two isopentenyl groups, one methylene [δ _H 2.41 (1H, ddd, J = 1.7, 7.4, 13.5 Hz, H-3a; 1.96 (1H, ddd, J = 2.0, 9.4, 12.4, 13.5 Hz, H-3b)], two oxygenated methines [δ _H 4.92 (1H, m, H-4; 4.91 (1H, m, H-2)], one olefinic proton [δ _H 5.31 (s, 1H, H-6)], one methylenedioxy group [δ _H 6.02 (s, 2H)], and one methoxy group [δ _H 3.84 (s, 3H)]. The NMR spectroscopic data ([Table 1]) of 4 were similar to those of pongaflavanol (29) [31], except for the presence of a methylenedioxy group [δ _H 6.02 (s, 2H)] on the B-ring, which was deduced to be located at C-3′, C-4′ by HMBC correlations. The relative configuration at C-2 and C-4 was assigned as cis according to the coupling constant between H-2 and H _β -3 (J = 12.4 Hz), H-4 and H _β -3 (J = 9.4 Hz) [34]. The absolute configuration of 4 was determined using Mosherʼs reagents to yield the diastereomeric (S)- and (R)-MPA esters ([Fig. 2]). Thus, the absolute configuration of compound 4 was assigned as (2S, 4S), and the structure of 4 was identified as (2S, 4S)-3′,4′-methylenedioxypongaflavanol.

Compound 5 was obtained as a light yellow oil. Its molecular formula, C₂₇H₃₄O₄, was determined on the basis of the molecular ion observed in the positive ion HRESIMS at m/z 423.2535 [M + H]⁺ (calcd. 423.2535), which corresponds to 11 indices of hydrogen deficiency. Comparison of the NMR data of 5 with those of 4 suggested that the hydroxy group in 4 was replaced by a methoxy group in 5 and the methylenedioxy group was absent from the B-ring in 5. The 2,4-trans relative configuration was identified based on the J_2,3β value of 12.4 Hz and J_4,3β value of 2.9 Hz [34], [35], [36]. In flavans, the CE between 270 nm and 300 nm and the specific optical rotation value depend on the absolute configuration at C-2 [35], [37]. Thus, the absolute configuration at C-2 of 5 was determined to be 2S from its positive CE at 304 nm in the ECD spectrum and its specific optical rotation value ([α]_D ²⁵ + 12), which was similar to that of compound 4. Therefore, the absolute configuration of compound 5 was confirmed as (2S, 4R), and the structure of 5 was defined as (2S, 4R)-4,5-dimethoxy-8,8-bis(3-methylbut-2-enyl)-2-phenyl-3,4-dihydro-2H-chromen-(8H)-one.

Compound 6 was isolated as a yellow oil and its molecular formula was assigned as C₁₉H₁₆O₅ based on its ¹³C NMR spectroscopic data and the HRESIMS [M + Na]⁺ ion peak at m/z 347.0897 (calcd. 347.0895). Analysis of the ¹H and ¹³C NMR data revealed the presence of a monosubstituted benzene ring [δ _H 7.65 (2H, m, H-2′, 6′), 7.47 (2H, t, J = 7.4 Hz, H-3′, 5′), 7.41 (1H, t, J = 7.4 Hz, H-4′)], an aromatic singlet [δ _H 7.01 (1H, s, H-8)], a furan ring [δ _H 7.51 (1H, d, J = 2.2 Hz, H-2″), 6.98 (1H, d, J = 2.2 Hz, H-3″)], and a methylene group [δ _H 3.03 (2H, dd, J = 16.2 Hz, H-3)] ([Table 2]). The HMBC correlations of the aromatic singlet proton (H-8) with C-6/C-7/C-4a/C-8a ([Fig. 4]), the protons of CH₂-3 with C-2/C-4/C-1′, and the two methoxy groups with C-2 and C-5 were used to locate the furan ring at C6/C7 and the methoxy groups at C-2 and C-5. The C-2 absolute configuration was assigned by comparing the calculated ECD spectra of the 2R- and 2S-enantiomers with the experimental spectrum. The experimental ECD spectrum of 6 exhibited positive CEs at 243, 280, and 317 nm and a negative CE at 353 nm in methanol. The good agreement between the experimental and calculated ECD spectra of the (2R)-enantiomer led to the unequivocal assignment of the absolute configuration as 2R ([Fig. 3]). Hence, the structure of 6 was assigned as (2R)-2, 5-dimethoxy-[2″,3″:6,7]-furanoflavanone.

Compound 7 was obtained as white crystals. Its molecular formula, C₁₉H₁₆O₅, was deduced from the HRESIMS (m/z 325.1077 [M + H]⁺, calcd. 325.1076) and ¹³C NMR spectroscopic data. Comparison of the NMR spectroscopic data of 7 with those of 6 indicated that the furan ring in 7 was located at C-7/C-8, which was also supported by the HMBC correlations between H-6 and C-5/C-7/C-8/C-4a, H-3″ and C-7/C-8 ([Fig. 4]). The absolute configuration of 7 at C-2 was determined as 2R from the positive CEs at 245, 276, and 325 nm and a negative CE at 355 nm in the ECD spectrum, which were found to be consistent with those of 6 ([Fig. 5]). Accordingly, the structure of 7 was defined as (2R)-2,5-dimethoxy-[2″,3″:7,8]-furanoflavanone.

All of the isolated compounds (1–29) were evaluated for their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells [38], [39], consistent with the traditional anti-inflammation utilization of P. pinnata. To exclude cytotoxic effects, inhibition of the compounds on cell proliferation was measured using the MTT method. None of the compounds showed obvious cytotoxicity at a concentration of 50 µM. As shown in [Table 3], 26 compounds showed anti-inflammatory activities (IC₅₀ < 50 µM). The observed effects were comparable to the activity of the reference anti-inflammatory compound dexamethasone (IC₅₀ of 9.5 µM). Flavanone 26, with an IC₅₀ value of 9.6 µM, displayed the strongest anti-inflammatory activity among the isolated compounds, and most of the compounds exhibited moderate NO inhibition, with IC₅₀ values ranging from 10.0 µM to 43.4 µM. In addition, the IC₅₀ values of 1, 22, and 24, which shared a 6,8-diisopentenyl group as common structural feature, were obviously lower than those of 20, 21, and 23, which contained an 8-isopentenyl group in the A-ring. The weaker anti-inflammatory activity of 5 compared with that of 4, 28, and 29 indicated that the 4-methoxy substituent on the C-ring reduced its anti-inflammatory effects. The flavanones possessing a pyran ring on the A-ring (8–16) showed moderate inhibitory effects on NO production, while flavanones without a pyran ring (17–19) exhibited weaker anti-inflammatory activities.

Materials and Methods

General experimental procedures

UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. NMR spectra were recorded on a Varian INOVA-500 NMR spectrometer in CDCl₃ or CD₃COCD₃ (Jinouxiang Medicinal Inc.). The solvent peaks were used as an internal reference. ECD spectra were obtained using a J-810 spectrometer (Jasco). Optical rotations were measured with an Autopol VI polarimeter (Rudolph). HRMS data were acquired on a Waters Xevo G2 Q-TOF spectrometer fitted with an electrospray ionization source. CC was performed on silica gel (100 – 200 mesh or 200 – 300 mesh; Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Pharmacia). Semipreparative RP-HPLC was performed on an Agilent 1260 system with an Atlantis T₃ OBD prep column (10 mm × 250 mm, 5 µm) at a flow rate of 3.0 mL/min. TLC analysis and preparative TLC were carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Inc.) using n-hexane-EtOAc mixtures (4 : 1 – 2 : 1, v/v) as solvents. Spots were visualized under UV light (254 and 365 nm) or by heating after spraying with a 2% vanillin-H₂SO₄ solution. All solvents used were of analytical grade.

Plant material

The roots of P. pinnata (L.) Pierre were collected from Hainan Province, Peopleʼs Republic of China in April 2016. The plant material was identified by one of the authors (P.-F. Tu). A voucher specimen (no. PP201604) has been deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine.

Extraction and isolation

The roots of P. pinnata (10.0 kg) were extracted three times with 95% aqueous EtOH (100 L × 2 h). After evaporation under reduced pressure, the residue (625 g) was suspended in H₂O and partitioned with CH₂Cl₂. The CH₂Cl₂-soluble fraction (150 g) was subjected to silica gel CC eluted with petroleum ether-EtOAc (1 : 0 – 0 : 1, v/v, step gradient) to give nine fractions (A – I). Fraction A (14 g) was chromatographed over Sephadex LH-20 eluted with CH₂Cl₂-MeOH (5 : 5, v/v) to give six subfractions (A1 – A6). Compound 13 (176.2 mg, t _R 12.2 min) was purified from Fr. A6 by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (80 : 20, v/v) as the mobile phase. Fraction B (20 g) was chromatographed over Sephadex LH-20, eluted with CH₂Cl₂-MeOH (5 : 5, v/v) to give five subfractions (B1 – B5). Subfractions B2, B4, and B5 were then subjected to silica gel CC eluted with a stepwise gradient of n-hexane-EtOAc (from 6 : 1 to 0 : 1, v/v) to afford five fractions (Frs. B2a-B2e), four fractions (Frs. B4a-B4d), and three fractions (Frs. B5a-B5c), respectively. Fr. B2a was further purified by semipreparative RP-HPLC using MeCN-H₂O containing 0.1% HCOOH (90 : 10, v/v) to afford 16 (186 mg, t _R 12.6 min) and 27 (152 mg, t _R 13.8 min). Fr. B4b was further purified by semipreparative RP-HPLC using MeCN-H₂O containing 0.1% HCOOH (80 : 20, v/v) to afford 3 (26 mg, t _R 22.6 min) and 22 (180 mg, t _R 15.2 min). Compounds 23 (4.1 mg, t _R 9.3 min) and 24 (450 mg, t _R 8.0 min) were purified from Frs. B2b and B4c, respectively, by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (90 : 10, v/v) as the mobile phase. Compounds 14 (28.5 mg, t _R 29.1 min) and 20 (32.2 mg, t _R 4.5 min) were purified from Frs. B4a and B5c, respectively, by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (80 : 20, v/v) as the mobile phase. Frs. B5a and B5b were further purified by semipreparative RP-HPLC using MeCN-H₂O containing 0.1% HCOOH (70 : 30, v/v) to afford 18 (12.5 mg, t _R 12.6 min) and 9 (65.0 mg, t _R 16.9 min), respectively. Fraction C (25 g) was purified by Sephadex LH-20 eluted with CH₂Cl₂-MeOH (5 : 5, v/v) to give four subfractions (C1 – C4). Subfractions C1 and C3 were then subjected to silica gel CC and eluted with a stepwise gradient of n-hexane-EtOAc (from 6 : 1 to 0 : 1, v/v) to afford five fractions (Frs. C1a-C1e) and four fractions (Frs. C3a-C3d), respectively. Compound 12 (6.2 mg, t _R 9.9 min) was purified from Fr. C1c by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (80 : 20, v/v) as the mobile phase. Fr. C1d was further purified by semipreparative RP-HPLC using MeCN-H₂O containing 0.1% HCOOH (80 : 20, v/v) to afford 6 (6.3 mg, t _R 8.4 min), 8 (7.3 mg, t _R 9.2 min), 2 (16 mg, t _R 20.4 min), and 15 (26.3 mg, t _R 27.1 min). Compound 1 (9.5 mg, t _R 19.4 min) was purified from Fr. C1e by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH along a gradient from 70 to 80% over 35 min as the mobile phase. Compound 21 (72.3 mg, t _R 9.9 min) was purified from Fr. C3c by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (70 : 30, v/v) as the mobile phase. Fraction D (22 g) was subjected to Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (5 : 5, v/v) to give four subfractions, D1 – D4. Subfraction D1 was then subjected to silica gel CC eluted with a stepwise gradient of n-hexane-EtOAc (from 6 : 1 to 0 : 1, v/v) to afford seven fractions (Frs. D1a-D1g). Fr. D1e was purified by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (70 : 30, v/v) as the mobile phase to afford 7 (4.8 mg, t _R 10.1 min) and 11 (32.8 mg, t _R 14.1 min). Fraction F (18 g) was subjected to Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (5 : 5, v/v) to give four subfractions (F1 – F4). F1, F2, and F4 were fractioned on silica gel CC eluted with n-hexane-EtOAc (from 6 : 1 to 0 : 1, v/v) to afford four fractions (F1a-F1d), five fractions (F2a-F2e), and three fractions (F4a-F4c), respectively. Compound 26 (8.9 mg, t _R 16.2 min) was purified from Fr. F1a by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH along a gradient from 70 to 90% over 35 min as the mobile phase. Separation of Fr. F1d by semipreparative RP-HPLC (70% MeCN in H₂O containing 0.1% HCOOH) yielded 4 (19.5 mg, t _R 12.1 min) and 29 (218 mg, t _R 14.8 min). Separation of Fr. F2c by semipreparative RP-HPLC (70% MeCN in H₂O containing 0.1% HCOOH) yielded 17 (13.5 mg, t _R 7.9 min) and 28 (25.8 mg, t _R 18.2 min). Compound 10 (19.3 mg, t _R 12.4 min) was purified from Fr. F2d by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (70 : 30, v/v) as the mobile phase. Compound 19 (14.9 mg, t _R 12.7 min) was purified from Fr. F4c by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH along a gradient from 35 to 70% over 40 min as the mobile phase. Fraction G (17 g) was subjected to Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (5 : 5, v/v) to give four subfractions (G1 – G4). G4 was fractioned on silica gel CC eluted with n-hexane-EtOAc (from 6 : 1 to 0 : 1, v/v) to afford five fractions, G4a-G4e. Separation of Fr. G4c by semipreparative RP-HPLC (MeCN in H₂O containing 0.1% HCOOH along a gradient from 45 to 70% over 40 min) yielded 25 (45.2 mg, t _R 35.8 min). Fraction I (19 g) was chromatographed over Sephadex LH-20 eluted with CH₂Cl₂-MeOH (5 : 5, v/v) to give seven subfractions (I1 – I7). Compound 5 (5.2 mg, t _R 13.0 min) was purified from Fr. I2 by semipreparative RP-HPLC using MeCN in H₂O containing 0.1% HCOOH (60 : 40, v/v) as the mobile phase.

Compound 1 : Yellow oil; [α]_D ²⁵ − 8 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 203 (3.89), 224 (3.64), 284 (3.35) nm; IR (KBr) ν _max 3285, 2927, 1713, 1604, 1464, 1365, 1251, 1118, 1020, 816, and 580 cm⁻¹; CD (MeOH) λ _max (Δε): 219 (+ 2.46), 307 (− 1.46), 336 (+ 0.52) nm; ¹H and ¹³C NMR data are shown in [Table 1]; HRESIMS (negative ion mode) m/z 435.2163 [M – H]⁻ (calcd. 435.2171).

Compound 2 : Yellow oil; [α]_D ²⁵ − 18 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 203 (4.39), 220 (4.16), 281 (3.86) nm; IR (KBr) ν _max 3420, 2961, 2930, 1712, 1681, 1596, 1517, 1442, 1267, 1139, 1026, 809, and 579 cm⁻¹; CD (MeOH) λ _max (Δε): 218 (+ 5.63), 306 (− 3.42), 337 (+ 1.69) nm; ¹H and ¹³C NMR data are shown in [Table 1]; HRESIMS (negative ion mode) m/z 435.2162 [M – H]⁻ (calcd. 435.2171).

Compound 3 : Brown oil; [α]_D ²⁵ − 34 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 203 (4.54), 217 (4.32), 284 (4.14) nm; IR (KBr) ν _max 3422, 2976, 2919, 1681, 1595, 1490, 1444, 1253, 1037, 931, 810, and 578 cm⁻¹; CD (MeOH) λ _max (Δε): 220 (+ 9.57), 302 (− 7.61), 337 (+ 3.99) nm; ¹H and ¹³C NMR data are shown in [Table 1]; HRESIMS (positive ion mode) m/z 421.2016 [M + H]⁺ (calcd. 421.2015).

Compound 4 : Red brown oil; [α]_D ²⁵ + 114 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 204 (4.59), 224 (4.30), 285 (3.66), 344 (3.68) nm; IR (KBr) ν _max 3447, 2970, 2920, 1715, 1650, 1544, 1446, 1364, 1219, 1115, 1037, 930, and 529 cm⁻¹; CD (MeOH) λ _max (Δε): 205 (+ 7.53), 229 (− 6.57), 307 (+ 6.44) nm; ¹H and ¹³C NMR data are shown in [Table 1]; HRESIMS (positive ion mode) m/z 453.2280 [M + H]⁺ (calcd. 453.2277).

Compound 5 : Light yellow oil; [α]_D ²⁵ + 12 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 218 (4.45), 283 (3.72), 329 (3.60) nm; IR (KBr) ν _max 3386, 2922, 2851, 1724, 1617, 1467, 1352, 1275, 1218, 1126, 1039, and 577 cm⁻¹; CD (MeOH) λ _max (Δε): 216 (+ 7.85), 234 (− 1.59), 304 (+ 2.12), 342 (− 1.30) nm; ¹H and ¹³C NMR data are shown in [Table 1]; HRESIMS (positive ion mode) m/z 423.2525 [M + H]⁺ (calcd. 423.2535).

Compound 6 : Yellow oil; [α]_D ²⁵ + 114 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 203 (4.25), 241 (4.39), 305 (3.46) nm; IR (KBr) ν _max 3357, 2939, 1686, 1618, 1468, 1433, 1351, 1276, 1158, 1126, 1042, 742, and 702 cm⁻¹; CD (MeOH) λ _max (Δε): 243 (+ 7.32), 280 (+ 4.27), 317 (+ 4.48), 353 (− 2.05) nm; ¹H and ¹³C NMR data are shown in [Table 2]; HRESIMS (positive ion mode) m/z 347.0897 [M + Na]⁺ (calcd. 347.0895).

Compound 7 : White needle crystals; [α]_D ²⁵ + 46 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 202 (4.09), 239 (4.10), 245 (4.12), 261 (3.75), 276 (3.65) nm; IR (KBr) ν _max 3440, 2917, 2850, 1721, 1594, 1470, 1383, 1273, 1217, 1041, 731, and 581 cm⁻¹; CD (MeOH) λ _max (Δε): 245 (+ 3.99), 276 (+ 2.34), 325 (+ 0.39) nm; ¹H and ¹³C NMR data are shown in [Table 2]; HRESIMS (positive ion mode) m/z 325.1077 [M + H]⁺ (calcd. 325.1076).

Computational methods

The relative configurations of the compounds were initially established on the basis of their NOESY spectra, optimized by the MM2 force field using ChemOffice 2014 software, and then submitted to random conformational analysis with the MMFF94s force field using the Sybyl-X 2.0 software package. The generated lowest-energy conformers were subjected to geometry optimization using the DFT method at the B3LYP/6-31G(d) level, and the frequency was calculated at the same level of theory. The stable conformers without imaginary frequencies were subjected to ECD calculation by the TDDFT method at the B3LYP/6 – 31+G(d) level using the CPCM model in MeOH. The ECD spectra of different conformers were simulated using SpecDis v1.51 with a half-band width of 0.3 eV, and the final ECD spectra were obtained according to the Boltzmann-calculated contribution of each conformer. The calculated ECD spectra were compared with the experimental data. All calculations were performed with the Gaussian 09 program package [40], [41].

Cell culture, viability assay, and measurement of nitric oxide production

BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell Bank. The cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (HyClone), penicillin (Macgene Biotech., 100 U/mL), and streptomycin (100 µg/mL; Macgene Biotech.) in a humidified incubator containing 95% air and 5% CO₂ at 37 °C. Accumulation of NO in the culture medium was determined using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturerʼs instructions. Briefly, BV-2 cells were seeded into 48-well plates at a density of 1 × 10⁵ cells/well and stimulated with 1.0 µg/mL of LPS in the presence or absence of test compounds at 37 °C for 24 h. Cell culture supernatants (300 µL) were collected and mixed with 200 µL of reagent 1 and 100 µL of reagent 2 for 10 min at room temperature. After centrifugation of the mixture for 3 min at 7489 g, 160 µL of each of the supernatants were collected into a 96-well plate and 80 µL of the chromogenic agent were added. The optical density was measured at 570 nm using a microplate reader (Tecan Trading AG). Sodium nitrite was used to prepare a calibration curve for the assay. The experiments were performed in parallel three times, and the 50% inhibition concentration (IC₅₀ value) was determined by curve fitting with the software GraphPad Prism 5 (n = 3). Dexamethasone (98%; Adamas) was used as a positive control. Cell viability was evaluated using an MTT assay.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was financially supported by National Key Technology R&D Program “New Drug Innovation” of China (No. 2018ZX09711001-008-003).

• References

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Correspondence

• References

• 1 Satish KBN. Phytochemistry and pharmacological studies of Pongamia pinnata (L.) Pierre. Int J Pharm Sci Rev Res 2011; 2: 12-19
  PubMedSearch in Google Scholar
• 2 Muthu C, Ayyanar M, Raja N, Ignacimuthu S. Medicinal plants used by traditional healers in Kancheepuram District of Tamil Nadu. India J Ethnobiol Ethnomed 2006; 2: 43-52
  PubMedSearch in Google Scholar
• 3 Al Muqarrabun LMR, Ahmat N, Ruzaina SAS, Ismail NH, Sahidin I. Medicinal uses, phytochemistry and pharmacology of Pongamia pinnata (L.) Pierre: a review. J Ethnopharmacol 2013; 150: 395-420
  CrossrefPubMedSearch in Google Scholar
• 4 Di Majo D, Giammanco M, Guardia M, Tripoli E, Giammanco S, Finotti E. Flavanones in citrus fruits: structure-antioxidant activity relationships. Food Res Int 2005; 38: 1161-1166
  CrossrefPubMedSearch in Google Scholar
• 5 Sakata K, Hirose Y, Qiao Z, Tanaka T, Mori H. Inhibition of inducible isoforms of cyclooxygenase and nitric oxide synthase by flavonoid hesperidin in mouse macrophage cell line. Cancer Lett 2003; 199: 139-145
  CrossrefPubMedSearch in Google Scholar
• 6 Frydoonfar HR, McGrath DR, Spigelman AD. The variable effect on proliferation of a colon cancer cell line by the citrus fruit flavonoid naringenin. Colorectal Dis 2003; 5: 149-152
  CrossrefPubMedSearch in Google Scholar
• 7 Orallo F, Camina M, Alvarez E, Basaran H, Lugnier C. Implication of cyclic nucleotide phosphodiesterase inhibition in the vasorelaxant activity of the citrus-fruits flavonoid (±)-naringenin. Planta Med 2005; 71: 99-107
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Prachyawarakorn V, Sangpetsiripan S, Surawatanawong P, Mahidol C, Ruchirawatacd S, Kittakoop P. Flavans from Desmos cochinchinensis as potent aromatase inhibitors. Med Chem Commun 2013; 4: 1590-1596
  CrossrefPubMedSearch in Google Scholar
• 9 Tabanca N, Pawar RS, Ferreira D, Marais JPJ, Khan SI, Joshi V, Wedge DE, Khan IA. Flavan-3-ol-phenylpropanoid conjugates from Anemopaegma arvense and their antioxidant activities. Planta Med 2007; 73: 1107-1111
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Zhan G, Zhou J, Liu T, Zheng G, Aisa HA, Yao G. Flavans with potential anti-inflammatory activities from Zephyranthes candida . Bioorg Med Chem Lett 2016; 26: 5967-5970
  CrossrefPubMedSearch in Google Scholar
• 11 Emim JA, Oliveira AB, Lapa AJ. Pharmacological evaluation of the antiinflammatory activity of a citrus bioflavonoid, hesperidin, and the isoflavonoids, duartin and claussequinone, in rats and mice. J Pharm Pharmacol 1994; 46: 118-122
  CrossrefPubMedSearch in Google Scholar
• 12 Bodet C, La VD, Epifano F, Grenier D. Naringenin has anti-inflammatory properties in macrophage and ex vivo human whole-blood models. J Periodontal Res 2008; 43: 400-407
  CrossrefPubMedSearch in Google Scholar
• 13 Vafeiadou K, Vauzour D, Lee HY, Rodriguez-Mateos A, Williams RJ, Spencer JPE. The citrus flavanone naringenin inhibits inflammatory signalling in glial cells and protects against neuroinflammatory injury. Arch Biochem Biophys 2009; 484: 100-109
  CrossrefPubMedSearch in Google Scholar
• 14 Magalhães AF, Azevedo Tozzi AMG, Noronha Sales BHL, Magalhaes EG. Twenty-three flavonoids from Lonchocarpus subglaucescens . Phytochemistry 1996; 42: 1459-1471
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 16 Andrei CC, Ferreira DT, Faccione M, de Moraes LAB, de Carvalho MG, Braz-Filho R. C-prenylflavonoids from roots of Tephrosia tunicate . Phytochemistry 2000; 55: 799-804
  CrossrefPubMedSearch in Google Scholar
• 17 Wang WL, Wang J, Li N, Zhang XR, Zhao WH, Li JY, Si YY. Chemopreventive flavonoids from Millettia pulchra Kurz var-laxior (Dunn) Z.Wei (Yulangsan) function as Michael reaction acceptors. Bioorg Med Chem Lett 2015; 25: 1078-1081
  CrossrefPubMedSearch in Google Scholar
• 18 Rao EV, Prasad YR, Murthy MSR. A prenylated flavanone from Tephrosia maxima . Phytochemistry 1994; 37: 111-112
  CrossrefPubMedSearch in Google Scholar
• 19 Fang N, Casida JE. New bioactive flavonoids and stilbenes in cube resin insecticide. J Nat Prod 1999; 62: 205-210
  CrossrefPubMedSearch in Google Scholar
• 20 Li LY, Li X, Shi C, Deng ZW, Fu HZ, Proksch P, Lin WH. Pongamone A–E, five flavonoids from the stems of a mangrove plant, Pongamia pinnata . Phytochemistry 2006; 67: 1347-1352
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• 21 Pouget C, Fagnere C, Basly JP, Besson AE, Champavier Y, Habrioux G, Chulia AJ. Synthesis and aromatase inhibitory activity of flavanones. Pharm Res 2002; 19: 286-291
  CrossrefPubMedSearch in Google Scholar
• 22 Dzoyem JP, Nkuete AHL, Kuete V, Tala MF, Wabo HK, Guru SK, Rajput VS, Sharma A, Tane P, Khan IA, Saxena AK, Laatsch H, Tan NH. Cytotoxicity and antimicrobial activity of the methanol extract and compounds from Polygonum limbatum . Planta Med 2012; 78: 787-792
  Thieme ConnectPubMedSearch in Google Scholar
• 23 Chokchaisiri R, Suaisom C, Sriphota S, Chindaduang A, Chuprajob T, Suksamrarn A. Bioactive flavonoids of the flowers of Butea monosperma . Chem Pharm Bull 2009; 57: 428-432
  CrossrefPubMedSearch in Google Scholar
• 24 Sakamoto Y, Ohmoto T, Nikaido T, Koike K, Tomimori T, Miyaichi Y, Shirataki Y, Monache FD, Botta B, Yokoe I, Komatsu M, Watanabe S, Ando I. On the relationship between the chemical structure and the cyclic AMP phosphodiesterase inhibitory activity of flavonoids as studied by ¹³C NMR. B Chem Soc JPN 1989; 62: 2450-2454
  CrossrefPubMedSearch in Google Scholar
• 25 Islam A, Gupta RK, Krishnamurti M. Furano chalcone and prenylated flavanones from Milletia ovalifolia seeds. Phytochemistry 1980; 19: 1558-1559
  CrossrefPubMedSearch in Google Scholar
• 26 Hossain MA, Salehuddin SM. Synthesis of 7-hydroxy-6, 8-di-C-prenyl-flavanone. Indian J Chem B 1999; 38: 593-595
  PubMedSearch in Google Scholar
• 27 Garcez FR, Scramin S, Nascimento MC, Mors WB. Prenylated flavonoids as evolutionary indicators in the genus Dahlstedtia. Phytochemistry 1988; 27: 1079-1083
  CrossrefPubMedSearch in Google Scholar
• 28 Decharchoochart P, Suthiwong J, Samatiwat P, Kukongviriyapan V, Yenjai C. Cytotoxicity of compounds from the fruits of Derris indica against cholangiocarcinoma and HepG2 cell lines. J Nat Med 2014; 68: 730-736
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• 29 Rao EV, Raju NR. Two flavonoids from Tephrosia purpurea . Phytochemistry 1984; 23: 2339-2342
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• 30 Tang H, Pei HY, Wang TJ, Chen K, Wu B, Yang QN, Zhang Q, Yang JH, Wang XY, Tang MH, Peng AH, Ye HY, Chen LJ. Flavonoids and biphenylneolignans with anti-inflammatory activity from the stems of Millettia griffithii . Bioorg Med Chem Lett 2016; 26: 4417-4422
  CrossrefPubMedSearch in Google Scholar
• 31 Yin H, Zhang S, Wu J, Nan HH, Long LJ, Yang J, Li QX. Pongaflavanol: a prenylated flavonoid from Pongamia pinnata with a modified ring A. Molecules 2006; 11: 786-791
  CrossrefPubMedSearch in Google Scholar
• 32 Gaffield W. Circular dichroism, optical rotatory dispersion and absolute configuration of flavanones, 3-hydroxyflavanones and their glycosides: determination of aglycone chirality in flavanone glycosides. Tetrahedron 1970; 26: 4093-4108
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• 33 Yenesew A, Midiwo JO, Miessner M, Heydenreich M, Peter MG. Two prenylated flavanones from stem bark of Erythrina burttii . Phytochemistry 1998; 48: 1439-1443
  CrossrefPubMedSearch in Google Scholar
• 34 Pouget C, Fagnere C, Basly J, Leveque H, Chulia A. Synthesis and structure of flavan-4-ols and 4-methoxyflavans as new potential anticancer drugs. Tetrahedron 2000; 56: 6047-6052
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• 35 Janeczko T, Dymarska M, Siepka M, Gniłka R, Lesniak A, Popłonski J, Kostrzewa-Susłow E. Enantioselective reduction of flavanone and oxidation of cis- and trans-flavan-4-ol by selected yeast cultures. J Mol Catal B Enzym 2014; 109: 47-52
  CrossrefPubMedSearch in Google Scholar
• 36 Ramadas S, Krupadanam GLD. Enantioselective acylation of (±)-cis-flavan-4-ols catalyzed by lipase from Candida cylindracea (CCL) and the synthesis of enantiopure flavan-4-ones. Tetrahedron Asymmetry 2004; 15: 3381-3391
  CrossrefPubMedSearch in Google Scholar
• 37 Won D, Shin BK, Kang S, Hur HG, Kimc M, Han J. Absolute configurations of isoflavan-4-ol stereoisomers. Bioorg Med Chem Lett 2008; 18: 1952-1957
  CrossrefPubMedSearch in Google Scholar
• 38 Zeng KW, Li J, Dong X, Wang YH, Ma ZZ, Jiang Y, Jin HW, Tu PF. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways. Toxicol Appl Pharmacol 2013; 273: 159-171
  CrossrefPubMedSearch in Google Scholar
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