Flavonoids From the Flowers of Sophora davidii and Their Anti-Tobacco Mosaic Virus Activities

Abstract

A new pterocarpan, named davidicarpan A (1), together with 9 known flavonoids, including 3 pterocarpans (2-4) and 6 isoflavones (5-10), were isolated from the flowers of Sophora davidii (France.) Pavol. Their structures were confirmed by high-resolution electrospray ionization-mass spectrometry, one-dimensional (1D) and 2D nuclear magnetic resonance spectroscopy, and comparison with reported literature values. Compounds 1 and 4 exhibited anti-tobacco mosaic virus (anti-TMV) activities with inhibition rates of 27.4% and 26.2%, which was higher than that of the positive control, ningnamycin.

The genus Sophora, belonging to the family Leguminosae, comprises approximately 70 species of shrubs or small trees that are widely distributed in tropical and temperate zones, with 21 species occurring in China. ¹ Among them, Sophora davidii (France.) Pavol., a deciduous shrub, occurs on hill slopes and sandy places in valleys in the Hubei, Sichuan, Henan, and Yunnan provinces. As reported previously, S. davidii contains a diversity of chemical constituents, including alkaloids, ² flavones, ³ and stilbene oligomers. ^4,5 It also exhibits various biological activities, such as antitumor, ⁶ anti-inflammatory, ⁷ and antiallergic ⁷ activities. In the current study, we report the isolation and structural identification of a new pterocarpan, davidicarpan A (1), which have a hydroxyl at C-6a as well as a β-D-glucose moiety at C-3 in the structure, along with 9 known compounds, including pterocarpin, ^2,8,9 kushenin, ^3,10 trifolirhizin, ^4,11 calycosin, ^5,11 pseudobaptigenin, ^6,12 sophoricoside, ^7,13 daidzein, ^8,11 formononetin, ^9,14 and biochanin A ^10,15 (Figure 1). Compounds 2, 3, and 8 to 10 were isolated from S. davidii for the first time in this study. Since some flavonoids ¹⁶ and the isolates from the plants of genus Sophora ¹⁷ exhibit potential anti-tobacco mosaic virus (TMV) activities, the anti-TMV activities of compounds 1 to 10 were also assessed.

Figure 1

Compounds 1-10 isolated from Sophora davidii (France.) Pavol.

Compound 1 was isolated as a yellow amorphous powder with a molecular formula of C₂₂H₂₂O₁₁, implying 12 degrees of unsaturation, which was determined based on the high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS) and ¹³C-nuclear magnetic resonance (NMR) spectra. The infrared (IR) spectrum displayed characteristic absorptions of hydroxy (3374 cm⁻¹) and aromatic ring (1619 and 1463 cm⁻¹) groups. The ¹H-NMR data (Table 1) of compound 1 displayed the signals readily corresponding to 1 set of the ABX system at δ _H 7.37 (1H, d, J = 8.5 Hz, H-1), 6.71 (1H, dd, J = 8.5, 2.5 Hz, H-6'), and 6.53 (1H, d, J = 2.5 Hz, H-4), belonging to a tri-substituted benzene ring, and 2 isolated aromatic protons at δ _H 6.96 (1H, s, H-7) and 6.51 (1H, s, H-10), which indicated the presence of a 1,2,4,5-tetrasubstituted aromatic ring. In the upfield region, the protons at δ _H 4.05 (2H, m, H-6) and an oxymethine signal at δ _H 5.26 (1H, s, H-11a) were also observed. In combination, the above NMR data suggested the existence of a pterocarpan structure. ¹⁶ The assignments for the pterocarpan skeleton were confirmed by the heteronuclear multiple bond correlation (HMBC) correlations (Figure 2) from H-11a to C-1, C-4a, C-6, C-6a, and C-6b and from H-7 to C-6a. Moreover, protons of a β-glucose moiety, for which the anomeric proton resonated at δ _H 4.83 (1H, d, J = 7.5 Hz, H-1′), were found in the high-field region, and the moiety was attached at C-3 as deduced by the HMBC cross-peak between H-1′ and C-3. The D-configuration of the glucose moiety was assessed via gas chromatography analysis of its chiral derivatives following snailase hydrolysis. Excluding the aforementioned signals, methylenedioxy group protons were found at δ _H 5.97 (1H, d, J = 1.0 Hz, H-12α) and 5.93 (1H, d, J = 1.0 Hz, H-12β) upfield of ¹H-NMR and at δ _C 101.7 (C-12) of ¹³C-NMR, the location of which was confirmed by the HMBC correlations from H-12 to C-8 and C-9. According to biogenetical regulations, the conformation at B/C ring junction has always been regarded as cis because these compounds are presumably derived from pterocarpan in which the cis fusion has been proven by X-ray crystallography. Furthermore, the absolute configuration at C-6a and C-11a was S, as indicated by the negative optical rotation value ([α]20 D‒142) and the negative cotton effect at 214 nm (Supplementary Material). ¹⁸ The structure of compound 1 was, therefore, established as (6aS,11aS)−3,6a-dihydroxy-8,9-methylenedioxy-pterocarpan-3-O-β-D-glucopyranoside (Figure 1). It is here named davidicarpan A.

Figure 2

Characteristic correlations observed by heteronuclear multiple bond correlation spectrum for compound 1.

Table 1

¹H and ¹³C-NMR Data of Compound 1 in DMSO-D₆ (δ, ppm; J, Hz).

No.	δ H	δ C	No.	δ H	δ C
1	7.37 (1H, d, J = 8.5 Hz)	131.9	10a		153.7
2	6.71 (1H, dd, J = 8.5, 2.5 Hz)	110.6	11a	5.26 (1H, s)	84.3
3		158.4	11b		114.5
4	6.53 (1H, d, J = 2.5 Hz)	103.9	12α	5.97 (1H, d, J = 1.0 Hz)	101.2
4a		155.5	12β	5.93 (1H, d, J = 1.0 Hz)
6	4.05 (2H, m)	69.2	1′	4.83 (1H, d, J = 7.5 Hz)	100.3
6a		75.6	2′	3.20 (1H, m)	73.1
6b		120.6	3′	3.24 (1H, m)	76.5
7	6.96 (1H, s)	103.8	4′	3.13 (1H, m)	69.7
8		148.7	5′	3.30 (1H, m)	77.0
9		141.4	6′	3.68 (1H, m); 3.43 (1H, m)	60.6
10	6.51 (1H, s)	93.4

As some flavonoids exhibit potential anti-TMV activities, all the isolates were evaluated for their protective effects against TMV replication at the concentration of 20 µM using the half-half method. ¹⁹ Ningnanmycin, a commercial product for plant disease in China, was used as a positive control. The results showed that compounds 1 and 4 exhibited anti-TMV activity with inhibition rates of 27.4% and 26.2%, respectively, compared with that of the positive control ningnanmycin (24.8%). Compounds 2 and 6 showed moderate activity with inhibition rates of 21.2% and 19.8%, respectively. The preliminary results suggested that the presence of 1,3-benzodioxole ring could lead to an increase of protective effects against TMV replication. Moreover, in contrast with compounds 1 and 4, compounds 2 and 6 have no β-D-glucose moiety on position C-3. This result implied that the β-D-glucose substitute at C-3 is necessary for the anti-TMV activity.

Experimental

General Experimental Procedures

Optical rotation, Jasco P-2000 polarimeter; UV, JASCO V-650 spectrophotometer; ECD, Jasco J-815 spectrometer; IR, Nicolet 5700 spectrometer; NMR spectra, Bruker AM500 FT-NMR spectrometer; HR-ESI-MS, AB SCIEX TripleTOF 6600 mass spectrometer; preparative high-performance liquid chromatography (HPLC), Waters Prep 150 LC system using a YMC-Pack ODS-column. Column chromatography, Diaion HP-20 macroporous resin and Sephadex LH-20 columns.

Plant Material

The flowers of S. davidii in the present study were collected from Yiliang City, Yunnan Province, China, in October 2018. The plant was authenticated by one of the authors (Sheng Huang) and the voucher specimens were deposited at the Department of Plant Resources, School of Food and Bioengineering, Zhengzhou University of Light Industry, China.

Extraction and Isolation

Dried flowers of S. davidii (10 kg) were extracted 3 times with 80% EtOH (3 × 20 L, 3 hours) under reflux. All of the EtOH solution was evaporated under reduced pressure, following which the dark brown residue (1.1 kg) was suspended in H₂O (3 L) and fractionated sequentially with petroleum ether (5 × 3 L), EtOAc (5 × 3 L), and n-BuOH (6 × 3 L). The obtained n-BuOH-soluble extract (0.4 kg) was subjected to chromatography on an HP-20 macroporous absorption resin column (200 × 15 cm i.d.) and eluted successively with 0%, 15%, 30%, 50%, 75%, and 95% ethanol (15 L each) to yield 6 fractions (fractions A-F). Fraction C (72 g) was separated on a Sephadex LH-20 column (120 cm × 10 cm i.d.) and eluted with a MeOH/H₂O (0%-100%, with 10% stepwise increase of MeOH, 2 L each) mixture to give fractions C1-C9 under guidance of liquid chromatography with a photodiode array detector. Fraction C4 was further chromatographed on a Sephadex LH-20 column with MeOH/H₂O (0%, 10%, 20%, 40%, 60%, 85%, and 100%, v/v, 1 L each) to generate fractions C4-1 to C4-18, of which fraction C4-9 was purified by reverse semipreparative HPLC, using MeOH/H₂O/HOA_C (45:55:0.1, v/v) as the mobile phase at 3 mL/min to afford compounds 1 (7 mg), 2 (24 mg), 3 (16 mg), and 4 (9 mg). Fraction C4-8 was chromatographed on a Sephadex LH-20 column with a gradient of MeOH/H₂O (0%, 10%, 30%, 50%, 70%, and 100%, v/v, 1 L each) to generate fractions C4-8A to C4-8H. Fraction C4-8D was fractionated by semipreparative HPLC using MeOH/H₂O/HOAc (40:60:0.1, v/v) at 3 mL/min to produce compounds 5 (28 mg), 6 (16 mg), and 7 (14 mg). Compounds 8 (9 mg), 9 (11 mg), and 10 (5 mg) were obtained from fraction C4-8F using MeOH/H₂O/HOA_C (65:35:0.1, v/v) as the mobile phase at 3 mL/min. All of the isolates were dried under reduced pressure, and comprehensive spectroscopic data were collected.

Anti-TMV Assays

The protective effects of all isolated compounds against TMV replication were examined at the concentration of 20 µM using the half-half method. TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry, Yunnan Academy of Tobacco Science. The virus was multiplied in Nicotiana tabacum cv. K326. The concentration of TMV was adjusted to 20 mg/mL as determined by UV absorption [virus concentration = (A260 × dilution ratio)/ $E_{1\mathrm{c}\mathrm{m}}^{0.1\mathrm{\%},260\mathrm{n}\mathrm{m}}$ ]. The purified virus was kept at −20°C and was diluted to 32 µg/mL with 0.01 M phosphate-buffered saline before use. Nicotiana glutinosa plants were cultivated in an insect-free greenhouse. N. glutinosa was used as a local lesion host. The experiments were conducted when the plants grew to the 5-6-leaf stage. The tested compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled H₂O to the required concentrations. A solution of equal concentration of DMSO was used as a negative control. The commercial antiviral agent ningnanmycin was used as a positive control. For the half-leaf method, the virus was inhibited by mixing with the solution of compound. After 30 minutes, the mixture was inoculated on the left side of the leaves of N. glutinosa, whereas the right side of the leaves was inoculated with the mixture of DMSO solution and the virus as control. The local lesion numbers were recorded 3 or 4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula inhibition rate (%) = [(C − T)/C] ×100%, where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment.

Davidicarpan A (1)

${\displaystyle [\alpha {]}_D^{20}}$ : ‒142 (c 0.1, MeOH)

ECD (c 3.1 × 10^‒4, MeOH) λ _max (Δε): 214 (−24.6), 307 (4.5) nm

IR (KBr) ν _max: 3374,1619, 1502, 1463, 828 cm⁻¹;

NMR data see Table 1;

HR-ESIMS: m/z 485.1069 [M + Na]⁺ (calculated for 485.1058, C₂₂H₂₂O₁₁Na)