New bakuchiol dimers from Psoraleae Fructus and their inhibitory activities on nitric oxide production

Background Dried fruits of Psoralea corylifolia L. (Psoraleae Fructus) is one of the most popular traditional Chinese medicine with treatment for nephritis, spermatorrhea, pollakiuria, asthma, and various inflammatory diseases. Bakuchiol is main meroterpenoid with bioactive diversity from Psoraleae Fructus. This study was designed to seek structural diverse bakuchiol derivants with anti-inflammatory activities from this plant. Methods Various column chromatography methods were used for isolation experiment. Structures and configurations of these compounds were determined by spectroscopic methods and single-crystal X-ray diffraction. Their inhibition on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were evaluated by the Griess reaction. Results Twelve unpresented bakuchiol dimmers, bisbakuchiols M–U (1–9) and bisbakuchiol ethers A–C (10–12), along with five known compounds (13–17), were isolated from the fruits of Psoralea corylifolia L. Compounds 1–3, 10–12, 16 and 17 exhibited inhibitory activities against LPS-induced NO production in RAW264.7 macrophages, and the inhibition of compound 1 (half maximal inhibitory concentration (IC50) value = 11.47 ± 1.57 μM) was equal to that of L-N(6)-(1-iminoethyl)-lysine (IC50 = 10.29 ± 1.10 μM) as a positive control. Conclusions Some compounds exhibited inhibitory activities against NO production, and the study of structure–activity relationship suggested that uncyclized compounds with oxygen substitution at C-12/12′ showed strong inhibitory activities, and carbonyl units contributed to enhanced activities. Supplementary Information The online version contains supplementary material available at 10.1186/s13020-021-00499-y.


Background
The higher plant, Psoralea corylifolia L. (Cullen corylifolia (L) Mefik) is an annual herb and belongs to family Leguminosae, distributed in China, India, Malay peninsula, and Indonesia [1]. Dried fruits of P. corylifolia (Psoraleae Fructus) is one of the most popular traditional Chinese medicine (TCM) and officially listed in Chinese Pharmacopoeia [2], and it is also a natural food additive [3]. It has been used for the treatment of nephritis, spermatorrhea, pollakiuria, asthma, and various inflammatory diseases [4]. Psoraleae Fructus contains approximately 110 compounds including coumarins, flavonoids, meroterpenoids, and benzofurans [5]. Among these, meroterpenoids are considered to be the one of characteristic and active components [6,7].

General experimental procedures
Infrared data were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. Ultraviolet data were acquired on a Mapada UV-6100 double beam spectrophotometer. HRESIMS data were collected using a Waters Xevo G2 QTOF spectrometer. NMR spectra were recorded on a Bruker AVANCE III HD 400 NMR spectrometer. Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter. X-ray data were collected by a Rigaku Micromax-003 X-ray single-crystal diffractometer with CuKα radiation. Open column chromatography (CC) was performed by packing silica gel (200-300 mesh, Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 gel (Pharmacia Biotek, Denmark). Thin layer chromatography (TLC) was carried out on silica gel GF254 plates (Merck, Darmstadt, Germany) with 10% H 2 SO 4 in 95% ethanol followed by heating. Reversed phase semipreparative HPLC (RP-SP-HPLC) was accomplished using an LC3000 system (Beijing Innovation Technology Co., Ltd), equipped with a phenomenon C 18 column (21.2 mm × 250 mm, 5 μm). Cells were cultured in Sanyo MCO-15 AC carbon dioxide (CO 2 ) incubator (Sanyo Electric Co., Ltd., Osaka, Japan).

Plant material
The mature fruits of Psoralea corylifolia L. were harvested from Yunnan province of People's Republic of China (GPS coordinates:23°32′N, 99°23′E) in October 2016, and authenticated by Prof. Xiu-Wei Yang of the School of Pharmaceutical Sciences, Peking University. A voucher specimen (accession number: BGZ201610) of the fruits was deposited at the State Key Laboratory of Natural Medicines and Biomimetic Drugs of Peking University.

Extraction and isolation
The dried mature fruits powder (47.9 kg) was extracted with 70% aqueous ethanol under reflux. After extracted for three times (first 479 kg for 2 h, and then 384 kg for 2 h two times), the crude extract (8.2 kg, yield 17.12%) was obtained. And then, part of the residue (6.0 kg) was suspended in H 2 O (8 L) and extracted with cHE (

X-ray crystallographic analysis
The X-ray crystallographic experiments were carried out on a XtaLAB Synergy R, HyPix diffractometer with CuKα radiation. Crystallographic data

ECD calculations
The calculation was performed by the Gaussian 16 software. Conformation analysis were proceeded with the MMFF94s molecular mechanics force field. Optimization of the stable conformers with a Boltzmann distribution over 1% was conducted by time-dependent density functional theory (TD-DFT) at the Cam-B3LYP/6-31 + G(d, p) level for compounds 8 and 9, with the CPCM model in MeOH. The ECD data was analysed by SpecDis v1.71 with the half-bandwidth no more than 0.3 eV. The final ECD spectra were obtained based on the Boltzmann-calculated contribution of each conformer.

Inhibition assay on NO production
RAW264.7 cells were maintained in DMEM containing 10% FBS, in a constant humidity atmosphere of 5% CO 2 and 95% air at 37 °C. The cells were cultivated at a density of 3 × 10 5 cells/mL for 24 h in 96-well culture plates. And then, the cells were stimulated with LPS (1 μg/mL) and treated with various concentrations (1.56-50.00 μM) of assay compounds. After exposure to the compounds for 24 h, MTT (20 μL, 5 mg/mL) was added to each well [14]. 4 h later, 100 μL of lysis solution (40 g SDS, 20 mL isopropanol, 0.4 mL concentrated HCl and 400 mL ddH 2 O) was added to dissolve the formazan crystals. Absorbances at 490 nm were measured after 10 h by a Multiskan MK3 Automated Microplate Reader (Thermo-Labsystems, Franklin, MA, USA).
The RAW264.7 cells were grown at a density of 3 × 10 5 cells/mL in 96-well culture plates. After 24 h, the cells were stimulated with LPS (1 μg/mL) and treated with various non-cytotoxic concentrations of assay compounds. And then, the cell culture supernatant (100 μL) was collected and reacted with the same volume of Griess reagent (100 μL) for 15 min at room temperature [15].
The absorbance was determined at 540 nm. The experiments were performed in parallel for three times, and L-NIL was used as a positive control. IC 50 (half maximal inhibitory concentration) value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO production.

Statistical analysis
Data were analyzed by SPSS statistics package v.20.0 (SPSS Inc., Chicago, IL, USA). Results were expressed as the mean ± SD. Students't-test was used for Statistical significances calculation, and p < 0.05 was considered to be statistically significant.

Results
Phytochemical investigation on cHE fraction of 70% ethanol extract of Psoraleae Fructus resulted in twelve unpresented bakuchiol dimmers (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and five known compounds (13-17) (Fig. 1). Structures of these new compounds were assigned by NMR spectra and single crystal X-ray diffraction. Compounds 1-3, 6-9, and 13-17 could be detected from ultrasonic extraction of Psoraleae Fructus by LC/MS analysis, suggesting that these compounds were natural products (Additional file 1: Fig.  S1). The presence of an α,β-unsaturated ketone group was revealed by the band at 1692 cm -1 in its IR spectrum, which was confirmed by the resonance at δ C 180.4(s) in its 13 C NMR spectrum. Comparison of the 13 C NMR spectrum of 1 with those of bakuchiol, the chemical shifts of C-3 and C-5 were shifted downfield to δ C 121.4, suggesting that this substituted group was connected to C-4 of bakuchiol moiety by an ether linkage. The full assignment of 1 H and 13 C NMR resonances was supported by 1 H-1 H COSY, DEPT, HSQC and HMBC spectral analyses. The X-ray structure was shown in Fig. 2 and confirmed the absolute configuration of 9S,9'S for 1. Thus, the structure of 1 was as shown in Fig. 1 and named bisbakuchiol M. The plausible biosynthetic pathway of bisbakuchiol M was proposed (Fig. 3). Hydroxylation reactions occurred at the positions of C-2 and C-5 in bakuchiol to form M1. Once the 4-hydroxyl group in M1 lost a proton to generate M2-1, migrations of the double bond would start. The double bond at C-7 and C-8 would attack C-12 to form a five-membered ring, along with the generation of carbanion at C-13 (M2-2). Subsequently, the carbanion at C-13 attacked C-6 to form six-membered ring (M2-3). The proton at C-6 left, which was accompanied by electron migrations of negative ion of oxygen to produce ketone carbonyl (M3). And then, the α-proton of double bond at C-8 was easily to be hydroxylated to generate M4. The elimination reaction would follow to the generation of M5. Similarly, the hydroxylation occurred at C-11 (M6). Subsequently, 11-hydroxyl group would be oxidized to ketone carbonyl (M7). Finally, M7 and bakuchiol were condensed to produce bisbakuchiol M. The IR spectrum of 3 showed absorption bands at 3373, 1604, 1507, 1454, 1238, 1007 cm -1 ascribable to hydroxyl group and ether functions and aromatic ring. Compared with the NMR data of bakuchiol, a side chain (3-ethenyl-3,7-dimethyl-1,6-octadienyl) and a p-disubstituted benzene ring in 3 were identical to that of bakuchiol, together with a set of remaining NMR signals, which were very similar to those of psoracorylifol F characterized from the fruits of P. corylifolia [17]. However, the NOE correlation between H-17' at δ H 6.43 and H-7' at δ H 2.89 was observed, which indicated that H-7' was α-oriented. The large coupling constant (J = 11.7 Hz) of H-7' and H-12' indicated a trans configuration of the two methine protons. Likewise, the configuration of H-8' was confirmed β-oriented on the basis of the large coupling constant (J = 10.6 Hz). Thus, the configuration was assigned as 7'S,8'S,9'S,12'S from the occurrence of (9S)bakuchiol only from nature [18,19]. Furthermore, the HMBC cross-peaks of H-8' at δ H 4.06 with aromatic C-4 at δ C 159.7 indicated that C-8' was connected to C-4 of bakuchiol moiety by an ether linkage (Fig. 4). According to the above data, the structure of compound 3, named bisbakuchiol O, was established as shown in Fig. 1.
Compound 4 was also isolated as yellowish oils with  (Tables 1 and 2) for compound 4 was comparable to those of compound 3. Compared with compound 3, H-7' at δ H 2.96 (1H, br dd, J = 11.7, 10.5 Hz) displayed a NOE correlation with 16'-CH 3 at δ H 1.30 (1H, s), indicating that they were cofacial, and H-7' was assigned in a β-configuration. And the large coupling constants (J = 11.7, 10.5 Hz) indicated that H-8' and H-12' were α-oriented. As a result, the configuration was confirmed as 7'R,8'R,9'S,12'R. According to the above data, compound 4 was a dimer, whose C-8' of psoracorylifol F was connected to aromatic C-4 of bakuchiol moiety by an ether linkage (Fig. 4). Thus, the structure of compound 4, named bisbakuchiol P, was established as shown in Fig. 1. Combined with NMR data, a set of bakuchiol unit signals except for downfield shift to δ C 157.0 for C-4, and a set of psoracorylifol A unit signals [20] except for downfield shift to δ C 79.9 for C-7' were observed. In the HMBC spectrum of 5 (Fig. 4), a psoracorylifol A unit located at C-4 of the bakuchiol unit was verified by correlations from H-7' at δ H 5.17 to C-4 at δ C 157. These features permitted assignment of the planar structure of compound 5 as shown in Fig. 1. In the NOESY spectrum (Fig. 5), correlations between H-7' at δ H 5.17 and CH 3 -16'β at δ H 1.10, H-8' at δ H 3.46 and CH 3 -16'β, indicated that H-7' and H-8' were β-oriented. Whereas H-12' at δ H 4.58 was α-oriented, which was verified by the NOE correlation from H-8' and CH 3 -15' at δ H 1.35. Thus, the absolute structure of compound 5, named bisbakuchiol Q, was established as 9S,7'S,8'S,9'S,12'S.
Compound 6 was isolated as white amorphous powders, and possessed the molecular formula of C 37 H 50 O 4 Similar NMR signals of bakuchiol and psoracorylifol A to 5 were observed in 6. In the HMBC experiment (Fig. 4), a characteristic methoxyl group at δ H 3.04 (3H, s) correlated with C-7' enabled us to attach this methoxyl group to the C-7' . In NMR spectra of 6, the signals of an exomethylene of psoracorylifol A unit had disappeared, while a new signal for characteristic methyl group at δ H 0.49 (3H, s) and an oxygenated quaternary carbon at δ C 82.0 had appeared. Combined with the downfield shift of C-3 and C-5 of bakuchiol unit, it was obvious that two units were attached together by C 4 -O-C 13' . In the NOESY spectrum (Fig. 5), correlations between H-7' at δ H 4.18 and CH 3 -16'β at δ H 1.10, H-8' at δ H 3.96 and CH 3 -16'β, indicated that they were cofacial and were β-oriented. Similarly, the NOE correlation between H-8' and CH 3 -15' at δ H 0.82 supported that H-12' at δ H 3.14 was α-oriented. Finally, the structural assignment of 6 was assigned as 9S,7'S,8'S,9'S,12'S, and compound 6 was named bisbakuchiol R.
Compound 9 was isolated as yellowish oils with [α]25 D + 10.0, and possessed the same molecular formula,  Tables 1 and 2) of 9 were quite superimposable with those of compound 8, which clearly indicated the same skeleton as that of 8. Likely, the NOE correlation between H-7' at δ H 4.91 and 16'-CH 3 at δ H 1.07, and the coupling constant (J = 6.1 Hz) between H-7' and H-8' , indicated that the configuration of 9 was 9S,7'R,8'R,9'S, which was consistent with ECD data (Fig. 6). Therefore, the structure of compound 9, named bisbakuchiol U, was established as shown in Fig. 1.
Compound 10 was also isolated as yellowish oils. Its HRESIMS data exhibited a sodium adduct ion at m/z 445.3080 [M + Na] + , establishing the molecular formula as C 29 H 42 O 2 . Comparison of NMR spectra of 10 (Table 3) and known bakuchiol, a side chain (3-ethenyl-3,7-dimethyl-1,6-octadienyl) and a p-disubstituted benzene ring of 10 were identical to that of bakuchiol. In addition, the COSY, HSQC and HMBC correlations showed the presence of 2-ethenyl-2-methyl-5-isopropanol-cyclopentan-1-ol (6-ethenyl-6-methyl-4-isopropanol-cyclopentan-5-ol) substituted group in 10. The chemical shifts of C-3 and C-5 in 10 were shifted downfield to δ C 124.5, suggesting that this substituted group was connected to C-4 of bakuchiol moiety by an ether linkage. The relative configuration was mainly assigned by NOESY spectrum. The signals of H-4' at δ H 2.31 and H-5' at δ H 4.01 showed a NOE correlation, whereas H-4' or H-5' and 11'-CH 3 showed no correlation in its NOESY spectrum, indicating that both H-4' and H-5' were α-oriented. Therefore, the structure of compound 10, named bakuchiol ether A, was defined as shown in Fig. 1.
Compound 11 showed a molecular formula of C 33 H 48 O 2 on the basis of the HRESIMS ion at m/z 477.3713 [M + H] + . Similarly, compound 11 possessed a bakuchiol moiety by its' NMR data. In addition, 2D NMR correlations in 11 showed the presence of clovane-2β,9αdiol [22,23] moiety with the exception of the resonances of C-1' , C-2' to downfield shifts and C-3' and C-4' to highfield shifts. In the key HMBC spectrum (Fig. 4), the key correlation between H-2' at δ H 4.24 and C-4 at δ C 158.2 revealed that C-4 of bakuchiol moiety was connected to C-2' of clovane-2β,9α-diol moiety by an ether linkage. Therefore, the structure of compound 11, named bakuchiol ether B, was defined as shown in Fig. 1.
Compound 12 was isolated as yellowish oils with [α]25 D + 30.0. It showed a molecular formula of C 33 H 48 O 2 . The COSY, HSQC and HMBC correlations showed the presences of one set of the bakuchiol signals and one set of the caryolane-1,9β-diol signals in compound 12 [23]. The chemical shifts of C-3 and C-5 were shifted downfield to δ C 121.6 and the chemical shifts of C-1' was shifted downfield to δ C 80.2 in 12, suggesting that C-1' of this caryolane-1,9β-diol moiety was connected to C-4 of bakuchiol moiety by an ether linkage. Therefore, the structure of compound 12, named bakuchiol ether C, was defined as shown in Fig. 1.
Interestingly, when the quaternary carbon from the other unit was connected to bakuchiol unit by C-O-C 4 , the chemical shifts of C-3 and C-5 would shift downfield (from115 to 121 or 123 ppm) as shown in compounds 1, 6, 7, 10, 12, 15, 16 and 17. Whereas, the link by CH-O-C 4 would not result in changes of chemical shifts at C-3 and C-5 as shown in compounds 3, 4, 5 and 11. Therefore, we could infer the connection position of the dimers by the carbon chemical shifts of C-3/5 in bakuchiol unit.
NO, an unstable biological free radical, comes of L-arginine under the action of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS). NO functions as a signaling molecule participating in neurotransmission and vasodilation. However, overproduction of NO is involved in inflammatory diseases, which can be treated by NO inhibitor. To evaluate their anti-inflammatory activities, compounds 1-17 (1.56-50.00 μM) were tested for inhibition effect on NO production in LPS-stimulated RAW264.7 macrophages using the Griess reaction [15]. L-NIL, a selective inhibitor of iNOS, was used for the positive control. Firstly, the cytotoxicity of these compounds at concentrations from 1.56 to 50 μM was assessed. The MTT tests demonstrated that compounds 4 and 16 showed cytotoxicity at the concentration of 50.00 μM, whereas other compounds were not cytotoxic. The IC 50 values of these compounds were calculated at nontoxic concentrations. As shown in Table 4, compound 1 exhibited significant inhibition of NO production with IC 50 value at the concentration of 11.47 ± 1.57 μM, which showed no significant difference with that of L-NIL (10.29 ± 1.10 μM). Compounds 2, 3, 10-12, 16 and 17 exhibited moderate inhibitory activities with IC 50 values at the range of 15.98-27.80 μM. The IC 50 values of the other compounds were more than 50.00 μM, and they showed weak inhibitory activities against NO production.

Discussion
In our previous researches, we have obtained fourteen meroterpenoids and seventeen heterodimers of bakuchiol and evaluated their cytotoxicity [12,13]. Further investigation on the cHE extract brought about 29 bakuchiol monomers and dimers, and their NO inhibition activities in LPS-stimulated RAW264.7 macrophages were studied. We have reported 9 monomers and 3 dimers in Chinese Traditional and Herbal Drugs [24]. In this research, seventeen bakuchiol dimmers, including twelve unpresented ones, were reported. Fortunately, a new skeleton bakuchiol dimer (1) was isolated, and it exhibited significant NO inhibition activities with IC 50 value of 11.47 μM. Compounds 2, 3, 10-12, 16 and 17 exhibited moderate inhibitory activities with IC 50 values at the range of 15.98-27.80 μM, and other compounds showed weak inhibitory activity with IC 50 values more than 50.00 μM.
In order to fully explore the relationship between structure and activity, results of 29 bakuchiol monomers and dimers were compared. Bakuchiol showed cytotoxicity at 12.50-50.00 μM, and exhibited weak activity with inhibitory rate of 32% at the concentration of 6.25 μM. Interestingly, structural changes at the side chain, including oxidation, cyclization and dimerization, reduced cytotoxicity. We found that the activities of uncyclized bakuchiol derivants seemed to be superior to cyclized ones. Notably, some uncyclized monomers and dimers with oxygen substitution at C-12/12′ showed stronger inhibitory activities than L-NIL, such as 12,13-dihydro-12,13-epoxybakuchiol, 12-oxobakuchiol and (12′S)bisbakuchiol C. Among dimers, compound 1 and (12′S)-bisbakuchiol C had excellent activities, which were mostly contributed by the 6/6/5 tricyclic ketone unit and the 12,13-dihydro-12,13-dihydroxybakuchiol unit respectively. And it was worth to mention that compounds (3, 16 and 17) with a psoracorylifol F unit possessed better inhibitory activities than ones (5-7) with a psoracorylifol A unit.

Conclusion
Seventeen bakuchiol dimers (1-17), including 12 undescribed dimers and 5 known compounds, were isolated from the fruits of Psoralea corylifolia L. and their structure were identified by spectral methods and X-ray single-crystal diffraction. Bisbakuchiol M (1), whose other bakuchiol unit was cyclized to form a 6/6/5 tricyclic system, was a new skeleton compound. And the plausible biosynthetic pathway of bisbakuchiol M was proposed. Their inhibition on NO production in LPS-stimulated RAW264.7 macrophages were evaluated by the Griess reaction. Compounds 2, 3, 10-12, 16 and 17 exhibited inhibitory activities, and the inhibition of compound 1 was equal to that of L-NIL. Their structure-activity relationship was discussed, showed that uncyclized monomers and dimers with oxygen substitution at C-12/12′ showed strong inhibitory activities. And carbonyl units contributed to enhanced activities. These findings suggested that Psoraleae Fructus provided natural antiinflammatory constituents and were of great significance in the design for anti-inflammatory drug.