Anti-Neuroinflammatory Components from Clausena lenis Drake

Clausena lenis Drake (C. lenis) is a folk medicinal herb to treat influenza, colds, bronchitis, and malaria. The 95% and 50% ethanol extract of C. lenis showed significant nitric oxide (NO) inhibition activity in BV-2 microglial cells stimulated by lipopolysaccharide (LPS). Bio-guided isolation of the active extract afforded five new compounds, including a chlorine-containing furoquinoline racemate, (±)-claulenine A (1), an amide alkaloid, claulenine B (2), a prenylated coumarin, claulenin A (3), a furocoumarin glucoside, clauleside A (4), and a multi-prenylated p-hydroxybenzaldehyde, claulenin B (5), along with 33 known ones. Their structures were determined via spectroscopic methods, and the absolute configurations of new compounds were assigned via the electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction analysis. Compounds 2, 23, 27, 28, 33, and 34 showed potent anti-neuroinflammatory effects on LPS-induced NO production in BV-2 microglial cells, with IC50 values in the range of 17.6–40.9 μM. The possible mechanism was deduced to interact with iNOS through molecular docking.


Introduction
Neuroinflammation generally refers to an inflammatory response in the brain or spinal cord and has a pivotal role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) [1] and Parkinson's disease (PD) [2,3]. This inflammation is mediated by cytokines, chemokines, reactive oxygen species, and secondary messengers produced by resident central glial cells (microglia and astrocytes), endothelial cells, and immune cells of peripheral origin [4]. Among them, microglia, as innate immune cells of the central nervous system, are major participants in neuroinflammation [1] and have various neuroimmunological functions in the central nervous system under normal and pathological conditions [5].

Structural Elucidation
(±)-Claulenine A (1) was obtained as a white solid, [α] 25 D 0 (c 0.12, MeOH). The HR-ESI-MS data indicated the presence of one chlorine in the molecule from the relative abundance (∼1/3) of the isotope peaks observed at m/z 366.1101 and 368.1077, corresponding to a molecular formula of C 18 H 20 NO 5 Cl (calcd for C 18 H 21 NO 5 Cl, 366.1108; mass error −1.9 ppm), with nine degrees of unsaturation. Analysis of the 1 H-NMR data (Table 1) revealed the presence of a trisubstituted furoquinoline, which was indicated by the typical adjacent protons of a furan moiety at δ H 7.59 (1H, d, J = 2.7 Hz, H-2) and 7.00 (1H, d, J = 2.7 Hz, H-3) [23] and two aromatic singlet signals at δ H 7.45 and 7.34. Meanwhile, two methoxys (δ H 4.03 and 4.00), two methyls (both δ H 1.77), an oxymethylene [δ H 4.93 (1H, dd, J = 9.8, 3.7 Hz) and 4.75 (1H, dd, J = 9.8, 6.8 Hz)], and an oxymethine [δ H 4.18 (1H, dd, J = 6.8, 3.7 Hz)] signals were also observed in the 1 H NMR data (Table 1). In the 13 C-NMR data of 1, there were 18 carbon resonances, comprising two methyls, two methoxyls, one methylene, five methines (four olefinic and one oxygenated aliphatic), and eight quaternary carbons, which were very similar to those of (2S)-1-[(6,7-dimethoxyfuro[2,3-b]quinolin-4-yl)oxy]-3-methylbutane-2,3-diol [24]. Considering the molecular formula of 1, one of the two hydroxy groups of (2S)-1-[(6,7-dimethoxyfuro[2,3-b]quinolin-4-yl)oxy]-3-methyl-butane-2,3-diol was deduced to be substituted by a chlorine atom in 1. The above deductions were further confirmed by 2D NMR experiments ( Figure 2). The two methoxy groups showed HMBC cross-peaks with the carbon at δ C 148.1 and 152.8, respectively, locating them at positions C-6 and C-7; H 2 -1 [δ H 4.93 (dd, J = 9.8, 3.7 Hz) and 4.75 (dd, J = 9.8, 6.8 Hz)] presented an HMBC correlation to C-4 (δ C 154.4), suggesting that the oxygenated prenyl unit was linked to C-4 of furoquinoline via an oxygen bridge. In addition, the chlorine at C-3 and the hydroxy group at C-2 were indicated by the key COSY correlations ( Figure S11 Figure 1) and denominated as claulenine A. were in the aromatic region (δH 6.2-7.7). The 13 C NMR data displayed 19 carbons including a signal of amide (δC 166.7). Careful analysis of 1 H and 13 C NMR data revealed that the signals of 2 resembled those of anhydromarmeline [25], except that the prenyl moiety was replaced by a methoxy group and an additional methyl positioned at the nitrogen atom    However, 1 was isolated as a raceme indicated by its zero specific rotation value and no Cotton effects in the electronic circular dichroism (ECD) spectrum. Further HPLC separation on chiral phase afforded the enantiomers of (+)-1a and (-)-1b ( Figure S14, Supporting Information). To clarify their absolute configurations, ECD calculations were utilized, and the results showed that the ECD spectrum of (R)-1 matched well with the experimental curve of (+)-1a ( Figure 3). This conclusion was finally confirmed by the single-crystal X-ray diffraction for (+)-1a using Cu Kα radiation ( Figure 4). Considering that there are fewer phytochemicals containing chlorine atoms, a freshly prepared methanol extract of the title plant was detected by using the multiple reaction monitoring (MRM) mode of UPLC/Qtrap-MS/MS, and the results suggested that 1 comes from natural source ( Figure S15, Supporting Information).   [26]. The 1 H NMR data of 3 (Table 1)   HSQC spectra displayed 19 carbons classified into four methyls, two methylenes (one aliphatic and one olefinic), five methines (one oxygenated aliphatic and four olefinic), and eight quaternary (one ester carbonyl, two aliphatic, and five olefinic, including two oxygenated) carbons. The 1 H and 13 C-NMR data of 3 bore a close resemblance to those of gravelliferone A [19], except that the methoxyl at C-7 in gravelliferone A was replaced by a hydroxy in 3, which was supported by the molecular formula of 3 and the chemical shift of C-7 (δC 162.4). Therefore, the planar structure of 3 was deduced.
To clarify the absolute configuration of the only chiral center in 3, ECD calculation was performed. The ECD experimental curve was in good agreement with the calculated one of (R)-3 ( Figure 3). Taken together, the structure of 3 was finally assigned and named claulenin A. The β-D-glucose was demonstrated via analysis of the coupling constant (7.8 Hz) of the anomeric proton [28] and the aryl thiocarbamate derivate of the hydrolyte by HPLC ( Figure S40, Supporting Information). The absolute configuration of aglycone moiety (4a) was assigned as (2′R) by comparison of the experimental and calculated ECD curves ( (Table 1)   Claulenine B (2) was obtained as a yellow oil, and its molecular formula was determined as C 19 H 19 NO 2 from the HR-ESI-MS ion at m/z 294.1490 [M + H] + (calcd for C 19 H 20 NO 2 , 294.1494; mass error −1.4 ppm) and 13 C-NMR data. In the 1 H NMR spectrum, except for the up-field shifts at δ H 3.78 (-OCH 3 ) and 3.10 (-NCH 3 ), the remaining signals were in the aromatic region (δ H 6.2-7.7). The 13 C NMR data displayed 19 carbons including a signal of amide (δ C 166.7). Careful analysis of 1 H and 13 C NMR data revealed that the signals of 2 resembled those of anhydromarmeline [25], except that the prenyl moiety was replaced by a methoxy group and an additional methyl positioned at the nitrogen atom in 2. The HMBC correlations ( Figure 2) of -OCH 3 /C-6 (δ C 159.5) and -NCH 3 /C-1 (δ C 127.1), C-9 (δ C 166.7) supported the deduction. Besides, the coupling constant of olefinic protons (J = 15.5 Hz for H-7 and H-8; J = 8.6 Hz for H-1 and H-2 ) pointed out that they were Eand Z-oriented, respectively. Hence, the gross structure of claulenine B (2) was depicted as given ( Figure 1 [26]. The and one olefinic), five methines (one oxygenated aliphatic and four olefinic), and eight quaternary (one ester carbonyl, two aliphatic, and five olefinic, including two oxygenated) carbons. The 1 H and 13 C-NMR data of 3 bore a close resemblance to those of gravelliferone A [19], except that the methoxyl at C-7 in gravelliferone A was replaced by a hydroxy in 3, which was supported by the molecular formula of 3 and the chemical shift of C-7 (δ C 162.4). Therefore, the planar structure of 3 was deduced.
To clarify the absolute configuration of the only chiral center in 3, ECD calculation was performed. The ECD experimental curve was in good agreement with the calculated one of (R)-3 ( Figure 3). Taken together, the structure of 3 was finally assigned and named claulenin A.
Clauleside A (4)  The β-D-glucose was demonstrated via analysis of the coupling constant (7.8 Hz) of the anomeric proton [28] and the aryl thiocarbamate derivate of the hydrolyte by HPLC ( Figure S40, Supporting Information). The absolute configuration of aglycone moiety (4a) was assigned as (2 R) by comparison of the experimental and calculated ECD curves ( Figure 3). Finally, 4 was established as scataccanol 4 -O-β-D-glucopyranoside and referred to as clauleside A.
The absolute configuration of C-2 was determined by the ECD calculation. The results (Figure 3) shown that the calculated ECD spectrum of (2 S)-5 agrees well with the experimental one, which allowed the absolute configuration of 5 to be specified as 2 S. As a result, the structure of claulenin B (5) was clarified.

Anti-Neuroinflammatory Activities
The 95% and 50% ethanol extract (CLT) of C. lenis, along with its three partitioned extracts with different polarity solvents, i.e., petroleum ether extract (CLPE), ethyl acetate extract (CLEA), and n-BuOH extract (CLnB) were evaluated for anti-neuroinflammatory activities based on reduction of NO production stimulated by LPS in BV-2 microglial cells. The results exhibited that NO production can be dose-dependently inhibited in the range of 10-80 µg/mL ( Figure 5). To further investigate which compounds are responsible for the effect, all of these isolates (1-38) were subjected to an evaluation of their antineuroinflammatory activities using the same method. Six compounds (2, 23, 27, 28, 33, and 34) inhibited NO production by more than 50% at 50 µM and their IC 50 values were finally determined to be from 17.6 to 40.9 µM (Table 2). Dexamethasone (DEX) was used as a positive control. Alongside this, no cytotoxicity was observed in BV-2 microglial cells treated by these test subjects at 50 µM (cell viability > 95%).

Interactions of Bioactive Compounds with iNOS
Nitric oxide synthases (NOSs) catalyze the NO production using oxygen and nitrogen derived from arginine. Inducible NOS (iNOS) is one of the isoenzymes of NOSs and plays a major part in NO production during inflammation [63]. In recent years, structurebased calculations have been widely used to predict the pharmacological mechanisms of active compounds, among which molecular docking is a commonly used method [64]. To explore the possible mechanism of those bioactive compounds against NO production, we investigated the interaction between iNOS and compounds 2, 23, 27, 28, 33, and 34 by molecular docking [65,66]. The results showed that compounds 27 and 33 had good affinities with iNOS (Glide Score < −5) (Table 3), and both of them have a hydrogen bond with the residues TYR341 of iNOS ( Figure 6). Therefore, the possible mechanism of NO inhibition of 27 and 33 is through interaction with iNOS by targeting the residues in the active pocket of iNOS.

Interactions of Bioactive Compounds with iNOS
Nitric oxide synthases (NOSs) catalyze the NO production using oxygen and nitrogen derived from arginine. Inducible NOS (iNOS) is one of the isoenzymes of NOSs and plays a major part in NO production during inflammation [63]. In recent years, structurebased calculations have been widely used to predict the pharmacological mechanisms of active compounds, among which molecular docking is a commonly used method [64]. To explore the possible mechanism of those bioactive compounds against NO production, we investigated the interaction between iNOS and compounds 2, 23, 27, 28, 33, and 34 by molecular docking [65,66]. The results showed that compounds 27 and 33 had good affinities with iNOS (Glide Score < −5) (Table 3), and both of them have a hydrogen bond with the residues TYR341 of iNOS ( Figure 6). Therefore, the possible mechanism of NO inhibition of 27 and 33 is through interaction with iNOS by targeting the residues in the active pocket of iNOS.

Discussion
Thirty-seven compounds were isolated and identified from C. lenis and five of them are new ones (1-5). The structures were elucidated based on MS, UV, IR, and NMR spectroscopic data and comparison with the data reported in literature. The absolute configurations of new compounds were characterized by using the ECD calculations and singlecrystal X-ray diffraction analysis. (±)-Claulenine A (1) is a chlorine-containing furoquinoline racemate. Chlorinated natural products are rare in terrestrial plants, but common in bacteria, marine animals, and macroalgae [67,68]. 1 could be an artifact produced in the isolation procedure, but could also be generated by Fe(II)-and 2-oxoglutarate-dependent halogenases (2ODHs) [69]. The LC/MS analysis of the fresh C. lenis proved 1 to be originated from a natural source, but the real chlorine source is not clear. Claulenine B (2) is a high conjugated amide alkaloid. Claulenin A (3) is a prenylated coumarin with an oxirane ring and clauleside A (4) is a furocoumarin glucoside. Claulenin B (5) is a p-hydroxybenzaldehyde with multi-prenyl substituents. Of the known compounds, 20 and 38 are the first reported natural products, and 20 and 28 compounds are obtained from Clausena species and C. lenis, respectively, for the first time. Most of these compounds are prenylated, so the prenyltransferases play an important role in their biosyntheses.
The ethanol extract and its partitioned extracts with different polarities from C. lenis have significant inhibitory effects on NO production in LPS-induced BV-2 microglial cells. In the subsequent bioactivity-guided fractionation, compounds 2, 23, 27, 28, 33, and 34 were disclosed to be the potentially active compounds with inhibition effects. Most of the bioactive compounds are coumarins, suggesting that coumarins might be the main bioactive substances for the anti-neuroinflammatory properties of the extract of C. lenis. Furthermore, the molecular docking results revealed that 27 and 33 had a good interaction with iNOS, which could be one of the mechanisms for their anti-inflammation effects.

General Experimental Procedures
Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). The electronic circular dichroism (ECD) and UV data were acquired on a JASCO, J-1500 CD spectrophotometer (JASCO, Tokyo, Japan). IR spectra were recorded on a Thermo Scientific Nicolet iS50 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). HR-ESI-MS data were measured on a Waters Xevo G2 Q-TOF mass spectrometer (Waters Co., Milford, MA, USA). The

Discussion
Thirty-seven compounds were isolated and identified from C. lenis and five of them are new ones (1-5). The structures were elucidated based on MS, UV, IR, and NMR spectroscopic data and comparison with the data reported in literature. The absolute configurations of new compounds were characterized by using the ECD calculations and single-crystal X-ray diffraction analysis. (±)-Claulenine A (1) is a chlorine-containing furoquinoline racemate. Chlorinated natural products are rare in terrestrial plants, but common in bacteria, marine animals, and macroalgae [67,68]. 1 could be an artifact produced in the isolation procedure, but could also be generated by Fe(II)-and 2-oxoglutarate-dependent halogenases (2ODHs) [69]. The LC/MS analysis of the fresh C. lenis proved 1 to be originated from a natural source, but the real chlorine source is not clear. Claulenine B (2) is a high conjugated amide alkaloid. Claulenin A (3) is a prenylated coumarin with an oxirane ring and clauleside A (4) is a furocoumarin glucoside. Claulenin B (5) is a p-hydroxybenzaldehyde with multi-prenyl substituents. Of the known compounds, 20 and 38 are the first reported natural products, and 20 and 28 compounds are obtained from Clausena species and C. lenis, respectively, for the first time. Most of these compounds are prenylated, so the prenyltransferases play an important role in their biosyntheses.
The ethanol extract and its partitioned extracts with different polarities from C. lenis have significant inhibitory effects on NO production in LPS-induced BV-2 microglial cells. In the subsequent bioactivity-guided fractionation, compounds 2, 23, 27, 28, 33, and 34 were disclosed to be the potentially active compounds with inhibition effects. Most of the bioactive compounds are coumarins, suggesting that coumarins might be the main bioactive substances for the anti-neuroinflammatory properties of the extract of C. lenis. Furthermore, the molecular docking results revealed that 27 and 33 had a good interaction with iNOS, which could be one of the mechanisms for their anti-inflammation effects.

Plant Material
In November 2019, dry leaves and stems of Clausena lenis Drake were collected from Baoting County, Hainan Province, People's Republic of China. Botanical identification was made by Prof. Pengfei Tu, one of the authors, and a voucher specimen (No. CL201911) was kept in the herbarium of Modern Research Center for Traditional Chinese Medicine, Peking University.
4.5. X-ray Crystallography of (+)-1a The single crystals of (+)-1a were collected from methanol solution at room temperature. An Agilent Gemini E X-ray single-crystal diffractometer with an Oxford Cryostream cooler was used to collect the single crystal data with Cu Kα radiation at T = 174.7 K. The structure was solved with the direct method using SHELXS-97 and refined anisotropically by full-matrix least-squares on F2 using SHELXL-97. The H atoms were placed in calculated positions and refined using a riding model. The absolute configuration was determined by refinement of the Flack parameter based on resonant scattering of the light atoms.

Absolute Configurations Determination of Sugar Moiety for 4
Compound 4 (1.0 mg) was hydrolyzed with 2 mol/L HCl (5 mL) for 5 h at 85 • C. The reaction product was extracted three times with CH 2 Cl 2 . After the aqueous layer was concentrated to dryness, 2 mL anhydrous pyridine containing 2.0 mg L-cysteine methyl ester hydrochloride was added and heated at 60 • C for 1 h. Subsequently, otolylisothiocyanate (10 µL) was added and heated at 60 • C for 1 h. D-glucose standard (2 mg) and L-glucose standard (2 mg) were respectively reacted in the same procedure. Then, each reaction mixture was filtered by a 0.22 µm membrane and analyzed directly by an Agilent Extended C18 column (250 mm × 4.6 mm, 5 µm) on an Agilent 1260 HPLC with a gradient elution of MeCN-H 2 O (5:95-30:70, v/v, 0-30 min, 1.0 mL/min) at 30 • C, and UV detection wavelength was 210 nm ( Figure S40, Supporting Information). The sugar moiety of 4 was detected as D-glucose by the same t R value with that of the D-glucose standard derivative [72].

Nitric Oxide (NO) Production Measurement and Cell Viability Assay
BV-2 cells (1 × 10 5 cells/well) were cultured in 48-well plates and stimulated with 1.0 µg/mL LPS (Escherichia coli 0111:B4, Sigma, St. Louis, MO, USA) with or without test extracts or compounds at 37 • C for 24 h. The production of NO was tested using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), according to the manufacturer's instructions. Cell culture supernatants (160 µL) were reacted with 80 µL of the Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diaminedihydrochloride, and 2% phosphoric acid) for 10 min in the dark at room temperature. The absorbance was measured at 540 nm using a microplate reader (Tecan Trading AG, Basel, Switzerland). The experiments were performed in triplicate, and the results are presented as the mean ± SD of three independent experiments. The cell viability was evaluated according to MTT assay. Dexamethasone was used as the positive control.

Molecular Docking
The crystal structure of iNOS (PDB ID: 3E6T) was obtained from the Protein Data Bank of RCSB (Research Collaboratory for Structural Bioinformatics). Docking simulations between bioactive compounds and iNOS were performed using the Maestro software suite 2015 (Schrodinger, New York, NY, USA). The ligand molecules were drawn with Chem3D Pro 14.0 (CambridgeSoft, Waltham, MA, USA) and optimized by the Ligprep module of Maestro. The protein receptor was prepared by deleting the ligand and water molecules and then was adopted for molecular docking with ligands. The reported inhibitor binding sites of iNOS was chosen as the binding pocket [73].