Eight Indole Alkaloids from the Roots of Maerua siamensis and Their Nitric Oxide Inhibitory Effects

Maerua siamensis (Capparaceae) roots are used for treating pain and inflammation in traditional Thai medicine. Eight new indole alkaloids, named maeruanitriles A and B, maeroximes A–C, and maeruabisindoles A–C, were isolated from them. Spectroscopic methods and computational analysis were applied to determine the structure of the isolated compounds. Maeroximes A–C possesses an unusual O-methyloxime moiety. The bisindole alkaloid maeruabisindoles A and B possess a rare azete ring, whereas maeruabisindole C is the first indolo[3,2-b]carbazole derivative found in this plant family. Five compounds [maeruanitriles A and B, maeroxime C, maeruabisindoles B, and C] displayed anti-inflammatory activity by inhibiting nitric oxide (NO) production in the lipopolysaccharide-induced RAW 264.7 cells. Maeruabisindole B was the most active inhibitor of NO production, with an IC50 of 31.1 ± 1.8 μM compared to indomethacin (IC50 = 150.0 ± 16.0 μM) as the positive control.


Introduction
Maerua is a genus in the family Capparaceae distributed in Africa, Arabia, South Asia, and Indo-China. Up to now, 69 accepted species have been discovered [1]. However, there are only a few reports on the phytochemicals of Maerua plants. For example, aminoguanidine derivatives were isolated from the leaves of Maerua edulis from southern Africa [2], whereas lupane triterpenoids were obtained from the aerial parts of M. oblongifolia collected in Saudi Arabia [3] and ionol and flavonoid glycosides were found in the aerial parts of M. crassifolia grown in Egypt [4]. The methanolic leaf extract of M. crassifolia has been reported to exhibit analgesic and anti-inflammatory activities in rodent models [5]. Moreover, extracts from M. subcordata were able to inhibit nitric oxide (NO) production in murine macrophage and human osteosarcoma cells [6]. In traditional Thai medicine, the roots of Maerua siamensis (Kurz) Pax are used in several anti-inflammatory and pain-relieving preparations [7]. The plant is the only recorded species of Maerua in Thailand [8]. Recently, the anti-inflammatory activity of its ethanolic root extract has been demonstrated in a protein denaturation assay [9]. Previous phytochemical studies on the leaves and twigs of M. siamensis revealed the presence of larvicidal 1H-indole-3 acetonitrile glycosides [10]. These indole derivatives are similar to the ones found in plants  Compound 3 was isolated as a reddish-brown amorphous solid. The molecular mula was deduced to be C13H16N2O2S (IHD 7), according to a pseudo-molecular [M + Compound 2 was isolated as a reddish-brown amorphous solid with the molecular formula of C 12 H 12 N 2 O 2 S (IHD 8), based on its [M + Na] + ion peak at m/z 271.0511 (calcd. for C 12 H 12 N 2 O 2 SNa, 271.0512). The IR spectrum showed absorption bands that were attributable to amine NH (3164 cm −1 , broad), nitrile (2250 cm −1 ), the aromatic ring (1627, 1451 cm −1 ), and sulfoxide (1022 cm −1 , strong) functionalities. The 1 H-NMR spectrum of 2 showed signals due to ABX coupling protons [δ H  and one methylsulfinyl group (δ H 2.16, 2-SOCH 3 ). Its 13 C-NMR spectrum showed twelve carbon signals of eight unsaturated carbons (δ c 160. 6, 140.3, 132.5, 121.4, 121.4, 113.4, 110.8, 95.3), one aliphatic methylene carbon (δ c 13.0, C-8), one methoxy carbon (δ c 55.9, 6-OCH 3 ), one nitrile carbon (δ c 118.9) and one methylsulfinyl carbon (δ c 40.4, 2-SOCH 3 ) (Table 1). These NMR data of 2 were similar to those of 1 except for the presence of an additional tetra-substituted double bond and a methylsulfinyl instead of a carbonyl group. The methylene signals (H-8a and H-8b) of the acetonitrile group appeared more downfield due to the anisotropic effect of a double bond between positions 2 and 3. This effect was also observed in indole-3-acetonitrile-2-S-β-glucopyranoside, which was isolated from the roots of I. indigotica [12]. The HMBC correlation of H-4 to a carbon signal at δ c 110.8 (C-3) helped assign the carbon signal at δ c 118.9 as that of the nitrile C-9. In addition, an HMBC correlations of H-8a and H-8b signals to C-2 (δ c 132.5), C-3, C-3a (δ c 121.4), and C-9 supported the indole-3-acetonitrile structure of 2, and a HMBC correlation of 2-SOCH 3 to C-2 signals, which established the position of the methylsulfinyl group ( Figure 1). Thus, the structure of compound 2 was determined as 2-(6-methoxy-2-(methylsulfinyl)-1H-indol-3-yl)acetonitrile and given the trivial name maeruanitrile B.
Compound 3 was isolated as a reddish-brown amorphous solid. The molecular formula was deduced to be C 13 3 ) and one methylthio group at δ H 2.17 (3H, s, SCH 3 ). The difference in the structure of this compound from compound 2 is the absence of a substituent on C-2, as supported by a 1 H-1 H COSY correlation of NH-1/H-2, and the presence of substituted imine, instead of nitrile, in its side chain. A methylthio group was located at C-9 on the imine double bond, based on an HMBC correlation between its methyl signal (δ H 2.17) to C-9 (δ C 157.8), and a methoxy group (δ H 3.86, δ C 61.5) was placed on the nitrogen atom of this imine bond as an O-methyloxime substructure based on the comparison of its chemical shift with that of 2-isopropyl-5-methylcyclohexanone O-methyloxime [16]. The methyl-Nmethoxyethanimidothioate-2-yl substitution at C-3 was confirmed by HMBC correlations of H-8 methylene signals (δ H 3.80) to C-2 (δ C 123.1), C-3 (δ C 107.8), C-3a (δ C 121.5), and C-9 ( Figure 2). The cis orientation between the N-OCH 3 and SCH 3 groups was suggested by the lowest relative energy computation based on a DFT calculation at a B3LYP/6-31g (d,p) level (Section 2.2). Therefore, the chemical structure of 3 was proposed to be methyl (Z)-N-methoxy-2-(6-methoxy-1H-indol-3-yl)ethanimidothioate and named maeroxime A. Sulfur-containing indole derivatives from M. siamensis could be phytoalexins similar to those found in Brassicaceae plants, and glutathione-S-transferase (GST) might be involved in the biosynthesis of these phytochemicals [17].  Compound 4 was obtained as an orange-brown amorphous solid. Its molecular formula of C 13 H 14 N 2 O 3 S (IHD 8), determined from the HR-ESI [M + H] + ion peak at m/z 279.0782 (calcd. for C 13 H 15 N 2 O 3 S, 279.0803), was 14 mass units higher than compound 3, and thus, suggested the presence of a carbonyl instead of a methylene group. This was supported by a conjugated keto carbonyl signal (δ C 182.5) in its 13 C-NMR spectrum. The 3,6-disubstituted 1H-indole nucleus of 4 is identical to that of 3, as evidenced by their 1 H-and 13 C-NMR data in Table 2. The difference was the presence of a carbonyl C-8 (δ C 182.5), which displayed an HMBC correlation with an H-2 signal (δ H 7.97) ( Figure 2). Similar to compound 3, the cis conformation between the N-OCH 3 (δ H 3.73, δ C 61.9) and SCH 3 groups (δ H 2.43, δ C 12.7) on the imine bond was proposed based on computational studies (Section 2.2). Thus, the chemical structure of 4 was established as methyl (Z)-Nmethoxy-2-(6-methoxy-1H-indol-3-yl)-2-oxoethanimidothioate and given the trivial name maeroxime B.

Computational Analysis
Configuration of compound 1 and conformational analysis of compounds 2-7 were optimized by the density of the functional theory (DFT) at a B3LYP/6-31G(d,p) level. Theoretical ECD spectra of maeruanitrile A (1) were computed using a time-dependent−density functional theory (TD-DFT) method at a B3LYP/6-311++G(d,p) level. The ECD spectrum of (S)-1 agreed with the experimental CD spectrum of 1 and supported the assignment of the S configuration on C-3 (see Supplementary materials A: Figure S4). Based on the lowest relative energy, the proposed conformational structures of maeruanitrile B (2) and maeruabisindoles A and B (6-7), and geometrical conformations of maeroximes A-C (3-5) were consistent with their NOESY interactions ( Figure 6).

Computational Analysis
Configuration of compound 1 and conformational analysis of compounds 2-7 were optimized by the density of the functional theory (DFT) at a B3LYP/6-31G(d,p) level. Theoretical ECD spectra of maeruanitrile A (1) were computed using a time-dependentdensity functional theory (TD-DFT) method at a B3LYP/6-311++G(d,p) level. The ECD spectrum of (S)-1 agreed with the experimental CD spectrum of 1 and supported the assignment of the S configuration on C-3 (see Supplementary materials A: Figure S4). Based on the lowest relative energy, the proposed conformational structures of maeruanitrile B (2) and maeruabisindoles A and B (6-7), and geometrical conformations of maeroximes A-C (3-5) were consistent with their NOESY interactions ( Figure 6).

Inhibition of Nitric Oxide Production of Isolated Compounds
Nitric oxide (NO) is an effector mediator in the immune system synthesized by nitric oxide syntheses (NOS). When infection or tissue injury occurs, macrophages are stimulated by pro-inflammatory cytokines [tumor necrosis factor α-(TNF-α), interleukin-1 (IL-1)], and/or lipopolysaccharide (LPS). The inducible NOS gene is subsequently expressed, and NO is produced [22,23]. In chronic inflammation, NO can stimulate cyclooxygenase-2 (COX-2) activity, resulting in increased prostaglandin production and the pathogenesis of inflammation [24]. Thus, screening for inhibitors of nitric oxide production is an important step to determine the potential anti-inflammatory activity of the test compounds.

Inhibition of Nitric Oxide Production of Isolated Compounds
Nitric oxide (NO) is an effector mediator in the immune system synthesized by nitric oxide syntheses (NOS). When infection or tissue injury occurs, macrophages are stimulated by pro-inflammatory cytokines [tumor necrosis factor α-(TNF-α), interleukin-1 (IL-1)], and/or lipopolysaccharide (LPS). The inducible NOS gene is subsequently expressed, and NO is produced [22,23]. In chronic inflammation, NO can stimulate cyclooxygenase-2 (COX-2) activity, resulting in increased prostaglandin production and the pathogenesis of inflammation [24]. Thus, screening for inhibitors of nitric oxide production is an important step to determine the potential anti-inflammatory activity of the test compounds.

Plant Material
The roots of Maerua siamensis (Kurz) Pax. were collected in the Sikhio district, Nakhon Ratchasima province, and identified by one of the authors (C.C.) according to the Botanical Garden Organization (BGO) plant database, Thailand. A voucher specimen (CC-MS-0419) has been deposited at the herbarium of the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand.

Extraction and Isolation
Dried roots were cut into small pieces and extracted with EtOAc (3 × 30 L) to yield the EtOAc extract (29.3 g). The marc was further extracted with MeOH (3 × 30 L) to obtain the MeOH extract (350 g). The MeOH extract was mixed with distilled water and partitioned with n-butanol (3 × 5 L) to give an n-butanol extract (50.8 g).

Computational Detail
For the theoretical ECD spectra of maeruanitrile A (1), the possible configurations were computed at a B3LYP/6-31G(d,p) level. The ECD spectra were calculated using the time-dependent density functional theory (TD-DFT) method with a B3LYP functional and 6-311++G(d,p) basis set. The geometry optimization and TD-DFT calculations were both performed with a polarizable continuum model (PCM) solvation model using methanol (MeOH). The rotary strengths of 70 excited states were calculated. All calculations were performed using the Gaussian16 program package [28]. The ECD spectra were simulated with overlapping Gaussian functions with a σ = 0.20 eV fitting parameter using the SpecDis1.64 program [29]. The more reliable length gauge representation was used for the ECD spectra.

Preparation of Test Solutions
The compounds were dissolved in DMSO to prepare their stock solutions at a concentration of 50 mM. Then, they were pipetted to mix in the culture medium to make the maximum tested concentration of 200 µM containing 0.4% DMSO. Then, the 200 µM tested solution was further diluted with a culture medium, giving the concentration series of 200, 100, 50, and 25 µM (2-fold dilution). The working concentrations of the test compounds [25,50,100, and 200 µM] were used to treat cells.

Stimulation of Inflammation in Raw264.7 Cells
Raw264.7 cells were seeded at a density of 5 × 10 4 cells/well in a 96-well plate. The cells were pre-treated with various concentrations of samples for 24 h. Cells were induced with 100 ng/mL LPS for 24 h. The culture supernatant was collected for NO production analysis, and cells were further examined for their viability.
The cell viability was measured by the MTT assay [30]. An MTT solution (1 mg/mL) was added to each well and incubated for 4 h at 37 • C. Then, the MTT solution was removed, and the formazan production was dissolved with DMSO. The absorbance was measured at 570 nm using a microplate reader (BioTeK, Santa Clara, CA, USA).

Measurement of NO Production
The NO production was measured using a Griess reagent kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) [31]. Ninety µL of culture media were mixed with 10 µL of Griess reagent and incubated at room temperature for 30 min, and then the NO concentration was measured at 540 nm using the microplate reader. The percentage of NO production was calculated as Equation (1). The NO inhibitory activity was expressed as half the maximum (IC 50 ) of the inhibitory concentration calculated using GraphPad Prism 9.

Statistical Analysis
The IC 50 values were expressed as the mean ± standard deviation (SD) from at least three independent experiments. The mean differences of the compounds vs. indomethacin (the positive control) were evaluated by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad Prism 9.3.1 software, San Diego, CA, USA). Statistical significance was defined as p < 0.05, p < 0.005, p < 0.001, and p < 0.0001.

Conclusions
Eight new indole alkaloids were isolated from the roots of Maerua siamensis (Capparaceae). Their structures were elucidated based on spectroscopic methods and computational analysis. Among them, maeruanitriles A (1) and B (2), maeroxime C (5), and maeruabisindoles B (7) and C (8) inhibited nitric oxide production in LPS-induced RAW 264.7 cells. This finding supports the traditional use of M. siamensis roots for analgesic and anti-inflammatory purposes in traditional Thai medicine. Author Contributions: S.N. performed the extraction, isolation, spectroscopic operation, and structure elucidation and prepared and edited the manuscript. A.J. performed the nitric oxide inhibition assay and data analysis and prepared the manuscript. P.P. performed the computational studies and prepared the manuscript. M.K. supervised the nitric oxide inhibition assay and provided comments on the preparation of the manuscript. T.R. supervised the computational studies and provided comments on the preparation of the manuscript. H.-S.C. supervised the extraction, isolation, and structure elucidation and provided comments on the preparation of the manuscript. R.S. provided comments and suggestions on structure elucidation and reviewed and edited the manuscript. C.C. advised the extraction and isolation, performed the NMR operation and structure elucidation, conceptualized, designed, and supervised the research, along with preparing, reviewing, and editing the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was financially supported by the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, the CU Graduate school thesis grant, and Natural Products and Nanoparticles Research Unit, Chulalongkorn University.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data are present in the article and supplementary data.