Bioactivity‐guided isolation of anti‐inflammatory limonins from Chukrasia tabularis

Abstract Chukrasia tabularis is an economically important tree and widely cultured in the southeast of China. Its barks, leaves, and fruits are consumed as a traditional medicine and perceived as a valuable source for bioactive limonin compounds. The extracts from root barks of C. tabularis showed significant anti‐inflammatory effect. The aim of this research was to explore the material basis of C. tabularis anti‐inflammatory activity, and to purify and identify anti‐inflammatory active ingredients. By a bioassay‐guided isolation of dichloromethane fraction obtained two novel phragmalin limonins, Chukrasitin D and E (1 and 2), together with 12 known limonins (3–14). The chemical structure of these compounds is determined on the basis of extensive spectral analysis and chemical reactivity. In addition, the activities of these isolated limonins on the production of nitric oxide (NO), tumor necrosis factor alpha (TNF‐α), and nuclear factor kappa B (NF‐κB) in RAW264.7 cells induced by lipopolysaccharide (LPS) were evaluated. Limonins 1 and 2 indicated significant anti‐inflammatory activity with IC50 values of 6.24 and 6.13 μM. Compound 1 notably inhibited the production of NF‐κB, TNF‐α and interleukin 6 (IL‐6) in macrophages. The present results suggest that the root barks of C. tabularis exhibited anti‐inflammatory effect and the limonins may be responsible for this activity.

Previous chemical research on this plant provided a series of phragmalin limonins. Limonin is a kind of nortriterpene with diversified structure, which has a wide range of bioactivities, like antifeeding insects, antimalarial and anticancer activities (Liao et al., 2009;Tan & Luo, 2011;Fang et al., 2011).
In the physiologic responses to infection or damage, macrophages have a special impact on the progress of inflammatory processes (Alivernini et al., 2020). Both the production of pro-inflammatory mediator and the aggravation of inflammation are impossible to separate from the action of macrophage (Eissa et al., 2018). Many pro-inflammatory cytokines, like tumor necrosis factor alpha (TNFα), interleukin 1β (IL-1β), and interleukin 6 (IL-6), are originated from macrophages. Nuclear factor kappa B (NF-κB) is an example of signal transduction and gene modulation associated with macrophages' immersion and activation (Sae- Tan et al., 2020). When activated, NF-κB causes the production of pro-inflammatory cytokines like TNFα, IL-6, and IL-1β. Given the potential relevance of inflammation and macrophages, it is important to find a way to modulate the expression of inflammatory cytokines and control the activation of macrophages.
Lipopolysaccharide (LPS) has been widely used to stimulate macrophages in inflammatory models in experiments on anti-inflammatory mechanisms. After LPS stimulation, NF-κB signaling cascade was activated, resulting in changes in related protein expression (Ren et al., 2020).
In recent years, the anti-inflammatory, antitumor, and antioxidant activities of Chukrasia tabularis have been widely reported (Kaur et al., 2011). In our studies on the anti-inflammatory constituents of Meliaceae plants, two new phragmalin limonoid orthoesters Chukrasitin D and E (1 and 2) (Figure 1) were isolated and identified from the root barks of C. tabularis, together with 12 known limonoids (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In this study, we report the separation, structure identification, and bioassay results of the extracts and isolated compounds. The in vitro anti-inflammatory assay of compounds 1-14 on LPS-mediated macrophages showed that limonins 1 and 2 displayed a significant inhibitory effect. In addition, the effects of limonin 1 on the production of nitric oxide, NF-κB, and TNFα in RAW 264.7 cells induced by LPS and their possible antiinflammatory mechanisms were also evaluated. Therefore, the current study focused on anti-inflammatory evaluation of C. tabularis extracts and isolated limonins.

| Reagents and materials
The optical rotation was obtained using a JASCO P-1020 polarimeter. Infrared (IR) spectra were measured on a Nicolet 170SX FT-IR spectrometer, ultraviolet (UV) spectra were detected on a 210A UV spectrum. The nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz Bruker spectrometer. Electrospray ionization mass spectrometry (ESIMS) and high-resolution electrospray ionization mass spectrometry (HRESIMS) were measured on a 2020 LCMS spectrum and Bruker APEX II mass spectrum, respectively.

| Preparation of extracts from C. tabularis and bioassay-guided separation
The anti-inflammatory test of xylene-induced ear edema in mice showed that the dichloromethane extract had significant antiinflammatory activity (Table 1), so the dichloromethane phase was selected for further separation. Subfractions of dichloromethane extracts Fr.C and Fr.D showed significant anti-inflammatory activity by mouse xylene auricle swelling experiments (Table 1), so isolation and purification focused on these two fractions.
The chipped root bark of C. tabularis (5.6 kg) was extracted three times with MeOH at room temperature for 7 days each (20 L). The obtained solution was evaporated in vacuo to gain a brownish extract (890 g). The residue was suspended in H 2 O and divided by petroleum ether (PE), dichloromethane (CH 2 Cl 2 ), ethyl acetate, and n-butanol.

| Laboratory animals
Male Institute of Cancer Research (ICR) mice (18 ± 2 g), specific pathogen free, were supplied by the experimental animal center of Fujian

| Xylene-induced ear edema in mice
The extracts were dissolved in 0.5% CMC-Na (sodium carboxymethyl cellulose) and Aspirin was applied as a positive control. After gavage of the extracts or control for 1 h, the right ear of each mouse was treated with 40 μl of xylene solution, and the left ear served as a control. One hour after xylene treatment, mouse was executed due to cervical dislocation. A circular part with a diameter of 6 mm of each ear was weighed with an electronic analytical balance, and its inhibitory activity on ear edema was calculated (Table 1).

| In vitro anti-inflammatory activities
RAW 264.7 cells obtained from the China Center for Cultivated Studies (Shanghai, China) were maintained in DMEM contained with 1% penicillin and streptomycin and 10% fetal bovine serum, and under 5% CO 2 at 37°C. Cells were stimulated with LPS. In brief, cells were placed on the 96-well plate (1 × 10 5 cells/well). After 2 h of preincubation, the LPS (2 μg/ml) and compounds were added and the samples incubated for 24 h. The supernatant of cell culture was collected 24 h later and NO was detected by the Griess reagent (Gasparotto et al., 2013).

| Measurement of NFκ B, IL-6, and TNFα production
The levels of NF-κB, TNFα, and IL-6 were determined by enzymelinked immunosorbent assay (ELISA) based on manufacturer's protocol. The standard solution and the antibody-bearing sample were placed at 37°C for 60 min, added to the working solution, incubated in 37°C for 30 min, and washed. Tetramethylbenzidine (TMB) was then added and the TMB termination solution was added after 20 min. In the end, the absorbance at 450 nm was recorded by ELISA.

| Statistical analysis
The data obtained were expressed as mean ± SD. All experiments had 3 replicates. The t-test was used to verify differences between groups by IBM SPSS Statistics 24.

| Bioactivity-guided abstraction and isolation of active components
The anti-inflammatory activities of methanolic, petroleum ether, dichloromethane (CH 2 Cl 2 ), EtOAc, and n-butanol extracts and fractions from the root barks of C. tabularis were assessed in vivo by xylene-induced ear edema in mice. The result showed that the dichloromethane extract displayed significant anti-inflammatory activities with an inhibition rate of 42.41% (400 mg/kg) ( Table 1).
The subfractions of dichloromethane extract Fr.C and Fr.D exhibited significant anti-inflammatory activities with inhibitory values of 43.65% and 42.93% (400 mg/kg). Two novel phragmalin limonins, Chukrasitin D (1) and E (2), together with 12 known limonins (3-14) were separated and identified from Fr.C and Fr.D (Figure 1). 10 methyl groups (three methoxys), seven methylene groups, five methane groups (two oxygenated), and 13 quaternary carbons (five oxygenated). In addition, a comprehensive analysis of its 1 HNMR (proton nuclear magnetic resonance) and 13 CNMR (carbon nuclear magnetic resonance) and data (Table 2) showed the presence of three methyl esters, one orthoacetate moiety, one propanoyl, and one 3-methylbutyryl group. In molecule 1, there are 11 unsaturates, of which 5 are occupied by 5 ester carbonyls, and the remaining 6

| Structural elucidation of isolated compounds
unsaturates require 1 to be hexacyclic in the center. The foregoing data indicated that 1 was a limonoid orthoester of phragmalin type (Lin et al., 2009).
Extensive 2DNMR (two-dimensional nuclear magnetic reso-  9, 14-, 8, 9, 30-, and 1, 8, 9-orthoacetate (Lin et al., 2011;Silva et al., 2008;Zhang, Fan, et al., 2008;. to the above results, the relative configuration of 1 was completed, as shown in Figure 1. By comparing experiments and computational ECD data, the absolute configuration of 1 was finally proved, which is a suitable method for solving the absolute configuration of natural TA B L E 2 1 H-NMR (proton nuclear magnetic resonance) (400 MHz) and 13 C-NMR (carbon nuclear magnetic resonance) (100 MHz) spectroscopic data for 1 and 2  Figure 2). Thus, the stereochemistry of 1 was constructed as shown in Figure 1 (Figure S1).
Chukrasitin E (2) was found to possess a quasimolecular ion peak was constructed as shown in Figure 1 (Figure S9).

| Anti-inflammatory effects of separated limonins from C. tabularis
Nitric oxide is a major bioinformatics molecule with dual roles of biological messenger and cytotoxic molecule. It is involved in the pathogenicity of inflammation, is overexpressed in LPS-mediated macrophages, and is an indicator of inflammation (Jeon et al., 2016;Keisuke et al., 2021). The in vitro anti-inflammatory effects of limonins (1-14) were determined by LPS-stimulated RAW 264.7 cells by evaluating the production of NO. Cell viability determination showed that limonins (1-14) have no toxicity to RAW 264.7 cells at a concentration of 100 μM. To determine whether limonins (1-14) suppressed NO production by LPS-mediated RAW 264.7 cells, the concentration of NO in medium containing these limonins was evaluated. Table 3, 14 limonins exhibited anti-inflammatory effects at the tested concentration. The result exhibited that D-ring-opened phragmalin limonoid orthoester (1-2) showed strong NO inhibitory activities, while limonins (3-14) showed potent to moderate activity. Limonins 1-2 displayed significant anti-inflammatory activities

TA B L E 2 (Continued)
F I G U R E 2 Calculated and experimental electronic circular dichroism (ECD) spectra of 1 with IC 50 values of 6.24 and 6.13 μM. Limonoids 3-14 showed effective anti-inflammatory effect with the inhibition rate between 12.30 and 50.19 μM. Considering that anti-inflammatory components are found in the root bark of C. tabularis, it can be determined that they are a source of natural anti-inflammatory molecules. It is worth noting that limonins 1 and 2 showed the strongest anti-inflammatory activity. Therefore, the potential anti-inflammatory activity and molecular mechanism of compound 1 were further studied.
Excessive production of pro-inflammatory cytokines exacerbates a variety of diseases, including allergies, autoimmune disease, and cancer (Benedetto et al., 2019;Guo et al., 2019). We investigated the activity of limonin 1 on LPS-stimulated pro-inflammatory cytokines in RAW 264.7 cells. The results in Figure 3 exhibited that the levels of NF-κB, IL-6, and TNFα in the LPS group were notably higher than those in the control group. As shown in Figure 3a-c, the addition of limonin 1 notably suppressed production of NF-κB, IL-6, and TNFα in a dose-dependent manner. The result indicated that limonin 1 could suppress the expression of NF-κB, IL-6, and TNFα in LPSstimulated macrophage, and achieved anti-inflammatory activity.
The regulation of anti-inflammatory activity on macrophages may be partly involved in the protective activity of Chukrasia tabularis on inflammatory diseases.

| DISCUSS ION
Macrophage is involved in most inflammatory responses, including LPS stimulation, and secretes pro-inflammatory cytokines like NF-κB, TNFα, and IL-6 (Kim et al., 2020). Modern studies have suggested that natural product may inhibit inflammation by modulating NO or inflammatory factor in macrophage (Fang et al., 2008).
Among these natural products, a thorough in-depth research on homologous medicinal and edible plants, it is found that limonin is the main anti-inflammatory active ingredient (Fan et al., 2019), mainly through inhibition of inflammatory mediators' NFκβ signaling cascade (Jin et al., 2018). C. tabularis bark and fruit extract was proven to have anti-inflammatory activities by inhibiting pro-inflammatory cytokines such as NO, TNFα, and IL-6 (Perianayagam et al., 2004).
However, few reports have focused on anti-inflammatory activities and mechanism of limonins in C. tabularis extracts (Yang et al., 2020).
In this study, a combination of octadecyl silica gel, Sephadex LH-20, and HPLC was used to separate anti-inflammatory limonins from   effective trigger for inflammatory responses (Pandher et al., 2021).
Inflammation is the main risk element for many diseases, and macrophages are the primary immune cells and the first line of defense against pathogen invasion (Leseigneur et al., 2020). In the process of inflammation, macrophages produce excess induced nitric oxide synthase as inflammatory mediators and pro-inflammatory cytokines such as TNFα, IL-6, and IL-1β (Huang et al., 2019). NO is a biological signal and effect or element that modulates the expression of proinflammatory cytokine (Zamora et al., 2000). Inflammatory damage is thought to be caused by the excessive production of NO-induced pro-inflammatory cytokine (Kany et al., 2019;Zhang et al., 2017).
Excessive production of nitric oxide occurs in inflammation and various diseases, where NO plays a cytotoxic role in the pathological process (Lea et al., 2020;Shao et al., 2013). Therefore, suppression of NO production is important for the prevention of inflammatory disease. Among inflammatory stimulants, LPS induces macrophage activation leading to the release of pro-inflammatory cytokine in the inflammatory response (Bonizzi & Karin, 2004;Ronchetti et al., 2017). Cytokines arouse fever, stun, and various inflammatory diseases. Therefore, it is essential to suppress the overproduction (2) have been identified in C. tabularis root bark, and they have been proved to have strong anti-inflammatory activities, providing a theoretical basis for the application of C. tabularis in anti-inflammatory activity.

| CON CLUS IONS
Screening for anti-inflammatory activity of Chukrasia tabularis root bark extracts led to the separation of 14 limonins, including two novel phragmalin limonoids (1-2), 12 known limonoids (3-14). All isolated compounds were determined for NO production by LPSmediated RAW 264.7 cells. The result exhibited that D-ring-opened phragmalin limonoid orthoester (1-2) showed strong NO inhibitory activities while limonins (3-14) showed potent to moderate activity. Limonins 1-2 displayed significant anti-inflammatory activities with IC 50 values of 6.24 and 6.13 μM. Compound 1 inhibited the production of NO and TNFα in stimulated cells and reduced the secretion of NO and TNFα levels during inflammatory processes.
These results provide basic information for further research on utilizing C. tabularis as natural anti-inflammatory resource.

ACK N OWLED G EM ENT
This work was financially supported by the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T.
The data presented in this study are available in the Supplementary Materials.