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Phytochemical Constituents of Aquilaria malaccensis Leaf Extract and Their Anti-Inflammatory Activity against LPS/IFN-γ-Stimulated RAW 264.7 Cell Line

This study aims to identify the major phytochemical constituents in Aquilaria malaccensis (Thymelaeaceae) ethanolic leaf extract (ALEX-M) and elucidate their ability to suppress nitric oxide (NO) production from a murine macrophage-like cell line (RAW 264.7) stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Dichloromethane (DCM) and ethyl acetate (EtOAc) fractions of ALEX-M were subjected to column chromatography. Eight known compounds were isolated for the first time from this species. Compounds were identified using spectroscopic techniques (IR, UV, HRESIMS, and 1D and 2D NMR). Anti-inflammatory activity of both extract and isolated compounds were investigated in vitro. The fractions offered the isolation of epifriedelanol (1), 5-hydroxy-7,4′-dimethoxyflavone (2), luteolin-7,3′,4′-trimethyl ether (3), luteolin-7,4′-dimethyl ether (4), acacetin (5), aquilarinenside E (6), iriflophenone-2-O-α-l-rhamnopyranoside (7), and iriflophenone-3-C-β-glucoside (8). The findings suggest the pharmacological potential of the crude extract (ALEX-M) and its isolates as natural anti-inflammatory agents, capable of suppressing NO production in RAW 264.7 cells stimulated by LPS/IFN-γ.


INTRODUCTION
Inflammation has become a major global health issue in recent years. Although inflammation is a normal physiological body response against a noxious stimulus to initiate tissue homeostasis, 1,2 it has been recognized among the causal pathophysiology of several chronic progressive diseases including cancer, 3 rheumatoid arthritis, atherosclerosis, and diabetes. 4 Synthetic medications used for treatment of inflammation such as the nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids may cause intolerable gastrointestinal, hepatic, and cardiovascular side effects. 1 Plant-derived products have become safe alternatives for alleviation of inflammation through interference with inflammatory pathways. 5 Aquilaria malaccensis (family Thymelaeaceae) was used traditionally as a tonic during pregnancy and after giving birth, as a carminative and to control heart palpitations. 6 It was also prescribed to alleviate jaundice, ulcer, edema, skin diseases, fever, and body aches. 6,7 In addition, the plant was involved in the treatment of central nervous system diseases such as epilepsy and schizophrenia as well as fumigation therapy for sterilization and personal hygiene. 8 Previous scientific research on A. malaccensis reported its potential pharmacological activities including anticancer, 9,10 antimicrobial, 10−13 antioxidant, 11,13−16 anti-inflammatory, 17,18 immunomodulatory, 19 antidiabetic, 13,20 embryogenesis, 21,22 and antitrypanosomal activity. 23 Research on the phytochemical constituents of Aquilaria species reported the presence of a number of compounds such as flavonoids, terpenoids, phenolic acids, benzophenones, xanthonoids, sesquiterpenes, chromones, fatty acids, phytosterols, and lignans. 24−26 Previous anti-inflammatory studies have been conducted on Aquilaria species. It has been reported that the ethanolic extract of Aquilaria sinensis leaves a demonstrated inhibitory effect on NO levels in LPS-induced peritoneal macrophages with an IC 50 = 80.4 μg/mL. 27 In addition, the flavonoid Aquisiflavoside, which was isolated from Aquilaria sinensis leaves, was recognized as a nitric oxide inhibitor in LPSinduced RAW 264.7 cells with an IC 50 = 39.95 μM. 28 Chen et. al reported the ability of different Aquilarones isolated from the resinous agarwood of Aquilaria sinensis to inhibit NO production in LPS-stimulated RAW 264.7 with IC 50 ranging between 5.95 μM and 22.26 μM. 29 Similarly, other chromones isolated from Aquilaria sinensis agarwood demonstrated antiinflammatory activity against LPS-induced NO production in the RAW 264.7 cell line as reported by several researchers. 30−32 Herein, as an extension of the phytochemical profiling studies carried on A. malaccensis species and their pharmacological activities, A. malaccensis ethanol leaf extract (ALEX-M) and its structurally identified compounds were investigated for their potential to inhibit NO production from LPS/IFN-γstimulated RAW 264.7 in the present study.

ACS Omega
http://pubs.acs.org/journal/acsodf Article Table 2 demonstrates the 1 H and 13 C NMR data of compound 5, while Figure 2 illustrates the 2D NMR correlations. Thus, compound 5 was identified as acacetin, and its spectroscopic data are identical with those obtained by. 44 Assignments were confirmed by COSY, HMQC, and HMBC. Compound 6 was collected as a white amorphous powder. An absorption band appeared at 285 nm on the UV spectrum. Compound 6 showed bands at 3275, 1709, and 1608 cm −1 in IR spectrum. The molecular formula of the compound (C 21 H 22 O 10 ) was in agreement with the molecular ion peak that occurred at m/z 434.1250 [M − H] − analyzed using the HRESIMS technique. Based on the spectroscopic data of the compound presented in Table 3, which are similar to aquilarinenside E previously isolated from Aquilaria sinensis leaves 34 and flower buds, 45 compound 6 is identified as aquilarinenside E. 40 The HRESIMS spectrum of compound 7 revealed a significant peak at m/z 391.1002 [M − H] − indicated to the molecular formula of C 19 H 20 O 9 , while the UV spectrum demonstrated a maximum absorption band at 285 nm. IR spectrum revealed absorption bands at 3326, 1635, and 1595 cm −1 recognized as OH, CO, and aromatic groups absorptions, respectively. The compound was isolated as white needle crystals. The data of 1 H and 13 C NMR of compound 7 are presented in Table 3. The comparison of the spectral data obtained with literature recommended that 7 is iriflophenone 2-O-α-L-rhamnopyranoside. 41 Compound 8, isolated as orange crystalline powder, had the molecular formula of C 19 H 20 O 10 according to the HRESIMS which revealed an [M − H] − peak at m/z 407.0997. The compound demonstrated strong absorption bands at 295 and 310 nm in the UV, while the IR spectrum exposed absorption bands of OH group (3296 cm −1 ) and CO group (1607 cm −1 ). The 1 H and 13 C NMR data of compound 8 are displayed in Table 3. Compound 8 was identified as iriflophenone 3-C-β-glucoside. 42,43 2.2. Cytotoxicity Assay. Before further studies were performed, the extract and the isolated compounds 1−8 were examined for their cytotoxicity against RAW 264.7 cell line using MTT assay. The consequence of the tested samples on the viability of RAW 264.7 is demonstrated in Figure 3. It was noted that the LPS/IFN-γ group showed no effect on cell viability. Safe concentrations of the extract and the compounds that are well tolerated by RAW 264.7 cell line and that showed cell viability above 85% were selected for the subsequent Griess anti-inflammatory assay, as it indicates the inhibition of NO levels is not a result of cell death but due to the inhibitory potential of NO production by the tested substance. 46 2.3. Anti-Inflammatory Assay. Macrophages can promote the production and release of nitric oxide (NO) when stimulated by the bacterial endotoxin lipopolysaccharide (LPS). 47,48 The subsidence of the NO levels reflects a potential anti-inflammatory effect. The cell lines were treated with the samples 1 h prior to stimulation with LPS/IFN-γ. After incubation for 12 h, the control group produced a basal level of NO of about 8.28 ± 0.43 μM in the culture medium, while the LPS/IFN-γ-treated group demonstrated a remarkable increase in NO of about 25.03 ± 1.66 μM. The extract and compounds groups demonstrated significant inhibition of NO production at different concentrations ranging between 200 and 6.25 μg/mL (P < 0.05) when compared to the LPS/IFNγ-stimulated group. Compounds 1−5, and 7 were able to significantly reduce NO production (P < 0.05) at low concentration (6.25 μg/mL). However, compounds 1 and 2 displayed significant toxicity toward RAW 264.7 cells when the concentration reached 100 μg/mL, as demonstrated in Figure  3. The inhibitory concentration that reduces NO production by 50% (IC 50 ) of the crude extract and the compounds was calculated and is presented in Table 4. The extract showed a moderate but significant NO inhibitory effect compared to the LPS/IFN-γ-treated cells, while compounds 2, 4, 5, and 7 showed the uppermost suppression of NO in cells stimulated by LPS/IFN-γ, followed by compounds 1 and 3. Compounds 6 and 8 inhibited NO levels by 45.44 ± 4.22% and 40.58 ± 5.55%, respectively, at a dose of 200 μg/mL. Ibuprofen was used as a reference standard in the present study. Figure 4 demonstrates the inhibitory effect on NO production by the extract and the compounds. The results of MTT cell viability assay revealed that the inhibitory effect of the samples was not caused by cell death (viability >85%).
3.5. Cytotoxicity Assay. In accordance with the protocol used by refs 54 and 55, the MTT assay was conducted. Concisely, RAW 264.7 cells were seeded at 1 × 10 5 cells/well into 96-well plates and were incubated for 12 h. After 24 h of exposure to samples dissolved in high glucose phenol red-free DMEM containing DMSO (0.1%) and complemented with 10% v/v FBS and penicillin-streptomycin (1% v/v), the cells were then stained with MTT solution (5 mg/mL) and were incubated for 4 h. The supernatants were afterward drawn, and where OD is the absorbance at λ = 540 nm. 3.6. Anti-Inflammatory Griess Assay. High-glucose DMEM incorporating FBS (10% v/v) and penicillinstreptomycin (1% v/v) was used to culture the murine RAW 264.7 cells. Approximately, 1 × 10 5 cells/well were seeded into 96-well plates and were incubated at 37°C overnight. ALEX-M and the compounds (1−8) were dissolved in media containing 0.1% DMSO. After the cells were exposed to different concentrations of samples after 2 h, the NO production was stimulated by adding 0.1 μg/mL of LPS and 10 U/mL of IFN-γ. Based on the results of the cytotoxicity assay, the minimum toxic dose (≥85% viability) was used as the maximum dose in the Griess assay.
Equal amounts (50 μL) of the supernatant and Griess reagent were mixed in another 96-well plate. Subsequent to incubation at room temperature for a duration of 15 min, the absorbance was determined at λ = 540 nm (Multiskan Sky Microplate Spectrophotometer, Thermo Fisher Scientific, Massachusetts, USA). The NO inhibition percentage was determined with reference to the standard sodium nitrite as per the following equation: 54,57 OD

LPS/IFN stimulated cells OD test OD LPS/IFN stimulated cells 100
where OD is the absorbance at λ = 540 nm. 3.7. Quantitative RT-PCR Analysis. One-tenth of the resulting cDNA was used as a template for real-time PCR amplification using Maxima SYBR Green qPCR master mix (Thermo Fisher Scientific, Massachusetts, USA). For each sample, the quantitative real-time PCR was performed in triplicates in StepOne Real-Time PCR System (Applied Bio Systems) using the SYBR green method. Specific primers for IL6 and TNF-α were used. GAPDH was amplified and used as the endogenous control to normalize for gene expression. A no template control was used as negative control using DEPCtreated water. The primers used for the amplification of the indicated genes were designed using the IDT Primer Quest Primer Design Tool and are listed in Table 5. Fold changes in gene expression were determined using the 2 −ΔΔCT method. 58 Thermal cycle parameters were: 1 cycle at 95°C for 15 min followed by 35 cycles at 95°C for 10 s, annealing at 61°C for 20 s and at 72°C for 20 s.
3.8. Statistical Analysis. All experiments were carried out in triplicate (n = 3) independently. Comparisons were carried out by analysis of variance (ANOVA) followed by Tukey's post-hoc test using p < 0.05 as the level of significance, using GraphPad Prism Software (San Diego, CA, version 5.0).

CONCLUSION
The current research resulted in the separation of one terpenoid, four flavonoids, and three benzophenones from noninoculated A. malaccensis leaf for the first time. By using detailed spectroscopic analysis and comparison with previously published data, the structures of the isolated compounds were elucidated. The anti-inflammatory activity of the extract and eight compounds was evaluated, and they demonstrated the ability to suppress NO levels in RAW 264.7 cells induced by LPS/IFN-γ. The study introduced the use of A. malaccensis leaves extract and the isolated bioactive compounds in the treatment of inflammatory-associated disorder. Currently, a study is in progress to elucidate the underlying immunomodulatory mechanisms of the extract. Further studies are ought to be dedicated toward identifying the inhibitory NO pathways of the active compounds.