*2.3. Effects of the Methanol Extract and Isolated Lignans on LPS-Induced Nitric Oxide Production*

Nitric oxide (NO) is a free-radical signaling molecule playing a role in many biological processes, including inflammation. NO is synthesized from L-arginine and oxygen by using NOS as catalysts. There are three main isoforms of NOS, endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The iNOS enzyme is a major isoform in the inflammatory process [3,21,22]. Thus, upregulation of iNOS might be reflected in NO production in the cells during inflammation. NO is immediately oxidized to generate nitrate and nitrite, both of which are used for measuring NO levels in the cells. Therefore, this study investigated the inhibitory effects of methanol extract and isolated lignans from *M. sirikitiae* leaves on LPS-mediated NO production by determining the levels of nitrate and nitrite in RAW 264.7 cells. Incubation of the cells with LPS (10 μg/mL) robustly increased nitrate and nitrite productions (Figure 5). Treatment with the methanol extract, (−)-epieudesmin (**1**), (−)-phylligenin (**2**), or 3 ,4-*O*-dimethylcedrusin (**6**) at a concentration of 10 μg/mL resulted in a significant reduction of LPS-induced nitrate and nitrite levels in RAW 264.7 cells (Figure 5A and Figure 5B, respectively). In contrast, treatment with 2-(3,4 dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (**3**), magnone A (**4**), and mitrephoran (**5**) (5 μg/mL) had no effect (Figure 5). These results indicated that the lignans found in *M. sirikitiae* leaves, including (−)-epieudesmin (**1**), (−)-phylligenin (**2**), and 3 ,4-*O*-dimethylcedrusin (**6**) exhibited anti-inflammatory activities by attenuating NO production in RAW 264.7 cells. Consistent with our present data, many previous studies have demonstrated that some lignans can suppress NO production during inflammation. For example, the methanol extract of *F. koreana* fruits and its isolated lignan, phylligenin, could inhibit NO synthesis in LPS-treated RAW 264.7 cells [15]. Furthermore, nine lignans isolated *Acanthopanax sessiliflorus* fruits [18] and two dimeric lignans isolated from *Zanthoxylum podocarpum* barks [19] have been reported to have inhibitory effects on

−

NO production in LPS-treated RAW 264.7 macrophages. Thus, natural phenols found in plants as lignans may serve as potential anti-inflammatory agents via inhibition of the NO signaling pathway.

**Figure 5.** Effects of the methanol extract and isolated lignans **1**–**6** from *M. sirikitiae* leaves on LPSinduced nitrate and nitrite productions in RAW 264.7 cells; Serum-starved cells were pretreated with crude methanol extract (C) (10 μg/mL) or lignans (5 or 10 μg/mL) for 3 h, and then stimulated with LPS (5 μg/mL) for 24 h at 37 ◦C. The levels of nitrate and nitrite in the medium were assessed by a total nitric oxide assay. The relative nitrate (**A**) and nitrite (**B**) productions were quantified using a standard curve and expressed as the mean ± SEM (*n* = 3). \*, *p* < 0.05 vs. vehicle; #, *p* < 0.05 vs. LPS.

### *2.4. Effects of the Methanol Extract and Isolated Lignans on LPS-Induced mRNA Expressions of Inflammatory Biomarkers*

According to the methanol extract of *M. sirikitiae* leaves and some isolated lignans representing the inhibitory effect on LPS-mediated PGE2, TNF-α, and nitric oxide secretions in RAW 264.7 cells, the mRNA expression of inflammatory biomarkers, including COX-2, iNOS, TNF-α, IL-6, IL-10, and NF-κB affected by the methanol extract and isolated lignans **1**–**6** was subsequently studied. In this study, incubation of the RAW 264.7 cells with LPS (5 μg/mL) markedly increased TNF-α, IL-6, IL-10, NF-κB, COX-2, and iNOS mRNA expression levels as compared with a control group (Figure 6A, Figure 6B, Figure 6C, Figure 6D, Figure 6E, and Figure 6F, respectively), indicating the induction of inflammation. The methanol extract of *M. sirikitiae* leaves was able to suppress LPS-induced mRNA expression of NF-κB, COX-2, and iNOS (Figure 6D, Figure 6E, and Figure 6F, respectively) while treatment of the cells with this methanol extract tended to inhibit LPS-induced TNF-α and IL-10 syntheses (Figure 6A and Figure 6C, respectively). However, the extract did not affect IL-6 mRNA expression (Figure 6B). These results demonstrated that the methanol extract of *M. sirikitiae* leaves possesses anti-inflammatory effects by suppressing the synthesis of several inflammatory mediators, indicating that the anti-inflammatory effects are derived from some active compounds contained in *M. sirikitiae* leaves.

**Figure 6.** Effects of the methanol extract and isolated lignans **1**–**6** from *M. sirikitiae* leaves on LPSinduced mRNA expressions of inflammatory biomarkers in RAW 264.7 cells; Serum-starved cells were pretreated with crude methanol extract (C) (10 μg/mL) or lignans (5 or 10 μg/mL) for 3 h, and then stimulated with LPS for 6 h at 37 ◦C. After treatment, the total RNA was extracted from the cells and the mRNA expressions of TNF-α (**A**), IL-6 (**B**), IL-10 (**C**), NF-κB (**D**), COX-2 (**E**), and iNOS (**F**) were analyzed by RT-qPCR with gene-specific primers. The relative mRNA levels were quantified and shown as the mean ± SEM (*n* = 3). \*, *p* < 0.05 vs. vehicle; #, *p* < 0.05 vs. LPS.

Moreover, all isolated lignans **1**–**6** were investigated for their effects on suppressing mRNA expressions of inflammatory mediators induced by LPS. As shown in Figure 6A, treatment of the cells with (−)-epieudesmin (**1**), (−)-phylligenin (**2**), magnone A (**4**), and 3 ,4-*O*-dimethylcedrusin (**6**) tended to inhibit LPS-induced mRNA expression of TNF-α. In addition, treatment of the cells with 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)- 3,7-dioxabicyclo[3.3.0]octane (**3**) and mitrephoran (**5**) significantly inhibited LPS-induced TNF-α mRNA expression. Macrophage cells are the main source of proinflammatory cytokine TNF-α, which is used as one of the major markers of inflammation. This study reported for the first time that isolated lignans **3** and **5** from *M. sirikitiae* leaves exhibited

the anti-inflammatory effects by suppressing LPS-induced TNF-α secretion (Figure 4B) and synthesis (Figure 6A) in RAW 264.7 cells.

However, lignans **1**–**6** did not show the inhibitory effects on LPS-induced mRNA expression of inflammatory cytokines, IL-6, IL-10, and NF-κB in RAW 264.7 cells (Figure 6B–D). After exposure to a pathogen (e.g., LPS), macrophages are activated to produce proinflammatory cytokines such as IL-1 and IL-6, which play an important role in the inflammatory response [23]. Although the lignans isolated from *M. sirikitiae* leaves did not show the inhibitory effects on LPS-induced IL synthesis, the previous study demonstrated that other types of lignans isolated from the caulis of *U. rosea* exhibited anti-inflammatory effects by reducing the LPS-induced IL-6 production in RAW 264.7 cells with the IC50 values ranging from 5 to 50 μM [17]. Thus, each lignan has its own anti-inflammatory properties and mechanisms.

NF-κB is one of the major regulators of inflammatory gene expression. The activation of NF-κB resulted in the upregulation of mRNA expression of cytokines such as TNF-α, IL-1*β*, IL-6, and IL-8, including COX-2 [24]. Even though the methanol extract of *M. sirikitiae* leaves was able to suppress LPS-induced NF-κB synthesis, all lignans isolated from the *M. sirikitiae* leaves had no effect on the inhibition of NF-κB synthesis induced by LPS (Figure 6D). Further studies are required to identify other constituents that have inhibitory effects on the synthesis of NF-κB.

Interestingly, treatment with (−)-phylligenin (**2**), magnone A (**4**), and 3 ,4-*O*dimethylcedrusin (**6**) significantly inhibited the mRNA expression of COX-2 in LPS-stimulated RAW 264.7 cells (Figure 6E). The effects of the methanol extract and (−)-phylligenin (**2**) on COX-2 mRNA expression were correlated with their inhibitory effects on LPS-induced PGE2 secretion in RAW 264.7 cells. Our data are in concordance with other studies that some lignans, such as koreanaside A isolated from *F. koreana* flowers, could downregulate COX-2 mRNA expression and attenuate PGE2 secretion in LPS-induced RAW 264.7 cells [16]. The methanol extracts of *Schisandra rubriflora* and *Schisandra chinensis* and their isolated lignans, including 6-obenzoylgomisin O, schisandrin, gomisin D, gomisin N, and schisantherin A, have been revealed to exhibit significant COX-2 inhibitory activity [14]. Our study demonstrated the inhibitory effects of magnone A (**4**), and 3 ,4-*O*-dimethylcedrusin (**6**) on LPS-induced COX-2 synthesis for the first time. During inflammation, arachidonic acid converses into PGE2 mediated through COX-2 catalytic reaction. Inhibition of COX-2 activity and synthesis has been proposed as a useful treatment for various inflammatory diseases [25]. Therefore, COX-2 is a well-known target of various anti-inflammatory drugs such as aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). In our present study, the methanol extract of *M. sirikitiae* leaves could inhibit the synthesis of COX-2 and reduce PGE2 secretion in LPS-treated macrophages. Thus, *M. sirikitiae* might be used as a medicinal plant for the prevention and restoration of inflammatory diseases.

The iNOS is an inducible isoform of NOS and upregulation of iNOS activity and synthesis occurred in response to inflammation [22]. Therefore, the inhibitory effect on iNOS enzyme, which subsequently suppresses NO generation was investigated. We found that treatments of the cells with the methanol extract of *M. sirikitiae* leaves, (−)-phylligenin (**2**), and 3 ,4-*O*-dimethylcedrusin (**6**) significantly inhibited LPS-induced iNOS mRNA expression in RAW 264.7 macrophages, while (−)-epieudesmin (**1**), 2-(3,4-dimethoxyphenyl)-6- (3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (**3**), magnone A (**4**), and mitrephoran (**5**) had no effects (Figure 6F). These inhibitory effects of the methanol extract, together with (−)-phylligenin (**2**), and 3 ,4-*O*-dimethylcedrusin (**6**) on iNOS mRNA expression (Figure 5F) were correlated with their inhibitory effects on LPS-induced NO production (Figure 4) in RAW 264.7 cells. Interestingly, phylligenin (10–100 μM) and koreanaside A (20–80 μM) isolated from *F. koreana* have previously reported the inhibitory effects on LPS-induced iNOS synthesis in RAW 264.7 cells [15,16]. These findings from ours and previous studies confirmed the anti-inflammatory effects of (−)-phylligenin (**2**) by inhibiting iNOS synthesis and subsequent reducing NO production. Moreover, our study reported the inhibitory effects on iNOS synthesis of 3 ,4-*O*-dimethylcedrusin (**6**), the lignan isolated from *M. sirikitiae* leaves, for the first time.

In this study, the structure–activity relationships (SARs) of the natural lignans for anti-inflammatory properties were considered in order to identify the specific structures and functional groups that play an important role in the activities. Based on a variety of chemical core skeletons and functional groups of lignans, isolated lignans **1**–**6** from *M. sirikitiae* leaves could be classified into three subgroups, including furofurans (lignans **1**–**3**), furans (lignans **4**–**5**), and benzofurans (lignan **6**) [26]. The obtained results from our present study indicated that the possible SARs of the anti-inflammatory lignans are different in each specific mechanism of action. For instance, substitution of a hydroxyl group on the phenyl ring and a furofuran moiety as found in lignan **2** might be important for the inhibitory effects against LPS-induced PGE2 secretion as well as COX-2 mRNA expression. Moreover, the furofuran with hydroxy-substituted phenyl (lignan **2**) and benzofuran (lignan **6**) skeletons played a determinant role against NO production and iNOS mRNA expression induced by LPS in cells. Meanwhile, furofuran with non-hydroxy-substituted phenyl (lignan **3**) and furan with hydroxy-substituted phenyl (lignan **5**) moieties were found to be essential for the inhibition of LPS-induced TNF-α secretion and TNF-α mRNA expression. Although the SARs of lignans for the downregulation of LPS-induced mRNA expression of IL-6, IL-10, and NF-κB could not be discussed in this study, some previous studies have disclosed that furofuran skeleton is possibly related to the suppression of NF-κB signaling pathway [27]. Moreover, the SARs of coumarinolignans have reported that the substitution of hydroxyl groups on the phenyl ring is important to anti-inflammatory activity [28]. In order to fully understand SARs of lignans for anti-inflammatory properties, a number of lignans with different skeletal types and functional groups are required for the biological testing.

It should be noted that our data were obtained from LPS-induced inflammation in RAW 264.7 cells, and therefore may not reflect the actual systemic anti-inflammatory effects in the human body. Thus, further studies will be necessary to evaluate the antiinflammatory activities in vivo studies.

#### **3. Materials and Methods**
