Essential Oils from the Leaves, Stem, and Roots of Blumea lanceolaria (Roxb.) Druce in Vietnam: Determination of Chemical Composition, and In Vitro, In Vivo, and In Silico Studies on Anti-Inflammatory Activity

Blumea lanceolaria (Roxb.) Druce, a flowering plant, is used for treating cancer and inflammatory diseases. In this study, we determined the chemical composition of the EOs extracted from the leaves (LBEO), stem (SBEO), and roots (RBEO) of B. lanceolaria and analyzed their anti-inflammation potential. Overall, 30 compounds representing 99.12%, 98.44%, and 96.89% of total EO constituents of the leaves, stem, and roots, respectively, were identified using GC-MS. ELISA, Western blotting, and qRT-PCR studies showed that LBEO, SBEO, and RBEO inhibited multiple steps in the inflammatory responses in the RAW 264.7 cell model, including NO production; TNF-α, IL-6, iNOS, and COX-2 transcription and translation; and phosphorylation of IκBα and p65 of the NF-κB pathway. In the carrageenan-induced paw edema model, all three EOs inhibited paw edema at both early and delayed phases. Molecular docking studies indicated that the main components of B. lanceolaria EOs (BEOs) targeted and inhibited major components of inflammation-related pathways, including the arachidonic acid metabolic pathway, NF-κB pathway, and MAPK pathway. We present the first study to characterize the chemical composition of BEOs and confirm their potent anti-inflammatory effects in in vitro, in vivo, and in silico analysis. These results can facilitate the development of effective anti-inflammatory drugs with limited side effects in the future.


Introduction
Inflammation is a protective response of the immune system to tissue damage or foreign stimuli, including human pathogens, allergens, toxic chemicals, or irradiation [1]. The vital functions of inflammation involve repair of damaged tissues, elimination of harmful Table 1 shows the chemical components of LBEO, RBEO, and SBEO including the relative content (%), retention time (RT), and retention index (RI) of each constituent. In total, 30 compounds ( Figure 1 and Table 1) were identified, representing 99.12%, 98.44%, and 95.62% of the total EO constituents of the leaves, stem, and roots, respectively. Among these, 5, 15, and 20 constituents were identified in the LBEO, SBEO, and RBEO, respectively, according to their mass spectra and relative RI (Table 1). Table 1. Chemical compositions of the BEOs from the leaves, stem, and roots analyzed using GC-MS/GC-flame ionization detection (FID).  The EOs obtained from the leaves and stem of B. lanceolaria harvested in December 2020 differed considerably from that published previously. In our plant samples, 0.24% and 0.36% thymol methyl ether (9) were present in the leaves and stem of B. lanceolaria EOs, respectively. However, Dung et al. reported that thymol methyl ether contributed to 94.96% of the content of EOs obtained from the leaves and stem of B. lanceolaria harvested in June 1989 [24]. The content of methyl carvacrol (10) was highest in our samples, with 58.28% in the leaves and 89.40% in the stem, whereas only 0.02% was reported by Dung et al. Similarly, β-caryophyllene (16) content was also high in our samples, with 0.26% in LBEO and 0.82% in SBEO, compared to 0.04% reported previously. o-Cymene (3) content was the second highest in our leaf (38.29%) and stem (3.35%) samples, although it was Among the five volatile constituents of LBEO, two major compounds were identified to be o-cymene (38.29%) and carvacrol methyl ether (58.28%), along with three other minor constituents, namely thymol methyl ether (0.24%), β-caryophyllene (0.26%), and hexadecanol (2.05%). The LBEO was found to be rich in monoterpene hydrocarbons (96.81%), followed by other alcoholic compounds (2.05%) and sesquiterpenes (0.26%) ( Figure S1).
Comparison of the major chemical components of the LBEO, SBEO, and RBEO revealed considerable variability among some of the major components in the samples. The content of carvacrol methyl ether (10), contributing to 58.28% of the components in LBEO, increased to 89.40% in SBEO (highest among all EOs), but disappeared in RBEO (0%). Similarly, β-caryophyllene (16) content increased from 0.26% in LBEO to 0.82% in SBEO and then decreased to 0.53% in RBEO. The contents of monoterpene o-cymene (from 38.29% to 3.35% and 0.92%) and hexadecanol (from 2.05% to 0.26% and 0%) tended to decrease from LBEO to SBEO and RBEO. However, α-pinene (2) was not found in LBEO, although its content increased to 2.28% and 33.36% in SBEO and RBEO, respectively. The second highest increase in content was observed for thymol methyl (9) ether; its content increased from 0.24% in LBEO to 0.36% in SBEO and 21.70% in RBEO. The levels of α-copaene (from 0% to 0.37% and 0.65%), α-humulene (from 0% to 0.17% and 0.24%), and δ-cadinene (from 0% to 0.18% and 0.26%) also increased. Among the 30 compounds identified from the three EO samples, three compounds (10% of the total) were only present in the stem (SBEO), including linalool (0.12%), α-cadinol (0.27%), and hexadecanal (0.25%). Two compounds, carvacrol methyl ether and hexadecanol, were present in the stem and leaf, but not in the root (Table 1), while fifteen compounds were found to be present only in RBEO, including thymol isobutyrate, which accounted for the greatest proportion of the components (29.23%).
The EOs obtained from the leaves and stem of B. lanceolaria harvested in December 2020 differed considerably from that published previously. In our plant samples, 0.24% and 0.36% thymol methyl ether (9) were present in the leaves and stem of B. lanceolaria EOs, respectively. However, Dung et al. reported that thymol methyl ether contributed to 94.96% of the content of EOs obtained from the leaves and stem of B. lanceolaria harvested in June 1989 [24]. The content of methyl carvacrol (10) was highest in our samples, with 58.28% in the leaves and 89.40% in the stem, whereas only 0.02% was reported by Dung et al. Similarly, β-caryophyllene (16) content was also high in our samples, with 0.26% in LBEO and 0.82% in SBEO, compared to 0.04% reported previously. o-Cymene (3) content was the second highest in our leaf (38.29%) and stem (3.35%) samples, although it was not reported by Dung et al. (1991). Furthermore, according to Dung et al., the contents of limonene (4) and α-thujene (1) were 0.12% and 0.04%; whereas we could not detect them in our leaf and stem samples, and only 0.14% was detected in the root sample. These differences in results can be attributed to differences in geographic factor, time of collection (December 2020 versus June 1989), and technique used (15 compounds were found in our leaf and stem samples prepared using steam distillation compared to 11 compounds reported in literature) [24]. In addition, these differences may be the result of differences in analytical method, Dung et al. used a 25 m × 0.25 mm I.D.-fused silica OV-1 (0.25 pm) column and the nitrogen gas was carried at a flow rate of 1.2 mL/min. The oven was programmed after 5 min at 60 • C, at 5 • C/min to 220 • C, with a final hold time of 20 min. In our analyses, the HP-5 MS column with a dimension of 60 m × 0.25 mm and film thickness of 0.25 µm was used for separation. Running conditions were set as follows: injector temperature at 250 • C; initial temperature started from 60 • C then increased to 240 • C with increasing step of 4 • C/min; the carrier gas was helium with the flowrate of 1 mL/min; full scan modes under electron ionization with voltage: 70 eV, emission current: 40 mA; and mass range scan: 35-450 a.m.u.

Effect of the BEOs on Macrophage Viability
We evaluated the toxicity of BEOs in RAW 264.7 using the CCK-8 kit (Abcam, Cambridge, UK). The results indicate that at concentrations of 0-50 µg/mL, none of the three essential oil samples were toxic to the RAW 264.7 cells (Figure 2). However, 100 µg/mL LBEO, SBEO, and RBEO were cytotoxic, causing 34.89%, 22.12%, and 37.28% cell death, respectively. When the concentration of the BEOs was increased to 150 µg/mL, the cell survival rate decreased to <50%. Thus, 50 µg/mL BEO is relatively safe for cells, with a survival rate of more than 95%. The calculated IC 50 values of LBEO, SBEO, and RBEO are 151.11, 335.60, and 123.05 µg/mL, respectively. Therefore, the optimal concentration range of 5-50 µg/mL, at which the EOs were not toxic but active, was selected for anti-inflammatory studies.
min. In our analyses, the HP-5 MS column with a dimension of 60 m × 0.25 mm and thickness of 0.25 μm was used for separation. Running conditions were set as follo injector temperature at 250 °C; initial temperature started from 60 °C then increase 240 °C with increasing step of 4 °C/min; the carrier gas was helium with the flowrate mL/min; full scan modes under electron ionization with voltage: 70 eV, emission curr 40 mA; and mass range scan: 35-450 a.m.u.

Effect of the BEOs on Macrophage Viability
We evaluated the toxicity of BEOs in RAW 264.7 using the CCK-8 kit (Abc Cambridge, UK). The results indicate that at concentrations of 0-50 µ g/mL, none of three essential oil samples were toxic to the RAW 264.7 cells (Figure 2). However, µ g/mL LBEO, SBEO, and RBEO were cytotoxic, causing 34.89%, 22.12%, and 37.28% death, respectively. When the concentration of the BEOs was increased to 150 µ g/mL, cell survival rate decreased to <50%. Thus, 50 µ g/mL BEO is relatively safe for cells, w a survival rate of more than 95%. The calculated IC50 values of LBEO, SBEO, and RB are 151.11, 335.60, and 123.05 µ g/mL, respectively. Therefore, the optimal concentra range of 5-50 µ g/mL, at which the EOs were not toxic but active, was selected for ti-inflammatory studies.

Inhibition of NO Production by BEOs
Inhibitory effects of BEOs on NO production in RAW 264.7 cells were assessed using the Griess reagent (Promega, Madison, WI, USA). As shown in Figure 3, all three EOs (BLEO, SBEO, and RBEO) remarkably inhibited NO production (p < 0.05) in a concentrationdependent manner. Among the EOs, LBEO showed the best NO inhibition ability at all the three tested concentrations (5, 10, and 50 µg /mL). Both 50 µg/mL LBEO and RBEO showed the highest NO inhibition ability (approximately 50%). Compared to LBEO and RBEO, 50 µg/mL SBEO showed the lowest NO inhibitory activity of 33.15 ± 1.01%. Thus, all three EOs markedly inhibited NO production in RAW 264.7 macrophages.

Effect of the BEOs on Transcription and Translation of TNF-α and IL-6
The ability to inhibit the production of TNF-α and IL-6 is an important indicator for evaluating the anti-inflammatory property of BEOs. ELISA results ( Figure 4) show that the level of TNF-α increased to more than 2000 pg/mL when stimulated by LPS. TNF-α production decreased from 2000 pg/mL to 61, 91, and 44 pg/mL in cells treated with 50 µg/mL BLEO, SBEO, and RBEO, respectively. These results indicate that all three BEOs strongly inhibit TNF-α production. Similarly, in the presence of BEOs, the level of IL-6 decreased significantly compared to that in the control sample (LPS-treated macrophages). The amount of IL-6 decreased from 250 pg/mL to 94.67, 92.67, and 88 pg/mL when treated with increasing concentrations (up to 50 µg/mL) of LBEO, SBEO, and RBEO, respectively (Figure 4d-f).
(BLEO, SBEO, and RBEO) remarkably inhibited NO production (p < 0.05) in a concentration-dependent manner. Among the EOs, LBEO showed the best NO inhibition ability at all the three tested concentrations (5, 10, and 50 µ g /mL). Both 50 µ g/mL LBEO and RBEO showed the highest NO inhibition ability (approximately 50%). Compared to LBEO and RBEO, 50 µ g/mL SBEO showed the lowest NO inhibitory activity of 33.15 ± 1.01%. Thus, all three EOs markedly inhibited NO production in RAW 264.7 macrophages. Figure 3. Effects of LBEO, SBEO, and RBEO on NO production in RAW 264.7 macrophages. NO secretion in the macrophages treated with 5, 10, and 50 µ g/mL BEOs was measured using the Griess reagent system. Data from three independent experiments were used to calculate mean values and standard deviation * p < 0.01 vs. positive control group (L-NMMA).

Effect of the BEOs on Transcription and Translation of TNF-α and IL-6
The ability to inhibit the production of TNF-α and IL-6 is an important indicator for evaluating the anti-inflammatory property of BEOs. ELISA results ( Figure 4) show that the level of TNF-α increased to more than 2000 pg/mL when stimulated by LPS. TNF-α production decreased from 2000 pg/mL to 61, 91, and 44 pg/mL in cells treated with 50 µ g/mL BLEO, SBEO, and RBEO, respectively. These results indicate that all three BEOs strongly inhibit TNF-α production. Similarly, in the presence of BEOs, the level of IL-6 decreased significantly compared to that in the control sample (LPS-treated macrophages). The amount of IL-6 decreased from 250 pg/mL to 94.67, 92.67, and 88 pg/mL when treated with increasing concentrations (up to 50 µ g/mL) of LBEO, SBEO, and RBEO, respectively (Figure 4d-f).    The expression of the TNF-α mRNA in LPS-treated cells (Figure 5a-c) was approximately 17.71-fold higher than that in cells not stimulated by LPS; this decreased to 5.76-, 8.09-, and 1.44-fold when the cells were incubated with 50 µ g/mL of LBEO, SBEO and RBEO, respectively. The IL-6 mRNA level increased by approximately 11.6-fold in the LPS-supplemented samples, which decreased to 5.73-, 4.75-, and 2.02-fold in the presence of 50 µ g/mL of LBEO, SBEO, and RBEO, respectively (Figure 5d-f). These results suggest that LBEO, SBEO, and RBEO acted as anti-inflammatory agents in LPS-induced RAW 264.7 cells by suppressing the production of TNF-α and IL-6. Among the three EOs, RBEO inhibited the transcription and translation of the pro-inflammatory cytokines the most (Figures 4 and 5).   COX-2 is an important enzyme that controls the production of PGE2 in the inflammatory response [25,26]. Figure 6 shows the effect of BEOs on COX-2 expression at both transcription and translation levels. The COX-2 protein expression increased significantly in response to LPS stimulation. However, in the presence of BEOs (5, 10, or 50 µg/mL), the COX-2 protein expression decreased in a concentration-dependent manner (Figure 6d

Inhibitory Effect of BEOs on the NF-κB Pathway in RAW 264.7 Macrophages
The NF-κB pathway regulates many genes involved in the inflammatory response [15,27,28]. To further investigate the inhibitory targets of EOs, we analyzed whether BEOs can affect the phosphorylation of two key proteins (IkBα and p65) in NF-κB pathway by monitoring the levels of IκBα, phosphorylated IκBα (p-IκBα), p65, and phosphorylated p65 (p-p65) in RAW 264.7 macrophages using Western blotting. In the presence of increasing concentrations of LBEO, SBEO, and RBEO (Figure 7a-f), the level of phosphorylated IκBα in LPS-stimulated macrophages decreased gradually to that observed in the unstimulated stage. The levels of p-p65 gradually decreased to the level in the unstimulated state when the EO concentration reached 50 µg/mL (Figure 7a-c,g-i). These results suggest that BEOs inhibited the NF-κB pathway by inhibiting p65 and IκBα phosphorylation. ence of increasing concentrations of LBEO, SBEO, and RBEO (Figure 7a-f), the level of phosphorylated IκBα in LPS-stimulated macrophages decreased gradually to that observed in the unstimulated stage. The levels of p-p65 gradually decreased to the level in the unstimulated state when the EO concentration reached 50 µ g/mL (Figure 7a-c,g-i). These results suggest that BEOs inhibited the NF-κB pathway by inhibiting p65 and IκBα phosphorylation.

In Vivo Anti-Inflammatory Assay
To evaluate the anti-inflammatory effect of BEOs at the in vivo level, we used Swiss mice as an animal model for the carrageenan-induced paw edema test. As shown in

In Vivo Anti-Inflammatory Assay
To evaluate the anti-inflammatory effect of BEOs at the in vivo level, we used Swiss mice as an animal model for the carrageenan-induced paw edema test. As shown in Figure 8a, all the EOs (LBEO, SBEO, and RBEO) inhibited paw edema in the Swiss mice model compared to that observed after treatment with indomethacin, a common nonsteroidal anti-inflammatory drug. Among the four samples (LBEO, SBEO, RBEO, and indomethacin), LBEO showed the maximum inhibition of paw edema throughout the trial period of 1-6 h after carrageenan-induced inflammation, with percentage inhibition of paw edema being 35.66 ± 8.29%, 43.11 ± 5.12%, 67.22 ± 3.31%, 70.74 ± 1.40%, and 55.45 ± 6.61% after 1, 2, 3, 4, and 6 h, respectively. The edema inhibition abilities of SBEO and RBEO were similar to that of indomethacin from 2 to 6 h after carrageenan induction. Compared to the other time points, at about 3 h after carrageenan induction, all three essential oils showed the highest inhibition (p < 0.001) of paw edema in mice; LBEO showed 67.22 ± 3.31% inhibition, while, LBEO, SBEO, and the positive control (indomethacin) showed 48.03 ± 5.05%, 47.01 ± 2.87%, and 46.38 ± 6.59% inhibition, respectively. The percentage increase in paw edema in Figure 8b and the images of the mouse paw edema (Figure 8c) at 3 h also indicated that the inhibitory effects of LBEO, SBEO, and RBEO on paw edema were similar or even better than that of indomethacin.
involved the release of NO and pro-inflammatory cytokines, such as IL-6 and TNF-α. Our in vivo results show that all three EOs inhibited paw edema at both phases. This agrees with our in vitro results showing that LBEO, SBEO, and RBEO can inhibit multiple steps in the inflammatory responses of the RAW 264.7 cell model, including (1) NO production; (2) TNF-α, IL-6, iNOS, and COX-2 expression at both mRNA and protein levels; and (3) IκBα and p65 phosphorylation in the NF-κB pathway.

Molecular Docking of 12 Main Compounds with Anti-Inflammatory Protein Targets
We showed that BEOs possessed anti-inflammatory activities and also revealed some of the important targets of these activities using Western blotting, ELISA, and RT-PCR. However, the underlying molecular mechanisms and interaction affinities of the EO components with inflammatory protein targets remain unknown. Hence, to further investigate the potential interaction affinity of BEO compounds with inflammatory pro- Carrageenan-induced paw edema model is a well-known in vivo model for studying acute inflammation. In this model, carrageenan induces edema in two-phase responses, namely, the early and delayed phases. The early phase (0−2 h) involves the production of histamines, serotonin, and bradykinin, while the delayed phase (3−6 h) involved the release of NO and pro-inflammatory cytokines, such as IL-6 and TNF-α. Our in vivo results show that all three EOs inhibited paw edema at both phases. This agrees with our in vitro results showing that LBEO, SBEO, and RBEO can inhibit multiple steps in the inflammatory responses of the RAW 264.7 cell model, including (1) NO production; (2) TNF-α, IL-6, iNOS, and COX-2 expression at both mRNA and protein levels; and (3) IκBα and p65 phosphorylation in the NF-κB pathway.

Molecular Docking of 12 Main Compounds with Anti-Inflammatory Protein Targets
We showed that BEOs possessed anti-inflammatory activities and also revealed some of the important targets of these activities using Western blotting, ELISA, and RT-PCR. However, the underlying molecular mechanisms and interaction affinities of the EO components with inflammatory protein targets remain unknown. Hence, to further investigate the potential interaction affinity of BEO compounds with inflammatory protein targets, we selected 12 main compounds ( Table 2) present in BEOs, which were then docked with inflammatory protein targets [28][29][30] of the AA, NF-κB, and MAPK pathways. The results of molecular docking are summarized in Table 2.  These results indicate that our docking method provided reliable results and can be used to further analyze the interaction of other potential ligands with the target protein structures.

NF-κB Signaling Pathway
The NF-κB pathway is also associated with inflammation [15,27,28]. Therefore, important components of the NF-κB signaling pathway (TLR, NF-κB p50p65, TNF-α, and iNOS) were selected for studying their interactions with components of BEOs using molecular docking analysis. No co-crystal structure of inhibitor/NF-κB complex is reported so far. Therefore, the re-dock strategy cannot be used to validate docking methods for

NF-κB Signaling Pathway
The NF-κB pathway is also associated with inflammation [15,27,28]. Therefore, important components of the NF-κB signaling pathway (TLR, NF-κB p50p65, TNF-α, and iNOS) were selected for studying their interactions with components of BEOs using molecular docking analysis. No co-crystal structure of inhibitor/NF-κB complex is reported so far. Therefore, the re-dock strategy cannot be used to validate docking methods for NF-κB. However, a docking method that was confirmed to be effective for NF-κB proteins in previous studies [33,34] can be used, in which the binding region of NF-κB with DNA fragment was used as the binding site for docking. The grid box was set at 50 × 50 × 50 Å with a 0.375 Å space to cover the DNA binding pocket of the most prevalent form of NF-κB, p50-p60 (PDB ID: 1VKX), and all other docking parameters were set to default values. The docking results (Table 2) show that compounds 10, 11, and 16 have good binding affinity for the NF-κB p50-p65 homodimer complex, with the calculated binding values of −6.2, −6.1, and −6.7 kcal/mol, respectively. Compounds 20, 23, and 27 showed strong binding affinities for NF-κB p50-p65, with the calculated binding values of −7.0, −7.6, and −7.1 kcal/mol, respectively. Interestingly, these compounds interacted with the cavity near the cysteine 359 of p50 (Figure 11a-c). This result agrees with that of a previous study showing that the binding region of the p50 inhibitor (andrographolide) is near the cysteine 359 of p50 [35]. This suggests that many compounds of BEOs can interact and inhibit NF-κB p50. −7.6, and −7.1 kcal/mol, respectively. Interestingly, these compounds interacted with the cavity near the cysteine 359 of p50 (Figure 11a-c). This result agrees with that of a previous study showing that the binding region of the p50 inhibitor (andrographolide) is near the cysteine 359 of p50 [35]. This suggests that many compounds of BEOs can interact and inhibit NF-κB p50.
Another important component of the NF-κB pathway is iNOS. Compounds 10, 11,  14, 16, 19, 20, 23, and 27 showed good interaction with iNOS, with binding affinity values lower than -6.0 kcal/mol . Compounds 14, 16, 19, 20, 23, and 27 also showed good interaction with TNF-α, with binding affinity values lower than -6.0 kcal/mol. However, TLR did not appear to be the inhibitory target of BEO components when all the calculated binding values ranged from -3.1 to -5.2 kcal/mol (Table 2). These results suggest that NF-κB, TNF-α, and iNOS are the main targets of the bioactive compounds in BEOs. Another important component of the NF-κB pathway is iNOS. Compounds 10, 11, 14,  16, 19, 20, 23, and 27 showed good interaction with iNOS, with binding affinity values lower than -6.0 kcal/mol . Compounds 14, 16, 19, 20, 23, and 27 also showed good interaction with TNF-α, with binding affinity values lower than -6.0 kcal/mol. However, TLR did not appear to be the inhibitory target of BEO components when all the calculated binding values ranged from -3.1 to -5.2 kcal/mol (Table 2). These results suggest that NF-κB, TNF-α, and iNOS are the main targets of the bioactive compounds in BEOs.

MAPK Signaling Pathway
The MAPK signaling pathway is one of the key pathways for inducing inflammatory responses [15,27,28]. In this study, important components of the MAPK pathway mediating inflammatory responses, including JNK, p38 MAPK, ERK5, EGFR [36], and ERBB2 [37], were selected for the docking study with the twelve main components of BEOs. The docking results (Table 2) suggest that almost all compounds (except for compound 30) docked well with five target proteins of the MAPK pathway, with calculated binding affinities lower than −6.0 kcal/mol . Compounds 14, 16, 19, 23, and 27 showed strong binding affinities for all the five target proteins (p38MAPK, JNK, ERK5, EGFR, and ERBB2), as their calculated binding affinities were lower than -7.0 kcal/mol. Compounds 16 and 23 showed strong binding ability with p38 MAPK, with the binding affinity values of −8.2 and −8.5 kcal/mol, respectively, which are better than that of a reference compound (SB0) (−7.8 kcal/mol) for p38 MAPK. The binding sites of compounds 16 and 23 with p38 MAPK (Figure 12) are similar to that of SB0 (Figure 9), indicating the reliability of our docking method. Compound 16 showed very high binding with p38MAPK, JNK, ERK5, and ERBB2, with calculated binding affinity values of −8.2, −8.0, −7.2, and −7.7 kcal/mol, respectively. These results suggest that many bioactive compounds of BEOs can interact and inhibit multiple important targets of the MAPK pathway.

MAPK Signaling Pathway
The MAPK signaling pathway is one of the key pathways for inducing inflammatory responses [15,27,28]. In this study, important components of the MAPK pathway mediating inflammatory responses, including JNK, p38 MAPK, ERK5, EGFR [36], and ERBB2 [37], were selected for the docking study with the twelve main components of BEOs. The docking results (Table 2) suggest that almost all compounds (except for compound 30) docked well with five target proteins of the MAPK pathway, with calculated binding affinities lower than −6.0 kcal/mol . Compounds 14, 16, 19, 23, and 27 showed strong binding affinities for all the five target proteins (p38MAPK, JNK, ERK5, EGFR, and ERBB2), as their calculated binding affinities were lower than -7.0 kcal/mol. Compounds 16 and 23 showed strong binding ability with p38 MAPK, with the binding affinity values of -8.2 and −8.5 kcal/mol, respectively, which are better than that of a reference compound (SB0) (−7.8 kcal/mol) for p38 MAPK. The binding sites of compounds 16 and 23 with p38 MAPK (Figure 12) are similar to that of SB0 (Figure 9), indicating the reliability of our docking method. Compound 16 showed very high binding with p38MAPK, JNK, ERK5, and ERBB2, with calculated binding affinity values of −8.2, −8.0, −7.2, and −7.7 kcal/mol, respectively. These results suggest that many bioactive compounds of BEOs can interact and inhibit multiple important targets of the MAPK pathway.

Inflammatory Interleukins (IL-6 and IL-23R)
Inflammatory interleukins also play essential roles in inflammatory responses [29,38]. In this study, IL-6 and IL-23 receptors (IL-23R) were selected for molecular docking studies. Almost all the 12 selected compounds showed low binding affinity for IL-6 and IL-23R (ranging from −3.6 to 5.9). Only compound 27 showed good binding affinity for IL-6 and IL-23R, with binding values of −6.2, and −6.1 kcal/mol, respectively. Compound 16 showed good binding affinity for IL-6, with a binding affinity value of −6.1 kcal/mol. Overall, the binding abilities of the selected compounds of BEOs with inflammatory cytokines (IL-6, IL-23R) were not as high as those of other inflammatory targets, such as COX-1, COX-2, ALONX15, NF-κB, p38MAPK, JNK, and ERBB2. These results suggest that IL-6 and IL-23R are not the key anti-inflammatory targets of BEOs.
Although the 12 main compounds of BEOs have good docking scores with important anti-inflammatory protein targets, the anti-inflammatory effects of BEOs may also be due to interactions with other inflammatory protein targets. The in silico anti-inflammatory effects of the 18 other compounds of low abundance in the BEOs (especially RBEO) have to be analyzed using molecular docking in the future.

Experimental Animals and Ethics Statement
Swiss mice (female, 8-12-week-old, 20-25 g) from the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam were maintained at the Animal Facility, Faculty of Biology, HUS, Vietnam National University, Vietnam, using standard conditions with commercial food and water ad libitum. Ethical approval for animal studies was obtained from the Ethic Committee of the Dinh Tien Hoang Institute of Medicine (certificate number: IRB-A-2102).

Plant Material
The whole plant, containing leaves, stem, and roots of B. lanceolaria were collected from Hanoi city (

Preparation of EOs
The plant materials were separated into three parts: leaves, stem, and roots. These materials were then sliced into small pieces before being steam-distilled to obtain three essential oils, namely, LBEO (0.1677%), SBEO (0.0111%), and RBEO (0.0177%).

GC/MS Analyses
The GC-MS/GC-flame ionization detection (FID) comprising of an HP7890A model GC and HP5975C MS detector (Agilent Technologies, Santa Clara, CA, USA) was used for analyzing the chemical constituents of LBEO, SBEO, and RBEO. A HP-5 MS column with a dimension of 60 m × 0.25 mm and film thickness of 0.25 µm was used for separation. The running condition was set as follows: injector temperature at 250 • C; initial temperature started at 60 • C then increased to 240 • C with an increasing step of 4 • C/min; the carrier gas was used of helium and the flowrate was set as 1 mL/min; the split ratio was 100:1; full scan modes under electron ionization with voltage: 70 eV, emission current: 40 mA; mass range scan: 35-450 a.m.u. Chemical constituents in each essential oil were identified by analysis of RI and MS values in comparing with standard compounds in the NIST database and literature [39].

Cell Culture
The RAW 264.7 cells were obtained from the Animal Cell Culture Lab, Faculty of Biology, University of Science, Vietnam National University. The cells were grown in DMEM medium at 37 • C with 10% FBS, 50 µg/mL streptomycin/penicillin, and 5% CO 2 .

Cell Viability Assay
The toxicity of the EO samples toward RAW 246.7 macrophages was determined using CCK-8 (ab228554, Abcam, Cambridge, UK). The macrophages were maintained at 37 • C for 24 h in a 96-well plate (2 × 10 4 cells in 100 µL culture per well) before being treated with various concentrations of EOs from B. lanceolaria (BEOs) for another 24 h. Each sample was incubated with 10 µL of CCK-8 solution for 2 h at 37 • C before being measured at the OD of 450 nm.

Measuring NO Production
RAW 264.7 macrophages (2 × 10 5 cells/well) were cultured in FBS-free DMEM for 3 h before being incubated with EOs (0, 5, 10, and 50 µg/mL) for 2 h. The cells were then incubated with LPS (1 µg/mL) for 24 h to stimulate NO production. NG-methyl-L-arginine acetate (L-NMMA) was used as the positive control. Cell culture medium (100 µL) was mixed with 100 µL Griess reagent (Promega, Madison, WI, USA) at 25 • C for 10 min and the absorbance at 540 nm was then recorded using the SpectraMax Plus 384 microplate reader (Molecular Devices, California, USA).
The inhibition of NO production was determined using the formula:

ELISA
The macrophages (2 × 10 5 cells/well) were maintained for 24 h at 37 • C before being treated to 5, 10, and 50 µg/mL BEOs for 2 h. The cells were then stimulated by LPS (1 µg/mL) for 24 h. The supernatant was used to measure the concentrations of TNF-α and IL-6 produced using ELISA kits (Invitrogen, Vienna, Austria).

Western Blotting
The effects of BEOs on the expression of important proteins involved in the inflammatory response in RAW 264.7 macrophages were assessed by Western blotting. The cells (4 × 10 5 cells/well in 6-wells plate) were cultured for 24 h before incubating with BEOs for 2 h. The cells were then stimulated by LPS (1 µg/mL) for 24 h before being harvested and resuspended in radioimmunoprecipitation assay lysis buffer (Thermo Scientific, Rockford, IL, USA) to obtain the total protein. Total protein samples (20 µg each) were analyzed using SDS-PAGE before being transferred to a PVDF membrane. The membrane was blocked with T-TBS (Tris-buffered saline, 0.1% Tween 20) containing 2% bovine serum albumin (Biobasic, Markham, Ontario, Canada) for 1 h. The membrane was then incubated with primary antibodies (1:1000 dilutions) for detecting β-actin, iNOS, COX-2, IκBα, p-IκBα, NF-κB p65, and p-NF-κBp65 for 16 h at 4 • C. The membranes were then washed thrice with T-TBS before being incubated with HRP-conjugated goat anti-rabbit IgG for 1 h at 25 • C. The membranes were washed thrice and then incubated with Clarity Max TM Western ECL substrate (Bio-Rad, Milan, Italy) and the signals were detected using a ChemmiDoc TM imaging system (Bio-Rad, Hercules, CA, USA). Protein quantities from Western blot results were calculated by Image Lab software Version 6.1 (Bio-Rad, Hercules, CA, USA).

qRT-PCR
To determine the effect of BEOs on the mRNA production of IL-6, TNF-α, iNOS, and COX-2, the RAW 264.7 macrophages (4 × 10 5 cells/well) were cultured for 24 h before being treated with LBEO, SBEO, and RBEO (5, 10, and 50 µg/mL) for 2 h. Inflammatory responses were stimulated by LPS (1 µg/mL) for 16 h. Total RNA (5 µg/mL) purified from each treated sample using the TRIzol™ reagent (Invitrogen, Rockford, Illinois, USA) was converted to cDNA using M-MLV reverse transcriptase (Thermo Fisher Scientific). The qPCR reaction (10 µL in total volume) contained 2 µL cDNA templates, 5 µL GoTaq ® qPCR master mix (Promega, Madison, WI, USA), 0.25 µL forward/reverse primers (Table 3), and 2.5 µL H 2 O. All reactions were run with 40 cycles; each cycle included DNA denaturation step at 95 • C for 30 s, primer annealing step at 60 • C for 30 s, and DNA strand extension step at 72 • C for 30 s. Each reaction was run in triplicate. The relative mRNA levels were calculated using the 2 -∆∆Ct method [40]; β-actin mRNA level was used as an internal standard for normalizing expression data.

Anti-Inflammation Assay Using Carrageenan-Induced Edema Model
The effects of BEOs on acute inflammation in an animal model were evaluated using the carrageenan-induced paw edema model [44]. Swiss mice (21-24 g) were stabilized under laboratory conditions for at least 7 days prior to testing. Five groups of mice (n = 6 mice/group) were prepared, including: (1) negative control group (dimethyl sulfoxide or DMSO, 10 mL/kg), (2) positive control group (indomethacin, 10 mg/kg), (3) LBEO (50 mg/kg), (4) SBEO (50 mg/kg), and (5) RBEO (50 mg/kg). Mice were orally administered with indomethacin, or BEOs, and DMSO (the vehicle control). After 60 min, 0.025 mL of 2% carrageenan suspension (Sigma-Aldrich) was injected under the soles of the right hind paws to induce inflammation. The changes in the right hind paw thickness of the mice at 0, 1, 2, 3, 4, and 6 h after carrageenan injection was measured using a micrometer (Mitutoyo, Japan). The changes in the hind paw thickness in the BEO-treated groups were compared to those in the control group (DMSO) at the same time to evaluate the anti-inflammatory effect of the tested samples. The BEOs or the reference drug (indomethacin) were considered to show an acute anti-inflammatory effect if the extent of reduction in paw edema was statistically significant compared to that in the negative control group (DMSO). The percentage inhibition of inflammation was calculated using the following formulae: where I (%): Percentage inhibition at t h. ∆C C : Percentage increase in the thickness of the hind paw in the negative control group at t h compared to 0 h. ∆Ct: Percentage increase in the thickness of the hind paw in the positive control group or treated groups at t h compared to 0 h.

Statistical Analysis
The mean values were statistically compared using one-way analysis of variance (ANOVA) with Tukey's test. The differences were considered significant for p < 0.05. The statistical tests were applied using OriginPro, version 8.5.1 (OriginLab Corp, Northampton, MA, USA).

Conclusions
In this study, the chemical components and their concentrations in the EOs from the leaf, stem, and root samples of B. lanceolaria, collected from Vietnam, were successfully elucidated. GC-MS/GC-FID analysis identified 30 compounds in the BEOs, among which, LBEO, SBEO, and RBEO contained 5, 15, and 20 compounds, respectively. Despite the variability among some of the major components in all three types of EOs, all of them showed remarkable anti-inflammatory effects in in vitro and in vivo models of inflammation. LBEO, SBEO, and RBEO inhibited multiple steps in the inflammatory responses of the RAW 264.7 cell model, including (1) NO production, (2) TNF-α, IL-6, iNOS, and COX-2 expression at both mRNA and protein levels, and (3) IκBα and p65 phosphorylation in the NF-κB pathway. In the carrageenan-induced paw edema model, all three EOs inhibited paw edema at both early and delayed phases. These in vivo results are in agreement with the in vitro results, as the inhibition of paw edema at both phases is also associated with the inhibition of key inflammatory components, including COX-1, iNOS production, nitric oxide (NO), and pro-inflammatory cytokines (TNF-α and IL-6). Furthermore, our molecular docking simulation suggested that the chemical components of B. lanceolaria EOs target and bind very strongly with many protein components of all three important signaling pathways related to inflammation, including the AA metabolic pathway (COX-1, COX-2, and ALOX15), NF-κB pathway (NF-κB, TNF-α, and iNOS), and MAPK pathway (p38MAPK, JNK, ERK5, EGFR, and ERBB2).
Overall, our results show, for the first time, the detailed chemical composition of BEOs and confirmed their potent anti-inflammatory effects using an in vitro cell model (RAW 264.7), in vivo animal model (carrageenan-induced mouse model of edema) and in silico model (molecular docking of key inflammatory components). These results demonstrate the promising anti-inflammatory potential of BEOs, which can be further utilized to develop effective anti-inflammatory drugs with limited side effects in the future.