The following technologies were used in this research: NMR spectra were performed on Bruker AM-400 instruments with TMS as the internal standard (Bruker, Bremerhaven, Germany); column chromatography was performed by silica gel (200–300 and 300–400 mesh, Qingdao Marine Chemical Inc., Qingdao, China); Rp-18 gel (40–63 μm, Merck, Darmstadt, Germany); Sephadex LH-20 (20–150 μm, Amersham Biosciences, Uppsala, Sweden); automatic microplate reader (Thermo Fisher Scientific, Shanghai, China); QuanStudio 3 Real-Time PCR (Thermo); NanoDrop2000c Spectrophotometer (Thermo); Fluor Chem M (Protein simple); flow cytometer (BD Facsverse, BD Biosciences, Franklin Lakes, NJ, USA).

The following reagents were used in this research: RAW264.7 cells were purchased from the Typical Culture Preservation Committee Cell Bank, Chinese Academy of Sciences; Dulbecco's modified Eagle's medium (DMEM) (ProCell, Wuhan, China); a penicillin-streptomycin mixture, nucleoprotein extraction kit, BCA protein quantitative kit, and protease phosphatase inhibitor (SolarBio, Beijing, China); fetal bovine serum (FBS) (Gibco, Grand Island, NE USA); LPS (Sigma-Aldrich, St. Louis, MO, USA); NO kit and ROS kit (Beyotime Biotechnology, Shanghai, China); mouse TNF-α ELISA kit and mouse IL-6 ELISA kit (4A Biotech Co, Ltd., Beijing, China); Evo M-MLV RT Kit with gDNA Clean and SYBG Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology, Hunan, China); antibody NF-κBP65, p-NF-κBP65, P38, p-P38, SAPK/JNK, p-SAPK/JNK, P44/P22 ERK, and p-P44/P22 ERK (Cell Signaling Technology, Danvers, MA, USA); primers were designed and synthesized by Thermo Fisher Scientific (Shanghai, China). Primer sequences are shown in Table 1.

The dried aerial parts of O. majorana (4.9 kg) were extracted with a petroleum ether at room temperature for 72 h to remove the volatile oil. The filter residue was soaked and extracted three times (each time for 7 days) with 70% ethanol at room temperature and then filtered and evaporated under reduced pressure to obtain a crude extract (1,360 g).

The crude extract was chromatographed on D101 macroporous resin and eluted with water, 20% ethanol, 40% ethanol, 60% ethanol, and 95% ethanol, respectively. Then, five fractions were obtained (Fr. A–Fr. E). The 40% ethanol fraction, Fr. B (115 g), was subjected to silica gel column chromatography, eluting with a gradient of CHCl₃-MeOH (40:1~1:1) to obtain eight fractions (Fr. B-1~Fr. B-8) by a TLC plate analysis.

Fr. B-2 (2.01 g) was subjected to silica gel column chromatography, eluted with CHCl₃-EtOAc (6:1, v/v), to obtain four fractions (Fraction 1 to Fraction 4). Fraction 2 (725 mg) was separated by Sephadex LH-20 column chromatography (MeOH) and then purified by semi-preparative HPLC (MeOH-H₂O, 57:43, v/v) to obtain compound 1 (3.4 mg) and compound 2 (9.5 mg).

Fr. B-3 (900 mg) was separated by Sephadex LH-20 column chromatography (MeOH) to obtain six fractions (Fraction 1 to Fraction 6). Fraction 2 (385 mg) was subjected to silica gel column chromatography, eluted with CHCl₃-MeOH (6:1, v/v), and then purified by semi-preparative HPLC (MeOH-H₂O, 65:35, v/v) to obtain compound 3 (33 mg).

Fr. B-4 (3.1 g) was separated by an automatic preparation of liquid chromatography (MeOH-H₂O, 55:45, v/v) to obtain four fractions (Fraction 1 to Fraction 4). Fraction 1 (1.04 g) was subjected to silica gel column chromatography, eluted with a system of EtOAc-MeOH (10:1, v/v), and then purified by Sephadex LH-20 (MeOH) to obtain compound 4 (237 mg).

Compound 1: 1H-indole-2-carboxylic acid, pale yellow crystal; ¹H-NMR (CD₃OD, 400 MHz) δ: 7.95 (1H, s, H-3), 8.08 (1H, dd, J = 6.4, 2.3 Hz, H-4), 7.21~7.14 (2H, m, H-5, 6), 7.43 (1H, m, H-7). ¹³C-NMR (CD₃OD, 100 MHz) δ: 133.3 (C-2), 108.8 (C-3), 122. (C-4), 123.6 (C-5), 122.3 (C-6), 112.9 (C-7), 138.2 (C-8), and 127.6 (C-9). Its data were in good accordance with those of 1H- indole-2-carboxylic acid (25).

Compound 2: (+)-lariciresinol, pale yellow jelly; ¹H-NMR (CD₃OD, 400 MHz) δ: 6. 91 (1H, d, H-2), 6.77 (2H, d, H-5, H-6), 4.75 (1H, d, J = 6.9 Hz, H-7), 2.38 (1H, m, H-8), 3.63 (1H, dd, J = 11, 6.5 Hz, Ha-9), 3.81 (1H, overlapped, Hb-9), 6.80 (1H, d, J = 1.9 Hz, H-2′), 6.70 (1H, m, H-5′), 6.64 (1H, dd, J = 8., 1.9 Hz, H-6′), 2.49 (1H, dd, J = 13.4, 11.3 Hz, Ha-7′), 2.93 (1H, dd, J = 13.4, 4.8 Hz, Hb-7′), 2.73 (1H, m, H-8′), 3.72 (1H, dd, J = 8.4, 5.8 Hz, Ha-9′), 3. 98 (1H, dd, J = 8.4, 6.4 Hz, Hb-9′), 3.83 (3H, s, 3-OMe), 3.84 (3H, s, 3′-OMe); ¹³C-NMR (CD₃OD, 100 MHz) δ: 135.7 (C-1), 110.6 (C-2), 149. (C-3), 147. (C-4), 116. (C-5), 119.8 (C-6), 84. (C-7), 54.1 (C-8), 60.4 (C-9), 133.5 (C-1′), 113.4 (C-2′), 149. (C-3, C-3′), 145.8 (C-4′), 116.2 (C-5′), 122.2 (C-6′), 33.7 (C-7′), 43.9 (C-8′), 73.5 (C-9′), 56.3 (3-OMe), and 56.3 (3′-OMe). Its data were in good accordance with those of (+)-lariciresinol (26).

Compound 3: (+)-isolariciresinol, pale yellow jelly; ¹H-NMR (CD₃OD, 400MHz) δ: 6. 68 (1H, d, J = 1.5 Hz, H-2), 6.74 (1H, d, J = 8. Hz, H-5), 6.61 (1H, dd, J = 8., 1.7Hz, H-6), 3.82 (1H, overlapped, H-7), 1.77 (1H, m, H-8), 3.68 (3H, m, Ha-9, H-9′), 3.40 (1H, dd, J = 11.2, 4. Hz, Hb-9), 6.65 (1H, s, H-2′), 6.18 (1H, s, H-5′), 2.78 (2H, d, J = 7.7 Hz, H-7′), 2.00 (1H, m, H-8′), 3.80 (3H, s, 3′-OMe), 3.79 (3H, s, 3-OMe); ¹³C-NMR (CD₃OD, 100 MHz) δ: 138.6 (C-1), 113.7 (C-2), 149. (C-3), 145.9 (C-4), 116. (C-5), 123.2 (C-6), 48. (C-7), 48. (C-8), 62.2 (C-9), 129., (C-1′), 112.3 (C-2′), 147.1 (C-3′), 145.2 (C-4′), 117.3 (C-5′), 134.1 (C-6′), 33.6 (C-7′), 39.9 (C-8′), 65.9 (C-9′), 56.3 (3-OMe), and 56.4 (3′-OMe). Its data were in good accordance with those of (+)-isolariciresinol (26).

Compound 4: procumboside B, amorphous white powder; ¹H-NMR (CD₃OD, 400 MHz) δ: 696. (1H, d, H-2), 6.94 (1H, d, H-3), 6.66 (1H, d, H-5), 6.64 (1H, d, H-6), 4.73 (1H, d, J = 7.3 Hz, H-1′), 3.49~3.39 (3H, m, H-2′, 3′,4′), 3.65 (1H, m, H-5′), 4.54 (1H, dd, J = 11.6, 2.1 Hz, H-6a), 4.34 (1H, dd, J = 11.6, 5.6 Hz, H-6b), 7.18 (1H, d, H-2″), 6.83 (1H, d, H-5″), 7.08 (1H, dd, H-6″), 7.64 (1H, d, J = 15.9 Hz, H-7″), 6.39 (1H, d, J = 15.9 Hz, H-8″), 3.90 (3H, s, 3″-OCH₃) 0.1³C-NMR (CD₃OD, 100 MHz) δ: 153.9 (C-1), 119.5 (C-2), 116.6 (C-3), 152.3 (C-4), 116.6 (C-5), 119.6 (C-6), 103.7 (C-1′), 74.9 (C-2′), 77.9 (C-3′), 71.8 (C-4′), 75.4 (C-5′), 64.7 (C-6′), 127.7 (C-1″), 111.6 (C-2″), 150.7 (C-3″), 149.4 (C-4″), 116.5 (C-5″), 124.2 (C-6″), 147.1 (C-7″), 115.2 (C-8″), 119. (C-9″), 56.4 (3″-OCH₃). Its data were in good accordance with those of procumboside B (27).

RAW264.7 cells were cultured by the assay that was the same as that of Junya Wang (28). RAW264.7 cells with logarithmic growth were inoculated in 96-well plates at a density of 1 × 10⁴ cells per well and incubated at 37°C and 5% CO₂. When the cell fusion reached about 70–80%, the cell culture medium was removed, and the cells were treated with 100-μL DMEM, containing different concentrations of pB (6.25, 12.5, 25, 50, 100, and 200 μM) for 24 h. The blank group was given the same amount of complete DMEM without pB. Approximately 10-μL of MTT (0.5 mg/mL) were added to each well, followed by further incubation for 4 h at 37°C. Then, the cell culture medium was discarded and added to 100-μL of DMSO. The absorbance was measured at 490 nm to calculate the survival rate of cells.

RAW264.7 cells with logarithmic growth were inoculated in 24-well plates at a density of 1 × 10⁴ cells per well and incubated with 500-μL DMEM at 37°C and 5% CO₂. When the cell fusion reached about 70–80%, the cell culture medium was discarded. The LPS positive group was stimulated with 1-μg/mL LPS, and the pB group was stimulated with pB. After further incubation for 24 h, the cell culture medium was collected, and the levels of NO were determined according to the instructions of the manufacturer.

Cell treatment was the same as above. The cell supernatants were collected, the dilution ratio was adjusted, and 100 μL of the sample was absorbed for content determination. The levels of TNF-α and IL-6 in RAW264.7 cells were measured by ELISA assay kits according to the instructions of the manufacturer.

Cell treatment was the same as above. The content of ROS was determined by flow cytometry according to the experimental method in the literature of Honglin Wang (15). The cell culture medium was discarded, and 1-mL DCFH-DA (10 μM) was added to each well. Then, cells were incubated for 30 min at 37°C in the dark and washed three times with PBS to completely remove the DCFH-DA from the cells. The intracellular ROS levels were detected by detecting the changes in fluorescence intensity caused by the oxidation of the fluorescent probe DCFH-DA.

Cell treatment was the same as above. The cells were collected and the cell density was adjusted to 1 × 10⁶ cells per 100 μL, then a 0.125-μg/100 mL antibody was added, and the cells were incubated for 30 min at 37°C in dark. After washing the cells with PBS three times, the expression of CD80, CD86, and MHC II on the cell surface was detected by flow cytometry.

Cell treatment was the same as above. Cells were collected; total protein was extracted with the appropriate volume of a weak radio-immune precipitation assay (RIPA) lysis buffer depending on the density of the cultured cells (about 120 μL per well); and protein concentration was determined by the BCA method. Proteins (30 μg) were isolated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder and hybridized with phosphorylated and non-phosphorylated antibodies overnight at 4°C. After being washed three times with Tris-buffered saline-Tween 20 (0.1%), the membranes were incubated with respective secondary antibodies conjugated with horseradish peroxidase for 2 h. Blots were visualized using enhanced chemiluminescence (ECL) detection kits and analyzed using the Image J software.

Cell treatment was the same as above. RAW264.7 cells were treated with LPS or pB for 12 h, and the cells were washed three times by PBS. Total RNA was isolated using Trizol reagent and reversed to cDNA by using Evo M-MLV RT Kit with gDNA Clean according to the instructions of the manufacturer. The RNA concentration and A260/A280 value of each sample were measured by NanoDrop2000 spectrophotometer (Thermo). Then, the expression of mRNAs was determined by the SYBR Green Pro Taq HS Premix qPCR kit. According to the ratio of the Ct value of the target gene to the Ct value of reference gene β-actin, the mRNA-relative expression level of the target gene in the samples was calculated by the 2^−ΔΔCt method.

The experiments were repeated independently at least three times and analyzed statistically by SPSS 20 software. Data were expressed as mean ± SD. Statistical significance was calculated by a one-way ANOVA analysis.

To determine the non-toxic concentration range of pB, the effects of six concentrations of pB (200, 100, 50, 25, 12.5, and 6.25 μM) on the viability of RAW 264.7 cells were detected by an MTT assay. In Figure 2, compared with the blank group, pB had no significant effects on cell viability in the concentration range of 200–6.25 μM. Therefore, 100, 50, and 25 μM were selected as the study doses for this study.

Macrophage-derived NO plays an important role in immunity and maintaining homeostasis. Nitric oxide synthase (iNOS), as a pivotal enzyme in the inflammatory response of macrophages, has catalytic effects on the production of NO (29, 30). Therefore, we evaluated the immune efficacy of pB by detecting NO content and the relative mRNA expression level of iNOS in different groups. In Figure 3, the content of NO secreted in the control group was low, while the content of NO was significantly increased after LPS stimulation (p < 0.001). After pB stimulation, the release of NO was significantly higher than that of the control group (p < 0.001) with a dose-dependent manner, indicating that pB could activate NO release. The increased mRNA expression level of iNOS detected by RT-PCR further verified these results (Figure 3B). Thus, it was found that LPS and pB (100 μM) increased the mRNA expression levels of iNOS at 12 h of treatment (p < 0.001).

When macrophages are activated, more cytokines will be secreted, including TNF-α and IL-6, which all exert immunomodulatory effect. TNF-α can increase the production of a series of inflammatory cytokines and enhance the immune response of monocytes and macrophages. IL-6 is a pleiotropic pro-inflammatory cytokine whose release is associated with chronic inflammation and multiple factors of autoimmune disorders, which can promote the proliferation and differentiation of B cells and T cells (31–33). Therefore, the contents of TNF-α and IL-6 in the supernatant of different groups of cells were detected by ELISA. In Figures 4A–D, the levels of TNF-α and IL-6 and the expression of their corresponding mRNAs were significantly increased after LPS stimulation (1 μg/mL). After pB (100 μM) stimulation, the contents of TNF-α and IL-6 and the expression of the corresponding mRNA were also significantly increased compared with the control group (p < 0.001). These results suggested that pB could induce the corresponding mRNA expression and thus increase the secretion of cytokines.

In macrophages, NF-κB and MAPKs-signaling pathways are the main signaling pathways that control the immune response. The key proteins in the signaling pathway, as markers of signaling pathway activation, were selected, such as IκBα and P65 and their corresponding phosphorylation forms in the NF-κB-signaling pathway, extracellular signal–related kinase (ERK)-1/2, P38, c-Jun NH2-terminal kinase (JNK), and corresponding phosphorylation forms in the MAPKs-signaling pathway (15). In Figure 5, compared with the control group, the expression levels of phosphorylated P65 and phosphorylated IκBα were significantly increased in the LPS positive group. By calculation, the ratios of p-P65/P65 and p-IκBα/IκBα in the LPS positive group were significantly higher than those of the control group (p < 0.001). After pB stimulation, the ratios of p-P65/P65 and p-IκB-α/IκBα were also significantly higher than those of the control group. The results indicated that pB could induce immune response through the activation of the NF-κB-signaling pathway. In Figure 6, the ratios of p-P38/P38, p-ERK, and p-JNK in the LPS positive group were significantly higher than those in the control group (p < 0.001). After pB stimulation, the ratios of p-P38/P38, p-ERK/ERK, and p-JNK/JNK were significantly higher than those in the control group. The results indicated that pB could activate the MAPKs-signaling pathway to induce an immune response.

Reactive oxygen species act as immunomodulatory-signaling molecules in macrophages, as other pro-inflammatory cytokines do (34). Therefore, we used flow cytometry to detect the content of ROS. In Figure 7, compared with the control group, the peak shape of the LPS positive group moved to the right, which indicated that the ROS content in RAW264.7 cells was significantly increased after LPS stimulation. After pB stimulation, the peak shape also shifted to the right compared with the control group, indicating that ROS production increased significantly. This suggested that pB could further affect the immune response by producing ROS.

Macrophages act as antigen-presenting cells that load peptides into the MHC-II molecule, subsequently internalizing and processing the antigen, thereby allowing interaction with specific T cell receptors (TCR). However, T cell activation requires that costimulatory molecules CD80 and CD86 bind to CD 28 or CD 152 on the surface of the T cell to complete the immune synapse (35). To explore the effects of pB on the presentation of antigens, we examined the expression of CD86, CD80, and MHC II on the cell surface. In Figure 8, compared with the control group, the expressions of CD86, CD80, and MHC II were significantly increased in the LPS positive group. After pB stimulation, the peaks of CD86 and CD80 shifted significantly to the right, which was significantly different from the blank group (p < 0.001). Unfortunately, the peak value of MHC after PB stimulation did not shift to the right, and there was no significant difference compared with the control group. This suggested that pB could upregulate the expression of CD86 and CD80, thus enhancing the antigen-presenting effects of cells.