Fresh Bulbophyllum drymoglossum plants were collected, rinsed with deionized water (dH₂O), and cut into 2–3 cm segments. A total of 100 g plant material was extracted in 300 mL dH₂O at 110°C for 15 min using an autoclave (WAC-60; Daihan Scientific). The extract was filtered through a 0.2 μm sterile cellulose acetate filter (Minisart; Sartorius) and stored at −80°C. The final concentration of BDAE was determined by dry weight measurement following lyophilization.

Porcine alveolar macrophages (3D4/31; ATCC CRL-2844) and mouse embryonic fibroblasts (NIH3T3; ATCC CRL-1658) were maintained at 37°C in a 5% CO₂ humidified incubator. 3D4/31 cells were cultured in a 4:6 (v/v) mixture of DMEM (10-013-CVR; Corning) and RPMI-1640 (10-040-CVR; Corning), while NIH3T3 in DMEM only. All media contained 10% heat-inactivated fetal bovine serum (FBS; TMS-013; Merck Millipore) and 1% penicillin-streptomycin (LS202-02; Welgene). For anti-inflammatory studies, cells were pretreated for 12 h with various BDAE concentrations prior to stimulation with 2 nM PMA (P1585-1MG; Sigma-Aldrich) in fresh medium containing the same BDAE.

Cell viability and proliferation were assessed using a WST-8 assay kit (QM2500; BIOMAX). Cells were incubated with 10% (v/v) WST-8 solution for 2 h, and the absorbance at 450 nm was measured with a FilterMax F3 microplate reader (Molecular Devices). Cell proliferation was evaluated via trypan blue exclusion and manual cell counting with a hemocytometer.

BDAE composition was characterized by gas chromatography–mass spectrometry (GC-MS; 7890B GC/5977B MSD; Agilent Technologies) with an HP-5MS column (30 m×0.25 mm, 0.25 μm). Helium was used as the carrier gas at 1.0 mL/min. The oven temperature was held at 60°C for 2 min, raised at 10°C/min to 300°C, then held for 10 min. Injector, transfer line, and ion source were maintained at 250°C, 280°C, and 230°C, respectively. 1 μL of derivatized sample (BSTFA+1% TCMS, 70°C, 30 min) was injected in splitless mode. EI mass spectra (70 eV, m/z 50–600) were acquired. Compounds were identified using the NIST/EPA/NIH library (ver. 2.3), accepting reverse similarity >700. Relative abundance: peak area/total ion chromatogram [14,15].

ROS were quantified after incubation with 1 μM H₂DCFDA (Sigma-Aldrich) for 30 min at 37°C. Mitochondrial parameters were assessed using Mito Tracker Red (100 nM; Thermo Fisher Scientific) and JC-1 dye (2 μM; Thermo Fisher Scientific), respectively. Following staining, cells were washed with PBS and analyzed by flow cytometry (Guava easyCyte; Merck Millipore). A total of 3,000–5,000 events per sample were acquired, and data analysis was performed using FlowJo software ver. 10.6.2 (Tree Star).

Lipid droplets were quantified using Nile Red staining. Cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, and then washed three times with PBS. Cells were then stained with a 1 mg/mL Nile Red (72485-100MG; Sigma-Aldrich) solution in PBS for 15 minutes. After washing twice with 1 mL of PBS, the solution was completely removed. Fresh PBS was added and images were taken using a fluorescence microscope. Imaging was performed with a Leica DMi8 microscope, and lipid content was quantified densitometrically using LAS X software and Adobe Photoshop CC 2024.

Cells were treated with 50 μM 2-NBDG (Cayman Chemical) for 30 min, washed with PBS, and analyzed by flow cytometry. Analysis was performed with FlowJo 10.6.2.

Total RNA was extracted (TRIzol; Invitrogen), reverse-transcribed (WizScript cDNA Kit; Wizbiosolutions), and subjected to quantitative real-time polymerase chain reaction (qRT-PCR; SYBR Green; BioFACT) using a StepOnePlus System (Applied Biosystems). Expression was normalized to β-actin using the 2^(−ΔΔCt) method (Table 1).

Innate immune responses were quantified using the Cellytics platform (MetaImmuneTech) equipped with label-free lens-free shadow imaging technology (LSIT). After activation (IL-2, IL-12, PMA, ionomycin), the following parameters were analyzed: Polarization and Protrusion Dynamics (PPD), Weighted Shape Metric SD (WSM_SD), and Composite Structural Parameter (CSP = PPD×WSM_SD). The innate immunity index (I³) was calculated as (CSP_ASC/CSP_Vehicle)×100. All data were service-provided (Supplement 1) [16].

Results are expressed as mean±standard deviation (n≥3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison using GraphPad Prism v10.2.3. p-values: ns, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

To evaluate the cellular compatibility and compositional profile of BDAE, a series of in vitro cytotoxicity assays and chemical analyses were performed. Fresh Bulbophyllum drymoglossum plants were subjected to aqueous extraction at 110°C for 15 minutes, and the resulting BDAE was prepared as described (Figure 1A). Viability assays were conducted in NIH/3T3 fibroblasts and porcine alveolar macrophages (3D4/31) following 12-hour exposure to a concentration range of BDAE (0.00009–0.09 μg/mL). No significant reduction in cell viability was observed in NIH/3T3 fibroblasts at any tested dose, while a modest but statistically significant decrease was noted in 3D4/31 cells at the highest concentration (**** p<0.0001) (Figure 1B). Four-day proliferation assays indicated that BDAE treatment did not alter cell growth kinetics compared to the vehicle control for either cell type, confirming that BDAE is not cytotoxic under these conditions (Figure 1C). Compositional analysis by GC-MS demonstrated that BDAE is primarily comprised of carbohydrate derivatives and polyols, with D-allose (30.07%), myo-inositol (23.62%), and sucrose (17.24%) as major constituents, and additional metabolites such as β-D-glucopyranose, D-fructose, and D-mannitol (Figures 1D, 1E). Minor levels of fatty acid derivatives, including octadecanoic, eicosanoic, docosanoic, and tetracosanoic acids, were detected. The predominance of these metabolites suggests that BDAE may modulate cellular energy metabolism and redox homeostasis [5]. Collectively, these results demonstrate that BDAE shows minimal cytotoxicity in both fibroblast and macrophage cell models at the tested concentrations and possesses a diverse metabolite profile, thereby supporting its suitability for further biological evaluation in line with Animal Bioscience reporting standards.

The effect of BDAE on intracellular ROS production was evaluated in porcine alveolar macrophages (3D4/31 cells) using flow cytometry and fluorescence imaging. Dose-dependent analysis showed that increasing concentrations of BDAE markedly suppressed basal ROS levels, with significant reductions observed at 0.09 μg/mL and 0.009 μg/mL, as determined by H₂DCFDA fluorescence (Figure 2A; ** p<0.01, **** p< 0.0001). To assess BDAE’s effect under inflammatory conditions, cells were pretreated with BDAE and subsequently stimulated with PMA, a potent ROS inducer. PMA treatment greatly increased ROS levels compared to control and vehicle groups. Notably, BDAE pretreatment substantially attenuated PMA-induced ROS generation, as confirmed by a significant decrease in H₂DCFDA mean fluorescence intensity (Figure 2B; ** p<0.0001, *** p<0.001).

Fluorescence microscopy corroborated these findings, showing elevated DCF fluorescence in PMA-treated cells and marked suppression in the BDAE+PMA group (Figure 2C). Quantification of DCF-positive cells revealed that co-treatment with BDAE significantly reduced the proportion of ROS-high cells compared to PMA alone (* p<0.0001). Taken together, these findings indicate that BDAE exhibits potent antioxidant properties, significantly reducing both basal and PMA-induced oxidative stress in macrophages.

The impact of BDAE on mitochondrial integrity was assessed in 3D4/31 macrophages using flow cytometric analysis of mitochondrial mass and membrane potential. Dose-dependent evaluation with MitoTracker Red staining revealed that BDAE treatment maintained mitochondrial mass, with only the highest concentration (0.0009 μg/mL) showing a modest but statistically significant reduction compared to control; lower concentrations did not significantly alter mitochondrial content (Figure 3A, * p<0.05). Assessment of mitochondrial membrane potential using the JC-1 dye demonstrated that PMA stimulation caused a marked decrease in the JC-1 red/green fluorescence ratio, indicative of mitochondrial depolarization and dysfunction. In contrast, cells pretreated with BDAE prior to PMA exposure exhibited a significantly higher JC-1 ratio compared to the PMA-only group, suggesting partial preservation of mitochondrial membrane potential (Figure 3B, * p<0.05, ** p<0.01). Altogether, these results show that BDAE helps maintain mitochondrial mass and prevents PMA-induced mitochondrial membrane depolarization in macrophages, underscoring its protective effect on mitochondrial function under oxidative stress conditions.

The metabolic effects of BDAE were investigated in 3D4/31 macrophages. Flow cytometric glucose uptake assays using 2-NBDG demonstrated that PMA stimulation significantly reduced glucose uptake, whereas BDAE pretreatment alleviated this effect, leading to a moderate restoration of glucose uptake capacity (Figure 4A, * p<0.05). Gene expression analysis via quantitative RT-PCR revealed that PMA drastically suppressed mRNA levels of genes involved in fatty acid uptake and β-oxidation (CPT1A, CD36, FFAR2, ACADVL, ACADL), while BDAE pretreatment substantially restored their expression (Figure 4B, * p<0.05 to **** p<0.0001). Similarly, BDAE reversed PMA-induced downregulation of lipid synthesis and lipolysis genes, including FASN, ACACB, ATGL, ACLY, and SUCLG1 (Figure 4C). Notably, BDAE also restored the expression of genes encoding mitochondrial respiratory chain complexes I–V (NDUFB8, uqcrc1, COX4I1, ATP5F1A), with statistical significance observed in most targets (Figure 4D). Lipid content staining using Nile Red indicated that PMA-induced lipid accumulation was markedly attenuated by BDAE pretreatment, as reflected by reduced fluorescence intensity compared to the PMA group (Figure 4D, *** p<0.001, **** p< 0.0001). These findings indicate that BDAE counteracts the metabolic reprogramming induced by inflammatory stimuli, supporting energy metabolism, mitochondrial function, and preventing excessive lipid accumulation in activated macrophages [17,18].

A conceptual summary is presented to illustrate the integrated effects of BDAE in porcine alveolar macrophages (3D4/31) under inflammatory stress (Figure 5). Upon stimulation with PMA, macrophages experience increased oxidative stress, mitochondrial dysfunction, and excessive lipid accumulation, as evidenced by elevated ROS generation (DCF-DA), impaired mitochondrial membrane potential (JC-1), and increased lipogenesis (Nile Red) [19,20]. Pre-treatment with BDAE exerts protective effects by attenuating PMA-induced oxidative stress, maintaining mitochondrial function, and reducing both lipogenesis and lipotoxicity. These collective actions demonstrate that BDAE mitigates key features of stress and inflammation at the cellular level, supporting its potential as a modulator of macrophage metabolic and oxidative responses.