n-Hexane (F2124114GC > 99%) was obtained from Aladdin Biotechnology (Shanghai, China). Butyl acetate (RH269812GC ≥ 97.7%) was obtained from Shanghai Yien Chemical Technology Co., Ltd. (Shanghai, China). Anhydrous sodium sulfate (1804081) was procured from Xilong Scientific (Guangdong, China).

Lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and dexamethasone (Dex) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Cell culture medium RPMI 1640, DMEM, phosphate buffer saline solution (PBS), and trypsin were obtained from Solarbio Life Sciences (Beijing, China). Fetal bovine serum (FBS) was obtained from Gibco BRL Life Technology (Gibco BRL, Gaithersburg, MD). A cell counting kit 8 (CCK-8) was obtained from Meilunbio (Dalian, China). Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) kits were purchased from Meimian Biotechnology (Jiangsu, China). Superoxide dismutase (SOD) assay kits were procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Solarbio Life Sciences (Beijing, China). Annexin V-FITC apoptosis detection kit, mitochondrial membrane potential assay kit, and reactive oxygen species (ROS) assay kit were obtained from Beyotime Biotechnology (Shanghai, China).

Asarum (jk20220118008) was purchased from Chengdu and authenticated as genuine Chinese herbal medicine by Professor Zhang Puzhao of the School of Pharmacy, Jiangxi University of Chinese Medicine. AEO was obtained by steam distillation (Asarum was cut into 1 cm, 12 times the amount of water was added, and extracted for 5 h), with an oil yield of approximately 1%. The volatile oil was stored in a refrigerated cabinet at 4°C.

AEO was spread on a thin film on a heated surface to ensure even heating and improve evaporation efficiency and shorten the distillation time. The feed flow rate and scraping speed had a relatively minor impact on the separation results, unlike the evaporation temperature and vacuum degree, which significantly affected the degree of evaporation (Wang et al., 2023). The optimum experimental conditions were determined by pre-experimentation using a short-path MD (F417-9078, UIC GmbH Company, Germany). The optimized conditions were as follows: evaporation temperature of 50°C, vacuum of 8 mbar, primary condensation temperature of 15°C, and secondary condensation temperature of −15°C. Finally, the crude oil (C) was separated, and the fractions were obtained as a heavy fraction (H), a middle fraction (M), and a light fraction (L).

PC12 (Rat adrenal pheochromocytoma) cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (ZQ 0150, Shanghai, China). The cells were cultured in RPMI 1,640 medium containing 1% penicillin/streptomycin and 10% FBS in a 5% CO₂ incubator at 37°C. The cells were harvested using 0.25% trypsin/EDTA when they reached 80%–90% confluency. The cells were treated with different concentrations of the previously obtained four distillates for the following experiments.

RAW264.7 murine macrophage cells were kindly given by the Research and Experiment Center, College of Traditional Chinese Medicine, Jiangxi University of Chinese Medicine. The cell medium was prepared with DMEM (containing 1% penicillin/streptomycin) and 10% FBS and was cultured in a humidified 5% CO₂ incubator at 37°C. After the cells reached 80%–90% confluency, they were collected using 0.25% trypsin/EDTA for the subsequent experiments. Then, the cells were treated with 0.5, 0.25, 0.125, and 0.0625 μL/mL concentrations of each of the four distillates.

LPS was diluted to 1 mg/mL in sterile PBS stored at −20°C and then diluted to 100 μg/mL in DMEM medium. Dex was diluted to 4 μg/mL in PBS. LPS and Dex were used in the anti-inflammatory experiment with RAW264.7 cells.

PC12 and RAW264.7 cells were harvested by centrifugation and seeded in 96-well plates at 1 × 10⁴ per well. Then, the cells were treated with eight different concentrations (2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625 μL/mL) of four distillates (C, H, M, and L) in an incubator for 24 h. According to the kit instructions, RPMI 1,640 containing 10% CCK-8 reagent was added to each well. After incubation at 37°C for 2 h, the optical density (OD) value of each well was measured at 450 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, United States).

A total of 4 × 10⁴ RAW264.7 cells per well were cultured in 24-well plates. Four concentrations (0.5, 0.25, 0.125, and 0.0625 μL/mL) of distillates C, H, M, and L were added for intervention on the second day. After 2 h of incubation, the cells were induced by LPS (100 μg/mL). The untreated RAW264.7 cells served as the control group, the cells induced by LPS without dosing were used as the model group, and dexamethasone was added for the Dex group. After 24 h of incubation, cell supernatants were collected for further experiments with commercial IL-6 and TNF-α kits. Then, after rinsing the plate five times, the kit reagents were added to the reaction plate and incubated at 37°C for 30 min. After washing the samples five times, reagents were added following the manufacturer’s instructions and reacted for 10 min. The OD values of the wells were detected at 450 nm via a microplate reader, and the anti-inflammatory activity of the cells was calculated.

PC12 cells were collected by trypsin and seeded in 6-well plates with 4 × 10⁵ cells per well. The cells were cultured overnight and exposed to 1, 0.5, 0.25, and 0.125 μL/mL of the four distillates (C, H, M, and L). Then, the cells were incubated in an incubator for 24 h. The cells and cell supernatants were centrifuged and collected in 2 mL EP tubes. Thereafter, the cells were broken using a cell disruptor and stored at −80°C for subsequent assays for the determination of oxidative enzymes. The SOD levels in PC12 cells were measured using the 96-well plates in the commercial assay kits with five replicate wells per sample. The OD values were measured via a microplate reader.

DAPI and Annexin V-FITC were used to detect the apoptosis of PC12 cells. For DAPI staining, 4 × 10⁵ PC12 cells were cultured in each well of the 6-well plates. Four different concentrations (1, 0.5, 0.25, and 0.125 μL/mL) of distillates C, H, M, and L were separately added to the cells and incubated for 24 h. After incubation, the cell supernatants were discarded. The cells were washed twice in PBS, and fixed with 4% paraformaldehyde (PFA) (DF0135, Leagene Biotechnology, China) for 15 min. Thereafter, the DAPI reagent was added for 5–10 min at room temperature (RT), followed by washing with PBS twice. The stained cells were observed under a fluorescence microscope (Nikon, Japan).

Annexin V-FITC assay was performed according to the manufacturer’s instructions. PC12 cells were collected after trypsin digestion and centrifuged (1,000 rpm, 5 min). After resuspending with PBS, the cells were centrifuged again, and the supernatants were discarded. Next, 195 μL Annexin V-FITC binding reagent was added. The cells were resuspended and 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added. The samples were incubated for 10–20 min at RT under a dark condition. A flow cytometer (Becton, Dickinson, and Company, United States) was used to analyze the stained cells.

The ROS levels in PC12 cells were determined using the fluorescent probe DCFH-DA. Approximately 4 × 10⁵ cells were seeded in each well of a 6-well plate, and the ROS level was measured after treatment with the distillates (C, H, M, and L) for 24 h. To each well, 2 mL of DCFH-DA was added, followed by incubation at 37°C for 20 min. Thereafter, the cells were rinsed thrice with serum-free RPMI 1,640 medium. The cells were then studied using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

JC-1 was used as a fluorescent probe in the Mitochondrial Membrane Potential Assay Kit (JC-1) for early apoptosis detection. PC12 cells (4 × 10⁵ per well) were seeded in 6-well plates and incubated with four concentrations (1, 0.5, 0.25, and 0.125 μL/mL) of the four distillates (C, H, M, and L) for dosing intervention. After 24 h of intervention, the experiment was performed according to the manufacturer’s instructions. JC-1 staining solution was added to the cells and cultured at 37°C for 20 min. A fluorescence microscope was used to observe the fluorescence of the cells.

A total of 90 SPF KM mice (6–8 weeks old; 18–22 g each), half male and half female, were obtained from the Experimental Animal Center of the Jiangxi University of Chinese Medicine. This experiment was approved by the Animal Ethics Committee of the university (Animal Ethics Number: 20220306029) and strictly followed the Guidelines for the Management and Use of Laboratory Animals. The acute toxicity test was performed to assess the toxicity of AEO, and the LD ₅₀ value was used as the assessment index. The mice were divided into 22 groups: blank (saline), control (20% Tween-80), and 20 experimental groups (four distillates, five dose groups for each distillate, six mice in each group, half male and half female). According to the pre-experimental results, the doses of both crude oil and H distillate were 1.0792, 1.3489, 1.6862, 2.1077, and 2.6347 g/Kg; the concentrations of M were 2.1077, 2.6347, 3.2865, 4.1080, 5.1349 g/Kg, and the administration concentrations of L distillate were 2.6347, 3.2865, 4.1080, 5.1349, 6.4185 g/Kg. The mice fasted without water for 12 h before the experiment. After 12 h, the four distillates were administered by gavage at 0.2 mL/10 g per mouse.

Data were processed using SPSS 21.0 (IBM, New York, NY, United States) for plotting and statistical analysis. Graphs were constructed using GraphPad Prism 9.0 (GraphPad Software, CA, United States) and SIMCA 14.1 (Umetrics, Sweden). Data were expressed as mean ± standard deviation (‾X ± SD) and compared using one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

Asarum volatile crude oil (C) was divided into three distillates, heavy oil (H), medium oil (M), and light oil (L) via MD. A total of 34 components were identified and isolated from the Asarum crude oil, among which methyl eugenol had the highest content (24.02%), and the others were mainly safrole (17.08%), 3,5-dimethoxytoluene (10.62%), myristyl ether (8.20%), 3,4,5-trimethoxytoluene (7.04%), 3-carene (4.77%), β-pinene (4.51%), eugenolone (3.00%), lobenol (1.21%), and levulinic-limonene (1.05%) (Figure 1, S4). The primary components of medium oil and light oil were also analyzed through distillation. The results showed that the main components of distillates M and L were (1S) -(−)-alpha-Pinene (10.64%, 19.84%), β-pinenelaevo (12.51%, 19.69%), and (1S) -(+)-3-Carene (13.96%, 19.63%). Unlike the light fraction, the M fraction contained safrole (12.55%) and 3,5-dimethoxytoluene (7.93%). The main components of the heavy fraction were methyleugenol (31.36%), safrole (19.42%), and 3,5-dimethoxytoluene (12.01%). From the aforementioned results, it is evident that the components in different distillates have significant differences in mass fraction. Therefore, by using MD, the separation and enrichment of AEO was achieved (Table 1).

From these results, the contents of monoterpenes in crude oil, H, M, and L were 13.58, 1.31, 53.72, and 77.25%, respectively, whereas the contents of ethers in C, H, M, and L were 69.44, 86.21, 27.86, and 5.83%, respectively. The monoterpenes, with boiling points generally in the range of 140°C–180°C, exhibit analgesic, antibacterial, detoxification, and diuretic effects. For example, the boiling point of β-pinenelaevo is 155°C–156°C, and (+) -3-caren has a boiling point of 168°C–169°C. However, the ethers in AEO had higher boiling points, such as safrole 232°C–234°C and methyl eugenol 254°C–255°C. These ether components exhibited neuroleptic effects and were also the primary toxic components of AEO. From these results, it can be concluded that the light distillate is enriched in low-boiling-point components, while the heavy distillate is enriched in high-boiling-point components.

To investigate the safety concentrations of the four distillates C, H, M, and L on RAW264.7 cells and PC12 cells, eight different concentrations (2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625 μL/mL) of these distillates were added to the RAW264.7 cells and PC12 cells to detect cell viability for 24 h.

The cell viability results of RAW264.7 cells showed that the survival rate was close to 0 at a concentration of 2 μL/mL. However, the survival rates increased with the decrease in the distillate concentration for all the distillates. Especially at 1 μL/mL concentrations, the cell viability with heavy, medium, and light distillates was significantly higher than that with crude oil. Meanwhile, when the concentrations of crude oil, heavy distillate, medium distillate, and light distillate were all less than or equal to 0.5 μL/mL, the cell viability of RAW264.7 cells was greater than 85%. Therefore, four concentrations (0.5, 0.25, 0.125, and 0.0625 μL/mL) of C, H, M, and L were selected for the following experiments on RAW264.7 cells (Figure 2A).

Furthermore, the cell viability results from PC12 cells showed an increase in survival rate with the decrease of concentrations of C, H, M, and L. At 2 μL/mL concentration of the four distillates, the survival rates of PC12 cells were almost 0, while at a concentration of 1 μL/mL of C, H, M, and L, the cell viability of the C group was lower than that of the H, M, and L groups. Notably, the survival rates of PC12 cells were greater than 85% at a concentration of 0.5 μL/mL for all four distillates. This indicates that 0.5 μL/mL was a safe distillate concentration for PC12 cells (Figure 2B).

The IL-6 level in RAW264.7 cells was determined via the commercial ELISA kit after treatment with AEO and its different distillates. First, the IL-6 levels at different concentrations (0.5, 0.25, 0.125, and 0.0625 μL/mL) of crude oil, heavy distillate, medium distillate, and light distillate were compared. The results showed that compared with the LPS-induced model group, there were decreasing trends in IL-6 levels in the Dex groups and four concentration groups of H, M, and L. Furthermore, the IL-6 levels in 0.5 and 0.25 μL/mL groups of C were decreased (p < 0.05, vs. model group). These results indicated that distillates H, M, and L, obtained from AEP by MD, could reduce the contents of pro-inflammatory cytokine IL-6 compared with that of the model group (Figures 3A–D).

Then, the levels of IL-6 in C, H, M, and L were analyzed at the concentration (0.5, 0.25, 0.125, and 0.0625 μL/mL)of distillates. Compared with the C group, the IL-6 levels were significantly lower in M and L groups for 0.5 L/mL distillate concentration (p < 0.05). When the concentrations were 0.25, 0.12,5, and 0.0625 μL/mL, the IL-6 levels IL-6 in H, M, and L groups were clearly decreased (p < 0.05, vs. C group). This indicated that distillates H, M, and L showed a relative decrease in the content of pro-inflammatory cytokine IL-6 at the same concentrations compared to crude oil in a dose-dependent manner (Figures 3E–H).

AEO also exhibits pro-inflammatory effects. Therefore, we investigated the TNF-α levels in RAW264.7 cells after AEO, and H, M, and L intervention. Similarly, the TNF-α levels in the four concentrations of C, H, M, and L were measured first. Compared with the LPS-induced model group, the TNF-α levels in the Dex groups and four concentrations (0.5, 0.25, 0.125, and 0.0625 μL/mL) of the H, M, and L groups showed a decreasing trend (p < 0.05) (Figures 4A–D). Further, the TNF-α levels in C, H, M, and L at the same concentration were compared. The results showed that at 0.5, 0.125, and 0.0625 μL/mL, the TNF-α levels in H, M, and L were notably lower than that of C group. Only the TNF-α level in the L group was decreased (p < 0.05, vs. C group) when the concentration was 0.25 μL/mL (Figures 4E–H). These results illustrated that the three distillates (H, M, and L) could reduce the release of TNF-α in RAW264.7 cells and exert anti-inflammatory effects compared with crude oil.

Principal component analysis was used to analyze the relationship between AEO and the IL-6 and TNF-α levels via SIMCA14.1software. The results are shown in Figure 5. The data were downscaled and simplified by OPLS analysis, with Q2 (cum) of the TNF-α model being 0.832 and that of (cum) Q2 (cum) of the IL-6 model being 0.586, which showed good overall predictive ability. Further correlation analysis was performed on the two data sets to screen for compounds (Figure 6).

The correlation analysis between anti-inflammatory activity and components of AEO revealed that the anti-inflammatory activity was primarily associated with methyl eugenol, α-pinene, β-pinene, α-phellandrene, 1,8 eucalyptol, camphorene, and β-laurelene. Therefore, the strong anti-inflammatory activity of L may be related to the high relative percentage content of α-pinene and β-pinene.

Next, the effect of crude oil and its distillates on intracellular ROS in PC12 cells was investigated via flow cytometry. A1 to A4 were ROS level expressions under C stimulation, B1 to B4 were under H, C1 to C4 were under M, and D1 to D4 were under L intervention. These findings from this experiment demonstrated that both the intracellular ROS levels and fluorescence intensity significantly changed after C, H, M, and L intervention compared to those of the normal group (blank group). Moreover, the fluorescence intensity tended to be normal as the drug concentration decreased. In addition, the statistical results showed that under C intervention, the fluorescence intensities of A2 (0.5 μL/mL), A3 (0.25 μL/mL), and A4 (0.125 μL/mL) were significantly changed compared with that of the normal group (p < 0.01). Fluorescence intensities corresponding to all four concentrations of H were statistically significant, while only C4 (0.125 μL/mL) had a significant change in M distillate (p < 0.01). Finally, all four concentrations (1, 0.5, 0.25, and 0.125 μL/mL) of H distillate caused significant changes in the cellular oxidative stress levels compared to the normal group (p < 0.01) (Figure 7).

The Annexin V-FITC assay kits were used to investigate the cell apoptosis rates of crude oil and distillates with PC12 cells. The results of this study showed that the high concentration (1 μL/mL) of these distillates (H, M, L) obtained after MD exhibited a higher apoptosis rates compared to the normal group (blank group). Furthermore, the apoptosis rate varied in a concentration-dependent manner. Lower concentration resulted in lower cell apoptosis rate. In addition, the statistical findings revealed that A2 (0.5 μL/mL) in distillate C and B1 (1 μL/mL) in distillate H exhibited a significant increase in apoptosis rate (p < 0.01, compared with that with the normal group). In M groups, the apoptosis rates of 1, 0.5, and 0.25 μL/mL were significantly different from those of the normal group. In distillate L groups, the apoptosis rates were significantly changed for the concentrations of 1, 0.25, and 0.125 μL/mL (p < 0.05, compared with the normal group) (Figure 8).

The effects of Asarum volatile crude oil and the different distillates on PC12 cell morphology by DAPI staining were observed. Cells in 1 μL/mL of each of C, H, M, and L (A1, B1, C1, and D1) groups demonstrated significant morphological changes with abnormal nuclei and increased apoptotic cells. However, as the concentration of the added distillate decreased, the morphology of cells in C, H, M, and L (A4, B4, C4, and D4) groups at 0.12 5 μL/mL of C, H, M, and L cells demonstrated similar morphology to that of the normal group (blank group), with intact nuclei and significantly more viable cells. These results suggested thatthat the survival rates of PC12 cells is concentration-responsively changed following treatment with AEO and its different distillates. (Figure 9).

Oxidative damage can induce apoptosis. The expression levels of superoxide dismutase (SOD) in the different distillates of AEO at different concentrations are shown in. The SOD levels were significantly decreased at high concentrations of AEO, suggesting that AEO affects the oxidative stress system at high concentrations. Notably, the SOD expression of H was significantly lower than that of the other three groups (B2 < A2, C2, D2) when the concentration was 0.25 μL/mL, which may be related to the higher safrole content in H (Figure 10 ).

Acute toxicity experiments on mice were performed to investigate the toxicity of AEO and three AEO distillates. Mice mortality was investigated and determined after AEO administration. The results showed that the LD ₅₀ of C, H, and M was 1.8852, 1.9566, and 3.6741 g/Kg, respectively, and the toxicity of the M group was lower than that of the other two groups. The mortality rate of mice was only 16.67% when the administration concentration of L was 6.4185 g/Kg, indicating that the toxicity of L was significantly lower than the other distillates (Table 2).