The roots of L. communis were collected from Tengchong in Yunnan Province, China, in June 2022. A voucher specimen was expertly identified by Dr. Chunlin Long and has been archived in the Herbarium at MUC.

Briefly, the air-dried and powdered roots of L. communis (1.2 kg) were extracted with 95% EtOH (30 L × 3) at room temperature. The solvent was evaporated under reduced pressure, resulting in a brown residue (LCR, 276.2 g), which was then suspended in ddH₂O (3 L) and subjected to sequential extractions with n-hexane (3 L × 3) and EtOAc (3 L × 3), ultimately yielding hexane-soluble fractions: the n-hexane-soluble (LCH) fraction (101.2 g), EtOAc-soluble (LCE) fraction (87.1 g) and H₂O -soluble (LCW) fraction (67.3 g).

Sample (100 ± 2 mg) was put into the 20 mL headspace bottle, and 10 μL of 2-Octanol (10 mg/L stock in dH₂O) was added as internal standard. All samples were analyzed by gas chromatograph system coupled with a spectrometer (GC-MS) and GC-MS analysis of each biological replicate was replicated three times. In SPME cycle of PAL rail system, the incubate temperature was controlled at 60°C. Preheat time is 15 min; Incubate time is 30 min; Desorption time is 4 min. GC-MS analysis was performed using an Agilent 7,890 gas chromatograph system coupled with a 5977B mass spectrometer. The system utilized a DB-Wax. Injected in Splitless Mode. Helium was used as the carrier gas, the front inlet purge flow was 3 mL min⁻¹, and the gas flow rate through the column was 1 mL min⁻¹. The initial temperature was kept at 40°C for 4 min, then raised to 245°C at a rate of 5°C min⁻¹, kept for 5 min. The injection, transfer line, ion source, and quad temperatures were 250, 250, 230, and 150°C, respectively. The energy was −70 eV in electron impact mode. The mass spectrometry data were acquired in scan mode with the m/z range of 20–400, a solvent delay of 2.37 min. Chroma TOF 4.3X software of LECO Corporation and Nist database were used for raw peaks exacting, the data baselines filtering and calibration of the baseline, peak alignment, deconvolution analysis, peak identification, integration, and spectrum match of the peak area.

The LCH was diluted in MHB medium at 37°C and incubated in a 96-well microtiter plate format for the MIC of LCH against E. faecalis (Enterococcus faecalis 102668), S. epidermidis (Staphylococcus epidermidis 102555), S. aureus (Staphylococcus aureus 3406520), E. coli (Escherichia coli 133264), S. para-typhi β (Salmonella para-typhi β 103169), and, Methicillin-resistant Staphylococcus aureus MRSA JCSC 3063 were cultured under aerobic condition. Through previous experimental validation, it was confirmed that dimethyl sulfoxide (DMSO), used as the solvent, exhibited no significant toxic effects on methicillin-resistant Staphylococcus aureus (MRSA) at a concentration of 2.5%. Different concentrations of LCH groups were solubilized using 2.5% DMSO, while the control group received an equivalent volume of 2.5% DMSO without LCH. MRSA was cultured in MHB at 37°C until at an OD₆₀₀ of 0.3 and then treated with LCH at 0.1, 0.2, 0.4, and 0.8 mg/mL (1, 2, 4, and 8 MIC) (Epoch full-wavelength microplate reader, BioTek Company, United States). To ensure the consistency of experimental conditions, in addition to maintaining uniform drug administration protocols, we rigorously controlled other factors that could influence bacterial growth, such as temperature, humidity, shaker speed, and incubation time.

MRSA (logarithmic growth phase) was subjected to three washes with 5 mM HEPES. Following this, the bacterial were adjusted to achieve an OD₆₀₀ of 0.5, thoroughly mixed, and subsequently incubated with DIOC2(3) (30 μM) (Thermo Fisher Scientific 66, Massachusetts, United States) for 20 min at 37°C. Following an incubation period, the bacterial cells were exposed to various concentrations of LCH (0.1, 0.2, 0.4, and 0.8 mg/mL) as well as CCCP at a concentration of 100 µM. Measurements were conducted in fluorimeter equipped with 530 nm excitation and 630 nm emission filters (Multifunctional enzyme marker, Espire, PerkinElmer, United States).

MRSA was cultured at 37°C until reaching an OD₆₀₀ of 0.3 and subjected to treatment with LCH at 0.1, 0.2, 0.4, and 0.8 mg/mL, or 100 µM CCCP. Samples were collected at 10, and 30 min, diluted to an OD ₆₀₀ of 0.15, and then incubate with resazurin at 30°C. Fluorescence intensities were measured at 540 and 630 nm (Multifunctional enzyme marker, Enspire, PerkinElmer, United States).

The MRSA (OD₆₀₀ = 0.4) was treated with the LCH for 4 h, then incubated with 10 μg/mL PI until the fluorescence reading stabilized. The fluorescence intensity was measured at 535 nm (excitation) and 617 nm (emission) of (Multifunctional enzyme marker, Enspire, PerkinElmer, United States).

The MRSA suspension (logarithmic growth phase, OD₆₀₀ = 0.4) was washed three times with 0.85% NaCl, then treated with 4 MIC of LCH and melittin for 3 hours, and incubated with 3 µM of SYTOX green. Finally, the suspension was fixed on a 6-well plate and then observed using a fluorescence microscope (IX81 electric inverted microscope, Olympus, Japan) with an excitation wavelength of 485 nm.

Scanning electron microscopy (SEM) was used to observe the morphological changes in MRSA. MRSA were treated with 0.2 mg/mL LCH for 4 h, collected by centrifuge at 5,000 g for 10 min, and then fixed with 2%–5% glutaraldehyde overnight at 4°C. A gradient dehydration protocol was employed, sequentially immersing the samples in ethanol solutions with concentrations of 30%, 50%, 70%, 85%, 90%, 95% and finally, absolute ethanol (100%). The sample underwent drying, followed by deposition of a 10 nm-thick coating using an ion sputtering device. Subsequently, it was stored in a desiccator until examination under a scanning electron microscope operating at 20–0 kV (Hitachi S-4800, Tokyo, Japan).

The MRSA was washed three times with centrifuge, and the cell was adjusted to 10⁶ CFU/mL. Then, the bacterial was supplemented with LCH at 0.1, 0.2, 0.4, and 0.8 mg/mL and subjected to incubation at 37°C for a duration of 10 h. Following bacterial collected by centrifuge at 8,000 g for 10 min, and alkaline phosphatase was tested by AKP assay kit (Beyotime, Shanghai, China).

The cell viability of LCH on HaCaT were assessed using the Cell Counting Kit-8 (CCK-8). Briefly, HaCaT cells were seeded in 96-well plates with 100 μL (2 × 10⁵ cells/well). Following incubation with varying concentrations of the tested substance, then tested with CCK-8 assay kit. Finally, we measured the OD value at 450 nm using a Microplate Reader (Beyotime, Shanghai, China).

A total of 32 SD rats were procured from the Department of Experimental Animal Science, Peking University Health Science Center (SCXK(JING)2021-0013) and acclimated for 1 week prior to the experiment. The animals were housed in 22° ± 2° with natural light/dark cycles and provided clean water and chow. The rats with excision wounds were divided into four groups, each consisting of eight rats: the model M group (wounds + no treatment), the control (C) group (no wounds), the positive group (wounds + treated with 1% SSD (Silver sulfadiazine)), and the LCH group (excision wounds + treated with 10 mg LCH (red oil)). After 24 h of wound creation, LCH (red oil) 10 mg, 1% silver sulfadiazine (SSD) 0.1 g were topically applied to gently cover the wounded area once daily until complete wound healing. Wound area and wound contraction were monitored every 48 h until complete wound healing was achieved for all groups. All the wounds received daily standard wound cleansing with 0.9% normal saline prior to the topical application of treatments (0.1 g/unit wounded area [4 cm²]/day). The experimental procedure was carried out in compliance with the ethics requirements (ECMUC2021012AO).

Upon full wound healing, rats were euthanized approved by the ethical review of MUC. The sections of tissue were prepared using an established method (George et al., 1995). In brief, the healed tissues were initially fixed in 10% formalin, followed by progressive dehydration by graded ethanol, clearing in xylene, and subsequent embedding in paraffin wax for histological assessment. The tissue samples were sectioned into four-mm slices using a microtome, followed by staining with Harris hematoxylin and eosin (HE). The stained sections were then mounted onto microscopic slides, covered with coverslips, and subsequently examined using a light microscope. Images were acquired using Ultra View ERS confocal laser scanning microscopy (Nikon/OLYMPUS, E100/CX23, Japan).

All the data were subjected to ANOVA for multiple groups using GraphPad Prism 10.0 software (Prism GraphPad) (P < 0.05).

The phytochemical components present with their name, molecular formula, compound types, retention time (RT), as well as their relative abundance (Aera), which was quantified as the percentage of peak area detected by GC-MS. The results revealed that 49 compounds, based on comparisons with the NIST library. These compounds belong to different classes, including fatty acids, terpenes and other compounds. The chemical composition of the LCH is shown in Table 1, accounting for 85.68% of the total content. The volatile oils in the root hexane Extract of L. communis included characteristic detected components such as humulene-type sesquiterpenes, elemane-type sesquiterpenes, guaiane-type sesquiterpenes and Decanoic acid, ethyl ester (lauric acid), which is consistent with the phytoconstituents reported the review of Lindera species (Cao et al., 2015).

The roots of L. communis were extracted using 95% ethanol, and the resulting crude ethanol extracts was fractionated to obtain n-hexane, EtOAc, and water fractions. As shown in Table 2, all of the extracts presented bactericidal activity against bacteria. The antimicrobial activity of LCH against MRSA JCSC 3063 (MIC: 0.1 mg/mL). The fractions exhibited limited efficacy against Gram-negative bacteria.

As shown in Figure 1A, the impact of LCH on the growth of MRSA is detected. Arrow marks are the time point of LCH addition. The bactericidal activity of LCH was equivocal after 1 h exposure at concentrations of 1× MIC and 2× MIC, while a pronounced bactericidal effect was observed with a marked reduction in CFU following 1 h exposures to doses of different multiples of MIC values.

DIOC2(3) is a potentiometric probe known for its affinity to visualized the change of membrane permeability. Disruption of membrane permeability results in the release of the probe into the surrounding medium, thereby inducing a substantial enhancement in fluorescence. DIOC2(3) is a widely acknowledged membrane potential probe that specifically localizes within the bacterial membrane to detect alterations in membrane potential (Minderman et al., 1996; Aqawi et al., 2021). We investigated whether LCH disrupts the membrane potential using the probe DiOC2(3) (Figure 1B). Indeed, our results revealed hyperpolarization of cells, in contrast to the depolarization induced by the proton ionophore CCCP (Figure 1B), showing that LCH may give an ion channel, a membrane pore, or even potentially influence the respiratory chain. And further studies will be conducted to confirm this hypothesis (Saeloh et al., 2018).

To ascertain the impact of LCH on respiratory chain, we evaluated the decrease of resazurin, a widely used indicator for quantifying cellular reductive capacity (Abu-Amero and Bosley, 2005; Müller et al., 2016). Certainly, after incubation with LCH, there was only a slight reduction in the reductive capacity of MRSA, suggesting a relatively modest impact on the electron transport chain (Figure 1C). The observed effect exhibited a comparable magnitude to that elicited by CCCP, known for its role in decoupling the electron transport chain, and was closer to the control group (Figure 1C). Consequently, the influence of LCH on the maintenance and generation of the proton motive force can be deduced.

PI was employed to further investigate and probe the membrane-disrupting effects of LCH, which was utilized to assess the integrity of the cell membrane through binding to exposed nucleic acids in a fluorescent fashion (Lin et al., 2017). Melittin, used as a positive control, resulted in substantial increases in fluorescence intensity, signifying the disruption of cell membranes. This effect was further validated through the use of the fluorescent indicator PI is capable of cellular entry through pores or in cases of severe membrane breaches (Wenzel et al., 2013). After treatment with LCH, a notable PI influx was observed at concentrations of 1×, 2×, 4× and 8× MIC, closely resembling the results obtained with Melittin (Figure 1D). This indicated that LCH may either disrupt bacterial cell membranes or create small breaches in them. Thus, the PI assay corroborates the findings from the membrane potential study, where significant fluorescence intensity was observed across different concentrations. It is speculated that LCH exerts its antibacterial effects by affecting the membrane.

Most satisfyingly, the SYTOX Green assay yielded consistent results with the fluorescence intensity, which was observed in the PI assay (Figure 1E). When LCH was treated with 1 mg/mL LCH for 4 h, the fluorescence was clearly observed in the SYTOX Green assay. The control group, in contrast, exhibited no change in fluorescence, whereas the melittin group induced a sharp increase in fluorescence intensity under identical conditions. This result aligns with the findings of the PI assay, further confirming that LCH disrupts the cell membrane (Lebaron et al., 1998).

Alkaline phosphatase (AKP), located within the space between the cellular membrane and wall, is released into the extracellular milieu upon compromise or damage to the cell wall (Diao et al., 2018). Therefore, the AKP activity serves as an indicative measure of the cell wall’s integrity. As depicted in Figure 1F, AKP activity in the C group and the LCH-treated groups at 1×, 2×, 4×, and 8× MIC remained relatively stable even after a 10 h incubation period, whereas the standard group exhibited a significant 5.39-fold increase in AKP activity, which indicates that the permeability of the MRSA cell wall remained largely unchanged following treatment with LCH.

The morphological alterations in MRSA induced by LCH were observed using SEM. As illustrated in Figure 2, the cell surface of the control group appeared smooth and round, with well-defined edges, intact structure, and uniform short rods. After a 4 h incubation with 0.2 mg/mL of LCH, significant morphological alterations were observed, characterized by distinct ruptured membranes and the collapse of cell surface structures. The observed alterations in cell morphology strongly indicate that the potent biological activities of LCH primarily result from its ability to disrupt membrane integrity.

An assessment of the toxicities of LCH was conducted on the HaCaT cell lines (Human immortal keratinocyte lines). The IC₅₀ value against HaCaT cells was 1.83 ± 0.21 mg/mL, Which is far higher than the MIC agaisnst MRSA and safety with an appropriate dosage.

After establishing wounds model in rats, receiving daily treatments subsequently, it was observed that the untreated excisional-wounded rats had a longer period of epithelialization compared to 1% SSD group. Additionally, LCH treatment shortened the period of epithelialization compared to the M group (Figure 3). The wound contraction rate was observed to be slower in the Mrats compared to the 1% SSD group. However, treatment with LCH group significantly improved the wound contraction rate compared to the model group (Figure 3). Meanwhile, the control group exhibited noticeable pus formation from day 6–12, while the LCH-treated group did not. Previous studies have demonstrated the potential of Lindera extracts as analgesic agents due to their remarkable anti-inflammatory effects and pain-relieving properties in traditional medicine (Cao et al., 2015). It showed that antibacterial, noninflammatory and antinociceptive effects have resulted in the wounding healing.

The control group showed variable-sized hair follicles, large sebaceous glands, and loose granulation tissue with limited inflammatory cell infiltration (Figure 4A). The positive control group showed a thin epidermis and loose granulation dermis with widespread eosinophilic collagen fibers, moderate inflammatory cell infiltration, coagulation within the hair follicle, fewer hair follicles, and sebaceous glands (Figure 4B). The model group exhibited a thickened epidermis and compact fibrotic granulation dermis with mild eosinophilia of collagen fibers, accompanied by significant infiltration of inflammatory cells and minimal presence of sebaceous glands (Figure 4D). The LCH group exhibited a thickened epidermis and a loosely arranged granulation dermis with moderate inflammation and sparse sebaceous glands (Figure 4C). The histomicrographs indicate that LCH demonstrates a significant decrease in the infiltration of inflammatory cells compared to the model, suggesting its potential as an effective anti-inflammatory and antibacterial agent.