Whole lotus flowers were used for extraction using an absolute extraction method from Tropicalife Co., Ltd., according to the manufacturer's protocol. The organic compounds of the lotus essential oil (LEO) were analyzed using GC-MS by Scientific and Technological Instruments Center of Mae Fah Luang University, Thailand. The operating conditions were as follows: The capillary column (HP-5 ms) was used with a flow rate of 1 ml/min. The gas chromatograph oven was set at 40-240˚C. Hexane was used as the solvent. The temperature of the injector and detector were set at 250 and 230˚C, respectively. The mass spectrometer was set to determine a molecular weight range of 20-300 Dalton. The organic compounds were identified by comparing their retention times and mass spectra with the data from the National Institute of Standards and Technology Mass Spectra database (NIST08). The relative quantity of each compound was presented by area %.

LEO antioxidant activity was evaluated by assessing the radical scavenging effect of the stable DPPH free radical. According to the method described by Kumaran et al (15), 50 µl LEO at various concentrations (5, 10, 20, 40, 80, 160, 320 and 640 µg/ml) was added to 200 µl 0.1 mM DPPH-ethanol solution in a 96-well plate. After 30 min of incubation in the dark at 25˚C, the absorbance was determined at 520 nm using a Cytation5 microplate reader (BioTek Instruments, Inc.) and the DPPH radical scavenging activity was calculated as the IC₅₀ using the formula: % inhibition = [(A_control - A_sample)/A_control]x100; where A_control is the absorbance of DPPH without sample and A_sample is the absorbance of DPPH with LEO. The experiment was performed three times for each sample.

The dilution of essential oil in ethanol was performed as described by Shinomiya et al (16) was used. Briefly, LEO was diluted 50% (v/v) in 70% ethanol before treatment. The final concentration of ethanol was 0.01% in RPMI medium. DRG cell culture was performed as described by Hausott et al (17). Briefly, male Wistar rats were sacrificed using CO₂ at a volume displacement rate of 30-70% of the chamber per min for 5 min.

Death was confirmed by cardiac arrest, the spinal cord was exposed and ~45 DRGs were collected in cold RPMI medium (Invitrogen; Thermo Fisher Scientific, Inc.). Thereafter, DRGs were incubated in collagenase type I (5,000 U/ml, Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at 37˚C followed by 0.25% trypsin/EDTA (Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at 37˚C. DRGs were next washed 3 times in RPMI medium containing 10% horse serum and 5% fetal bovine serum (Sigma-Aldrich; Merck KGaA), and then transferred to culture medium (RPMI supplemented with B27; Invitrogen; Thermo Fisher Scientific, Inc.) and dissociated using fire-polished Pasteur pipettes. The cell suspension was seeded onto plastic dishes coated with poly-D-lysine hydrobromide (100 µg/ml, Sigma-Aldrich; Merck KGaA) at 37˚C overnight and then coated with laminin (5 µg/ml; Sigma-Aldrich ; Merck KGaA) at 37˚C for 4 h. A total of 1 h after seeding the medium was changed and the cells were treated with 0.15, 0.3 or 0.6 µl/ml LEO in RPMI medium supplemented with B27 supplement and an antibiotic-antimycotic (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were incubated in a humidified incubator at 37˚C with 5% CO₂ for 24 h. The DRG neuron cultures were performed three times.

Male Wistar rats with an initial weight of 200-220 g (6-8 weeks old) were obtained from Nomura Siam International Co., Ltd. The rats were housed in a temperature (25±2˚C) and humidity (35-60%) controlled room with a 12 h light/dark cycle, and provided ad libitum access to food and water. The rats were randomly divided into a control, sham, sciatic nerve injury (SNI), SNI + 50 mg/kg LEO or SNI + 100 mg/kg LEO group (6 rats/group). To induce SNI, the rats were anesthetized using 50 mg/kg intraperitoneal injection of sodium pentobarbital. The nerve crush procedure was performed on the left hind limb. An incision was made in the middle of the thigh, and the muscles were carefully incised at the intermuscular septum without cutting the muscle fibers to expose the sciatic nerve. The nerve was clamped at 1 cm proximal to the bifurcation using artery forceps (straight 12 cm for 30 sec. The sham rats underwent the same surgery without clamping of the nerve. Thereafter, the skin was sutured with a nylon suture to close the wound without stitching up muscles to avoid muscle damage (18,19). After surgery, all rats were an intraperitoneal injection of 10 mg/kg meloxicam to provide pain relief and reduce inflammation for 3 consecutive days.

Thermal stimulation was applied to determine recovery of sensory function on days 7, 14, 21 and 28 after surgery (20). Prior to the test, the rats were placed on a non-heated plate for 10 min. Subsequently, the paw withdrawal latency (PWL) in response to a heat stimulus (50˚C) using a hot plate at the plantar side of the hind paw was recorded. The rats were not allowed contact with the heated plate for more than 20 sec. PWL was recorded on the left paw three times and the time interval between tests was 5 min. The experiment was repeated three times with each rat.

The recovery of motor function was assessed on days 7, 14, 21 and 28 after surgery using walking tract analysis. Briefly, the hind paws of trained rats were pressed down onto a stamp pad soaked with water-soluble black ink. The rats were allowed to walk along a confined box (30x90x20 cm), which was placed on a white paper (21x90 cm). The footprints were recorded to calculate the sciatic functional index (SFI) as described by Bain et al (21). The experiment was repeated three times with each rat.

After 24 h, DRG neurons were fixed with 4% paraformaldehyde at 25˚C for 30 min. Cells were washed three times with PBS and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) at 25˚C for 5 min, and blocked with 10% goat serum (Thermo Fisher Scientific, Inc.) in PBS at 25˚C for 30 min. Cells were incubated with primary antibodies against neuron specific β-III tubulin (Thermo Fisher Scientific, Inc.; cat. no. MA1-118; 1:1,000) and phospho- (p-)ERK (Cell Signaling Technology, Inc.; cat. no. 3179; 1:250) at 4˚C overnight. Secondary antibodies (Alexa-594 chicken anti-mouse; cat. no. A-21201) for β-III tubulin staining and Alexa-488 goat anti-rabbit (cat. no. A32731) for p-ERK staining; both at 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) were applied for 30 min at 25˚C. Immunofluorescence labeled neurons were observed using an inverted fluorescence microscope (Zeiss Axiovert 100, Carl Zeiss AG; magnification, x20 objective lens). p-ERK fluorescence intensity measurements and neurite outgrowth were determined using Metamorph^© version 7. Evaluation of the average fluorescence intensity was performed after background subtraction. The longest neurite from the cell body to the growth cone was measured and taken as the maximal distance (22).

Histological analysis was performed on day 28 after nerve injury. The rats were sacrificed using CO₂ (30-70% volume displacement rate for 5 min), the sciatic nerve and L4-L6 DRG were fixed in 4% PFA at 4˚C for 48 h followed by 15% sucrose in PBS for 24 h and then 30% sucrose in PBS for 48 h. Sections were cut (20 µm thick) using a cryostat (Leica Microsystems, Inc., CM1950) for hematoxylin & eosin (H&E) (Sigma-Aldrich; Merck KGaA) staining. The sections were stained for 10 min with hematoxylin followed by 20 sec with eosin both at 25˚C. DRG neurons were counted in area of 80,000 µm² using a 40x objective lens, and axon diameters were measured under a 100x oil immersion lens. Tissue morphology was determined using the ECLIPSE Ni-U | Upright Microscopes (Nikon Corporation) and analyzed using NIS Elements imaging software version 5 (Nikon Corporation). All histological experiments were performed three times.

Data are presented as the mean ± SD for DPPH analysis or SEM for DRG cell culture, tissue morphological analysis and behavioral tests. A one-way ANOVA followed by a Tukey's post-hoc test was used to analyze the data in GraphPad Prism version 9 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

There were three primary compounds that individually accounted for >10% of the total content of LEO: Palmitic acid ethyl ester (25.12%), linoleic acid ethyl ester (18.17%) and methyl 8,11,14-heptadecatrienoate (10.45%) (Table I). DPPH is widely used to evaluate radical scavenging activity (23-25). Ascorbic acid and Trolox (both from Sigma-Aldrich; Merck KGaA) were used as positive controls antioxidants. LEO had ability to inhibit DPPH radical activity with an IC₅₀ value of 29.01±2.93 µg/ml (Table II).

DRG neurons were cultured and treated with various concentration of LEO. The length of the longest neurite of DRG neurons was measured as the maximal distance to indicate the effect of LEO on axon elongation. Neurite outgrowth was observed after 24 h of treatment, LEO at concentrations of 0.3 and 0.6 µl/ml showed a significant increase in maximal distance when compared with the negative control and vehicle control groups (both P<0.001, Fig. 1). LEO-mediated promotion of neurite outgrowth may be mediated via ERK activation. Elevation of the p-ERK/ERK ratio plays a crucial role in axonal elongation in vivo (22). Phosphorylation of ERK was investigated by immunofluorescence staining of DRG neurons after 24 h of LEO treatment. Average fluorescence intensity of the p-ERK/ERK ratio in DRG neurons treated with 0.3 or 0.6 µl/ml LEO was significantly higher compared with the negative control and vehicle control groups (P<0.01 and P<0.001, respectively; Fig. 2).

To study the effect of LEO on PNI, sciatic nerve and L4-L6 DRG were cut and stained with H&E. After 28 days of SNI, injured rats orally administered 50 or 100 mg/kg LEO showed a significant increase in the number of DRG neurons compared with the SNI group (P<0.001 and P<0.05, respectively; Fig. 3). Axon diameters were measured to assess the effect of LEO on nerve regeneration. On day 28 after SNI, axon diameters were decreased in all injured animals. However, oral administration of 50 or 100 mg/kg LEO significantly increased axon diameter when compared to the SNI group (P<0.05, Fig. 4).

The in vitro and in vivo data indicated that LEO enhanced neurite elongation, and promoted DRG neuron survival and nerve regeneration. Therefore, whether oral administration of LEO exerted a therapeutic effect on sensory and motor function recovery was next assessed in vivo. Prior to establishment of the SNI model, all animals were tested behaviorally to obtain baseline values of locomotor function using walking tract analysis and sensory function using a thermo-withdrawal test. The baseline values did not differ between animals. After SNI, injured animals exhibited sensory and motor function deficits. Behaviors were assessed over a 4-week period. Sensory function recovery was improved in mice treated with 50 or 100 mg/kg LEO by day 21 after SNI (P<0.05). The differences became more pronounced by day 28 (P<0.001) (Fig. 5A).

Motor function recovery was assessed by walking tract analysis. The SFI gradually improved with time. Significant differences between SNI alone vs. SNI + 50 or 100 mg/kg LEO were observed on week 4 after lesion (P<0.01 and P<0.001, respectively; Fig. 5B). Overall, the results indicated that administration of LEO improved sensory and motor functions in a rat model of SNI, and this corresponded with enhanced nerve regeneration.