The peel of dried Z. schinifolium was purchased from Sichuan Neautus Traditional Chinese Medicine Co., Ltd. (Chengdu, China) and authenticated by Xu Runchun (Associate Professor of Chengdu University of Traditional Chinese Medicine). A voucher specimen (ID 20191006) has been preserved in the School of Pharmacy, Chengdu University of Traditional Chinese Medicine.

The supercritical CO₂ extraction device used to extract the volatile oil of Z. schinifolium was produced in Haian Huada Petroleum Instrument Co., Ltd. (Jiangsu, China). Nitroglycerin (NTG) injection was purchased from Beijing Yimin Pharmaceutical Co., Ltd. (Beijing, China). Reserpine was purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Zolmitriptan tablets were purchased from Venturepharm Pharmaceutical (Hainan) Co., Ltd. (Hainan, China). The commercial enzyme-linked immunosorbent assay (ELISA) kits for NO, 5-HT, CGRP, and ET-1 were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). IL-1β was purchased from Abcam (United Kingdom). NF-κB p65 was purchased from Immunoway (United States). GAPDH, BCA Protein Quantitative Detection Kit, HRP-labeled goat anti-rabbit, HRP-labeled goat anti-mouse, and HRP-labeled goat anti-rat were purchased from Multisciences (Lianke) Biotech Co., Ltd. (Hangzhou, China). IκBα, SDS-PAGE gel preparation kit, and phosphorylated protease inhibitor were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China).

Sprague–Dawley rats and Kunming mice used in this study were obtained from the Chengdu Dossy Experimental Animals Co., Ltd., and the animal license number was SCXK (Chuan) 2020-030. They were maintained at room temperature (25 ± 1°C) and humidity (60 ± 5%) with a 12-h light/dark cycle and free access to chow and water. This study was conducted in strict accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The protocol and experimental designs were approved by the Ethical Committee of Hospital of Chengdu University of Traditional Chinese Medicine (Approval ID: 2019KY-082).

Two kilograms of Z. schinifolium was crushed by a pulverizer and separated by No. 10 screen, and the passing part was used to extract ZSE. The extraction pressure of the supercritical CO₂ extraction device was 26 MPa, the temperature was 60°C, the analytical pressure was 7 MPa, the temperature was 55°C, the CO₂ flow rate was 3 L·h⁻¹, and the extraction time was 6 h. The calculation formula of the CO₂-ZSE yield is given as follows: C O 2 - ZSE   y i e l d / % = M 1 / g M 2 / g × 100. (1)

M1 represents the weight of CO₂-ZSE and M2 represents the weight of Z. schinifolium.

1 ml of CO₂-ZSE was put into a 10-ml volumetric flask, fixed with ethyl acetate, and then ultrasonically mixed. After 1 ml of the solution was taken out of the volumetric flask, it was filtered with a 0.45 μm microporous filter membrane, and the filtrate was used for GC-MS analysis.

The samples (1 µL each) were injected into the gas chromatograph system with a split inlet equipped with an HP-5 capillary column (30 m × 250 µm inner diameter and 0.25 µm film thickness) under the following conditions: initial oven temperature was set at 60 °C for 2 min, increased to 200°C at a rate of 5°C min⁻¹ for 5 min, and then increased to 260°C at a rate of 10°C·min⁻¹. Helium was applied as the carrier gas at a constant flow rate of 1 ml min⁻¹, the injector temperature was set at 280°C, and the split ratio was 100:1.

The mass spectrometry conditions included a standard electron ionization (IE) source (70eV), an ion source temperature of 230°C, and an interface temperature of 150°C. The quadrupole mass analyzer had a scan range of 35–550 amu.

The components of CO₂-ZSE were positively identified using the National Institute of Standards and Technology (NIST) 14.0 Mass Spectral Database. The semi-quantitative analysis of CO₂-ZSE was performed by comparing their peak areas in the GC-MS total ion chromatogram. The percentage compositions of the compounds were calculated using the area normalization method.

Sixty adult Sprague–Dawley rats weighing 220–250 g, half male and half female, were randomly divided into six groups according to the weight, including control, model, positive zolmitriptan (0.25 mg·kg⁻¹), high dose (250 mg·kg⁻¹), medium dose (125 mg·kg⁻¹), and low dose (62.5 mg·kg⁻¹) of CO₂-ZSE, respectively. On the day before the experiment, all the rats were depilated 2 cm × 3 cm on the back. The dosages of CO₂-ZSE were determined according to the results of the preliminary experiments. Normal saline was applied on the back of the rats in the control group. NTG was injected subcutaneously into the rats (10 mg·kg⁻¹) in other groups to copy the migraine models (Ni et al., 2019). CO₂-ZSE was applied on the back of rats at 30 and 120 min after modeling. The positive group was administered zolmitriptan solution orally. The occurrence time of head scratching was recorded within 3 h after modeling, and the number of head scratching was recorded every 30 min. The occurrence time of head scratching was marked by more than five consecutive scratches. Four hours after the establishment of the migraine model, the rats in all groups were anesthetized by an intraperitoneal injection of 0.2 g·ml⁻¹ urethane (1.5 g·kg⁻¹). Subsequently, blood was taken from the abdominal aorta, and the brain tissue was rapidly excised to separate the TCC and then stored at −80°C until further processing.

Sixty Kunming mice weighing 18–22 g, half male and half female, were randomly divided into six groups according to body weight, which were control, model, positive zolmitriptan (0.36 mg·kg⁻¹), high dose (360 mg·kg⁻¹), medium dose (180 mg·kg⁻¹), and low dose (90 mg·kg⁻¹) of CO₂-ZSE. On the day before the experiment, all the mice were depilated 2 cm × 3 cm on the back. Except the control group, the mice were injected subcutaneously with reserpine at a dose of 1.0 mg·kg⁻¹ once a day for 10 days, with the mice in the control group receiving injections of isovolumic saline (Pu et al., 2019). After the establishment of the migraine model, RCXTA(in the CO2-ZSE groups), zolmitriptan (in the positive group), or tap water (in the control and model groups) were applied on the back of the mice for 5 days, One hour after the last administration, the eyeballs of the mice were taken and blood samples were collected. Then, the mice were euthanized, and the brain tissues were placed on ice and rapidly dissected while the isolated midbrain was immediately frozen and thereafter stored at −80°C until homogenization.

Thirty adult Sprague–Dawley rats weighing 220–250 g, half male and half female, were randomly divided into five groups according to body weight, including control, model, high dose (185 mg·kg⁻¹), medium dose (92.5 mg·kg⁻¹), and low dose (46.5 mg·kg⁻¹) of linalool. The dosage of linalool was based on the amount of CO₂-ZSE multiplied by the percentage of linalool. On the day before the experiment, all the rats were depilated 2 cm × 3 cm on the back. Except for the control group, NTG was injected subcutaneously into the rats (10 mg·kg⁻¹) in each group to copy migraine models. Linalool was applied on the back of rats at 30 and 120 min after modeling, and normal saline was applied on the back of rats in the control group. The occurrence time of head scratching was recorded within 3 h after modeling, and the number of head scratching was recorded every 30 min. The occurrence time of head scratching was marked by more than five consecutive scratches.

Sixty adult Sprague–Dawley rats weighting 220–250 g, half male and half female, were randomly divided into six groups according to body weight, which were the normal, model, positive, linalool oral administration, linalool transdermal administration, and linalool inhalation groups. On the day before the experiment, all the rats were depilated 2 cm × 3 cm on the back. The migraine model was copied by subcutaneous injection of 10 mg·kg⁻¹ NTG into the back of the neck of each group. The positive group was given 0.25 mg·kg⁻¹ zolmitriptan, the control group was given the same amount of normal saline, and the linalool oral administration group was given linalool by gavage at 30 and 120 min. Linalool was applied on the back of the rats in the linalool transdermal administration group at the same time, and the rats in the linalool inhalation group were placed in the sniffing device; the dosage of linalool was 185 mg·kg⁻¹. The occurrence time of head scratching was recorded within 3 h after modeling, and the number of head scratching was recorded every 30 min. The occurrence time of head scratching was marked by more than five consecutive scratches. Four hours after the establishment of the migraine model, the rats in all groups were anesthetized by an intraperitoneal injection of 0.2 g·ml⁻¹ urethane (1.5 g·kg⁻¹). Subsequently, blood was taken from the abdominal aorta until further processing.

The levels of 5-HT in the brainstem tissue and NO, CGRP, and ET-1 in serum were determined by ELISA. The tissue was mixed with nine times the homogenate medium and ground. Then the ground solution was centrifuged at 3,000–4,000 r·min⁻¹ for 10 min. The supernatant was prepared into 10% tissue homogenate and used immediately or directly frozen, and stored at −80 C. The serum was separated by centrifugation at 3,000 r·min⁻¹ for 10 min at 4°C. Each multiplex assay was performed in accordance with the manufacturer’s instructions. The absorbance was measured in the microplate spectrophotometer.

The tissue was thoroughly homogenized with 10 times the tissue volume of the RIPA lysate on ice and centrifuged at 4°C at 12000 r·min⁻¹ for 10 min, and the supernatant was separated and collected. The total protein was extracted according to the manufacturer’s instructions, and the protein concentration was determined by the BCA method. The protein was separated by SDS-PAGE and transferred onto the PVDF membrane, and the transformed membrane was placed on a decolorizing shaker at room temperature and sealed with 5% skimmed milk (0.5%TBST) for 1 h. The diluted primary antibody (5% skim milk dissolved by TBST, phosphorylated protein using 5% BSA dissolved by TBST) was incubated overnight at 4°C, and then incubated with the secondary antibody at room temperature for 30 min. ChemiScope Capture software filmed and quantified the samples. The intensity of the GAPDH protein band was used as the internal control.

The experimental data were expressed as mean ± standard error of the mean (SEM) (‾x ± s). The experimental data were statistically analyzed and plotted with SPSS 21.0. One-way analysis of variance (ANOVA) was used for comparison between groups. A p value < 0.05 indicates a significant difference, and a p value < 0.01 indicates a very significant difference.

The results showed that the extraction rate of ZSE by supercritical CO₂ extraction was 12.3%.

The ZSE extracted by supercritical CO₂ extraction showed a total of 125 peaks by GC-MS analysis. Thirty-seven chemical constituents were identified. As shown in Figure 1 and Table 1, the main component of CO₂-ZSE is linalool, accounting for 74.16% of the total volatile oil, followed by D-limonene and sabinene, with contents of 5.45 and 3.19%, respectively.

After the rats were injected with NTG, the behavioral characteristics were consistent with those described in “3.3.1 behavioral investment.” The rats in the normal group had no red ear symptoms and occasionally scratched their heads to climb the cage within 3 h of behavioral monitoring. As shown in Figure 7, after the intervention of linalool, the high and medium doses of linalool could significantly reduce the number of head scratching in rats within 30 min, and this effect lasted at least 150 min, especially in the high-dose group. The number of head scratching in the low-dose linalool group suddenly increased 30 min after administration, even higher than that in the model group, and decreased slowly after 60 min of administration, indicating that different doses of linalool may have different regulatory effects on NTG-induced migraine rats. Low dose of linalool aggravates migraine symptoms, while high dose of linalool can relieve migraine symptoms. In addition, the effects of linalool and CO₂-ZSE with the same amount of linalool on the number of head scratching in migraine rat models induced by NTG were basically the same, indicating that linalool is, indeed, the main active component of CO₂-ZSE against migraine.