2.5.3 Formalin and capsaicin tests

The method for the formalin test was same as before (Chen S., et al., 2018). The formalin test was made by subcutaneous injection of 5% formalin (20 μL/paw) into the right posterior leg. The nociceptive response produced by mice after capsaicin injection was mainly licking the injection site, so the time spent licking the paw was recorded as the pain threshold. The negative control group was injected with isotonic saline and the positive control group was injected with TSS (20 mg/kg intraperitoneally). C10 (20 μM) and C9 (4 μM) were injected respectively in the experimental group with a content of 0.2 mL/10 g. The mice were pretreated with the above different drugs 30 min before formalin injection. Formalin injection can cause two stages of pain sensation, the first stage lasting for about 5 min immediately after injection, and the second stage lasting for about 15–45 min after injection. The mice were placed in a glass cylinder and the licking times of mice in the two stages were recorded separately.

The capsaicin test was made by subcutaneous injection of 200 μM capsaicin (20 μL/paw) into the right posterior leg. The pain threshold was the time of licking paw after capsaicin injection. The negative control group was injected with isotonic saline and the positive control group was injected with TSS (20 mg/kg intraperitoneally). C10 (20 μM) and C9 (4 μM) were injected respectively in the experimental group with a content of 0.2 mL/10 g. The mice were pretreated with the above different drugs 30 min before capsaicin injection. The mice were placed in a glass cylinder and the licking time of mice within 5 min from the injection of capsaicin was recorded.