(-)-Naringenin 4',7-dimethyl Ether Isolated from Nardostachys jatamansi Relieves Pain through Inhibition of Multiple Channels

Abstract

(-)-Naringenin 4',7-dimethyl ether ((-)-NRG-DM) was isolated for the first time by our lab from Nardostachys jatamansi DC, a traditional medicinal plant frequently used to attenuate pain in Asia. As a natural derivative of analgesic, the current study was designed to test the potential analgesic activity of (-)-NRG-DM and its implicated mechanism. The analgesic activity of (-)-NRG-DM was assessed in a formalin-induced mouse inflammatory pain model and mustard oil-induced mouse colorectal pain model, in which the mice were intraperitoneally administrated with vehicle or (-)-NRG-DM (30 or 50 mg/kg) (n = 10 for each group). Our data showed that (-)-NRG-DM can dose dependently (30~50 mg/kg) relieve the pain behaviors. Notably, (-)-NRG-DM did not affect motor coordination in mice evaluated by the rotarod test, in which the animals were intraperitoneally injected with vehicle or (-)-NRG-DM (100, 200, or 400 mg/kg) (n = 10 for each group). In acutely isolated mouse dorsal root ganglion neurons, (-)-NRG-DM (1~30 μM) potently dampened the stimulated firing, reduced the action potential threshold and amplitude. In addition, the neuronal delayed rectifier potassium currents (I_K) and voltage-gated sodium currents (I_Na) were significantly suppressed. Consistently, (-)-NRG-DM dramatically inhibited heterologously expressed Kv2.1 and Nav1.8 channels which represent the major components of the endogenous I_K and I_Na. A pharmacokinetic study revealed the plasma concentration of (-)-NRG-DM is around 7 µM, which was higher than the effective concentrations for the I_K and I_Na. Taken together, our study showed that (-)-NRG-DM is a potential analgesic candidate with inhibition of multiple neuronal channels (mediating I_K and I_Na).

Keywords: (−)-Naringenin 4′,7-dimethyl ether; analgesic candidate; delayed rectifier potassium currents; ion channels; mechanism study.

Figure 1

The structure of (−)-naringenin 4′,7-dimethyl ether ((−)-NRG-DM).

Figure 2

Analgesic effects of (−)-NRG-DM in the formalin-induced mouse inflammatory pain model. (A) 30 mg/kg compound (−)-NRG-DM attenuated the biphasic pain responses, including both licking time (left) and score (right) throughout the 60 min trial. (B) 50 mg/kg (−)-NRG-DM attenuated the biphasic pain responses, including both licking time (left) and score (right) throughout the 60 min trial. Bar graph showing the effects of vehicle (white), 30 mg/kg and 50 mg/kg compound (−)-NRG-DM (grey) on the pain behaviors during phase I (C) and phase II (D) in the formalin-induced mouse inflammatory pain model. In all groups, n = 10 animals. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Analgesic effects of (−)-NRG-DM in the mustard oil-induced mouse colorectal pain model. (A) 30 mg/kg and 50 mg/kg (−)-NRG-DM attenuated the acute pain-related behaviors throughout the 30 min trial. (B) Bar graph showing the effects of vehicle (white), 30 mg/kg and 50 mg/kg (−)-NRG-DM (grey) on the writhing number caused by pain during a 30 min period in the mustard oil-induced mouse colorectal pain model. In all groups, n = 10 animals. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

(−)-NRG-DM inhibited the neuronal excitability of acutely isolated mouse dorsal root ganglion neurons. (A) Representative traces of action potentials following 200 pA current injection with or without (−)-NRG-DM at indicated concentrations in DRG neurons. Bar graph showing the effects of (−)-NRG-DM on the firing frequency (B), amplitudes of the first action potential (C) before (control, white) and after the application of (−)-NRG-DM (grey) at indicated concentrations (n ≥ 5). (D) Responses of representative DRG neurons with or without 30 µM (−)-NRG-DM to 500 ms depolarization current steps for the generation of all-or-none action potential. (E) The averaged number of action potentials of DRG neurons before and after application of 30 µM (−)-NRG-DM (n = 5). Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

Inhibitory effects of (−)-NRG-DM on the potassium currents of DRG neurons in mice. (A) Typical traces of the transient outward potassium currents I_A and the delayed rectifier potassium currents I_K in the presence of (−)-NRG-DM at indicated concentrations. (B) The dose–response curve of (−)-NRG-DM on I_K and I_A currents. The IC₅₀ values were 5.10 ± 0.04 μM and 119.90 ± 0.03 μM, respectively (n = 5). (C) Representative activation current traces of Kv channels before and after 10 μM (−)-NRG-DM. (D) Activation curves of Kv currents before and after 10 μM of (−)-NRG-DM (n = 6).

Figure 6

Characterization of (−)-NRG-DM inhibition on Nav currents of DRG neurons in mice. (A) Representative traces of Nav currents in the absence or presence of (−)-NRG-DM at indicated concentrations. (B) Current–voltage relationship of Nav currents with or without 30 μM (−)-NRG-DM (n = 8). (C) The dose–response curve of (−)-NRG-DM on native Nav currents. The IC₅₀ value was 34.78 ± 0.14 μM (n = 5). Typical activation current traces (D) and steady-state activation curves (E) of total Nav currents before and after application of 30 µM (−)-NRG-DM (n = 6). Representative inactivation traces (F) and steady-state inactivation curves (G) of total Nav currents before and after perfusion of 30 μM (−)-NRG-DM (n = 6). Typical activation current traces (H) and steady-state activation curves (J) of TTX-R currents before and after perfusion of 30 µM (−)-NRG-DM (n = 7). Representative inactivation traces (I) and steady-state inactivation curves (K) of TTX-R currents before and after perfusion of 30 μM (−)-NRG-DM (n = 7). Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 7

Inhibitory effects of (−)-NRG-DM on Kv2.1 channels heterologously expressed in CHO cells. (A) Representative Kv2.1 current traces in the absence and presence of 20 µM (−)-NRG-DM (Inset) The recording protocol. (B) Time course of peak and end-pulse currents of Kv2.1 channels before and after perfusion of 20 µM (−)-NRG-DM. (C) The dose–response curve of (−)-NRG-DM on Kv2.1 channel. The IC₅₀ value was 21.17 ± 0.11 µM (n = 5). (D) Representative activation current traces of Kv2.1 channels in absence and presence of 20 µM (−)-NRG-DM (Inset) The recording protocol. (E) Activation curves of Kv2.1 channels with or without 20 µM (−)-NRG-DM (n = 5). Statistical significance: * p ≤ 0.05.

Figure 8

Inhibitory effect of (−)-NRG-DM on Nav currents. (A) Representative Nav1.7 and Nav1.8 current traces in the absence and presence of 30 µM (−)-NRG-DM recorded with the depicted protocol. (B) Summarized data showing the suppression effect of (−)-NRG-DM (30 µM) on Nav1.7 and Nav1.8 channels (n = 5). (C) Representative Nav1.8 current traces in absence and presence of 30 µM (−)-NRG-DM recorded with the protocol shown inset. (D) Activation curves obtained in the absence and presence of 30 µM (−)-NRG-DM (n = 13). (E) Representative activation current traces of Nav1.8 currents before and after 30 μM (−)-NRG-DM. (F) Steady-state inactivation curve of Nav1.8 currents before and after 30 μM (−)-NRG-DM (n = 13). Statistical significance: *** p ≤ 0.001.