Application of Licoisoflavone A

Abstract

The invention provides an application of Licoisoflavone A in preparation of a reagent for passivating pain perception of human or animals, and relates to the technical field of biomedicine. Experiments on model animal mice show that intraperitoneal injection of LFA can passivate the perception ability of mice on pain, including acute nociceptive pain, chronic inflammatory pain and neuropathic pain. Therefore, the LFA can enhance the resistance of mammals such as human beings or mice to the pain, and can be developed into analgesic to be applied to the human beings and other mammals to relieve the pain. Meanwhile, experiments show that the Licoisoflavone A acts on a receptor Nav1.8 in a targeting manner, and the Licoisoflavone A can be used for remarkably inhibiting activation of a Nav1.8 voltage-gated ion channel of a mammal, so that the Licoisoflavone A can be applied to research on Nav1.8 related ion channel diseases and can be used as a potential therapeutic drug.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of Licoisoflavone A. Background Art

[0002] According to statistics from the World Health Organization, about 20% of the world's population is experiencing moderate to severe chronic pain. This phenomenon has driven the rapid development of the analgesic drug market. The number of chronic pain patients in China has exceeded 300 million, with 10 million to 20 million new patients each year. The aging population is further driving the demand for analgesics. It is estimated that by 2025, the scale of China's analgesic market will exceed 20 billion yuan, with an annual compound growth rate of more than 10%. Pain is a complex subjective experience that includes not only unpleasant feelings but also emotional feelings. Studies have shown that pain is usually associated with existing or potential tissue damage. Although it is a protective warning mechanism of the human body, persistent or severe pain can disrupt normal physiological functions and seriously affect the patient's quality of life. From a physiological point of view, pain signals originate from nociceptors in the peripheral nervous system. These special receptors are distributed in the skin, muscles, joints and visceral tissues, and can convert thermal, mechanical or chemical stimuli into nerve impulses. Through afferent nerve fibers, these signals are first transmitted to the neuronal cell bodies of the dorsal root ganglion (DRG), and then uploaded to the higher centers of the brain, ultimately forming pain. The key link in this process is the generation of neuronal action potentials, which mainly depends on the activation of voltage-gated sodium ion channels (NaV) on the cell membrane. When the cell membrane is depolarized, the sodium ion channels open and a large amount of sodium ions flow in, triggering action potentials. Therefore, regulating abnormal sodium ion channel activity has become an important strategy for pain treatment.

[0003] The human voltage-gated sodium channel is a transmembrane protein complex composed of an α subunit with a molecular weight of about 260 kD and a β subunit with a molecular weight of 30-40 kD. According to the different characteristics of the α subunit, 9 subtypes (Nav1.1-Nav1.9) have been identified. Among them, Nav1.5, Nav1.8 and Nav1.9 are resistant to tetrodotoxin (TTX). Especially in the field of pain, Nav1.8 has attracted much attention due to its specific distribution in the peripheral nervous system. This subtype not only plays an important role in diseases such as chronic pain and atrial fibrillation, but is also considered to be an ideal target for the development of highly selective analgesics. Nav1.8 is encoded by the SCN10A gene located in the human chromosome 3p21-22 region and is mainly expressed in neurons of the trigeminal ganglion and dorsal root ganglion. Studies have shown that this channel has unique electrophysiological characteristics of slow inactivation and rapid recovery. In the neuropathic pain model, nerve injury leads to a significant upregulation of Nav1.8 expression in axons and neuronal cell bodies. Experimental data confirmed that reducing Nav1.8 expression through antisense oligonucleotide technology can effectively relieve pain symptoms. In addition, Nav1.8 gene knockout mice showed obvious loss of visceral inflammatory pain, while gain-of-function mutations in the human Nav1.8 gene were closely related to peripheral neuropathy.

[0004] Licoisoflavone A (LFA), also known as Licorice Isoflavone A or Licorice Isoflavone A, has a CAS number of 66056-19-7 and a molecular formula of C 20 H 18 O6, chemical name is 4H-1-Benzopyran-4-one,3-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-5,7-dihydroxy, molecular weight 354.35. LicoisoflavoneA is a flavonoid compound with a typical isoflavone skeleton mainly derived from Glycyrrhiza glabra, Glycyrrhiza inflata, and Glycyrrhiza uralensis. Existing studies have shown that LicoisoflavoneA has antiviral effects. Bioactive compounds with antiviral effects were screened from Q-14. LicoisoflavoneA has significant antiviral activity, with an inhibition rate of >90% at a concentration of 100μM. Studies have found that licoisoflavoneA in Tongmai Yangxin Pills (TMYX) can effectively reduce myocardial hypertrophy by upregulating the expression of Sirt 3. However, there is currently no research on the relationship between LicoisoflavoneA and pain perception. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide the use of LicoisoflavoneA in the preparation of an agent for passivating the perception of pain in humans or animals. The LicoisoflavoneA can significantly inhibit the activation of the Nav1.8 voltage-gated ion channel of mammals, and can be used as an inhibitor of Nav1.8 and for the treatment of diseases related to the Nav1.8 channel; and can passivate the induction of external pain stimuli within a certain period of time, and can be developed into an analgesic for use in humans or other mammals to relieve pain.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention protects the use of Licoisoflavone A in the preparation of an agent for blunting the perception of pain in humans or animals. The structural formula of the Licoisoflavone A is shown in Formula I:

[0008]

[0009] Furthermore, the pain includes acute nociceptive pain, chronic inflammatory pain and neuropathic pain.

[0010] The present invention also protects the use of LicoisoflavoneA in preparing Nav1.8 inhibitors.

[0011] Furthermore, the Nav1.8 inhibitor is used in the preparation of drugs for treating Nav1.8 ion channel related diseases.

[0012] The present invention also provides an analgesic, wherein the active ingredient of the analgesic comprises LicoisoflavoneA.

[0013] Compared with the prior art, the present invention has the following technical effects:

[0014] The present invention proves for the first time that LicoisoflavoneA can significantly inhibit the activation of Nav1.8 (Voltage-Gated Sodium Channel Subtype 1.8) voltage-gated sodium ion channels in mammals such as humans or mice. Electrophysiological experiments show that LicoisoflavoneA can act on Nav1.8 receptors of other species including humans. Nav1.8 is mainly expressed in mammalian sensory neurons and participates in the transmission of pain signals. The receptors closely related to pain are inhibited, which will blunt the induction of pain for a certain period of time. Experiments on model animals such as mice showed that intraperitoneal injection of LicoisoflavoneA can enhance the tolerance of mice to acute noxious pain induced by acute heat stimulation in a dose-dependent manner, and increase the pain threshold of mice to acute noxious pain; it can also enhance the tolerance to simulated visceral pain induced by glacial acetic acid and inhibit the writhing behavior of mice in a dose-dependent manner, significantly enhancing the resistance of mice to visceral inflammatory pain; intraperitoneal injection of LicoisoflavoneA can dose-dependently inhibit chronic inflammatory and neuropathic hyperalgesia induced by complete Freund's adjuvant and paclitaxel, and significantly increase the pain threshold of mice quantified by mechanical force and thermal stimulation, relieve the pain sensitization induced by complete Freund's adjuvant and paclitaxel in mice, and blunt the pain perception of mice.

[0015] In summary, Licoisoflavone A enhances the resistance of mammals such as mice to pain and can be developed into an analgesic for use in humans or other mammals to relieve pain. Secondly, Licoisoflavone A targets the receptor Nav1.8 and can be used in the study of Nav1.8-related ion channel diseases and as a potential therapeutic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of Licoisoflavone A on hNav1.8 channel, where A and B are the voltage-current curves of Licoisoflavone A on hNav1.8; C represents the dose-effect curve of Licoisoflavone A on hNav1.8 fitted by Hill equation (n=6);

[0017] Figure 2 The simulated structure of Nav1.8 and the action site of LicoisoflavoneA, where A represents the side view of multiple LicoisoflavoneAs docking at Nav1.8 channels; B represents the enlarged view of one LicoisoflavoneA binding to a Nav1.8 subunit, with Nav1.8 marked in green, the side chain of the Leu1323 residue marked in blue, and LicoisoflavoneA marked in red;

[0018] Figure 3 The analgesic effect of Licoisoflavone A on the visceral pain model simulated by acetic acid and the acute heat injury pain model in mice, wherein A and B are the analgesic effects of Licoisoflavone A on the visceral pain model simulated by acetic acid and the acute heat injury pain model in mice, respectively (n=6);

[0019] Figure 4 The analgesic effect of LicoisoflavoneA on the chronic inflammatory pain model induced by complete Freund's adjuvant (CFA) in model mice, wherein A represents the quantification of pain threshold using the hot plate test within 24 hours after intraperitoneal injection of Licoisoflavone A; B represents the quantification of pain threshold using the Von frey tactile measurement kit within 24 hours after intraperitoneal injection of Licoisoflavone A (n=6);

[0020] Figure 5 The analgesic effect of Licoisoflavone A on the neuropathic pain model induced by paclitaxel (PTX) in mice, wherein A represents the quantification of pain threshold using the hot plate test within 24 hours after intraperitoneal injection of Licoisoflavone A; B represents the quantification of pain threshold using the Von frey tactile measurement kit within 24 hours after intraperitoneal injection of Licoisoflavone A (n=6);

[0021] Figure 6 The effect of Licoisoflavone A on the safety evaluation of mice. A represents the effect of Licoisoflavone A on the viability of ND7 / 23 cells in vitro (n=4); B represents the effect of Licoisoflavone A on the movement and exploration ability of mice after 30 minutes of intraperitoneal pre-administration (n=6); C represents the effect of Licoisoflavone A on the balance ability of mice after 30 minutes of intraperitoneal pre-administration (n=6). DETAILED DESCRIPTION

[0022] The present invention provides the use of Licoisoflavone A in preparing an agent for blunting the perception of pain in humans or animals. The structural formula of the Licoisoflavone A is shown in Formula I:

[0023]

[0024] Licoisoflavone A of the present invention is preferably a flavonoid compound derived from Glycyrrhiza uralensis, a plant of the genus Glycyrrhiza. Preferably, the flavonoid compound is extracted by an ethanol extraction method or the like: tubers of Glycyrrhiza uralensis are identified and collected from Panlong District, Kunming City, the tubers are air-dried and crushed into powder, then extracted with ethanol, and the solvent is evaporated by vacuum freeze drying to dry the extract.

[0025] The Licoisoflavone A of the present invention can significantly inhibit the activation of voltage-gated ion channels of Nav1.8 (Voltage-Gated Sodium Channel Subtype 1.8) in different mammals, and Nav1.8 is inhibited as a receptor closely related to diseases such as chronic pain in humans or other mammals, which will blunt the induction of pain within a certain period of time, thereby affecting the perception of pain in humans and animals. Specifically, a mouse model experiment is provided in the embodiment of the present invention. Whether it is acute nociceptive pain, chronic inflammatory pain, or neuropathic pain, after intraperitoneal injection of the Licoisoflavone A, its resistance to pain can be improved, so it can be prepared into an agent that affects or improves the perception of pain in humans and animals.

[0026] LicoisoflavoneA described in the present invention can specifically target the receptor Nav1.8, which is a type of voltage-gated sodium ion channel, which is expressed in large quantities in sensory neuron cells of humans or other mammals, especially in dorsal root ganglia, trigeminal ganglia and peripheral sensory neurons. It is precisely because Nav1.8 plays a key role in the generation and conduction of action potentials that Nav1.8 is found to be closely related to acute nociceptive pain, chronic inflammatory pain and neuropathic pain. The present invention verifies the pain relief effect of LicoisoflavoneA on mice through experiments on acute nociceptive pain, chronic inflammatory pain models and neuropathic pain models induced by heat in mice. Patch clamp technology is used to verify that Licoisoflavone A inhibits the activation of hNav1.8, confirming the universality of LicoisoflavoneA in different mammals such as mice and humans.

[0027] The present invention provides an analgesic, wherein the active ingredient of the analgesic comprises LicoisoflavoneA.

[0028] The analgesic of the present invention is preferably applied to humans and animals by injection or the like to relieve pain, overcome chronic pain in the short term, etc.

[0029] The invention provides application of LicoisoflavoneA in preparing a reagent for inhibiting Nav1.8 ion channel.

[0030] In the present invention, the Licoisoflavone A can specifically target the receptor Nav1.8, which is a type of voltage-gated sodium ion channel and is abundantly expressed in sensory neuron cells of humans or other mammals, especially in dorsal root ganglia, trigeminal ganglia and peripheral sensory neurons. It is precisely because Nav1.8 plays a key role in the generation and conduction of action potentials that Nav1.8 is found to be closely related to chronic pain and neuropathic pain. Therefore, the Licoisoflavone A can be used to prepare an agent for inhibiting the Nav1.8 ion channel.

[0031] The invention provides an inhibitor of Nav1.8 ion channel, wherein the effective component of the inhibitor comprises LicoisoflavoneA.

[0032] The present invention overexpresses human Nav1.8 receptor in ND7 / 23 cell line, and finds through patch clamp technology that the target of LicoisoflavoneA is Nav1.8 receptor, and can inhibit the activation of Nav1.8 receptor; the molecular structure of human Nav1.8 is simulated by AutoDockTools molecular modeling software, and it is found that LicoisoflavoneA binds to Leu1323 site, so the compound can inhibit Nav1.8 channel.

[0033] The present invention provides the use of the above inhibitor in the preparation of a drug for treating Nav1.8 ion channel related diseases. The Nav1.8 ion channel related diseases of the present invention preferably include thermal acute nociceptive pain, chronic inflammatory pain or neuropathic pain.

[0034] The present invention provides a medicine for treating diseases related to Nav1.8 ion channel, wherein the effective ingredients of the medicine include the above inhibitor.

[0035] The application of Licoisoflavone A provided by the present invention in the preparation of agents that affect the pain perception of humans and animals is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 Electrophysiological experiment of targeting effect of LicoisoflavoneA on hNav1.8 pain-related receptors

[0037] Human Nav1.8 and other plasmids were transiently overexpressed in ND7 / 23 cell lines, and the above plasmids were synthesized by Shanghai Sangon Biotechnology Co., Ltd. All ND7 / 23 cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2.

[0038] Under an inverted microscope, cells with smooth cell membranes and uniform cytoplasm were selected for patch clamp experiments at room temperature of 20-25°C. WPI 0.86mm thin-walled borosilicate glass capillaries were selected as glass electrode materials. The glass electrodes were drawn on a drawing instrument (P-97, Shutter) in 5 steps. The diameter of the tip of the glass electrode was 1.5-3.0μm after thermal polishing. After drawing, the glass electrode was filled with intracellular fluid. The initial resistance of the glass electrode was 1.5-2.5MΩ. After a high-impedance GΩ seal was formed between the electrode and the cell membrane, the fast capacitance of the electrode was compensated and the cell was clamped to -80mV. Then a short and strong negative pressure was applied to quickly break the cell membrane clamped in the electrode, and then the slow capacitance of the cell was compensated. The recording method was clamping voltage -100mV, 50ms, test voltage 0mV, 100ms, and clamping voltage -100mV. . The drugs were perfused using Biolab RS200, and the switching speed between drugs was 50ms. The series resistance (Rs) was always kept within the range of 5-8MΩ during the experiment, and the system series resistance compensation was generally between 30% and 60%. The experimental data were analyzed using PatchMaster software, and the data were further analyzed using Igor software. n represents the number of experimental data.

[0039] The cells are slowly lifted up to the bottom edge of the drug-adding port of the rapid drug-adding switching system (RSC-200) by the electric micromanipulation system. The drug-adding switching system is directly connected to the amplifier, and all experimental data are recorded in the computer software PatchMaster through the amplifier. Cells are stimulated and treated with drugs through Patch Master and the switching drug-adding system, and the changes in channel current are recorded to detect the activity of different drugs on the channel. To ensure the accuracy of the experimental data, the sealing resistance and series resistance must be kept stable throughout the recording process.

[0040] Sodium ion channel internal solution: 5mM NaCl, 135mM CsF, 2mM MgCl2, 10mM EGTA, 2mM Na-ATP, 10mM HEPES, and the pH was adjusted to 7.4 with concentrated CsOH solution.

[0041] Sodium ion channel external solution: 140mM NaCl, 4mM KCl, 10mM D-glucose, 2mMMgCl2, 1.5mM CaCl, 10mM HEPES, pH adjusted to 7.4 with concentrated NaOH solution.

[0042] The results are as follows Figure 1 A. Figure 1 B. Figure 1 As shown in C, Licoisoflavone A inhibits the activation of Nav1.8 channels in a concentration-dependent manner.

[0043] Example 2 Structural simulation experiment of hNav1.8

[0044] hNav1.8 structural simulation

[0045] from https: / / www.uniprot.org / After downloading Nav1.8 (SCN10A), the macromolecular protein 3D structure was downloaded from https: / / www.rcsb.org / (PDB). The Nav1.8 channel and Licoisoflavone A were molecularly docked using AutoDockTools-1.5.6 software. 10,000 models were generated in each round. Among these models, the top 10 models with the lowest energy were selected as input for the next round of closed-loop modeling. After several rounds of KIC loop modeling, the top ten models merged well. Finally, the lowest energy model was selected for three-dimensional visualization analysis using Pymol software. The lowest energy model shows that there is a stable interaction between Licoisoflavone A and the Nav1.8 channel, which provides a structural basis for further studying the functional effects of Licoisoflavone A on the Nav1.8 channel ( Figure 2 ).

[0046] Example 3 Analgesic Activity of Licoisoflavone A

[0047] 1. LicoisoflavoneA helps mice resist pain

[0048] Experiment on visceral pain model induced by glacial acetic acid in mice: 30 adult male C57BL / 6J mice (20-23g), 18 of which were intraperitoneally injected with different concentrations of LicoisoflavoneA (0.5, 1, 5 mg / kg), the control group (6 mice) were intraperitoneally injected with an equal volume of vehicle solution (0.625% DMSO, 5.625% castor oil, 93.75% saline, the same below), and the positive control group (6 mice) were intraperitoneally injected with 5 mg / kg of morphine. The pain threshold of the mice was expressed and quantified by counting the number of mouse twisting times.

[0049] As expected, normal mice injected with the vehicle solution had a lower pain threshold, and the number of positive reactions (writhing) was significantly higher than that of the experimental group injected with Licoisoflavone A. The pain threshold of mice treated with Licoisoflavone A increased in a dose-dependent manner, and the positive reactions decreased. Compared with the vehicle group, the analgesic rate of the 5mg / kg LicoisoflavoneA group injected with the vehicle solution reached 83.84%, which was comparable to the effect of the 5mg / kg morphine group ( Figure 3 A) It proves that intraperitoneal injection of LicoisoflavoneA can successfully inhibit the visceral pain induced by glacial acetic acid in mice and help mice resist inflammatory visceral pain.

[0050] Hot water tail immersion test: 30 adult male C57BL / 6J mice (20-23g) were randomly divided into groups based on the basal pain threshold. The control group (6 mice) were intraperitoneally injected with vehicle solution, the positive control group (6 mice) were intraperitoneally injected with 5 mg / kg of morphine, and the test group (18 mice) were intraperitoneally injected with solutions containing different concentrations of Licoisoflavone A (0.5, 1, 5 mg / kg). Half an hour after intraperitoneal administration of the drug, the tail immersion test was performed (the water temperature was kept constant at 53±0.5℃). The tail tip of the mouse was immersed in hot water and the timing was started until a positive reaction (quickly shaking the tail and popping out of the water) appeared, and the pain threshold was quantified in the form of time numbers.

[0051] The results showed that the pain threshold of mice in each group injected with Licoisoflavone A was increased and dose-dependent. The analgesic rate of acute heat pain in mice injected with 5 mg / kg Licoisoflavone A reached 70.93%. The pain threshold of mice in the vehicle group injected with intraperitoneal injection was consistent with the basal pain threshold. Compared with the mice in the positive control group given Licoisoflavone A and morphine, the time of positive reaction was shorter, indicating a lower pain threshold. This proves that intraperitoneal injection of Licoisoflavone A can successfully inhibit the heat pain simulated by acute heat stimulation in mice and help mice resist acute heat injury pain ( Figure 3 B).

[0052] CFA-induced chronic inflammatory pain model experiment: 30 adult male C57BL / 6J mice (20-23g) were injected with 20μL CFA on the plantar surface of the right foot of the mice to create a chronic inflammatory pain model. In the test phase, the control group (6 mice) were intraperitoneally injected with vehicle solution, the positive control group (6 mice) were intraperitoneally injected with 5mg / kg of morphine, and the test group (18 mice) were intraperitoneally injected with different concentrations of LicoisoflavoneA solution (0.5, 1, 5mg / kg). Half an hour after intraperitoneal administration of the drug, the hot plate test and Von frey test were performed to quantify the pain threshold.

[0053] The results showed that the pain thresholds of mice in each group injected with Licoisoflavone A were significantly increased by the hot plate test and the Vonfrey test, and showed a certain dose-dependency. The pain thresholds of mice in each group injected with Licoisoflavone A were significantly increased compared with the pain hypersensitivity state of the control group within 0-4 hours, and reached a peak value half an hour to two hours after the administration of Licoisoflavone A. Half an hour after the administration of Licoisoflavone A, the pain inhibition rates of the 5 mg / kg Licoisoflavone A group mice caused by chronic inflammation evaluated by the hot plate test and the Von frey test reached 96.26% and 92.85%, respectively, while the mice in the vehicle group showed a state of pain hypersensitivity induced by CFA, which proved that the chronic inflammation simulated by CFA induced a decrease in the pain threshold of mice ( Figure 4 A. Figure 4 B).

[0054] Paclitaxel-induced simulated neuropathic pain model experiment: 30 adult male C57BL / 6J mice (20-23g) were modeled by low-dose multiple administration. Paclitaxel was intraperitoneally injected into the mice four times with an interval of one day each time to create a paclitaxel-induced neuropathic pain model. After the modeling, the experiment was carried out, in which the control group (6 mice) was intraperitoneally injected with a vehicle solution, the positive control group (6 mice) was intraperitoneally injected with 5 mg / kg of morphine, and the test group (18 mice) was intraperitoneally injected with different concentrations of LicoisoflavoneA solution (0.5, 1, 5 mg / kg). Half an hour after intraperitoneal administration of the drug, the hot plate test and Vonfrey test were performed to quantify the pain threshold.

[0055] The results showed that the pain threshold of mice in each group injected with Licoisoflavone A was increased and showed a certain dose-dependency. The pain threshold of mice in each group injected with Licoisoflavone A was significantly increased in 0-4h compared with the pain hypersensitivity state of the control group, and the pain threshold reached a peak half an hour to two hours after the administration of Licoisoflavone A. Half an hour after the administration of Licoisoflavone A, the pain inhibition rate of 5mg / kg Licoisoflavone A group mice caused by neuropathological lesions evaluated by hot plate test and Vonfrey test reached 68.86% and 60.00%, while the control group mice showed a state of hyperalgesia induced by paclitaxel, paclitaxel-induced activation of glial cells to release inflammatory factors, increase neuronal excitability, and degeneration of sensory nerve fibers, resulting in a state of pain sensitization ( Figure 5 A. Figure 5 B).

[0056] Example 4 Safety Evaluation of Licoisoflavone A

[0057] Cell viability detection (CCK-8 method): CCK-8 method was used for 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt, which is a compound similar to MTT. In the presence of electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-MethoxyPMS), it is reduced by dehydrogenase in mitochondria to a highly water-soluble orange-yellow (measured at A450) formazan product. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is proportional to the number of living cells. Therefore, this characteristic is used to directly analyze cell proliferation and toxicity. The ND7 / 23 cell line was selected and the IC value of Licoisoflavone A was used. 50 Based on the value, four experimental gradients of 0, 1, 10, 50, and 100 μm were set.

[0058] The results showed that Licoisoflavone A met the safety requirements in the dose range of 0.5-5 mg / kg in model mice ( Figure 6 A).

[0059] Open field experiment: The open field experiment was conducted to evaluate the effects of LFA on the exploratory behavior and motor ability of mice. Before the formal experiment, each mouse was tested to determine the baseline and randomly divided into groups. The mice were placed in the test room 60 minutes before the start of the experiment to adapt to the test room. The mice were intraperitoneally injected with 100 μL LicoisoflavoneA (0.5, 1 and 5 mg / kg), and the negative control group was given an equal volume of vehicle solution. After administering the corresponding drugs to each group of mice, each mouse was placed in the center of an empty field (40×40×40 cm) and allowed to explore freely for 60 minutes. The effect of LicoisoflavoneA on the exploration and motor ability of mice was evaluated by analyzing the total movement distance of the mice.

[0060] The results showed that the control group mice (6 mice) were intraperitoneally injected with an equal volume of carrier solution, and the experimental group mice (18 mice) showed that within the dose range of 0.5-5 mg / kg, Licoisoflavone A had no significant effect on the exploration ability and motor ability of mice, which was in line with the safety evaluation ( Figure 6 B).

[0061] Rotarod test: A rotarod apparatus (SA102 rotarod fatigue instrument, Saiangsi Biotechnology Co., Ltd., Jiangsu) was used to evaluate the motor coordination of mice after administration (intraperitoneal injection) of different concentrations of Licoisoflavone A. Before the drug administration test, all mice received two days of training, once a day (5rpm). The test process used a rotation speed of 24rpm, and the device automatically recorded the total delay of the mouse on each analysis test rod on the rod to evaluate the effect of LicoisoflavoneA on the motor coordination of mice.

[0062] The results showed that the control group mice (6 mice) were intraperitoneally injected with an equal volume of carrier solution, and the experimental group mice (18 mice) showed that within the dose range of 0.5-5 mg / kg, Licoisoflavone A had no significant effect on the motor coordination of mice, which was in line with the safety evaluation ( Figure 6 C).

[0063] In summary, the relevant behavioral experiments in mice have shown that by intraperitoneally injecting physiological concentrations of Licoisoflavone A into animals, animals can relieve pain. Licoisoflavone A enhances the resistance of mammals such as mice to pain and can be developed as a potential analgesic or pain prognosis aid for use in humans and other animals to relieve pain. Secondly, Licoisoflavone A targets the receptor Nav1.8 and can be used in the study of related ion channel diseases and as a potential therapeutic drug.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Licoisoflavone A in the preparation of an agent for blunting the perception of pain in humans or animals, characterized in that: The structural formula of Licoisoflavone A is shown in Formula I:

2. The use according to claim 1, characterized in that: The pain includes acute nociceptive pain, chronic inflammatory pain and neuropathic pain.

3. Application of Licoisoflavone A in the preparation of Nav1.8 inhibitors.

4. The use according to claim 3, wherein the Nav1.8 inhibitor is used in the preparation of a drug for treating Nav1.8 ion channel-related diseases.

5. A painkiller, characterized in that: The active ingredient of the analgesic includes Licoisoflavone A.

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