Antinociceptive Substances From Anoectochilus Elatus L. And Their Mechanisms

Pain is a feeling produced when the human body is subjected to a variety of harmful stimuli.It is a common symptom of many diseases and trauma.Military personnel are very vulnerable to various injuries such as weapon injuries and training injuries during combat missions or training exercises.Various kinds of acute and chronic pain have reduced the operational ability and quality of life of the military personnel,and seriously affected their normal training and combat ability.Therefore,development of analgesic drugs with high efficacy but little side effects is of great significance for the military personnel to relieve pain,promote the rehabilitation of the wounded and improve the operation ability of troops in peacetime and war time.Anoectochilus roxburghii is an invaluable Chinese herbal medicine featured in Fujian and Taiwan.It belongs to the family Orchidaceae and the genus Anoectochilus Blume.The whole herb is collected for both medicinal and edible use,which has the functions of clearing heat and cooling blood,dispelling wind and dampness,and detoxifying.Due to the exhaustive exploitation,the wild resources of A.roxburghii have decreased dramatically,so it is of practical significance to search for alternative sources from the same genus.In 2018,our group found a special plant of the genus Anoectochilus during the resource investigation in Yunnan,which was identified as Anoectochilus elatus L.and was a new recorded species in China.Its tissue culture has been successfully achieved in our laboratory.Through literature review to explore its traditional medicinal value,we found that it is commonly used in the folk medicine for the treatment of thoracic and abdominal pain in other Asian countries,but there has been no literature report on its pharmacological activity and potential active ingredients.Therefore,this project intends to use modern pharmacological and chemical techniques to systematically investigate the analgesic bioactivities and chemical constituents of A.elatus,in order to elucidate the material basis and mechanisms of its antinociceptive activity,and provide scientific basis for the traditional application and rational utilization of A.elatus.Using the plant samples derived from tissue cultures of A.elatus as our research material,the antinociceptive activity of crude extracts(ethanol extract EE and aqueous extract AE)from A.elatus was investigated by adopting different animal models of pain,including acetic acid-induced writhing,tail flick and formalin-induced foot licking in mice.Further combined with activity-guided isolation,the extracts were separated and purified by water extraction and ethanol precipitation,gel chromatography,preparative high performance liquid chromatography(HPLC),and the main components were structurally characterized by nuclear magnetic NMR,methylation,monosaccharide composition analysis and other spectroscopic and chromatographic methods.The above-mentioned animal models of pain were again used to evaluate the analgesic activities of isolated monomers and polysaccharides,and the possible analgesic mechanism of the main active components was preliminarily explored by using different types of antagonists in the animal models.Finally,western blot,patch clamp and gut microbiome were used to elucidate the analgesic mechanism of the active ingredients.The main research results are as follows:(1)The ethanol extract(EE)was obtained from the planting samples derived from tissue cultures of A.elatus by 95% ethanol extraction,and aqueous extract(AE)was obtained from the ethanol residue after drying by hydrothermal extraction.The analgesic activities of different extracts were evaluated by acetic-induced writhing,tail flick and formalin-induced foot licking in mice.The results showed that EE and AE could significantly inhibit the aceticinduced writhing times,prolong the tail flick tolerance time,and reduce formalin-induced foot licking time in late phase in mice,which revealed that these extracts from A.elatus had good analgesic activity.(2)The chemical composition of EE was analyzed by high performance liquid chromatography.A main peak was found in the chromatogram,the monomer compound corresponding to which were then purified and obtained by preparative HPLC.Based on NMR and mass spectrometry analysis,the major constituent in EE was confirmed to be goodyeroside A(GA),and the content of GA was up to 60% in EE.After further ethanol precipitation,AE was divided into supernatant and precipitated polysaccharide.The precipitated polysaccharide from AE could significantly inhibit the number of writhing induced by acetic acid in mice,while the supernatant had no analgesic activity,suggesting that the analgesic activity of the AE was mainly derived from the crude polysaccharide.The crude polysaccharide was further separated and purified on DEAE-52 anion exchange column and Sephacryl S-100 gel column chromatography.Combined with bioactivityguided isolation,a homogeneous polysaccharide GJXL-1 with molecular weight of10.28 k Da was obtained.The monosaccharide composition analysis showed that GJXL-1only contained glucose,indicating that GJXL-1 was glucan.Further methylation and 1D &2D NMR analysis indicated that GJXL-1 contains four types of residues: terminal Glcp-(1→,→4)-glcp-(1→,→3,4)-glcp-(1→,and →4,6)-glcp-(1→.The molar ratio was 0.095: 0.800:0.026: 0.079,respectively,with →4)-glcp-(1)as the main chain and a few branch chains at the O-3 and O-6 positions of the main chain glucose,and residues of 1,3,4-α-D-glcp were inα-D-(1→4)-glucan.(3)Further in vivo efficacy evaluation was conducted on GA from EE and GJXL-1from AE.The effects of GA and GJXL-1 on autonomic activity and nervous system were also investigated by open field test and sodium pentobarbitone-induced sleeping test.The results showed that both of GA and GJXL-1 displayed good analgesic activities.They could significantly inhibit the acetic-induced writhing times,prolong the tail flick tolerance time,and reduce the foot licking time in both early and late phase in mice,and did not prolong the sleep time induced by pentobarbital sodium and affect the locomotor activity in mice.In antagonistic experiments,it was found that the analgesic effect of GA and GJXL-1 could be partially antagonized by L-NAME,which suggested that the analgesic activity of GA and GJXL-1 was related to the regulation on NO system.In addition,GA could also effectively inhibit the chronic neuralgia simulated in rat in CCI(chronic constriction injury of the sciatic nerve)model,significantly increasing the threshold of mechanical pain in the hind limbs.(4)Mechanism studies revealed that GA significantly inhibited the phosphorylation of p38 and JNK of the MAPK signaling pathway in Bv-2 cells,but had no effect on the phosphorylation of ERK.Further studies showed that GA inhibited the upstream phosphorylated expression of IRAK1,IRAK4 and TAK1 proteins.GA had no significant effect on the current of Nav 1.1-1.8 sodium ion channels in vitro,and preliminary pharmacokinetic experiments showed that the prototype of GA could not be detected in the blood sample collected half an hour after oral administration,suggesting that GA may not exert analgesic effects by its prototype and its mechanism needs to be further clarified.GJXL-1 significantly inhibited the activation of NF-κB signaling pathway and reduced the phosphorylation levels of IRAK4,TAK1 and p65 in LPS-stimulated RAW264.7 cells,but had no effect on IRAK1 expression.In addition,GJXL-1 can also significantly increase the contents of short-chain fatty acids(SCFA)in the intestinal flora.Further sequencing and analysis of the fecal microbiome of mice showed that the microbial community composition in GJXL-1-treated animals was quite different from the control group.GJXL-1administration reduced the percentage of Desulfobacterota in the intestinal flora.In summary,this study systematically analyzed the antinociceptive material basis of A.elatus for the first time.It was found that the main component in EE was goodyeroside A,GA,and the main component in AE was homogeneous polysaccharide GJXL-1,which was a novel α-D-(1→4)-glucan containing 1,3,4-α-D-glcp residues.GA and GJXL-1 had good analgesic activity,which were effective for pains caused by different chemical and thermal stimuli.The analgesic effects of these two active ingredients were not related to the hypnosis or destruction of motor nerve in mice.In addition,GA could relieve neuropathic pain induced by chronic constrictive injury(CCI)in rats.Studies on the mechanism of action showed that the analgesic mechanism of GA was related to the intervention of IRAK1&4/TAK1/MAPK signaling pathway,while the analgesic mechanism of GJXL-1 is related to the inhibition of IRAK4/TAK1/NF-κB signaling pathway and the regulation of intestinal flora.These results indicated that GA and GJXL-1 are safe,effective and promising analgesic candidates for various types of pain,and GJXL-1 can also be used as a preferred prebiotic for maintaining intestinal homeostasis and regulating intestinal health.