Investigation On The Effect Of Synthetic Coptisine Free Base On Anti-inflammation And Anti-gastric Ulcer

Objects:Inflammation is a common and important pathological process.When the body is infected or injured,it can cause the defense response against the damage factor induced by immune system.Moderate inflammatory responses can protect the body from invasive factors,but excessive inflammation can cause redness,swelling,heat,pain,and dysfunction.In order to reduce the impact of inflammation on the body,nonsteroidal anti-inflammatory drugs are used to treat this disease,but the frequent use of these drugs can lead to the occurrence of gastrointestinal ulcer.Therefore,it is of great clinical significance to reduce the side effects of drugs and prevent the ulcer from being produced.As for the treatment of gastric ulcer,proton pump inhibitors,such as omeprazole and lansoprazole,are commonly used to attenuate the effect,but these also bring obvious side effects.In addition,more than half of the world's population get infected with Helicobacter pylori?H.pylori?,the occurrence of gastric ulcer often accompanies with the appearance of H.pylori.Besides,H.pylori urease is an important colonization and pathogenic factor of H.pylori.Increasing intentions have been paid to find new therapeutic drugs that can exert anti-inflammatory effect as well as inhibit the growth of H.pylori and allevite side the effect of nonsteroidal anti-inflammatory drugs.Coptisine is one of the main alkaloids in Coptis chinensis,it has been reported that coptisine had anti-inflammatory and gastric ulcer effects.Its free bases?8-hydroxy-7,8-dihydrocoptisine,CFB?can coexist with coptisine and they can convert into each other to maintain the balance in vivo.Because of the lipophilic of CFB,it could get into the cell membrane easier than coptisine.Therefore,this study is aim to synthesis the CFB and to investigate the mechanism of CFB by three anti-inflammatory models,a model of gastric ulcer induced by indomethacin and the inhibition of H.pylori urease activity as well as enriching the medicinal substance base of Coptis chinensis.Methods:1.Synthesis and acute toxicity of CFBThis study used 2,3-benzaldehyde as raw material,after the reflux and stir,the mixture was evaporated and got the oil-like liquid 1.The hydrogenation was carry out by using the sodium borohydride,the oil-like liquid 2 was obtainedafter the evaporation.The acid and the inorganic salt were utilized to carry on the closed loop of the compound,CFB was obtained the in the weak alkali water under 80?.In addition,the maximum tolerance of mice was determined by using 1,10,100,400,1000 mg/kg in CFB mice.2.The effect of CFB on anti-inflammationThe model of mice ear swelling induced by xylene,the capillary permeability model induced by acetic acid and paw edema induced by carrageenan were used to evaluate the macroscopic anti-inflammatory effect of CFB.The content of IL-1?,IL-6,PGE2 and TNF-?were also measured by ELISA.The mRNA expression of IL-1?and TNF-?were determined by fluorescence quantitative PCR,and the effect of CFB on NF-?B and MAPK pathway was also included to explore the anti-inflammatory mechanism.3.The effects of CFB on gastric ulcerDue to the side effects caused by NSAIDs,Effects of CFB on gastric ulcer was also investigated in this study.The rat gastric ulcer model induced by indomethacin was used in this research,and the ulcer area was calculated by Image J.The macroscopic ulcer was evaluated by HE staining,and the contents of various factors in gastric tissue were measured?SOD,GSH,MDA,IL-1?,TNF-?,MPO,Ang II and PGE₂?;The expression of cyclooxygenase?COX-1 and COX-2?were determined by fluorescence quantitative PCR,and the effect of CFB on Nrf2 and p38 MAPK pathway were also utilized to detect the mechanism how CFB retard the level of gastric ulcer.Besides,different concentrations of CFB were used to investigate for the IC50 of H.pylori urease.Results:1.Synthesis of CFBThe purity of the compound was greater than 98%,and compared with NMR,the compound was 8-hydroxy-7,8-dihydrocoptisine.The result of acute toxicity shown that CFB did not display any toxic signs up to 1000 mg/kg.2.The effects of CHB on anti-inflammationThe results showed that CFB could significantly reduce the ear swelling caused by xylene,it could also alleviate the capillary permeability caused by acetic acid,and can weaken the paw edema caused by carrageenan;To some extent,CFB could reduce the level of IL-1?,IL-6,PGE₂ and TNF-?;CFB could also decrease the mRNA expression of IL-1?and TNF-?,and CFB could mitigate the phosphorylation of p65,JNK and p38and the transfer to the nucleus.3.The effects of CFB on gastric ulcerThe results showed that CFB could significantly reduce the area of gastric ulcer induced by indomethacin,and the assessment of tissue after HE staining also reflect this situation.CFB could increase the content of SOD,GSH and PGE2,but reduce the level of MDA,IL-1?,TNF-?,MPO and Ang?;CFB can also increased the mRNA expression of COX-1 and COX-2,and CFB could augment the content of Nrf2 into nucleus,but prevent the phosphorylation of p38.The IC50 of H.pylori in CFB was0.9170 mmol/L,and the IC50 of coptisine hydrochloride was 1.6681 mmol/L.Conclusion:1.CFB was synthesize and the purity was higher than 98%,it also shown a wide rang of a safety doses.2.The anti-inflammatory effect of CFB is comparable to that of berberine,but different in mechanism,which may be related to its substitution group in the skeleton.3.CFB showed a significant effect of anti-gastric ulcer,and it's better than berberine in some indicators.The anti-gastric ulcer effect of different doses of CFB is similar to berberine and lansoprazole.In addition,the mechanism of gastric ulcer is not the same,CFB can activate Nrf2 pathway,but not lansoprazole.In addition,the IC50 of H.pylori in CFB was in a millimole level.In summary,the CFB could inhibit the production of inflammation.Meanwhile,CFB could also protect the gastric mucosa from damage.In addition,CFB could inhibit the development of H.pylori urease.The results could contribute to elucidate the pharmacological effect and mechanism of CFB in treating inflammation and gastric ulcer,and to provide a new precursor compound for anti-inflammatory and gastric ulcer drug.