Study On Quality Standard Of Diploclisia Glaucescens(B1.) Diels And Effective Parts For Anti-Inflammatory And Analgesia

Objective:1.To study the chemical constituents of Diploclisia glaucescens（B1.）.2.To establish the quality standard of Diploclisia glaucescens（B1.）.3.Acute toxicity evaluation of Diploclisia glaucescens（B1.）.4.Compare the anti-inflammatory and analgesic effects of different extracts of Diploclisia glaucescens（B1.）.5.Explore the curative effect and mechanism of Diploclisia glaucescens（B1.）n-butanol extract in the treatment of adjuvant arthritis.Method:1.UPLC-Q-Exactive HRMS technology is used to screen the chemical composition of Diploclisia glaucescens（B1.）,Separation using ACQUITY UPLC?HSS T3 1.8μm（2.1×150 mm）column,Use positive ion 0.1%formic acid water（B2）-0.1%formic acid acetonitrile（A2）;negative ion 5 m M ammonium formate water（B1）-acetonitrile（A1）as mobile phase for gradient elution,Flow rate 0.25m L/min,column temperature 40℃,It adopts the simultaneous acquisition mode of positive and negative ions,full scan and automatic triggering of the secondary mass spectrometry scan mode,Analysis of the chemical composition of Diploclisia glaucescens（B1.）,The relative molecular mass and multi-level ion fragment information of the obtained compound are matched with multiple databases of HMDB,METLIN,Massbank,Lipid Maps and mz Clound,Screen out the compound with higher matching degree,and combined with relevant literature,Perform mass spectrometry derivation of the compound and guess the possible compound.2.Observe the morphology,character and microscopic identification characteristics of the Diploclisia glaucescens（B1.）.And perform a thin-layer identification check.Determine the water,ash and extract content of Diploclisia glaucescens（B1.）according to the method in the fourth general rule of the2020 edition of the Chinese Pharmacopoeia.The content of magnolin in the medicinal materials was determined by high performance liquid chromatography.3.LD₅₀ and maximum tolerated dose method were used to observe the poisoning reaction of mice within 14 days after administration of Diploclisia glaucescens（B1.）Water extract and alcohol extraction.Preliminary exploration of the safety of its clinical administration.4.Using xylene-induced mouse ear swelling,carrageenan-induced mouse foot plantar swelling,and glacial acetic acid-induced mouse abdominal capillary permeability inflammation models,to evaluate the inhibitory effects of different extracts of Diploclisia glaucescens（B1.）on inflammation;Using glacial acetic acid-induced writhing in mice and hot plate-induced pain in mice models,to evaluate the inhibitory effects of different extracts of Diploclisia glaucescens（B1.）on pain;The effect of the pallor extract of Diploclisia glaucescens（B1.）n-butanol on the content of TNF-α,IL-1βand PGE₂ in the inflammatory tissues of the plantars of mice inflamed by carrageenan was used to preliminarily investigate its anti-inflammatory mechanism.5.Take 60 male rats and random Ly divide them into 6 groups:Normal control group,model group,tripterygium wilfordii polyglycoside tablet group（9 mg/kg）,Diploclisia glaucescens（B1.）n-butanol high,medium,low dose groups（0.11,0.22,0.44 g/kg）,10 per group.Stimulate by injecting complete Freund’s adjuvant to construct an adjuvant arthritis model.After successful modeling,Tripterygium wilfordii polyglycosides tablets and n-butanol extract were administered by gavage.The model group and the normal control group were given an equal volume of 0.5%sodium carboxymethyl cellulose,1 time/d,lasting 30 d.Measure rat body mass,joint swelling degree,spleen organ coefficient,and HE stain to observe histopathological changes of ankle joint synovium.ELISA detects the expression of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）and prostaglandin（PGE2）in serum.Result:1.According to the obtained ion fragments,the library is matched,and a total of 15 compounds with higher matching degree are screened out.Including 5 flavonoids,1 lignin,3 alkaloids,2 terpenoids,2 steroids.Combined with related references,some compounds are fragmented and deduced by mass spectrometry.Found that the matching results are basically consistent with the library.2.Diploclisia glaucescens（B1.）powder is brownish yellow to brownish black.Calcium oxalate square crystals can be seen under the microscope.The characteristics are more obvious and specific.TLC spots are clear,with good resolution.The moisture content of medicinal materials ranges from 4.66%to 10.65%,the ash content ranges from 2.10%to 9.74%,and the extract is from 5.95%to13.45%.Magnolin has a good linear relationship within 3.6364μg/m L～52.728μg/m L（r=0.99962）,and the average sample recovery rate is 101.4%.The content of magnolin in 14 batches of medicinal materials samples ranged from 0.0250%to 0.1975%.3.The 50%lethal dose LD50of the Water extract from Diploclisia glaucescens（B1.）is 107.44 g/kg,which is equivalent to 17 times the clinical daily dose of 60 kg,and the 95%confidence limit is 85.29～151.07 g/kg;The maximum tolerable dose of alcohol extract is 542.00 g/kg of crude drug,which is equivalent to 174times of the clinical daily dosage.4.All doses of Diploclisia glaucescens（B1.）n-butanol extract have obvious anti-inflammatory and analgesic effects.Among them,high-dose anti-inflammatory drugs are effective.The content of TNF-α,IL-1β,and PGE 2 in the inflammatory tissues of the foot plantar of mice in the high and medium dose groups of Diploclisia glaucescens（B1.）n-butanol extract were significantly reduced（P<0.05 or P<0.01）.5.Compared with the blank control group,the swelling degree of the foot plantar and the liver coefficient in the model group increased significantly,serum TNF-α,IL-1βand PGE₂ were significantly increased（P<0.05）.Compared with the model group,the plantar swelling degree and spleen coefficient of rats in each dose group of Diploclisia glaucescens（B1.）n-butanol extract were significantly reduced,and the serum levels of TNF-α,IL-1βand PGE2 were significantly reduced（P<0.05）.Conclusion:1.UPLC-Q-Exactive HRMS technology can identify many chemical components of Diploclisia glaucescens（B1.）quickly.2.The quality control method of Diploclisia glaucescens（B1.）.established in the experiment has good specificity and accuracy.The research results can provide scientific basis for the establishment of quality standard of Diploclisia glaucescens（B1.）.and its popularization and use.3.Pale said that the LD₅₀ of Diploclisia glaucescens（B1.）.water extract is 107.44 g/kg of crude drug.The maximum tolerance of its alcohol extract is 542.00 g/kg crude drug 4.Pale says that Diploclisia glaucescens（B1.）has anti-inflammatory and analgesic effects.The effective anti-inflammatory and analgesic extract is n-butanol extract.Its anti-inflammatory mechanism may be related to inhibiting the production of TNF-α,IL-1βand PGE₂ and reducing inflammation.5.It is pale that the high and medium doses of Diploclisia glaucescens（B1.）n-butanol extract may regulate the production of inflammatory factors.Then inhibit the inflammatory response of rheumatoid arthritis to reduce joint damage.