Study On Chemical Composition And Activity Of Active Parts Of Atalantia Buxifolia

Atalantia buxifolia?Poir?.Oliv.,the drying root of Rutaceae.The original recordis in"the private medicine recorded of Lingnan area",It has the functions of removing blood stasis and relieving pain and reducing phlegm.As a folk medicine,there is a great prospect in the treatment of chronic obstructive pulmonary disease?COPD?.In this paper,depart from the two major pathogenic factors of COPD:bacterial infection and smoking were taken as measures of activity tracking and activity test.Extracting Atalantia buxifolia into fractions of petroleum ether,chloroform,ethyl acetate and n-butanol for antimicrobial and anti-inflammatory experiments to find effective fractions and compounds through activity tracing.Then identify the compounds by UV and nuclear magnetic resonance spectrum.The antimicrobial and anti-inflammatory were used to study the activity of the compounds.The aim is to find possible effective partsand effective chemical compositions for the treatment of COPD.The experiment contents and results are as follows:?1?Antimicrobial and anti-inflammatory tests for selection of effective fractions.The extraction was extracted with 95%ethanol.Then concentrated it.Dispersed it with purified water and sequentially extracted with petroleum ether,chloroform,ethyl acetate and n-butanol to get four different fractions.Detect their inhibitory effect on pathogens like Klebsiella pneumoniae,Pseudomonas aeruginosa,and Escherichia coli,which can cause COPD.Disk diffusion test for antibacterial test.The results showed that chloroform,ethyl acetate and n-butanol fractions have inhibitory effect on three pathogens,and n-butanol fraction has the most obvious inhibitory effect on Klebsiella pneumoniae.The anti-inflammatory experiment is to stimulate human bronchial epithelial cells?16HBE?with cigarette smoke?CSE?which can induce inflammatory reaction.Observe the 16HBE cells by high magnification microscope and MTT assay for cell viability,measure the changes of inflammatory factors like TNF-?to research the effect of drugs on cigarette smoke-stimulated 16HBE cells.The results showed that the fractions of chloroform,ethyl acetate and n-butanol have a protective effect on cigarette smoke-stimulated 16HBE cells,which survival rate was significantly higher than the model group and the inflammatory factors were controlled,too.And which has a significantly different effects on is the high dose group of ethyl acetate fraction,high dose group of n-butanol fraction and middle dose group of n-butanol fraction.?2?Chemical composition of ethyl acetate and n-butanol fractions.Through pharmacological tests,we found that ethyl acetate and n-butanol have antibacterial and anti-inflammatory effects,they are effective fractions.Ethyl acetate and n-butanol fractions for the following separation.Anti-Klebsiella Pneumoniae test and MTT assay for the effects of drugs on 16HBE cells'death rate stimulated by CSE as guidence.Silica gel column chromatography,ODS,MCI,gel chromatography,HPLC,preparative HPLC were used on chemical separation.The chemical structures were identified by ¹H-NMR,¹³C-NMR spectroscopy.Sixteen monomer compounds were isolated and identified from them,respectively as:Buxifoliadine B?1?;Buxifoliadine A?2?;N-methylataphyline?3?;auraptene?4?;Atalaphylline?5?;Severinolide?6?;acetylisoepiatalantin?7?;trans-N-p-coumaroy tyramine?8?;Citrusinine II?9?;Citrusinine-I?10?;Atalafoline?11?;1,4,5-trihydroxy-3-methoxy-10-methyl-acrid one?12?;glycofolinine?13?Cycloseverinolide?14?;7-Hydroxycoumarin?15?;?-sitosterol?16?;?-D-Glu-3,4-dimethoxy-1-hydroxy-5,10-dimethoxy-acridone?17?.?3?Antimicrobial and anti-inflammatory activity test of monomer compounds.The antibacterial test of compound 1?16 were implemented through the disk diffusion test.Klebsiella pneumoniae,Pseudomonas aeruginosa and Escherichia coli were selected as pathogens.The results showed that compounds 2,3,9 and 13 inhibited Klebsiella pneumoniae;compounds 1,9,11,13 and 14 inhibited Pseudomonas aeruginosa;compounds 1,3,9,11 and13 inhibited Escherichia coli.Among them,the most effective compounds for the three kinds of bacteria is compound 9 and compound 13.It may be related to polyhydroxyl substituted acridone structure.The effects of compound 1?16 on cigarette smoke-induced human bronchial epithelial cell death were investigated by microscopy and MTT assay.The results showed that,compounds 1?13 and compound 15 have different degrees of protection against cigarette smoke-induced 16HBE damage.The effect of some monomer compounds on the inflammatory factors TNF-?,IL-8,and IL-10 was studied by the Elisa kits.As a result,compound 4and compound 8 can balance the inflammatory factors.Compound 4 is auraptene and compound 8 is coumaroy tyramine.