Study Of Chemical Constituents And Anti-inflammatory Activity Of Pollen Typhae

Pollen typhae is the dry pollen of the Typha angustifolia L.,Typha orientalis Presl.and same generic plants.Pollen Typhae produced in Jiangsu,Zhejiang,Jiangxi,Guangdong,Yunnan,Taiwan and other regions,mainly.It is used for Hematemesis,epistaxis,uterine bleeding,amenorrhea,dysmenorrhea,traumatic bleeding,abdominal pain,traumatic swelling,blood pouring and pain.In this experiment,pollen Typhae was extracted 3 times with methanol,and the extract was Separated with petroleum ether,ethyl acetate,n-butanol,orderly.By various chromatographic methods,22 compounds were isolated from the ethyl acetate extract and n-butanol extract,which include seven flavonoids,four alkaloids,four fatty acids glycerol esters,theer steroids,two long chain alcohols,two carboxylic acids.On the basis of physic-chemical characters and spectroscopic analysis such as NMR and MS,34 compounds were identified as Dotriacontane-5,10-diol(1),Tritriacontan-4-ol(2),Naringenin(3),Oleic acid(4),Kaempferol(5),Quercetin(6),Glyceryl oleate(7),Arachic acid triglyceride(8),Daucosterol-6'-dodecanoate(9),Tripalmitin(10),1,3-oleic acid diglyceride(11),Vanillic acid(12),Daucosterol(13),Daucosterol-6'-linoleate(14),2-hydroxy-N-((4E,13Z)-3-hydroxy-1-((3S,4R,5S,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)-tetrahydro-2H-pyran-3-yloxy)nonadeca-4,13-dien-2-yl)nonadecanamide(15),Narcissoside(16),Uridine(17),Zarzissine(18),6-amino-9-?-D-ribofuranosyl-9H-purine(19),Isorhamnetin-3-O-neohesperidoside(20),Typh-aneoside(21),Quercetin-3-O-(2G-?-L-rhamnosyl)-rutinoside(22).Compounds 1,2,4,7,8,9,11,14,17,19 are isolated from Pollen Typhae for the first time.The LPS can induce the peritoneal macrophages release NO.The compounds were prepared into four concentrations(6.25 ?M,12.5 ?M,25 ?M and 50 ?M).The Butein was used as positive control drug.Determination of absorbance will be done after Griess actions.Detected the inhibition rate on NO release.The results indicate that compounds 1,2,3,5,6,7,9,10,11,12,16,17,18,19,20,21 showed strong inhibitory activity and concentration dependent.The IC50 values were below 50 ?M.To develop a HPLC method HPLC for determining the Typhaneoside,Isorhamnetin-3-O-neohesperidoside and Narcissoside in Pollen Typhae,simultaneous-sly.Adopting LaChrom C18(4.6 mm×25 mm,5?m)chromatographic column.Aceton-itrile-0.05%phosphoric acid aqueous solution(15:85)as mobile phase.The detection wavelength was set at 254 nm.The flow rate was 1 ml/min.The column temperature was 30?.The results indicated that the system adaptability up to scratch,and,Typhaneoside,Isorhamnetin-3-O-neohesperidoside and Narcissoside showed good linearity.The results of precision test,reproducibility test and recovery test is meet a criterion.Results of determination show that The content of Typhaneoside and Isorhamnetin-3-O-neohesperidoside is 0.770%,which is Obviously higher than the standards.The volatile component in Pollen Typhae were extracted by methanol and handled by gas purge microsyringe extraction(GP-MSE),and were analyzed by gas chromatograph-mass spectrometer(GC-MS).Area normalization method was used for the detection of the volatile component.One hundred an one chromatographic peak were isolated,among them sixty-eight components were identified,which accounting 96.89%of total contents.The content of(9Z,12Z)-octadeca-9,12-dienoic acid is the highest(32.01%).And the others main components conclude methyl palmitate(7.46%),palmitic acid(25.19%),(9Z,12Z)-methyl octadeca-9,12-dienoate(7.89%),methyl oleate(3.12%),(Z)-tetradec-9-enal(5.8%),stearic acid(2.1%),et.Analysis the results revealed that the main volatile component of Pollen typhae belong to long chain fatty acid and their esters,long chain alkane and long chain alcohol,and alkaloids.