Fluorescence Property And Analytical Methods Of Flavonoids

In this thesis, fluorescence properties of 42 kinds of flavonoids are studied, influences of experimental conditions on fluorescence are investigated, fluorescence quantum yield of some flavonoids are measured, luminous mechanism of some flavonoids are discussed, empirical rules of fluorescence property vs. molecular structure are summarized, fluorimetric methods for determination of some flavonoid active constituents in certain medications are proposed.Five major parts are included in the thesis.1. Fluorescence properties of 14 kinds of flavonoids( having 2-phenylchromone), including 6-methylflavone, 6-methoxyflavone, 6-hydroxyflavone, 6-aminoflavone, luteolin, 7-methoxyflavone, 7-aminoflavone, 3-methylflavone-8-carboxylic acid, icariin, vaccarin, 7,4’-dihydroxyflavone, 5,7,3’,4’,5’-pentamethylflavone, flavoxate hydrochloride and apigenin, are studied. Comparison of the fluorescence properties and the molecular structure of the flavonoids, following empirical rules are obtained:(1) Under neutral condition, the molecular form of flavones have relative strong fluorescence, with excitation wavelength ?ex =300~350 nm, emission wavelength ?em = 400~460 nm, quantum yield Y = 0.01~0.2; in acidic condition, protonation effect leads to fluorescence weaken; in strong alkaline condition, cleavage reaction of γ-pyrone ring result in fluorescence quenching.(2) Under weak alkaline condition, 7-OH flavonoids generate fluorescence because of ionization.(3) Under strong alkaline condition and heating, 8-carboxyl or 8-ester flavonoids can produce strong fluorescence, since the cleavage reaction of γ-pyrone ring, quantum yield may as high as 0.50.(4) Most 5-OH flavonoids have no fluorescence, indicating that 5-OH has fluorescence quenching function.2. Fluorescence properties of 12 kinds of flavanone compounds, including flavanone, 6-methoxyflavanone, 6-hydroxyflavanone, 7-methoxyflavanone, 7-hydroxyflavanone, farrerol liquiritigenin, 2’-hydroxyflavanone, 4’-hydroxyflavanone, pinocembrin, naringenin and hesperitin, are studied. The molecular form of these compounds have fluorescence, ?ex = 270~360 nm, ?em = 375-480 nm, Y = 0.01~0.10; the ionic form of the compounds have weak fluorescence. Comparison of the fluorescence properties and the molecular structure of the flavanones, following empirical rules are obtained:(1) Flavanone with no substituent has relative strong fluorescence, ?ex/?em = 340/386 nm, Y = 0.057;(2) 7-donor substituents make flavanones??em blue shift, whereas 6-donor substituents make flavanones??em red shift;(3) The hydroxyl in B ring having fluorescence quenching effect;(4) Compare of flavone with flavanone which having the same substituent, fluorescence wavelength of flavanone is shorter.3. Fluorescence properties of 6 kinds of isoflavones, including formononetin, calycosin, ononin, calycosin-7-glucoside, genistein and 7-methoxy-4’-hydroxyisoflavone, are studied. The molecular form of isoflavones having 7-OH and without 5-OH substituent, have no fluorescence, whereas under weak alkaline conditions, the ionic form can produce fluorescence, with ?ex = 334~339 nm, ?em = 463~466 nm, Y = 0.01~0.04. Isoflavones with 7-glucoside substituent have no fluorescence under ordinary condition, but can produce fluorescence by heating in strong alkaline condition, due to the leavage reaction of γ-pyrone ring, with ?ex = 288-292 nm, ?em = 388-394 nm, Y = 0.01~0.02.4. Fluorescence properties of 7 kinds of flavonols, including 3-hydroxyflavone, 3-hydroxy-6-methoxyflavone, 3,7-dihydroxyflavone, galangin, kaempferol, isorhamnetin and quercetin, are studied. Fluorescence of flavonols is very weak, but they can react with Al3+ ion or Mg Ac2-Me OH to produce fluorescence. Some flavonols react with Al3+ ion to form fluorescent complex, with ?ex = 334~339 nm, ?em = 380~415 nm, Y = 0.05~0.95. The hydroxyl substituent on B ring reduces fluorescence. In methanol medium, some flavonols can react with Mg Ac2-Me OH, producing fluorescence, with??ex = 430~450 nm???em = 500~515 nm, Y ≈ 0.05, hydroxyl substituent on B ring gradually weaken the fluorescence.5. A fluorimetric method for determination of flavoxate hydrochloride in Flavoxate Hydrochloride Tablets is studied, and the content is detected to be 41.33%, which is consistent with the drug label. A fluorimetric method for detecting flavonols in traditional Chinese medicine materials Alpiniae Officinarum Rhizoma(Gaoliangjiang) is studied, and the total content of flavonols is detected to be 1.36%. A fluorimetric method for detecting flavonols in Chinese patent drug Ginkgo Leaf is studied, and the total content of flavonols is detected to be 1.01%. Based on three-dimensional fluorescence data combined with second order correction algorithm, a fluorimetric method for detecting flavonols in Chinese patent drug Ginkgo Leaf is studied, and the total content of flavonols is detected to be 1.05%. These studies provide sensitive and simple analytical methods for quality control of certain medications.