[Agr. Biol. Chem., Vol. 31, No. 9, p. 10291034, 1967] Flavones in Green Tea Part I. Isolation and Structures of Flavones Occurring in Green Tea Infusion By Yutaka SAKAMOTO Tea ResearchStation, Ministry of Agricultureand Forestry,Kanaya, Shizuoka ReceivedApril 24, 1967 Nineteen flavones were newly found in green tea infusion. Four pigments were isolated, and one of them was identified as vitexin (C-glycosyl flavone). As the remained pigments were obtained only in small quantities, their properties were studied spectrophotometrically. Similarity of their UV spectra and color reactions suggested that all of them have the same skeleton as 5, 7, 4'-trihydroxyflavone (apigenin). The major one has high water sol ubility and deep greenish yellow color in aqueous solution, so that it may be the important constituents related to the color of green tea infusion. The occurrence of flavonols and their gly cosides, which are regard as the important constituents related to the yellow color of tea infusion, has been reported in tea and tea leaves in many papers.'-" But there are no report about the occurrence of flavones in tea. However, four yellow pigments designated I, II, IIIa* and IIIb* in Fig. 1.) were isolated and other seventeen pigments (No. 1-17) were detected on the paper chromatograms from the green tea infusion, and they were identified as flavones except two spots (No. 7 and No. 12). Pigments I and II were obtained in small quantities and other seventeen minor pigments were finally purified by the method of pre parative paper chromatography using What- FIG. 1. Paper Chromatogram of Flavones Occurring man 3 MM after fractionation of each pigment in Green Tea Infusion. 1) Y. Takino, H. Imagawa and H. Yoshida, J. by the column chromatography using silica Agr. Chem. Soc. Japan, 27, 150 (1953); 28, 186, 190 (1954);36, 943 (1962). gel and water-saturated I-butanol as the sol- 2) Y. Oshima, and T. Nakabayashi, ibid., 27, 274, vent. Pigment III was purified as an a 754, 756, 759 (1953). 3) Y. Takino, H. Imagawa and H. Yoshida, This morphous yellow powder. However, it proved Journal, 26, 10 (1962). to be a mixture of IIIa and Tub which have * Y. Takino has presumed pigment III as ka empferol glycoside by preliminary test. This journal quite similar spectra (UV and IR) as shown 26, 10 (1962). in Figs. 2 and 3. 1030 Yutaka SAKAMOTO FIG. 3. IR Spectra of Pigments Ma and IIIb (KBr disc). Saponarin: Authentic preparation. ample, gave p-hydroxybenzoic acid and p- hydroxyacetophenone, but any other phenolic substances were not obtained. This indicates that B-ring of pigment III has a hydroxyl group in 4' position but A-ring seems to have FIG. 2. UV Spectra of Pigments Ma and Tub (in alkali-sensitive structure. EtOH). Pigments Ma and IIIb were not hydrolyzed by 10% hydrochloric acid or 5% sulfuric acid, All of these pigments exhibited strong in- nor by (ƒÀ-glucosidase. Therefore, whether tensity absorptions in 333-335 Daft region (Band these pigments are glycosides or not still re I) and 270.273 mp region (Band II). From mained unsettled, but by a comparison of the absorption spectral data on the aluminum their RF values with 2% acetic acid, it was chloride complex- and sodium acetate shifts, supposed that they are not such aglycone types and methyl ethers of them, indicated that as is apigenin or acacetin which scarcely these tea flavones, except two, have free moves from the starting point. On the other hydroxyl groups in 5, 7, and 4' positions, so hand, C-glycosyl flavones such as saponaretin that they have to belong to apigenin family. and vitexin, which are aglycones of saponarin, On a careful dissection of the alkaline move with the same solvent. (Table I). degradation products, pigment III, for ex- The infrared spectra of pigments IIIa and TABLE I. RF VALUES OF PIGMENTS I, II, IIIa, Mb AND AUTHENTIC SAMPLES * Authentic specimens ** n-Butanol-acetic acid-water (4 : 1 : 2 vol .%) Flavones in Green Tea. Part I. 1031 IIIb have displayed strong intensity absorptions EXPERIMENTL in 3400cm-1 region indicating the presence of Isolation of pigments from the green tea in- a number of alcoholic hydroxyl groups, and fusion. Green tea treated previously with ethyl the absorptions diminished very much by ether, was extracted with ten times its weight of acetylation. boiling water. After removal of precipitated formed The molecular weights of pigments IIIa and by an addition of lead acetate into the extract by suction filtration, the clear filtrate was treated with IIIb calculated from the absorbances at 335 mp ammonium hydroxide and lead acetate solution. The were 712 and 850, within the error of logo, yellow precipitate there formed was filtered by suction, respectively. Therefore, the differences be washed repeatedly with distilled water. This precipi tween the molecular weight of them and that tate was then suspended in aqueous methanol and of apigenin (MW=270) were approximately decomposed by means of 10% sulfuric acid and equal to 2 mol. and 3 mol. of hexose (MW= hydrogen sulfide. After removal of black lead sulfide 180), respectively. by filtration, the filtrate was concentrated under re These results described above suggested that duced pressure and dried in vacuo. (Yield: about IIIa and IIIb belong to C-glycosyl flavones. 5% of green tea). On paper chromatogram of this Moreover, I and II have coincided with fraction, the spots of I, II, III and others were detect- saponaretin and vitexin on comparison of their ed, besides already known spots of flavonols, that is, rutin, kaempfetrin, quertrin and myricetin glycosides. RF values with those of the authentic prepara Preliminary test showed that pigments I, II, and III tions. (Table I). were not hydrolyzed on such condition at that con By comparison of its spectra (UV and IR), dition flavonol glycosides were completely decom and elementary analysis with those of authentic posed. Thus, after hydrolysis with 3.5%o hydrochloric preparation obtained from the hydrolyzate of acid on the boiling water bath for an hour, the saponarin extracted from Saponaria officinalis examination of this fraction by the paper chromato L., pigment II was finally identified as vitexin. graphy revealed that only the spots of I, II, and III Similar behavior in the color reactions and remained as before, and those of kaempferol, quercetin UV spectra of these flavones in green tea in- and myricetin originated from their glycosides came fusion suggested that they are very closely out. Gallic acid, probably its parent compound related and also belong to C-glycosyl flavones. occurred in this fraction, was also detected. The hydrolyzate was at first shaken with ethyl ether to It is interesting that such flavones* contain- remove flavonol aglycones and gallic acid. And then ing C-glycosyl residue as vitexin, together it was shaken with ethyl acetate. Pigments I and II with other eighteen flavones which seem to moved into ethyl acetate layer, but pigment III re have very similar structure, were found in mained in the aqueous layer. green tea infusion. These flavones also seemed Separation of pigments I and II. The ethyl to be the important constituents related to acetate solution was concentrated under reduced pres- the color of green tea infusion, especially IIIa sure and then dried up in vacuo. The residue was and IIIb, for their high water solubility and devided into two parts by washing with ethanol, I deep greenish yellow color in an aqueous was soluble while II was sparingly soluble in ethanol. solution. Pigment II was repeatedly crystallized with a large Further studies on the structure of the volume of hot ethanol or pyridine-water and obtained unknown pigments are in progress. at bright-yellow plates. Pigment I could not be crystallized so that after development of banding on paper using Whatman 3 MM, the banding spot was eluted from the filter paper with 80% methanol and * As C-glycosyl flavones which belong to 5, 7, 4'- trihydroxy flavone derivative, oryzatin and homo then dried under reduced pressure. Thus it was oryzatinare also known besides saponarin, saponaretin obtained as a bright yellow powder. Total yield of and vitexin. (S. Kuwatsuka, "Biochemical Studies on the Polyphenols of Rice Plant." Report of Agr. Chem. I and II: about 20 mg. from 2 kg. of green tea. Insp. Lab., Kyushu Univ., May 1, 1962). Purification of pigment 111. Further fractiona- 1032 Yutaka SAKAMOTO tions of III were carried out by means of column Pigment I. Very soluble in methanol, ethanol , chromatography using silica gel and water-saturated ethyl acetate, and insoluble in cold water. n-butylalcohol as 4 solvent, or cellulose powder and Pigment II. Soluble in pyridine, sparingly soluble 2% acetic acid. Because III could not be crystallized in ethanol, acetic acid, and insoluble in water, m.p. from methanol or water, it was dissolved in a small 253°C. amount of hot methanol and poured into a large Both pigments above were dissolved in aqueous volume of ethanol. The resultant precipitate was sodium carbonate to give deep yellow solutions and then crystallized from a large volume of hot ethanol. II was crystallized out again by acidification of its Pigment III was finally obtained as an amorphous sodium carbonate solution with acetic acid. yellow powder. Yield: about 420 mg. from 2 kg. of Pigment III (IIIa and IIIb). M.p. more than 300°C green tea. very soluble in cold water, soluble in hot methanol, Crystalline acetyl derivatives were obtained in very sparlingly soluble in hot ethanol, insoluble in ethyl low yields by the use of acetic anhydride and one acetate, ethyl ether and acetone. Color reactions of drop of cone.