[Agr. Biol. Chem., Vol. 31, No. 9, p. 10291034, 1967]

Flavones in Green Tea

Part I. Isolation and Structures of 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 (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 (). 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 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 , 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. sulfuric acid, and use of acetic an- them on the paper chromatograms were as follows; hydride and sodium acetate under refluxing at 100°C pale yellow in visible light, reddish brown in ultra. for an hour. By refluxing III for more than three violet light, and change to yellow when exposed to hours with acetic anhydride and sodium acetate , ammonia, yellow with alkalis and aluminum chloride, acetyl derivatives were obtained as white opaque Reduction of these pigments with magnesium or zinc granules. However, III in the chromatograms develop- and cone. hydrochloric acid in ethanol gave pink ed with 2% acetic acid an n-butanol-acetic acid-water color. (4 : 1 : 2 vol. %) as solvents gave a single spot, when Ultraviolet spectra of the pigments.* Ultra developed with 75% phenol, it gave a tailing spot. violet spectra of the pigments were examined witl After separation of pigment III by preparative paper Beckman DB spectrophotometer with potentiometrii chromatography into three parts (111a, IIIa+IIIb and recorder. UV extinctions of I, II, IIIa and IIIb anc IIIb) according to RF values in 75% phenol, each part those of minor pigments were shown in Table II was separately purified. By paper chromatography These pigments exhibited strong absorptions in 333- of each part developing with different three solvents , 335 mp region (Band I) and 270273 mfr region (Band it was proved that III is a mixture of IIIa and IIIb II). The position of the Amax of Band I, 333•`335mƒÊ , (Table 1). indicated that they belong to flavones but not to Separation of seventeen minor pigments. When flavonols, confirmed also by bathochromic shifts and intensity in addition of aluminum chloride the hydrolyzate, after it was neutralized, was treated , that is. once more with neutral lead acetate and ammonium no free hydroxyl group exists in 3 position . The

hydroxide, small amount of brick-red precipitates was presence of 5-hydroxyl group was also confirmed by obtained accompanied with yellow ones. The brick- the bathochromic shifts of bands I and II in alumin red precipitates suspended in methanol were decom um complex. Fused sodium acetate gave bathochromic posed with hydrogen sulfide and then filtered by shift in the position of the peak of band II, indicat

suction. The filtrate was concentrated to a small ing the presence of free 7-hydroxyl group . Further- volume under reduced pressure. The paper chroma- more, the fact that ămax of band I in addition of sodium acetate did not exceed over 400 mfr togram is shown in Fig. 1. For separating these seven- , indicated teen pigments from each other, first the mixture was the presence of a free hydroxyl group only in 4'-posi subjected to partition chromatography using silica gel tion, and it was supposed by giving a single, well column in water-saturated n-butylacohol system , and defined peak of band II at 270-273 mg . then preparative paper chromatography using What- From the investigations of UV spectra, therefore man 3 MM with 2% acetic acid and n-butanol acetic nineteen of twenty one pigments separated from this acid-water (4: 1 : 2 vol. go), as solvents. The banding green tea infusion, that is, 1, 11, IIIa, IIIb and No spot was eluted with 80% aqueous methanol, and 1-,-17 (except 7 and 12), were presumed to have free then the eluate was dried in vacuo. In this method , hydroxyl groups in 5, 7, and 41 positions. By the these minor pigments were finally obtained as amor methylation of these pigments , both ămax in band. phous yellow-brown powders. * T . A. Geissman, "The Chemistry of Flavonoi_??_ General Properties of Pigments I, II and III Compounds," Pergamon Press, 1962 , p. 107-131. Flavones in Green Tea. Part I . 1033

TABLE II. ULTRAVIOLET EXTINCTION OF THE PIGMENTS ISOLATED FROM GREEN TEA

Beckman DB spectrophotometer with potentiometric recorder was used. s: shoulder *: added a few drops of 5% AlCl3 ethanol solution **: added a small quantity of fused sodium acetate ***: sample was too small amount to be measured #: authentic preparations

I and II showed hypochromic shifts, but in addition then by spectral identification on the alcoholic extracts

of aluminum chloride and sodium acetate to the of the spots with authentic preparations. It was found methylated pigments , no shifts in band II were ob that alkaline degradation products of III were p- served. Consequently, these results showed that free hydroxy acetophenone (_??_ 218 mƒÊ, 277 mƒÊ) and p- hydroxyl groups in 5, 7, and 4' positions were methy hydroxybenzoic acid (_??_ 207 mƒÊ, 254 mƒÊ), but no lated. other phenolic substances could be obtained.

Alkaline degradation of pigment III. (Mixture Hydrolysis of Pigment III

of IIIa and IIIb) Pigment III was fused with caustic Acid hydrolysis. By boiling under reflux with potash powder at 180°C for an hour. On the other 10% hydrochloric acid or 5% sulfuric acid for seven hand, it was refluxed with 35% potassium hydroxide hours, IIIa and IIIb were not hydrolyzed. solutionat 120°C for two hours . After cooling, the re Enzymic hydrolysis. The mixture consisted of action mixtures were diluted with water and acidified 5 mg. of III (IIIa and IIIb, respectively), 1 ml. of with dil. hydrochloric acid , and then ether extraction acetic acid-sodium acetate buffer at pH. 5.0, and was carried out. The resultant ketones, acids and 1 mg. of ƒÀ-glucosidase (Sigma Chem. Co.) was kept phenols were at first examined by co-chromatography at 30°C for more than 28 hours. On the other hand, on paper using six different developing solvents and instead of buffer solution described above, the pig- 1034 Yutaka SAKAMOTO ments were dissolved in water containing a small amount of tymol. Paper chromatographic studies showed that in both treatments any hydrolyzed pro ducts were not detected and III remained unchanged, whereas in the enzymic hydrolyzate of rutin which was used as a control, the spots of rhamnose and glucose were detected. Calculation of molecular weights of pigment III. Pigments IIIa and Tub did not completely dis solve in camphor at 180°C, so that Rast method could not be applied. Molecular weights were calculated from the absorbance* of them at 335 mp, supposing FIG. 4. IR Spectra of Pigment II. that their molecular extinction coefficients was ap- A: Pigment II isolated from green tea proximately equal to the average of those** of api B: Authentic vitexin. genin, saponarin, saponaretin and vitexin. The molecular weights of IIIa and IIIb were consequently estimated as 712 and 850 with the error of 10%, log s=4.174, 4.157 at 2,nax 271 mp and log e=4.153, respectively. 4.166 at 2.,, 335 mp, respectively. Identification of Pigment II with vitexin. RF As shown in Fig. 4, IR spectra of II and the value: As shown in Table I, the RF value of pigment authentic vitexin were completely superimposable. II was identical with that of authentic vitexin, which Anal. Found: C, 56.37; H, 4.80. Calcd. for was prepared by the hydrolysis of saponarin extract C21H20O10•EH2O: C, 56.00; H, 4.88%. ed from Saponaria officinalis L. The sample lost the weight equal to I mol. of H2O Melting point of pigment II was 253°C (measured when it was dried at 110°C for about 3 hours over by micro hot stage). No depression in m.p. by phosphorus pentoxide in vacuo. admixture with the authentic vitexin (m.p. 253°C) was observed. Acknowledgment. The author wishes to ex UV spectra and aluminum chloride- and sodium press his hearty thanks to Prof. Y. Oshima, acetate shifts of pigment II in ethanol were identical Kyushu Univ., Prof. Y. Takino, Tokyo Univ. with those of the authentic vitexin. (Table II). Mol of Educ., Dr. S. Kuwatsuka, The Institute of ecular extinction coefficients of II and vitexin were Phy. and Chem. Res., for their helpful gui dance and encouragement; to Emeritus Prof. * The absorbances of IIIa (18 .4 mg./I.) and IIIb T. Nakaoki, Toyama Univ. for a gift of (18.7 mg./l.) at 335 mƒÊ were 0.498 and 0.423 respec tively. authentic saponarin, and Prof. 0. Tamemasa, ** The absorbances of saponarin (11 .04 mg./l.), Shizuoka Univ. of Pharm. and Dr. K. Ina, saponaretin (10 mg./1.) and vitexin (20 mg./1.) at Shizuoka Univ. for elementary analysis and ƒÉmax,n, 335 mp were 0.399 (log e=4.346), 0.425 (log s=4.286), and 0.675 (log e=4.166), respectively. infrared analysis. The author also thanks Molecular extinction coefficient of apigenin at 336 mu was reported as log e=4.32 (L. Jurd., Arch. Biochem. Dr. H. Torii, Head of Div. of Techn., Tea 63, 276, (1956)). Res. Sta., M.A.F. for helpful advices.