Comparative Studies Several of Condensed Young Fruits Tannins
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J. Japan. Soc. Hort. Sci. 50(2) : 262-269. 1981. Comparative Studies of Condensed Tannins from Several Young Fruits Tomoaki MATSUO and Saburo IToo Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890 Summary Four polymeric tannins prepared from young fruits of persimmon, banana, carob bean and Chinese quince were shown, in addition to having some properties of con- densed tannins, to produce precipitate in methanolic solution by addition of K2HPO4, to form anthocyanidins on acid treatment, and to release the thioethers of flavan-3- ols by means of toluene-a-thiol treatment. It is suggested from these results that each tannin is a proanthocyanidin polymer and should be called flavanan or flavonan tannin instead of condensed tannin. Among the fruit tannins tested, loquat-tannin was an exception; it appeared to be a proanthocyanidin oligomer. Three methylated tannins from young fruits of banana, carob bean and Chinese quince were similar in molecular size (Mw=1.4-1.0 x 104, Mn=0.7-0.2 x 104), while methylated kaki-tannin is thought to be a slightly higher polymer. In comparison with kaki-tannin which mainly consists of catechin, catechin-3- gallate, gallocatechin and gallocatechin-3-gallate with the ratio of about 1 : 1.2 2.1 :2.2, banana-tannin consists of catechin and gallocatechin with the ratio of about 1 to 1.3 and carob bean-tannin consists of catechin-3-gallate, gallocatechin and gallocatechin-3-gallate with the ratio of about 1 : 3.3 : 4.6. Chinese quince- and loquat-tannin appear to be composed of only catechin. and recently established a simple and rapid Introduction purification method applicable to condensed In some species, immature or young fruits tannins in several astringent fruits (9). In have remarkable astringency, for example, addition, we found that toluene- x-thiol was banana and persimmon. It is generally known an excellent reagent for degradation of a poly- that astringency in fruits is caused by conde- meric proanthocyanidin such as kaki-tannin nsed or conjugated tannins (17, 20). These (8). terms, however, appear to be arbitrarily used Chemical comparison of four condensed without clear chemical definition. In some tannins prepared from different astringent reports, researchers described condensed tan- fruits (banana, Chinese quince, loquat and nins as immobile polyphenolic compounds on carob bean) with kaki-tannin from persimmon thin layer and paper chromatographies (10). fruit is described in the present paper. Technical problems in the past have impeded Materials and Methods researchers from developing a good purification Plant materials procedure for condensed tannins and adequate methods to degrade the purified tannins to Young persimmon fruits (Diospyros kaki their constituent (s) to elucidate their chemical Thunb., cv. "Hiratanenashi", astringent type) structures. were harvested from July to August in 1977 We have investigated chemical structures and 1978 at Toso Orchard of Kagoshima Uni- and properties of kaki-tannin, an astringent versity. Loquat fruits (Eriobotrya japonica component in immature persimmon fruit Lindl., cv. "Mogi") were harvested in March, known to be a typical condensed tannin (8), 1977 at Tarumizu Fruit Experiment Station of Kagoshima Prefecture. Carob beans (Cera- Received for publication March 23, 1981 tonia siliqua L.) were a gift from Mr. K. Y. 262 MATSUOAND IT00 : STUDIESOF C 0 NDE NSEDTANNINS FROM YOUNG FRUITS 263 Ogawa, Pasadena, California and were har- tribution were analyzed by gel permeation vested on April 9, 1978. Chinese quince (Chae- chromatography. High pressure liquid chro- nomeles sinensis Koehne) and banana (Musa matography analysis was performed under the sapientum L.) were harvested in Kagoshima following conditions; column; TSK G5, 4, 3. Prefecture, in June and August of 1978, re- 2 H 6, 7.5 X 600 mm, mobile phase; THF, flow spectively. All individual fruits were leaf- rate; 1.0 ml/min, detection; UV at 280 nm, green. The average fruit weights were the instrument; Waters LC. Polystylenes with same as those reported in our previous paper various polymerization degrees were used as (9). Fruits and tissues were stored in a standard markers for the molecular weight. freezer, or dipped in methanol after cutting Mw and Mn express weight average molecular into small pieces and stored at -20°C until weight and number average molecular weight, use. respectively. Analytical methods of condensed tannins Results The four condensed tannins were prepared by the same procedure described in the previous Preparation of condensed tannins from paper (9). Powders of each tannin obtained several young fruits were lyophilized and stored at -20°C until Condensed tannins were prepared from each chemical analysis. Preparation, acid degrada- of the five kinds of astringent young fruits by tion and toluene-a-thiol degradation of each means of the K2HPO4-precipitation method condensed tannin were carried out in the same described in the previous paper (9). The manner as that in the previous paper (8). For yield, purity and some properties were already identification of deep red pigments produced reported in the paper. Persimmon and carob by acid degradation of each tannin, ascending bean gave 2.0 and 1.9 percent of tannin yields. paper chromatography was performed using While banana and Chinese quince showed Toyo no. 51 filter paper. The developing sol- relatively lower contents of condensed tannin vents employed were; ( i ) acetic acid-HCl- (0.6 and 0.4 percent). Young loquat fruits water=30:3:10, v/v, AHW, (ii) n-butanol- were less astringent and differed from the acetic acid-water=4:1:5, v/v, upper phase, others in that it did not precipitate when K2 BAW, and (iii) 88% formic acid-HCl-water HPO4 was added to the methanolic extract. =5:2:3, v/v, FHW. The major component, proanthocyanidin, was Polyphenolic products released by toluene- detected in the butanol-soluble fraction using a-thiol treatment from each tannin were ana- the HCI-BuOH test. In this experiment the lyzed with thin layer chromatography using purified loquat-tannin was prepared by high precoated silica gel G-60 plates (Merk Co.). porous polymer chromatography with a eth- Solvents used here were: (A) CHCl3-EtOAc- anol-water gradient from 5% to 50%. The f ormic acid = 2 :1:1, v/v, (B) CHC13-EtOAc- yield was 0.48g per 100g of fresh tissue. AcOH=2:1:1, v/v, (C) benzene-AcOH-water Acid degradation of condensed tannins =10:10:1, v/v. The reagent sprays used for All of the five condensed tannins produced visualization were (a) Folin-Ciocalteu reagent some red pigment (s) when treated with 2 N and (b) 10% vanillin in ethanol-HCI. The HCl in a boiling water bath for 60min. The quantitative ratio of pigments or polyphenolics pigment products were examined by paper formed from each tannin was determined from chromatography and cellulose-TLC with three the surface area of the spots on chromato- different solvent systems (i, ii and iii) and grams. The area of each spot was determined identified by comparing their Rf values and by means of a Shimazu DB scanner using a spectrophotometric properties with the data of zigzag scanning mode, after coloration with Harborne (3). The quantitative ratio of the HCI-vanillin reagent (AR=700 nm, A5=530 or pigments produced from a tannin was calcu- 520 nm). Each tannin was methylated by the lated roughly by means of measuring the modified Hakomori's method twice or three surface area of the red spots on cellulose times. Their molecular weight and its dis- plates. 264 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE Table 1. Rf values and spectrophotometric properties of red pigments derived from several condensed tannins Table 2. Chromatographic identification of products given from several condensed tannins by toluene-a-thiol treatment Table3.Quantitativeratioofsomeflavan-3-olsconstitutedeachcondensedtannin Kaki-tannin obtained from young persim- one pigment, designated as Q-1 and L-1 in mon fruit produced two distinct pigments, Table 1. Banana-tannin produced two pig- which had already been identified as delphini- ments with a ratio of about 1 to 1, in addition din and cyanidin. The quantitative ratio lof to a trace of another pigment. They were both pigments was about 1 to 3 (8), Chinese designated as B-1, B-2 and B--3 in increasing quince-tannin and loquat-tannin yielded only order of amount. Carob bean-tannin gave MATSUO AND IT00 : STUDIES OF CONDENSE D TANNINS FROMYOUNG FRUITS 265 one major pigment and a trace of another, catechin and gallocatechin-3-gallate with the designated as C-1 and C-2. All five condensed ratio of about 1 to 3.3 to 4. 6, in addition to tannins also produced amorphous and red- a trace of catechin-like substance. brown phlobaphene-like substances as well as These results are very consistent with the deep red pigments, observed evidently as a results obtained from anthocyanidin examina- broad tailing line near the origin on cellulose- tion, considering the chemical properties of thin layer chromatograms. proanthocyanidin B groups (4) . That is, a The Rf values and spectrophotometric prop- tannin which produced cyanidin, an anthocya- erties of these pigments were examined indi- nidin having two hydroxyl groups on the B vidually and the results were compared in ring, released the thioether of catechin upon Table 1 with the data on anthocyanidins by toluene-a-thiol treatment. Similarly, other Harborne (3). The Table revealed the follow- which produced delphinidin, an anthocyanidin ing; Q-l, L-1 and B-2 were cyanidin, and having three hydroxyl groups on the B ring, C-2 and B-3 were delphinidin. In addition, released the thioether of gallocatechin upon B-1 appeared to be pelargonidin and C-1 was the same treatment. Table 2 indicates two a cyanidin-like compound.