[Agr. Biol. Chem., Vol. 31, No. 2, p. 133•`136, 1967]

Glycosides and Oligosaccharides in the L- Series

Part I. Enzymatic Partial Hydrolysis of Flavonoid-

By Shintaro KAMIYA,Sachiko ESAKIand Misao HAMA Laboratoryof FoodChemistry, Shizuoka Women's Junior College,Shizuoka ReceivedJuly 9, 1966

Naringinase, which was induced from Aspergillus niger, consisted ƒÀ-D-glucosidase and ƒ¿-L-rhamnosidase. The former was successfully inactivated by heating the crude enzyme

solution at 60•Ž and pH 6.4-,-6.8, whereas the latter was very stable under such treat ment. By using this enzyme solution flavonoid gycosides were partially hydrolyzed and

prunin from naringin, isosakuranin from poncirin, hesperetin-7-ƒÀ-D-glucoside from hesperidin and neohesperidin, isoquercitrin from , cosmociin from rhoifolin were obtained respec

tively in good yields. Furthermore kaempherol-3-robinobioside, a new flavonol , and were obtained from and kaempheritrin, respectively.

INTRODUCTION components ƒÀ-D glucosidase and ƒ¿-L-rhamno

The flavonoid-glycosides, which contain L- sidase. The authors have found that ƒÀ-D-

rhamnose, widely occur in nature. Rhamno glucosidase was inactivated by heating at glucoside rutin, hesperidin, neohesperidin, 60•Ž pH 6.4-6.8, though the latter was very naringin, poncirin and rhoifolin are the most stable under such treatment. readily available flavonoid compounds at Furthermore partial hydrolysis of robinin present. The glucosides corresponding to the and kaempheritrin by the same enzyme gave above rhamnoglucosides, which are desired kaempherol-3-robinobioside, a new flavonoid, for biological testing have not been available. and afzelin as the result. To our knowledge, partial hydrolysis of the EXPERIMENTAL rhamnoglucosides to remove only the rham nose and leave the glucose still attached to 1) The Preparation of Enzyme Solution the flavonoid portion has been very difficult. i) Naringinase. The naringinase solution was D. W. Fox, W. L. Savage and S. H. Wender1) prepared according to the preceeding procedure.2) ii) Rhamnosidase. To 1 ml of naringinase solu reported the method of partial hydrolysis of tion was added 2 ml of citrate-phosphate buffer (pH rutin, hesperidin, and naringin by employing 6.8) and heated for one hour at 60•Ž, and the mixture formic acid in cyclohexanol yielding the cor was employed as rhamnosidase solution. responding flavonoid-glucoside but the yields 2) Fractional Measurement of Glucosidase and were comparatively poor. The authors have Rhamnosidase described in other publications the enzymatic The reaction mixture composed of the following

properties of naringinase, which decomposed ingredients was kept for one hour at 50•Ž. the bitter substance of citrus Natsudaidai _??_ 0.5% Narigin 5ml

naringin, induced in Aspergillus niger and the Citrate-phosphate buffer solution (pH 5) 5ml enzyme was found to be composed of two Naringinase solution I ml

1) D. W. Fox, W. L. Savage and S. H. Wender. 2) S. Kamiya, Memoirs of Shizuoka Women's Col J. Am. Chem. Soc., 75, 2504 (1953). lege, 1962, 9. 134 Shintaro KAMIYA, Sachiko ESAKI and Misao HAMA

The reaction mixture was boiled and then a half spraying with aniline hydrogen phthalate solution.

volume of the solution was used to measure all RESULTS amount of the sugar liberated from narigin by Bert

rand method (expressed as mg of glucose). To the 1) The Change of Nariginase Activity by Heat residual volume of the solution was added 0.2g of ing at 60•Ž and at Various pH

Saccharomyces cereoisiae and the mixture was kept 2 A mixture of 1 ml of nariginase solution hours at 30•Ž in order to consume glucose, then the and 2 ml of citrate-phosphate buffer solution, residual sugar was measured and expressed as mg of pH of which varied between 4•`7.2, was glucose. From these result amount of glucose and heated at 60•Ž and glucosidase and rhamno rhamnose liberated from naringin was calculated. sidase activities were measured respectively. 3) Paper Chromatography The results were shown in Fig. 1. Paper chromatography of flavonoid and sugar com In the region of pH 4•`5, two enzymes pounds was carried out as follows: were stable but on the contrast at pH 7.2 Paper chromatograms of flavonoids were developed both were completely inactivated after 40 with the solvent systems of BuOH:AcOH:H2O (4:1:2) minutes. In the region of pH 6.4" 6.8, ƒÀ-D- and of AcOH:H2O (6:1) by ascending method and detected by spraying with diazotized sulfanilic acid glucosidase was inactivated after 40 minutes solution. while ƒ¿-L-rhamnosidase was stable.

Paper chromatograms of the sugar compounds were 2) Prunin from Naringin developed with the solvent system of BuOH:AcOH: To a solution of 5 g of naringin in 900ml H2O (4:1:2) by ascending method and detected by of water (pH 6.8) was added 645ml of

FIG. 1. The Change of ƒÀ-Glucosidase and ƒ¿-Rhamnosidase Activity at 60•Ž , pH 4•`7.2 Values. Glycosides and Oligosaccharides in the L-Rhamnose Series. Part I 135

rhamnosidase solution and the mixture was 3.5 g (91%), mp 210•Ž. Anal. Found: C, kept overnight at 45•Ž. 55.33; H, 5.18. Calcd. for C22H24O11H2O: C Prunin and rhamnose were detected by , 54.77; H, 5.02%.

paper chromatography but naringin and 5) Hesperetin-7-Glucoside from Neohesperidin glucose were not. Then the reaction mixture To a solution of 5 g of neohesperidin in was concentrated under reduced pressure to 250 ml of water, pH 6.6, was added 600 ml a small quantity and on cooling the crystals of rhamnosidase solution and the mixture was were deposited. After filtration it was re allowed to stand overnight. The reaction crystallized from aqueous alcohol to yield mixture was concentrated under reduced

3.5 g (9000) of prunin, mp 225•Ž, undepressed pressure, yielding the crystals on cooling. on admixture with authentic prunin. RF Recrystallization from ethanol gave 3.6 g value and UV spectrum were found to be (93%) of the glucoside, mp 210•Ž. Anal. identical with those of authentic prunin. Found: C, 55.12; H, 5.66. Calcd. for 3) Isosakuranin from Poncirin C22H24O11H2O: C, 54.77; H, 5.02%. RF value To a solution of 1 g of poncirin in 400ml and UV spectrum were identified as described of water, pH to 6.8 by dilute alkali, was above.

added 50 ml of rhamnosidase solution. After 6) Isoquercitrin from Rutin the mixture had been left to stand overnight To a solution of I g of rutin in 90 ml of at 45•Ž, the crystals were deposited. The water was added 70 ml of rhamnosidase solu crystals were collected by filtration, and were tion and the reaction mixture was kept over- washed with ether. Crystallization from hot night at 40•Ž. water and then from methanol gave 3.6g On removal of the solvent and cooling the (92%) of isosakuranin (mp 177•Ž), undepressed crystals were deposited. Recrystallization on admixture with authentic isosakuranin. from water gave the glucoside, 0.66g (96%), RF value and UV spectrum were also found mp 219•`220•Ž, undepressed on admixture to be indistinguishable from those of authentic with authentic isoquercitrin. UV spectrum specimen. and RF value were found to be indistinguish On hydrolysis with 5% sulphuric acid solu able with those of authentic specimen. tion it afforded glucose and isosakuranetin. On hydrolysis with 5% H2SO4 it afforded

4) Hesperetin-7-Glucoside from Hesperidin and D-glucose. To a suspension of 1 g of hesperidin in 7) Cosmociin from Rhoifolin 500 ml water was added 40% NaOH solution A suspension of 1 g of rhoifolin in 200 ml untill the pH of the suspension reached to of water was made pH 10 with 5% NaOH 9.5, whereupon hesperidin was dissolved solution, whereupon the materials dissolved completely. completely. After being adjusted the pH the Then pH of the solution was adjusted to solution of 6.7 with sulphuric acid the solution 6.8 by adding 5% HCl solution and to the was added to 50 ml of rhamnosidase solution solution was added 100 ml of rhamnosidase and the mixture was kept 5 hours at 60•Ž. solution and 0.4 g of methylcellulose in order The reaction mixture was concentrated under to inhibit the deposition of hesperidin during reduced pressure, whereupon the crystals were the reaction. deposited.

After being kept overnight at 45•Ž, the Recrystallization from water gave 0.7 g (93%) solution was concentrated and on cooling the of the product, mp 178•`180•Ž, Lit. 178•` crystals were deposited. Recrystallization 180•Ž. Anal. Found: C, 56.20; H, 5.12. Calcd. from ethanol several times gave the product, for C21H20O10•E2H2O: C, 56.00; H, 4.9000. 136 Shintaro KAMIYA, Sachiko ESAKI and Misao HAMA

8) Kaempherol-3-Robinobioside from Robinin After cooling of the reaction mixture in the To a solution of 4.6 g of robinin in 41 of ice-box overnight, a brownish substance sepa water 150 ml of rhamnosidase solution was rated was collected, washed with cold water added and the mixture was kept 6 hours at and recrystallized from aqueous methanol to

50•Ž. After boiling, the reaction mixture was yield slight yellow needles. It melted at 148- concentrated to 50 ml and allowed to stand in 150•Ž and showed no depression on admixture an ice-box for several days. The crystals with 3-hydroxy-5, 7, 4-trimethoxyflavone mp were deposited gradually. 149•Ž. Anal. Found: C, 65.58; H, 4.71. Calcd. After collection by filtration, they were for C15H7O3(OCH3)3: C, 65.85; H, 4.88%. dissolved in hot alcohol and filtrate was evapo Thus in the new glycoside, robinobiose rated under reduced pressure. residue is at the 3-position of kaempherol.

The residue was crystallized from hot water 9) Afzelin from Kaempheritrin to yield 1.6 g of the product. This new To a solution of 2 g of kaempheritin in 200 glycoside gave brownish-green colour with ml of water was added 50 ml of rhamnosidase FeCl3 and red colour by Mg-HCl and Zn-HCl solution and the mixture was allowed to stand test and was long needles of 'slight yellow overnight at 30•Ž. After filtration the filtrate colouration, mp 225.226•Ž (decomp. 228- was evaporated to about 20 ml under reduced 230•Ž). pressure. It was soluble in hot water, aqueous alkali, After being kept for several days in the ethanol, methanol, ethyl acetate and insoluble refrigerator crude yellow substance was de in ether, acetone, chloroform, benzene and posited. The material collected by filtration, and ligroin. RF and UV spectrum were found washed with cold water and recrystallized to be distinguishable with those of and from aqueous methanol to gave pale yellow afzelin. Anal. Found: C, 49.88; H, 5.5. Calcd. granules, mp 171-172•Ž undepressed on ad- for C27H30O15 3H2O: C, 50.00; H, 5.55%. mixture with authentic afzelin. Afzelin con Hydrolysis of 0.1 g of the glycoside with 5% tains 1(1/2) H2O of crystallization when it is sulphuric acid gave 43 mg of kaempherol (mp dried at 60-.-70•Ž. This water is lost on 276•Ž) and 51 mg of sugars which were found heating at 100•Ž in vacuo over P2O5. It is to be composed of and rhamnose easily soluble in acetone, difficulty soluble in by paper chromatography. hot methanol, ethanol and water and insoluble In order to determine the position of sugar in cold water, benzene and chloroform. RF attached to aglycon methylation was performed. value and UV spectrum were found to be To a supension of 0.2 g of the glycoside in indistinguishable from those of afzelin. Anal. 20 ml of acetone, 6 g of potassium carbonate Found: C, 54.86; H, 5.04. Calcd. for C21H20O10 and 2 ml of dimethyl sulphate were added 1(1/2)H2O: C, 54.90; H, 5.01%. and the mixture was refluxed. After 7 hours Hydrolysis of afzelin with 5% sulphuric acid 20 ml of acetone, 6 g of potassium carbonate solution yieled kaempherol and L-rhamnose, and 2 ml of dimethyl sulphate were added which were identified with authentic specimen. again to the reaction mixture and the whole

refluxed for 7 hours until colour reaction with Acknowledgement. The authors should like FeCl3 was quite negative. to express their thanks to Dr . Toshio Naka Then the solution was filtered from mineral bayashi of Shizuoka University for his kind salts and evaporated in vacuo. The residue supply of some flavonoid glycosides. They was dissolved in 30 ml of 5 % sulphuric acid are also grateful to Miss Yoshiko Saito of solution and heated on a boiling water bath Shizuoka Pharmaceutical college for element for 32 hours. ary analyses.