Carbohydrate structures of three new saponins from the root bark ofHovenia dulcis(Rhamnaceae)

摘要

1978 1289 Carbohydrate Structures of Three New Saponins from the Root Bark of Hovenia dufcis (Rhamnaceae) By Osamu Inoue, Tadahiro Takeda, and Yukio Ogihara,' Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabedori, Mizuhoku, Nagoya 467, Japan The carbohydrate sequences of two major saponins, hovenosides D (1) and G (2) and a minor saponin, hovenoside I (3), obtained from the root barks of Hovenia dulsisThunb (Rhamnaceae), have been determined mainly by g.1.c.- mass spectrometry, and the full molecular structures of these saponins have been assigned as 3-0-(2-0-p-D-xylo-pyranosyl) -3-0-(2-~-~-D-xylopyranosyI-6-~-~-D-glucopyranosy~-~-D-glucopyranosyl)-a-L-arabinopyranosyl-juju bog en in, 3-0-(2-0-p-D-xylo pyra nosy 1 ) -3-0-(2-0-p-D-xy lopyranosyl-p-D-g lu copyranosy I) -a-L-arabin0-pyranosyljujubogenin.and 3-0-(2-0-~-D-xylopyranosyl)-3-0-~-D-glucopyranosyl-a-~-arabinopyranosy~juju-bogenin respectively from additional chemical and spectroscopic evidence. Correlations among these saponins have been obtained by the action of enzymes. PREVIOUSpapers have described the isolation of the 187) in the mass spectrum. The methanolysis product new saponins hovenosides C, D, G, G', and H by droplet of (8) was deuteriomethylated with silver oxide and counter-current chromatography (d.c.c.) from the trideuteriomethyl iodide in dimethylformamide to give methanolic extracts of the root bark of Hovenia dulcis two kinds of methylated sugar. These were identified Thumb (Rhamnaceae) (the Japanese name is ' ken-by g.1.c.-mass spectrometry as methyl a-and P-pyrano- ponashi,' and the seeds are used as a folk medicine), and sides of 2,3,4,6-tetra-O-methylglucoseand 3-O-tri-some chemical studies of hovenoside G.This paper deuteriomethyl-2,4-di-O-methylarabinosea- and p-forms deals with a study of the carbohydrate moieties of are not separated from each other on g.1.c. and the lH hovenosides D (l),G (2), and I (3), the corresponding n.m.1. spectrum of this fraction shows two anomeric sapogenin of which is jujubogenin (6). proton signals at 6 4.03 (d, J 3.0Hz) and 4.16 (d, J 4.5 On Smith-de Mayo degradati~n,~ the saponins (1)-Hz). The structure of the latter was confirmed by (3) afforded the prosapogenin (5), C,,H,O,, which was analysis of the mass spectrum, in which the fragment hydrolysed with N-sulphuric acid to yield a secondary ions, CH,O-CH=CH-OCD, (m/e 91, 90) and CH,O- sapogenin, ebelin lactone (4),5 and L-arabinose (positive CH=CH-CHamp;H, (chief contribution to mle 101, base plane 0.r.d.curve 6). On a second Smith-de Mayo peak) lo were observed. From these data, the pro- degradation, (5) gave jujubogenin (6),' whose structure sapogenin (5) and compound (7) were determined to be was confirmed by X-ray crystallography. In the lH 3-0- (a-~-arabinop yranosyl) juj ubogenin and 3-0- (3-0- p-n.m.r. spectrum of (5),signals at 6 5.24 (1 H, d, J 9 Hz), D-glucopyranosyl-a-L-arabinopyranosyl)ebeh lactone,4.70 (1 H, m), and 4.01 (2 H, br,s) are assigned re- respectively.spectively to protons on C-23, C-22, and C-30 of the When hovenoside G (2) was incubated with jujubogenin moiety, and an anomeric proton signal at naringinase f (a mixture of naringinase, naringin-P-l,2- 6 4.36(d,J 5.5Hz) indicates the a-linkage of L-arabinose rhamnosidase, and p-glucosidase) , the glycoside (3) was (4C, conformation,)to the genin.A partial hydrolysis slowly produced. This was identical with natural hovenoside I (3). On acidic hydrolysis, hovenoside I (3)furnished (4)and L-arabinose, D-xylose, and D-glucose as sugar components (molar ratio 1 : 1: l), while (2) afforded 2 mol. equiv. of D-xylose. This indicated that a mole of D-xylose was liberated from (2) to produce (3). Its per-0-methyl derivative (9) of (3), prepared by the Hakomori method,ll exhibits fragment of hovenoside G (2), followed by repeated purification by column chromatography and preparative t .l.c., furnished a compound (7), which gave (4) and L-arabinose and D-glucose as sugar components (molar ratio 1: 1).Compound (7) exhibits five-membered lactonic (1 768 cm-l) absorption in the i.r. spectrum and strong U.V. absorption at 270, 278, and 290 nm characteristic of a conjugated triene, suggesting the existence of the ebelin lactone moiety in (7). Its per-0-methyl cleriva- tive (8), prepared by the Kuhn meth~d,~ exhibits a molecular ion (mle 832) and a peak due to a terminal permethylated hexose residue (m/e 219 coupled with ions due to the terminal permethylated pentose (m/e 175 and 143) and hexose (m/e219 and 187) residues in the mass spectrum and lH n.m.r.signals for three anomeric protons at 6 4.37 (d, J 5.5 Hz), 4.65 (d, J 7.5 Hz), and 4.69 (d, J 7.5 Hz), assigned respectively to a-L-arabinopyranose, p-D-glucopyranose (or xylose) in t Sigma Chem Co., Lot No. 4OC-1500-1. the 4C1 conformation, and p-D-xylopyranose (or glucose) K. Kawai, T. Akiyama, Y. Ogihara, and S. Shibata, Phyto-chemistry, 1974, 13,2829. 7 K. Kawai, Y. Iitaka, and S. Shibata, Acta Cryst. (B),1974, 2886. 8 R. Higuchi, K. Miyahara, and T. Kawasaki, Chem. and Pharm. Bull. Japan, 1972, 20, 1935. R. Kuhn, Angew. Chem., 1955, 67,32. lo N. K. Kochetkov and 0. S. Chizhov, (a) Tetrahed??on, 1965,21, 2029; (b) Adv. Carbohydrate Chem., 1966, 21, 39; (c) N.K. Kochetkov, N. S. Wulfson, 0. S. Chizhov, and B. M. Zolotarev, Tetrahedron, 1963, 19,2209. l1 S. Hakomori, J. Biochem. (Japan), 1964, 55, 205. Y. Ogihara, 0. Inoue, H. Otsuka, K. Kawai, T. Tanimura. and S.Shibata, J. Chromatog., 1976, 128,218. 3 M. Takai, Y. Ogihara, and S. Shibata, Phytochemistry, 1973,12, 2985. J. J. Dogan and P. de Mayo, Canad. J. Chem., 1965,43,2033. 6 R. A. Eade, L. P. Rossler, H. V. Simes, and J. J. H. Simes, Austral. J. Chem., 1965, 18,1451. I. Listowsky, G. Avigad, and S. England, J. Amer. Chem. Soc., 1965, 87,1765. 1290 J.C.S. Perkin I in the 4C, conformation. This conformational analysis identification of the last compounds was confirmed by is supported by application of Klyne's rule l2 (Table).the typical fragment ions, CD30-CH=CH-OCD, (m/e ROH2C OR (1) R = H (13)R= Me jujubogenin (6) ...litli (2) -0 R=H bsol; iH(5) (10) R= R'=Me iv (11) R =Me,R'=H RO OR ebelin lactone (4) OR lii OR OR (7') R = H Ro k?R Y (8) R= Me ROY OR (3) R=H (9) R=Me Reagents: i, snail enzyme or hesperidinase ; ii, naringinase; iii, Smith-de Mayo degradation ; iv, N-H,SO, at 100 "C for 5 h; v, O.~N-HCLat 100 "C for 6 h; vi, H,,Pt,O The methanolysis product of (9) was deuteriomethylated 94, 84 yo)and CH30-CH=CH-CH=6CH3 (m/e 104, base without further purification to give three kinds of peak). Therefore hovenoside I (3) is 3-0-(2-0-p-~-methylated sugar. These were identified by g.1.c.-mass xy~opyranosy~)-3-O-~-~-g~ucopyranosy~-a-~-arab~no-spectrometry as methyl a-and p-pyranosides of 2,3,4-pyranosyl jujubogenin.tri-O-methylxylose, 2,3,4,6-tetra-O-methylglucose,and Methylation of hovenoside G (2) by the methods of 2,3-bis-0-trideuteriomethyl-4-O-methylarabinose.The Hakomori and Kuhn gave the undeca-O-methyl deriva- l2 W. Klyne, Biochem. J., 1950, 47, xli. tive (10) and the deca-0-methyl derivative (ll), re- spectively. The lH n.m.r. spectrum of (10) exhibits an extra signal for the tertiary OCH, group at 6 3.16 (3 H, s). This indicates that all carbohydrate moieties Molecular rotations Saponins alD ("1 (") -AMlD (") (6) -36 -169.6" } +41.9" (5) -21.2 -128.0" } -89.3" (3) -24.2 -217.3" } -54.6" (2) -26.4 -271.9" } -80.9" (1) -29.9 -352.8" The following MID values were used: Me a-L-arabino- pyranoside +17.3"; Me p-L-arabinopyranoside +245"; Me a-D-glucopyranoside +11 1" ; Me p-D-glucopyranoside -66"; Me a-D-xylopyranoside +252"; Me p-D-xylopyranoside -108".of (2) are linked to the genin at the C-3 hydroxy group. The methanolysis product of (11) was treated as men- tioned above and the reaction mixture was examined by g.1.c.-mass spectrometry. This indicated the pre- sence of methyl a-and p-pyranosides of 2,3-di-O-tri-deuteriomethyl-4-O-methylarabinose, 2,3,4-tri-O-methyl-by Hakomori's method, revealed mass fragment ions due to the terminal permethylated pentose (m/e 175) and hexose (m/e 219) residues, and gave on methano- lysis four kinds of methylated sugar.They were identi- fied by g.1.c. (with authentic samples) as methyl a- and p-pyranosides of 2,3,4,6-tetra-O-methylglucose,2,3,4-tri-O-methylxylose, 4-O-methylarabinose, and 3,4-di-0-methylglucose. In the lH n.m.r. spectrum of (13), the anomeric proton signals appear at 6 4.25, 4.62 (2 H, br,d, J 7.5 Hz: accidental coincidence), 4.95, and 4.32 (d, J 7.5 Hz). The last anomeric proton signal is assigned to the second p-D-glucopyranose (4C1 conform- ation). Consequently hovenoside D (1) is identified as 3-0-(2-0- p-D-xylopyranosyl) -3-0- (2-0-p-~-xylopyranosyl-6-0-p-D-glucopyranosyl- p-D-glucopyranos yl) -a-L-arabino- pyranosyl jujubogenin. Other saponins (C, G', etc.) from the same materials whose sapogenin is different from jujubogenin are under investigation.EXPERIMENTAL (2-0-P-D-x ylop yranos yl- p-D-glucopyranosyl) -a-~-ara-binopyranos yl juj ubogenin. Generally speaking, the purification of highly polar saponins is extremely troublesome and it is also very difficult to obtain satisfactory evidence of purity. Repeated d.c.c. and silica gel column chromatography gave pure hovenoside D (1)as a colourless powder whose purity was proved by 13C n.m.r. spectroscopy.13 D.c.c. and 13C n.m.r. are very powerful weapons in this field. Acidic hydrolysis of the saponin (1) furnished (4) and L-arabinose, D-xylose, and D-glUCOSe as sugar com-ponents (molar ratio 1 :2 : 2). When (1) was incubated with snail intestinal juice (crude solution of p-glucuroni- dase) * or hesperidinase t (a mixture of hesperidinase, hesperidin- p-l,6-rhamnosidase, and p-glucosidase), the glycoside (2) was obtained almost quantitatively in a few days.The product (2) was identical with natural hovenoside G (2) in all respects including 13C n.m.r. spectrum. Its per-0-methyl derivative (13), prepared * SUC Helix Pomatia, Industrie Biogique Francaise F.F. 1828-6-70. t Tanabe Pharm. Ind. Co., Ltd., CW 0290M. crystals, a,determined to be 3-0-(2-0-p-~-xylopyranosyl)-3-0-xylose, and 2-O-trideuteriomethyl-3,4,6-tri-O-methyl-glucose. The identification of these last compounds was confirmed by the fragment ions, CD,O-CH=CH- OCH, (m/e 91, base peak in a-and p-forms) and CD,O-CH=CH-CH=amp;H, (m/e 104, 60 in a-form and 70 in p-form).In the lH n.m.r. spectrum, over-lapping of two anomeric and the C-23 proton signals obscured the analysis. So (11) was hydrogenated over Adams catalyst to afford the dihydro-derivative (12). The lH n.m.r. spectrum of (12) was clear, showing four sets of anomeric proton signals at 6 4.27, 4.64, 4.72, and 4.96. A new signal at 6 4.96 (d, J 7.5 Hz) is assigned to that of the second p-D-xylopyranose (4C, conform-ation). Thus the structure of hovenoside G (2) was M.p.s were measured with a Yanagimoto micro-apparatus. Unless otherwise stated, U.V. spectra were taken for solu- tions in ethanol, i.r. spectra for KBr discs, lH n.ni.r. spectra for solutions in hexadeuterioacetone, and optical rotations for solution in methanol. G.1.c.-mass spectrometry was carried out with a Shimadzu LKB-9000 instrument lo DEGS-Chromosorb W (60-80 mesh).D.c.c. experiments were carried out on home-made d.c.c. apparatus equipped with 500 columns (1.65 x 400 mm) by the ascending process moving phase, upper layer ; stationary phase, lower layer of (A) CHC1,-MeOH-H,O (35 : 65 : 40) or (B) CHC1,-MeOH-H,O (50 : 60 :40)l. Puri$cation of Saponins.-In the previous paper we have described the isolation of hovenosides by d.c.c. In that work only hove.pzoside G (2) was obtained as pure -26.4' (c 1.00) (Found: C, 55.2; H, 8.0. C,,H8,0,,*4H,0 requires C, 55.5; H, 8.2). The hoveno- side D fraction was chromatographed on silica gel lower layer of CHC1,-MeOH-H,O (7 : 3 : l) to afford pure hovenoside D (1) as a white powder from aqueous butanol, m.p.205-210', a, -29.6' (c 0.50), v,,,. 3 400 and 1 100-1 000 cm-l; no U.V. absorption above 210 nm (Found: C, 55.95; H, 7.9. C,,H,,0,,*2Hz0 requires C, 55.7; H, 7.85). On a prepacked column (Merck Kieselgel 60), the so-called hovenoside H fraction was separated into three pure compounds, hovenosides H,, H,, and I (3). Hovenoside 1 (3) was obtained from methanol as colourless needles, m.p. 272-274', a, -24.2' (c 0.50), v,,,. 3 400 and 1 100-1000 cm-l; no U.V. absorption above 210 nm (Found: C, 56.95; H, 8.45. C4,H,,01,-4H20 requires C, 56.9; H, 8.5). Acidic Hydrolysis of Suponins.-Hovenoside D (1) (5 mg) was dissolved in dioxan (1 nil), ~N-H,SO, (2 ml), and water (1 ml) and heated under reflux for 5 h.The solution was diluted with water and extracted with ether. From this layer, ebelin lactone (4) was obtained. The aqueous layer of the hydrolysate was neutralized with ion-exchange resin (IR-45) and evaporated. Trimethylsilylation followed by l3 0. Inoue, Y. Ogihara, and K. Yamasaki, J. Chem. Research, 1978(S), 144. g.1.c. 2 OV-1 on Chromosorb W (60-80 mesh) (3 mm x 2 m); column temperature 160"; N, gas flow rate 70 ml min-l showed the presence of arabinose, glucose, and xylose in the ratio 1 : 2 : 2. In the case of hovenosides G (2) and I (3), arabinose, glucose, and xylose were identified in the ratios 1: 1 : 2 and 1 : 1: 1, respectively. Smith-de Mayo Degradation of Hovenoside G (2).-To a cooled solution of hovenoside G (2) (500 mg) in water (50 ml) was gradually added sodium periodate (1 g) with stirring, and the mixture was stirred at room temperature for 4 days, then extracted with butan-1-01.The organic layer was washed with water and evaporated to dryness below 60 "C under reduced pressure. The residue was dissolved in aqueous 5 KOH (25 ml) and heated under reflux in argon for 2 h. The mixture was acidified to pH 3 by careful addition of 10 H3P04 and extracted with butanol. The butanol layer was washed with water and evaporated to dryness and the residue was purified on a silica gel column methanol-chloroform (1: 5) to give the J.C.S. Perkin I removal of catalyst, the filtrate was concentrated to dryness and the residue purified on silica gel n-hexane-acetone (5 : l) to afford the dihydro-derivative (12) (21 mg), colour- less needles from n-hexane-acetone, m.p.127-130", 6 4.27 (1 H, d, J 5.5 Hz), 4.64 (1 H, d, J 7.5 Hz), 4.72 (1 H, d, J 7.5 Hz), and 4.96 (1 H, d, J 7.5 Hz) (Found: C, 61.7; H, 9.1. C,1H1,40,1*H,0 requires C, 61.5; H, 8.95). Permethylation of Saponins by the Hakomori Method.- To a stirred solution of hovenoside I (3) (1 10 mg) in dimethyl sulphoxide (10 ml) under argon was added a solution (20 ml) of methylsulphinylmethanide and the mixture was stirred at room temperature for 5 h. Methyl iodide (10 ml) was then added and the reaction was continued for 24 h. The mixture was poured into water and extracted with chloroform.The organic layer was washed with aqueous 5 Na,S,O, and water and evaporated. The residue was chromatographed on silica gel n-hexane-acetone (5 : l) to give the nona-O-methyl derivative (9) (27 mg), colourless needles, m.p. 135-137"; no OH i.r. absorption (CCl,); prosapogenin (5), 3-0-(a-~-arabinopyranosyl)jujubogenin,G(CDC1.J 3.16 (3 H, S, 20s-OCHJ, 4.37 (1 H, d, J 5.5 Hz), obtained from methanol-chloroform (1: 2.5) as colourless 4.65 (1H, d, J 7.5 Hz), and 4.69 (1H, d, J 7.5 Hz) (Found: needles (45 mg), m.p. 237-241deg;, a, -21.2O (G 0.50), C, 63.6; H, 8.8. C,,H,,O,,*H,O requires C, 63.3; H, GCDCl,CI,OD (2.5: l) 4.36 (1 H, d, J 5.5 Hz) (Found; 9.1Yo).C, 67.8; H, 9.3. C,,H,,O,*H,O requires C, 67.5; H, Hovenoside G (2) (500 mg) was methylated and the 9.4).Hovenosides D (1) and I (3) also gave (5) by the product worked up as for (3) to give the undeca-O-methyl same procedure. derivative (10) (198 mg), colourless needles, m.p. 120-Isolation of L-Arabinose.-The prosapogenin (5) (30 mg) 123", a, -31.3" (G 1.00), m/e 379 (permethylated di-was hydrolysed in the same way as before and the resulting saccharide residue), G(CDC1,) 3.16 (3 H, s) and 5.24 (1 H, d, sugar portion was purified by d.c.c. solvent system (A) to J 9 Hz, olefinic) (Found: c, 62.6; H, 9.05. C62H1,4021 give 4.2 mg of L-arabinose (positive plane 0.r.d. curve in requires C, 62.8; H, 8.85). water). Hovenoside D (1) (30 mg) was methylated and the Partial Hydrolysis of Hovenoside G (2).-Hovenoside G product worked up as for (3) to give the tetradeca-O-methyl (2) (2 g) was dissolved in dioxan-0.h-HC1 (1 : 3; 60 ml) derivative (13) (10 mg), a colourless syrup, m/e 219, 187, and heated under reflux for 6 h.The mixture was diluted 175, and 143, 6 4.17 (3 H, s), 4.25 (1 H, d, J 5.5 Hz), 4.32 with water and extracted with butan-1-01. The organic (1 H, d, J 7.5 Hz), 4.62 (2 H, d, J 7.5 Hz), 4.95 (1 H, d, J layer was washed and evaporated to dryness and the 7.5 Hz), and 5.10 (1H, d, J 9 Hz). residue chromatographed on silica gel methanol-chloro-Methanolysis of Permethylated Saponins, followed by form (1 : 5). Further purification (preparative t.1.c.) gave DeuteriomethyZution.-A solution of the sample (5-10 mg) compound (7) (20 mg) , 3-0-(3-0-fi-~-glucopyranosyl-a-~-in 5 HC1-methanol was refluxed for 2 h and evaporated arabinopyranosyl)ebelin lactone, as a white powder, m.p.198-203" (decomp.), vnUx. 1768 cm-l, A,,,. 270, 278 (E 11 850), and 290 nm (Found: C, 62.8; H, 8.55. C41Hamp;,* 2H,O requires C, 62.75; H, 8.75). Compound (7) was hydrolysed in the usual way. From the organic layer, (4) was obtained, and L-arabinose and D-glucose were detected on g.1.c. in the ratio 1 : 1. Methylation of Compound (7) and Saponins by the Kuhn Method.-Compound (7) (85 mg) was methylated in di- methylformamide (2 ml) with freshly prepared silver oxide (430 mg) and methyl iodide (3 ml) at 100 "C for 15 h accord- ing to the Kuhn method. The precipitate was filtered off, and the filtrate was evaporated to dryness.The residue was chromatographed on silica gel n-hexane-acetone (5 : l) to give the per-O-methylpyranoside (8) (10 mg), white powder, m.p. 111-113", m/e 832 (AT'), 454 (ebelin lactone moiety ; C,,H4603+), and 21 9 (terminal permethylated hexose residue). Hovenoside G (2) (2 g) was methylated in the same way to give the deca-O-methyl derivative (1 1) ( 1 13 mg), colourless needles from n-hexane-acetone, m.p. 150-152", a, -33.0' (c 1.00), vmax.(CC14) 3 440w cm-l (Found: C, 61.3; H, 8.65. C,,H1,,O,,*H,O requires C, 61.55; H, 8.8). Hydrogenation of Compound (1 1) .-Compound (1 1) (90 mg) was hydrogenated over Adams catalyst in ethanol at room temperature overnight (uptake 1 mol. equiv.). After (hydrogen chloride removed under reduced pressure).The residue was deuteriomethylated in dimethylformamide (0.8 ml) with freshly prepared silver oxide (130 mg) and trideuteriomethyl iodide (0.4 ml) at 100 "C for 15 h. The precipitate was filtered off and the filtrate was evaporated to dryness. The residue was chromatographed on silica gel (2 g) n-hexane-acetone (5: l) to afford a sample for g.1.c.-mass spectrometry conditions for g.1.c. : 10 DEGS on Chroniosorb W (3 mni x 2 m) ; column temperature 16001. (a) In the sample from compound (euro;9, methyl a- and P-pyranosides of 2,3,4,6-tetra-O-methylglucoseand 3-O-tri- deuteriomethyl-2,4-di-O-methylarabinosem/e 179 (5y0), 178 (a),101 (looyo),and 91 (90y0) were identified. (b) In the sample from compound (9), methyl a-and P-pyranosides of 2,3,4-tri-O-methylxylose, 2,3,4,6-tetra-O-methylglucose, and 2,3-bis-O-trideuteriomethyl-4-O-methyl-arabinose m/e 182 (2), 181 (4), 104 (loo), and 94 (84y0) were identified.(c) In the sample from compound (lo), methyl a-and P-pyranosides of 2,3-bis-O-trideu teriomethyl-4-O-methyl- arabinose, 2,3,4-tri-O-methylxylose, and 2-O-trideuterio-methyl-3,4,6-tri-O-methylglucosem/e 222 (0.6y0), 208 (0.2y0), 104 (60y0), and 91 (looyo)in a-form; 222 (0.16y0), 208 (0.5), 104 (70y0), and 91 (looyo)in p-form were identified. (d) In the case of compound (13), methylated sugars, obtained by methanolysis, were identified as methyl a-and p-pyranosides of 2,3,4,6-tetra-O-methylglucose,2,3,4-tri-O-methylxylose, 4-O-methylarabinose, and 3,4-di-0-methylglucose on g.1.c.by comparison with authentic samples. Hydrolysis of Saponins with Crude Enzymes.-(a) Hoveno-side D (1) (60 mg) in distilled water (50 ml) was incubated with commercial snail juice (1 ml) at 30 "C for 24 11 and the hydrolysate was extracted with butan-1-01 saturated with water. The organic layer was concentrated to dryness and subjected to d.c.c. with solvent system (A) to give crystal- line hovenoside G (2) (22 mg). (b) Hovenoside D (1) (50 mg) in citrate-phosphate buffer solution (pH 3.9) (50 ml) was treated with commercial hesperidinase (55 mg) at 30 "C for 3 days and the product worked up as above to afford pure (2) ( 13 mg) . (c) Hovenoside G (2) (120 mg) in acetate buffer solution (pH 5.1) (50 ml) was incubated with commercial naringinase (300 mg) at 30 "C for 7 days and the product worked up as usual, but with solvent system (B),to furnish pure crystals of hovenoside I (3) (20 mg) and starting material (80 mg). We thank Professor S. Shibata, Meiji College of Pharmacy, for advice. We also thank Professors 0. Tanaka and K. Yamasaki, University of Hiroshima, for measurement of 13C n.1n.r. spectra, and I. Kitagawa, University of Osaka, and T. Kawasaki, University of Kyushu, for samples. Thanks are also due to Hoansha for a grant. 7/1768 Received, 7th October, 19771