[Agr. Biol. Chem., Vol. 36, No. 9, p. 1461-1466, 1972]

On Neocriocitrin and Veronicastroside in Citrus•õ

By Shintaro KAMIYA, Sachiko ESAKI and Fukuko KONISHI

Food Chemistry Laboratory, Shizuoka Women's College,

Shizuoka City, Japan

Received November 17, 1971

The sugar moiety of isonaringin (I) was confirmed as rutinose using NMR spectral data. Furthermore, the apigenin-7-rhamnoglucoside (IV), derived from compound I was successfully

converted to linarin by partial methylation with dimethyl sulfate, indicating that compound IV

has the structure of apigenin-7-ƒÀ-rutinoside. Thus, compound I was identical with narirutin. The optical rotatory dispersions of narirutin, didymin and neoeriocitrin were measured.

The former two compounds have the aglycones of 2 (S) configuration, and in the latter aglycone is racemized.

The, so called, lonicerin distributed in citrus plants was proven to be identical with veroni castroside.

This paper deals with supplemental material here the NMR spectra of acetyl and trimethyl to a series of papers'," already published. silyl derivatives of compounds I, II and IV, Isonaringin2) (I) contained in various kinds of and discuss their sugar structures. citrus plants could be converted to didymin For comparison, NMR spectra of the same

(II) and also to linarin (III), indicating that the derivatives of rutinose were also measured. sugar moiety of compound I is rutinose. In addition, a simplified preparation of ruti Based on these results, compound I is con nose (V) was also given. sidered to be identical with narirutin. In trimethylsilyl ethers of compounds I, Dehydrogenation of compound I, however, II, IV and V, the C-1 proton of afforded apigenin-7-ƒÀ-rhamnoglucoside (IV), appears as a doublet at ƒÂ4.35•`4.4 (J=2 Hz); mp 182•`183•Ž, the sugar portion of which whereas, the methyl proton signal occurs as must be rutinose. Contrary to our ex abroad peak in the range of 60.8---1.1. Data pectations, compound IV was not identical for compound I are shown in Fig. 2. with synthetic isorhoifolin, (mp 268•`270°C) On the other hand, spectra of their acetyl as reported by Wagner et al.,3) in its mp and derivatives exhibit signals for sugar protons IR spectra as shown in Fig. 1. in three distinct areas. The methyl protons

This sharp discrepancy prompted further of rhamnose come as a doublet (J=6 Hz) at investigation of the sugar moiety of compound 61.15-1.2. The signals integrating eight

I using NMR spectroscopy. protons, from the hydrogens at positions Rosier et al." reported that in NMR spectra 1, 2, 3 and 4 of and at 1, 2, 3 and 4 of acetyl- and trimethylsilyl derivatives of of rhamnose absorb around 64.7---5.5 while flavonoid rutinosides, signals for the sugar the signals at 63.1-4.2, integrating four protons are very chracteristic. We report protons, represent hydrogens at the 5 and 6

positions of glucose and the 5 position of •õ Flavonoids in Citrus and Related Genera (IV). rhamnose. The C-1 proton of rhamnose The previous paper, M. Nishiura, S. Kamiya and S. Esaki, Agr. Biol. Chem., 35, 1691 (1971). is characterized by a signal occurring near 1462 S. KAMIYA, S. ESAKI and F. KONISHI

FIG. 1. Comparison of Infrared Spectra of ƒ¿-Isorhoifolin (---) and ƒÀ-Isorhoifolin (-),

FIG. 2. The NMR Spectrum of Octatrimethylsilyl FIG. 3. The NMR Spectrum of Octaacetylnarirutin. narirutin. structure of apigenin-7-ƒÀ-rutinoside. ƒÂ4.70 as a sharp doublet, clearly separated Accrodingly, there must be two forms of

from the others in the lower field area. NMR isorhoifolin and we propose a-form as the data for the octaacetate of compound I is name for the compound obtained by Wagner

given in Fig. 3. These results are characteristic et al. and ƒÀ-form for the one we obtained. rutinose and are in accord with Rosler's The ƒÀ-form of isorhoifolin has three moles

report.4) of crystallization water. After drying it at

Thus, the sugar moiety of compound I was 150°C, the crystallization water was lost but shown to be rutinose from its NMR spectral its mp was raised slightly to 185•‹•`190•Ž.

data. The difference between the ƒ¿- and ƒÀ-forms of

Furthermore, compound IV could be con isorhoifolin can be ascribed to its polymor

verted to linarin by partial methylation with physm; the a-form consisting of minute needles dimethyl sulfate, indicating that it has the and the (3 form of long needles. On Narirutin, Neoeriocitrin and Veronicastroside in Citrus 1463

Some flavonoids have two melting points, i.e. naringin5) (mp 71°C, 171-172°C), querci trin6) (mp 182•`185•Ž, 250--252°C), robinin7)

(mp 195.198°C, 249.250°C) poncirin8) (mp 175180°C, 212213°C) etc. The difference in melting point between the first two com- pounds is due to the content of crystallization water. In the latter compounds differences may be ascribed to isomorphic modification. IR spectra of each of the different forms of the four flavonoids described as references, however, were identical in our experiment. Thus IR spectra of crystalline ƒ¿- and ƒÀ- forms of isorhoifolin as measured in potassium bromide pellets are different while their NMR spectra are identical.

The optical rotatory dispersion (ORD) and absolute configuration of narirutin, neo and didymin, which have been isolated from trifoliate orange," were studied. Gaffield and Waiss9) demonstrated that (-) flavanones of 2 (S) configuration and their exhibit a negative Cotton effect due to n-ƒÎ* transition (280-290 mp), while FIG. 4a. ORD Curves of Narirutin (---), Partially a positive Cotton effect appears in the same Racemized Narirutin (•\), Neoeriocitrin (---) region in (-_)- flavanones of 2 (R) configura and Didymin(•\•E•\•E). tion and their glycosides. On this basis they'll determined that the aglycones in naringin, neohesperidin and poncirin have the 2 (S) configuration.

We also studied the ORD of natural and partially racemized narirutin, neoeriocitrin R; OH Narirutin and didymin. Results are shown in Fig. 4. R ; OCH3 Didymin Narirutin and didymin showed negative Cotton FIG. 4b. Absolute Configuration of Narirutin and effects and their aglycones were determined to Didymin. have 2 (S) configurations and levorotatory activity. Neoeriocitrin (VI), however, showed compound VI is presumed to have a complete- a weak Cotton effect in the 250350 mƒÊ ly racemized aglycone, eriodictyol. region which was greatly reduced in magnitude Cyclization of chalcones with phloro similar to the ORD curves of racemized glucinol-type structures by Shimokoriyama's flavanones. According to Gaffield9) this re method,11) or modifications of it, gives com duction is probably related to asymmetric pletely racemized flavanones,9,11) but in the perturbation of the aromatic ring by optically narirutin chalcone described in a previous active carbon atoms of the glycosides. Thus, paper2) it particularly afforded corresponding 1464 S. KAMIYA, S. ESAKI and F. KONISHI

flavanones which exhibited fairly strong posi widely distributed in citrus1) plants is not tive Cotton effects. This may be the first lonicerin, but veronicastroside. report showing that there is asymmetric induc

tion with chalcone cyclization. EXPERIMENTAL Luteolin-7-rhamnoglucoside (VII), a flavone analogue of neoeriocitrin, was first isolated All melting points were determined with a micromelt ing point apparatus (Yanagimoto Mfg. Co.) and are from leaves of Loniceria japonica Thumb. and uncorrected.

named lonicerin by Nakaoki12) et al. in 1961. UV spectra were measured with a Hitachi EPI-S 2 Nakabayashi13) also isolated luteolin-7-rhamno spectrophotometer. glucoside (VIII) as yellow crystals, nip 216°C, IR spectra were recorded on a JASCO IRA-I spec. from young fruits of Citrus aurantiumt L. in trophotometer. All samples used were crystals and the same year. He considered it to be identical were measured in KBr pellets. NMR spectra were obtained on a JEOLG-60

with compound VII from comparisons of spectrometer in CC14 or CDCl3 using tetramethylsilane their mp, Rf values and UV spectra. The as the internal standard. sugar moiety of compound VIII was reported ORD spectra were measured with a JASCO Model as 4-O-L-rhamnosyl D-glucose by the same ORD UV-5 recorder. author. Recently Inouye14) et al. reported Chromatographic analysis for flavonoids was made the isolation of a new luteolin-7-rhamno- using a polyamide thin-layer chromatoplate with a solvent system consisting of nitromethane-methanol glucoside (IX), whose mp is 249.251°C and (5:2), and spots were detected under UV light. Simi. which is clearly different from lonicerin, from larly, sugar acetates were developed in a solvent system leaves of Veronicastrium sibiricum Pennel var. consisting of benzene and methanol (92: 8) on a silica Japonica Hara. He named it veronicastro gel plate, and were detected by spraying them with side. The sugar moiety in compound IX was sulfuric acid and successively heating them at 130°C. Trimethylsilylation of flavonoid glycosides was elucidated as neohesperidose by comparing carried out according to the procedure described by its nip, IR and NMR data with those of the Mabry et a1.15) synthetic compound. We think that the NMR spectral data for acetyl- and trimethylsilyl lonicerin obtained from citrus plants is pro derivatives of narirutin and its related compounds. bably identical with compound IX based on 1) Octaacetylnarirutin. NMR (20% in CDCl3) current knowledge of the biogenesis of o: acetoxyl group: 1.92-2.10 ppm (18 H); 2.32-2.40 flavonoids in citrus plants. This assumption (6 H, C-5-,4•L-acetoxyl); aglycone: H-3: 2.8-3.00 stimulated us to reexamine the sugar moiety (2H); H-2; 5.52 (quartet); H-6: 6.31 (doublet, J=3 of compound VIII. In a previous paper,1) Hz); H-8: 6.5 (doublet, J=3 Hz); H-3•L, H-5•L: 7.21 lonicerin, mp 215216°C, was isolated from (J=9Hz); H-2•L, H-6•L: 7.55 (doublet, J=9Hz); rhamnoglucosyl: rhamnose-CH3: 1.15 (doublet, J= leaves of Tanikawa-buntan, a variety of 6 Hz); glucose-H, 5,6,6 and rhamnose-H-5: 3.54.1 pummelo, according to the procedure reported (4H); glucose- H,1,2,3,4 and rhamnose-H, 2,3,4: by Nakabayashi.'" But after further purifica 5.0-5.31 (7H); rhamnose H-1: 4.72 (doublet, J= tion with polyamide column chromatography 1 Hz). the melting point of lonicerin was changed to 2) Heptaacetyldidyinin. NMR (20% in CDCl3) 249----251°C, and it was identical with com- 5: acetoxyl group: 1.94-2.08 (18 H); 2.38 (3 H, pound IX in its mp and IR spectrum. Com- C-5-acetoxyl); aglycone: H-3: 2.78-2.84 (2 H); H-2; pound VIII, isolated by Nakabayashi, was 5.52 (quartet); H-6: 6.25 (doublet, J=3 Hz); H-8: also identical with compound IX after purifica 6.44 (doublet, J=3 Hz); H-3•L,H-5•L: 6.9 (doublet, J=9 Hz); H-2', H-6': 7.35 (doublet, J=9 Hz); rhamno tion by the same procedure. Thus, we con glucosyl: rhamnose-CH3: 1.15 (doublet, J=6 Hz); cluded that the luteolin-7-rhamnoglucoside glucose-H-5,6,6 and rhamnose-H-5: 3.5---4.1 (414); On Narirutin, Neoeriocitrin and Veronicastroside in Citrus 1465

glucose-H-1,2,3,4 and rhamnose-H-2,3,4: 4.8-5.35 H-I : 4.4 (doublet, J=2 Hz).

(7H): rhamnose-H-1: 4.7 (doublet, J=1 Hz).

Preparation of rutinose 3) Octaacetylisorhoifolin (ƒÀ-form). NMR (20% in Although several synthetic methods16•`18) have CDCl3) S: acetoxyl group: 1.92.2 (18H); 2.35-2.48 been reported, they are relatively complicated. A (6H, C-5-, C-4•L-acetoxyl); aglycone: H-3: 6.55; H-6: simplified method is described below. To a solution 6.65 (doublet, J=3 Hz); H-8: 6.93 (doublet, J=3 Hz): of mercuric cyanide (13 g) and mercuric bromide (18 g) H-3•L, H-5•L: 7.22 (doublet, J=9 Hz); H-2•L, H-6•L: 7.83 in absolute acetonitrile (200 ml) were added crude (doublet, J=9 Hz); rhamnoglucosyl: rhamnose-CH3: ƒ¿-1 ,2,3,4-tetraacetyl D-glucose" (32 g) and aceto 1.15 (doublet, J=6 Hz); glucose- H-5, 6, 6 and rham bromorhamnose20) (32.6 g) with stirring until the solu nose-H-5: 3.6-4.2 (4H); glucose-H-1,2,3,4 and tion became clear. Then the reaction mixture was rhamnose-H-2,3,4: 5.0-5.3 (7H); rhamnose-H-l : treated as described in a previous paper.181 The free 4.75 (doublet, J=1 Hz). sugars obtained after deacetylation were acetylated

4) Heptaacetylrutinose. NMR (20% in CDCl3) again with a mixture of acetic anhydride (200 ml) and anhydrous sodium acetate (40 g) with heating at 100°C S: acetoxyl: 2.0-2.32 (18H); rhamnose-CH3 1.2 for 2 hr. No preliminary fractionation of free sugars (doublet, J=6 Hz); glucose-H-5,6,6 and rhamnose-H-5: by carbon column chromatography is necessary. After 3.14.06 (4H); glucose-H-1,2,3,4 and rhamnose-2,3,4: the reaction was over, the mixture was poured into a 4.95-5.50 (7H); rhamnose-H-1: 4.75 (doublet, J = Hz). large amount of ice-water and left overnight in a re frigerator. After filtration, the solid crystallized easily 5) Octatrimethylsilylnarirutin. NMR (200-0 in from absolute ethanol to afford colorless prisms in an

CCI.) S: aglycone: H-3 cis: 2.72 (doublet, J=6 Hz), H-3 almost pure state, trip 167•Ž, either alone or as an trans: 2.8 (doublet, J=9 Hz) ; H-2: 5.3 (quartet); H-6: admixture with an authentic sample of heptaacetylru 6.0 (doublet, H=1 Hz); H-8: 6.15 (doublet, J=1 Hz); tinose.18) Rf: 0.26.

H-3•L, H-5•L: 6.76 (doublet, J=9 Hz); H-2•L, H-6•L: 7.26 (doublet, 9 Hz); rhamnoglucosyl: glucose-H-1: Anal. Found: C, 50.09: H, 5.75: Calcd. for C26H36

4.75-5.05 (broad); rhamnose-H-1: 4.4 (doublet, J=2 017: C, 50.32; H, 5.08%. Hz), rhamnosylglucose (10 protons): 3.2-3.9: rham Deacetylation in the usual manner produced a hy

nose-CH3: 0.8--1.1 (broad). groscopic amorphous powder, rutinose.

6) Heptatriunethylsilvldidymin. NMR (20% in Conrcrsion of compound IV to linarin (III). CC1a) S: aglycone: H-3cis: 2.68 (doublet, J=3 Hz); Partial methylation of compound IV was carried H-3trans: 2.25 (doublet, J=10 Hz); H-2: 5.25 (quartet): out by the method of Wagner et a1.21) Compound IV H-6: 5.9 (doublet, J= 1.5 Hz); H-8: 6.09 (doublet, (100 mg) was methylated with dimethyl sulfate (0.02 ml) J=1.5 Hz); H-3•L, H-5•L: 6.75 (doublet, J=9 Hz); H-2•L, and potassium carbonate (350 mg) in dimethyl for H-6•L: 7.22 (doublet, J=9 Hz); rhamnoglucosyl: mamide (4 ml) under continuous stirring for three days glucose-H-1: 4.75-5.0 (broad); rhamnose-H-1: 4.35 at room temperature. After the reaction was over, (doublet, J=2 Hz), rhamnosylglucose (10 protons): the solution was diluted with water, then acidified with 3.2-3.75; rhamnose-CH3: 0.81.05 (broad). acetic acid. Minute colorless needles (III) were

deposited gradually. After being kept overnight in 7) Octatrimethylsilylisorhoifolin (;3 form). NMR a refrigerator, compound III was collected and recry (20% in CC14) 6: aglycone: H-3: 6.3 (singlet); H-6: stallized from pyridine-water (about I : 10) to afford 6.23 (doublet, J=1.5 Hz); H-8: 6.6 (doublet, 1.5 Hz); pure colorless needles, mp 265"C. Yield, 90 mg H-3•L, H-5•L: 7.80 (doublet, J=7.5 Hz); H-2•L, H-6•L: 7.7 (doublet, 7.5 Hz); rhamnoglucosyl: glucose-H-1 : (91.83%). UV) ƒÉ_??_mƒÊ: 269, 328. R/ 0.48. Anal. Found: C, 54.10; H, 5.45%. Calcd. for C,s H 4.85-51 (broad); rhamnose-H-1: 4.73 (doublet, 32 O 14•E11/2 H2O: C, 54.25; H, 5.64°n. J=2 Hz); rhamnosylglucose (10 protons): 3.2-3.90; Compound III was further acetylated with a mixture rhamnose-CH3: 0.8-1.1 (broad). of acetic anhydride and pyridine in the usual manner

8) Heptatrimethylsilyhzutirrose. NMR (20% in to give a heptaacetyl derivative (X). mp 125°C. (Lit.22)

CC14) 6: rhamnose-CH3: 1.0-1.4 (broad); glucose-H-2, 123•`125•Ž). final. Found: C, 56.72 H, 5.33%. 3,4,5,6,6 and rhamnose-H-2 ,3,4,5 (10 protons): 2.9- Calcd. for C,2 H46O21: C, 56.88: H, 5.19%. 4.0; glucose-H-1: 4.9 (doublet Compounds III and X were identified as linarin and , J=1.5 Hz); rhamnose- 1466 S. KAMIYA, S. ESAKI and F. KONISHI

its heptaacetate, respectively, by comparing their mp, 2) M. Nishiura, S. Kamiya, S. Esaki and F. Ito, mixed mp, UV, and IR with those of authentic samples. Agr. Biol. Chem., 35, 1683 (1971). 3) H. Wagner, G. Aurnhammer, L. Horhammer and L. Farkas, Chem. Ber., 102, 2083 (1969). Isolation of veronicastroside (IX) from C. grandis 4) H. Roster, T. J. Mabry, M. F. Crammer and J. Osbeck var. Tanikawana (Tanikawa-buntan). Kagen, J. Org. Chem., 30, 4346 (1965). Fresh leaves (240 g) collected at the beginning of 5) Y. Asahina and M. Inubuse, Yakugaku Zasshi, May were boiled with methanol three times, each time 49, 128 (1929). for 3 hr, after which they were treated as described in 6) S. Hattori, "The Chemistry of Flavonoid Com a previous paper to give lonicerin, mp 215---216"C,

as yellow needles: yield, 336 mg (0.12%), Crystals pounds," ed. by T. A. Geissman, Pergamon Press, 1962, p. 337. (78 mg) were dissolved in a small amount of methanol, 7) C. E. Sando, J. Biol. Chem., 94, 675 (1933). then poured onto a Woelm polyamide column (20 g, 8) H. Wagner, G. Aurnhammer, L. Horhammer, 2.5 38 cm) and eluted with a mixture of benzene- L. Farkas and M. Nogradi, Chem. Ber., 102,785 methanol (3: 1) leaving an unidentified yellow pigment on the top of the column. Fractions containing only (1969). 9) W. Gaffield and A. C. Waiss, Chem. Comn.,29, ( compound IX were combined and concentrated to 1968). dryness in vacuo. The solid was crystallized from 50 10) W. Gaffield, unpublished data, see the following methanol to afford yellow plates, mp 249-251°C. reference, R. M. Horowitz and B. Gentili, J. Yield, 35.4 mg. Rfvalue: 0.16. Agr. Food Chem., 17, 696 (1969). UV i, , "mp: 252, 266 sh, 346; _??_ mƒÊ: 11) M. Shimokoriyama, J. Am. Chem. Soc., 79,4199 266, 400; _??_ mƒÊ: 272, 296 sh, 427; (1957). _??_ mƒÊ; 263, 277 sh, 295 sh, 356, 384; 12) T. Nakaoki, N. Morita and A. Isetani, Yakugaku _??_mƒÊ. 404; _??_mƒÊ: 368. Zasshi, 81, 558 (1961). _??_ mƒÊ Anal. Found: C, 49.96; H, 5.85 %. Calcd, for C27 H;yo 13) T. Nakabayashi, Nippon Nogei Kagaku Kaishi, 015•3 H2O; C, 49.95; H, 5.55 00. 35, 945 (1961). 14) H. Inouye, Y. Aoki, H. Wagner, L. Horhammer, Purification of ‡€lonicerin‡€ (VIII). G. Aurnhammer and W. Budweg, Chem. Ber., Purification of compound VIII (78 mg) as described 102, 3009 (1969). above gave the pure compound IX as yellow plates 15) T. J. Mabry, J. Kagen and H. Roster, Phyto which was identical with veronicastroside in all re chernistry, 4, 177 (1965).

s pects. 16) G. Zemplen and A. Gerecs, Chem. Ber., 68,1320 (1935). Acknowledgment. We are indebted to Prof. H. 17) R. Bogner, F. F. Szabo, I. Farkas and H. Gross, Inouye of Kyoto University and Prof. T. Nakabayashi Carbohvd. Res., 5, 241 (1967). of Shizuoka University for the supply of veronicastro 18) S. Kamiya, S. Esaki and M. Hama, Agr. Biol. side. Thanks are also due to Prof. H. Wagner of Chem., 31, 261 (1967). Miunchen University for data on the IR and NMR 19) D. D. Reynolds and W. L. Evans, J. Am. Chem. spectra of isorhoifolin. We also wish to express our Soc., 60, 2559 (1938). sincere thanks to Mr. S. Katayama of Shizuoka College 20) E. Fischer, M. Bergman and A. Rabe, Chem. of Pharmacy for measuring the NMR spectra. Ber., 53, 2362 (1920). 21) H. Wagner, W. Budweg, L. Horhammer, B.

REFERENCES Vermes and L. Farkas, ibid., 104, 2118 (1971). 22) K. W. Merz and Y. H. Wu, Arch. Pharm., 274, 1) M. Nishiura, S. Esaki and S. Kamiya, Agr. Biol. 126(1936). Chem., 33, 1109 (1969).