No. 5 1817

Chem. Pharm. Bull. 35( 5 )1817-1822(1987)_

Tannins and Related of Rosaceous Medicinal Plants. IV."

Roxbins A and B from Rosa roxburghii Fruits

TAKASHI YOSHIDA," XIN-MIN CHEN,b TSUTOMU HATANO"

MAKOTO FUKUSHIMA` and TAKUO OKUDA"

Faculty of Pharmaceutical Sciences, Okayama University," Okayama 700, Japan, Chengdu Institute of Biology, The Chinese Academy of Sciences,b Chengdu, China and Laboratories, Pola Corporation, Takashimadai, Kanagawa-ku, Yokohama 221, Japan

(Received October 24, 1986)

From the unripe fruits of Rosa roxburghii TRATT., two new hydrolyzable , roxbin A (1)

(a dimeric ) and roxbin B (9), were isolated along with rugosin F (2), (3), (4), alnusiin (5), stachyurin (6), tellimagrandin 11 (7), 2,3-O-(S )-hexahydroxydiphenoyl- D- and ( + )-catechin. The structures of new tannins were elucidated on the basis of chemical and spectral evidence.

Keywords •\ Rosa roxburghii; Rosaceae; ; ellagitannin; roxbin A; roxbin B; centrifugal

partition chromatography

Many Rosaceous plants have been traditionally used as folk medicines for a variety of diseases in diverse areas of the world.2) Among them, the plants used as antidiahrreics, astringents and hemostatics are generally rich in tannins, which are believed to be the active principles. These medicinal plants include Agrimonia japonica (MIQ.) KOIDZ., Geum japo- nicum THUNB. and Rosa rugosa THUNB., from which we isolated oligomeric and related monomers, such as agrimoniin,3) gemins A-F1,4) and rugosins A-G.5) In a con- tinuation of the investigation on the Rosaceous medicinal plants, we have examined the polyphenolic constituents of Rosa roxburghii TRATT., the fruit of which is a Chinese fölk medicine used as a stomachic and a tonic. The fruit is also widely used to prepare a healthful drink in China, as it is a rich source of vitamin C. The present paper deals with the isolation and characterization of new tannins, named roxbin A (1) and roxbin B (9), along with eight known polyphenols, from the fruit extract of this plant. An aqueous acetone homogenate of the fruits was successively extracted with ether, ethyl acetate and n-butanol. The n-butanol soluble portion was subjected to centrifugal partition chromatography (CPC)6 ) and column chromatography over Toyopearl HW-40 and Sephadex LH-20 to afford a new tannin, roxbin A (1), along with rugosin F (2),5b) pedunculagin (3), 7) stachyurin (6)7) and 2,3-0-(S)-hexahydroxydiphenoyl-D-glucose.8) An additional new tannin, roxbin B (9), was isolated together with known tannins, casuarictin (4),7) alnusiin (5),9) tellimagrandin fl (7),7,10) and ( +)-catechin from the EtOAc-soluble portion by a combination of chromatographies over columns of Toyopearl HW-40 and Sephadex LH-20. Roxbin A (1), C75H50O48 •E 4H2O, was shown to be a dimeric ellagitannin by its similarities to rugosin F (2)5b)in the retention time on high-performance liquid chromatog- raphy (HPLC) (normal phase) and in the color reaction with the NaNO2--AcOH reagent. Acid hydrolysis of 1 gave , dilactone and glucose. The proton nuclear magnetic resonance (1H-NMR) spectrum exhibited double signals for most of the protons, indicating that 1 exists as a mixture ofα-and β-anomers in a ratio of ca 4:3. The signals of the 1818 Vol. 35 (1987)

3: R.RF1,0F1

2 4: R

5 6 7

Chart 1

anomeric protons were observed at (56.17 and 6.16 (each d, J= 9 Hz, 1H in total), and 5 5.43

(d, J= 3.5 Hz) and 5.13 (d, J=9 Hz) (1H, in total). The coupling pattern of the other pairs of glucose proton signals indicates that both glucose cores in 1 adopt the 4C, conformation. Thus the acyloxy group at the anomeric center of one of the glucose cores has the [3- configuration. The presence of a valoneoyl and three hexahydroxydiphenoyl (HHDP) groups in 1 was indicated in the aromatic region of the 111-NMR spectrum by the singlets at j 7.21, 7.20 (1H in total), 6.69 (1H), 6.62, 6.61 (1H in total), 6.55 (1H), 6.52, 6.46 (1H, in total), 6.39

(3H), and 6.25, 6.24 (1H, in total). Upon treatment with hot water, 1 gave two partial hydrolysates, which were identified as pedunculagin (3) and praecoxin A (8). Therefore, roxbin A is a dimer possessing a valoneoyl group, which is biogenetically" regarded as a product of intermolecular C-O oxidative coupling between the HHDP group at O-4 O-6 of 3, and the galloyl group at O-1 of 4. The proposed structure (1) is also consistent with the carbon-13 nuclear magnetic resonance ("C-NMR) spectrum: the glucose carbon resonances are in good agreement with those of 3 and 4, and hence were assigned as shown in Table I on the basis of the assignments of the peaks of the latter two compounds." ) Based on the data presented above, the structure 1 was assigned to roxbin A. Roxbin B (9), an off-white amorphous powder, C,H2,O26 •E 2H2O, is a monomeric hydrolyzable tannin as revealed by six sugar carbon signals (Table I) and five ester carbonyl carbon resonances in the 13C-NMR spectrum. The H-NMR spectrum showed a singlet due to a galloyl group at 67.27, and four 1H singlets at 67.14, 6.80, 6.73 and 6.60 which are ascribable to two HHDP groups. The presence of these groups was confirmed by acid hydrolysis of 9, which afforded and ellagic acid. The sugar obtained on the hydrolysis was identified as glucose by gas liquid chromatography (GLC) after trimethyl- silylation. The conformation of the glucose moiety in 9 was shown to be 4C, on the basis No. 5 1819

1

8 9

Chart 2

TABLE1. 13C Resonances of Glucose Moieties of Roxbin A (1), Roxbin B (9), Pedunculagin (3) and Casuarictin (4) in Acetone-d6 1820 Vol. 35 (1987)

of the large coupling constants (8.5 10 Hz) of the adjacent protons in the 1H-N MR spec- trum. The ƒÀ-configuration of an acyloxy group at C-1 was also demonstrated by the cou- pling constant (11,2 = 8.5 Hz) of the anomeric proton signal at ti5 6.08 (d). The circular dichroism (CD) spectrum of 9 exhibited a positive Cotton effect at 225 nm and a negative one at 255 nm which are indicative1.2) of the S-configurations of both HHDP groups in 9. These structural characteristics are similar to those of casuarictin (4) (1-O-galloy1- 2,3;4,6-bis-O-(S)-hexahydroxydiphenoyl-fl-D-glucose), and thus roxbin B should be an isomer of 4 concerning the positions of the aroyl groups. One of the C6 methylene protons of 9 resonates at lower field (6 5.30) than the other (6 3.80). Such a large difference in chemical shift between these protons is characteristic of ellagitannins in which the HHDP group is attached to O-6 and O-4 in the 4C1 glucose moiety.") The presence of the other HHDP group at O-1 O-3 of glucose core is excluded since it requires a boat conforma- tion, as in brevilagins.141 Therefore this HHDP group in 9 must be at O-1 O-2 as the 4C 1 glucose conformation permits the attachment of an HHDP group only at O-1--O-2 or O-2 O-3 besides O-4 O-6.13) Upon degalloylation of 9 with tannase, the reversed-phase HPLC of the reaction mixture showed a new single peak and a gallic acid peak. This result is also consistent with the assumption that the galloyl group in 9 is not located at 0-1, as degalloylation at C-1 of glucose should result in the formation of an a- and [3-anomer mixture which will show two peaks upon reversed-phase HPLC analysis, as found for pedunculagin (3),15) and roxbin A (1). Based on these observations, roxbin B was characterized as 3-O-galloy1-1,2;4,6-bis-O- (S)-hexahydroxydiphenoyl-fl-D-glucose (9) which is the first example of an ellagitannin possessing the HHDP group at O-1 O-2. It seems reasonable to presume that ascorbic acid in the fruits of R. roxburghii, is stabilized by the abundantly co-existing tannins which were found in the present study, as tannins and related polyphenols are effective inhibitors of the autoxidation of ascorbic acid.16)

Experimental

The 1H and 13C-NMR spectra were measured on a JEOL FX-270 spectrometer (270 MHz for 1H and 67.8 MHz for 13C) and on a Hitachi R-22FTS (90 MHz for 1I-1 and 22.6 MHz for 13C), with tetramethylsilane as an internal standard. The chemical shifts are given in d values. The normal phase HPLC was conducted on a Develosil 60-5 column (4 x 150 mm) using a solvent system of n-hexane-Me0H-THF-HCOOH (55 : 33 : 11 : 1) containing oxalic acid (450 mg/1), and the reversed-phase HPLC on a YMC A312 (ODS) column (6 x 150 mm) with 0.05 M H3PO4- 0.05 M KH2PO4-Et0H-Et0Ac (17 : 17 : 4 : 2). Thin layer chromatography (TLC) was carried out on microcrystalline cellulose (Avicel SF, Funakoshi) plates with 7% HOAc, and the spots were visualized by spraying the FeC13 or NaNO2-HOAc reagents. Column chromatography was performed using Toyopearl HW-40 (coarse and fine grades; Toyo Soda Mfg.) and Sephadex LH-20 (100 itm, Pharmacia Fine Chemicals). CPC was conducted on a CPC model L-90 (Sanki Engineering Ltd.) comprising a centrifuge equipped with twelve cartridges (the internal volume of each cartridge is ca. 15 m1).6) Isolation of Tannins •\ The dried unripe fruits (500 g) of R. roxburghii, collected in May in Chendgu, China, were homogenized in 70% aqueous acetone. After removal of the acetone, the aqueous solution was extracted with ether, EtOAc and water-saturated n-BuOH, successively. A part (5 g) of the EtOAc extract (20.95 g).was fractionated by chromatography on a Toyopearl HW-40C column using aqueous Me0H as the eluant. The 40% Me0H eluate afforded ( + )-catechin (0.16 g). The 70% Me0H eluate was rechromatographed over a Sephadex LH-20 with 70% Me0H, followed by purification on Toyopearl HW-40F with 60% Me0H to give roxbin B (9) (25 mg). The 80% Me0H fraction was further purified by chromatography on Sephadex LH-20 column using 70% EtOH to give

alnusiin (5) (154 mg) and casuarictin (4) (307 mg). Tellimagrandin 11 (7) (68 mg) was obtained from the Me0H eluate. A part (3.1 g) of the BuOH extract (19 g) was subjected to CPC (1000 rpm, flow rate 1 ml/min), by the ascending

method using the solvent system of n-BuOH-n-PrOH-H20 (4 : 1: 5), 10 ml per fraction being collected. This CPC was repeated three times. Fractions 38-80 of each development were combined and further purified by passage through a Toyopearl HW-40F column using the solvent system of Me0H-H20-acetone to afford 2,3-O-(S)-

hexahydroxydiphenoyl-D-glucose (46 mg), pedunculagin (3) (29 mg), stachyurin (6) (33 mg) and roxbin A (1)

(147 mg). In a separate experiment, a part (6 g) of the BuOH extract was subjected to chromatography on Toyopearl No. 5 1821

HW-40C using the solvent system of 70% EtOH-,MeOH. Fractions containing dimers (MeOH eluate) were collected and further purified by chromatography on Sephadex LH-20 and Toyopearl HW-40C to afford roxbin A (1) (18 mg) and rugosin F (4) ( 10 mg). Yields of tannin components from dried fruits were as follows: roxbin A (1), 0.06%; roxbin B (9), 0.02%; ( + )-catechin, 0.13%; alnusiin (5), 0.13%; casuarictin (4), 0.25%; tellimagrandin II (7), 0.06%; 2,3-0-(S)- hexahydroxydiphenoyl-D-glucose, 0.014%; pedunculagin (3), 0.007%; stachyurin (6), 0.08%; rugosin F (2), 0.002%. Roxbin A (1) •\ An off-white amorphous powder, TLC, Rf 0.32, HPLC (normal phase) tR 2.33 min,(reversed- phase) tR 5.29 and 5.91 min. [AD + 46.5 (c = 1.0, Me0H). Anal. Calcd for C75HSOO48 •E 4H20: C, 50.28; H, 3.26. Found: C, 50.17; H, 3.58. 1H-NMR (acetone-d6) 6: 6.17 (d, J=9 Hz), 6.16 (d, J=9 Hz), 5.43 (d, J-= 3.5 Hz), 5.13 (d, J=9 Hz, H-1), 5.11 (dd, J=9, 10 Hz), 5.05 (dd, J= 3.5, 9 Hz), 4.83 (dd, 9, 10 Hz, H-2), 5.22 (t, J=10 Hz), 5.45 (t, J= 10 Hz), 5.42 (t, J= 10 Hz, H-3), 4.99 (t, J=10 Hz), 4.98 (t, J=10 Hz), 5.14 (t, J=10 Hz), 5.10 (t, J= 10 Hz, H-4), 4.16 (dd, J=6, 10 Hz), 4.56 (dd, J=6, 10 Hz), 4.47 (dd, J=6, 10 Hz, H-5), 5.33 (dd, J=6, 13 Hz), 5.15 (dd, J-6, 13 Hz, H-6), 3.86 (d, J= 13 Hz), 3.73 (d, J= 13 Hz), 3.67 (d, J=13 Hz, H-6'), aromatic protons, see text. "C-NMR

(acetone-d6) 169.27, 169.21 (1C in total), 169.18, 169.12 (2C in total), 168.90, 168.79 (1C in total), 169.76 (1C), 167.77, 167.71, 167.61 (3C in total), 163.26, 163.16 (1C in total) (ester CO), Table II. CD (c =0.007, Me0H): [ƒÆ]232 +24.6 •~ 104, [01256 -10.2 •~ 104. Roxbin B (9) •\ An off-white amorphous powder, TLC, Rf 0.32, HPLC (normal phase), tR 2.94 min, (reversed- phase) tR 7.73 min. [ƒ¿] -22.8•‹ (c = 1.0, MeOH). Anal. Calcd for C41H28026.2H20: C, 50.62; H, 3.32. Found: C, 50.94; H, 3.65. 'H-NMR (acetone-d6) 6: 6.08 (d, J=8.5 Hz, H-1), 4.90 (dd, J=8.5, 9.7 Hz, H-2), 5.21 (t, J-9.7 Hz, H-3), 4.89 (dd, J-9.7 , 10 Hz, H-4), 4.40 (ddd, 1.5, 6.5, 10 Hz, H-5), 5.30 (dd, J=6.5, 13 Hz), 3.80 (dd, J-1.5, 13 Hz, H-6'), aromatic protons, see text. '3C-NMR (acetone-d6) 6: 168.26, 167.98, 167.81, 167.16, 164.85 (ester CO), 120.05 (galloyl, C-1), 110.51 (2C, galloyl, C-2, 6), 146.17 (2C, galloyl, C-3, 5), 139.84 (galloyl, C-4), 117.64, 117.44, 115.86, 115.70 (HHDP, C-1, 1'), 126.37, 126.11, 122.23 (2C, HHDP, C-2, 2'), 110.37, 110.16, 108.37, 108.03 (HHDP, C-3, 3'), 145.57, 145.33, 145.23, 145.08, 145.00, 144.86, 144.45, 144.29 (HHDP, C-4, 4', 6, 6'), 138.58, 137.24, 136.46, 136.44 (HHDP, C-5, 5'), Table I. cp (c =0.008, Me0H): [ƒÆ]236 + 2.2 x 104, [ƒÆ]255- 0.4x 104, [ƒÆ]270 +1.5 x 104, [ƒÆ]197

-1 .98×104. Acid Hydrolysis of Roxbin A (1) A solution of roxbin A (1) (2 mg) in 5% H2SO4 was kept at 90 •Ž for 6 h, then extracted with EtOAc. Analysis of the EtOAc extract by reversed-phase HPLC showed the presence of ellagic acid and . The aqueous layer was neutralized with Amberlite IRA-410 (OH form) and

evaporated, and glucose was detected as the trimethylsilyl ether by GLC (3% OV-1, column temperature, 170•Ž, N2 50 ml/min). Partial Hydrolysis of Roxbin A (1) A suspension of 1 (70 mg) in H20 (9 ml) was heated at 82-83 •Ž for 45 mm. The reaction mixture was concentrated and subjected to chromatography over Toyopearl HW-40F using 70% EtOH as the eluant to give pedunculagin (3) (9 mg), [a], +85 (c. =0.8, MeOH), and praecoxin A (8) (6 mg), ND +51•‹ (c= 0.6, Me0H). The identities of these products were confirmed by direct comparisons of the spectral data and chromatographic behavior with those of authentic samples. Acid Hydrolysis of Roxbin B (9) •\ Roxbin B (9) (2 mg) was hydrolyzed as described for 1, and gallic acid and ellagic acid were detected by HPLC and glucose by GLC after trimethylsilylation. Degalloylation of 9 with Tannase Roxbin B (9) (1 mg) was treated with tannase at 37 °C for 2 h. The reaction mixture showed upon HPLC (reversed-phase) two peaks of t, 2.3 and 3.1 min, and the former peak was identical with that of gallic acid.

Acknowledgements We thank Prof. Y. Asakawa, Associate Prof. M. Tori and Dr. T. Hashimoto, Faculty of Pharmaceutical Sciences, Tokushima Bunri University for the CD measuremen.ts.

References

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