2020 Volume 68 Issue 8 Pages 814-817
From the leaves of Zanthoxylum ailanthoides, four new phenolic glucosides, termed zanthosides A–D (1–4), were isolated. Their structures were elucidated by means of spectroscopic evidence. Zanthoside A was enzymatically hydrolyzed and thus the aglycone obtained was found to be (1′S,2′R)-(−)-trans-decursidinol, isolated from Angelica decursiva.
Rutaceous plants growing in the Okinawa Islands were inhibited from immigrating into the mainland of Japan until early 1980, due to the spread of the Oriental fruit fly, Bactrocera dorsalis. A mated female B. dorsalis punctures the skin of mature fruit, e.g., Hirami lemon, Citrus despressa, and deposits eggs underneath a fruit’s skin. Eggs hatch to larvae and molt twice while feeding on the flesh of the fruit. In 1982, B. dorsalis was exterminated from the Okinawa Archipelago using a pheromone, methyl eugenol. It attracted male B. dorsalis and the collected males were extinguished. After that, immigration rules were eased. However, some of the Rutaceous plants, for example Murraya paniculate and M. koengii etc., are still under the regulation. The genus Zanthoxylum belonging to the family Rutaceae comprises about 200 species and is found in tropical and moderate areas of both hemispheres. Z. piperitum De Candolle is a well-known medicinal plant, and its seed-depleted matured husks are used for an aromatic and pungent stomachic, anthelminthic, and so on. Young leaves and husks are also used for food seasonings. Z. ailanthoides Siebold et Zuccarini is a tall deciduous tree that grows in Japan (Honshu, Kyushu and Okinawa Islands), Taiwan, China, and the Philippines.1) In a previous paper, the isolation of an aliphatic glucoside, zanthoionic acid, and megastigmane glucosides named zanthoionosides A–E from the leaves of Z. ailanthoides, collected in the Okinawa Island, was reported.2) From further investigations of the same plant, one coumarin derivative (1, 4.7 mg), two prenylated phenylpropanoids (2, 42.6 mg) and (3, 9.8 mg) and a phenethyl alcohol glucoside (4, 21.1 mg), termed zanthosides A–D (Fig. 1), were isolated along with known glucosides, (Z)-6-(4-β-D-glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (5),3) skimmin (6),4) aesculin (7),5) mermesunin (8)6) nodakenin (9),7) decurcinol O-β-D-glucopyranoside (10),8) and picraquassioside B (11).9) This paper deals with the structural elucidation of the new compounds.
Four new compounds and seven known ones were obtained by extensive separation of the MeOH extract of the leaves of Z. ailanthoides using Diaion HP-20 column chromatography (CC), normal and reversed-phase silica gel CC, droplet counter-current chromatography (DCCC), and HPLC. The structures of new compounds were established mainly by NMR spectroscopic evidence (one- and two-dimensional spectroscopy), and known compounds were identified by comparison with data reported in the literature.
Zanthoside A (1), [α]D22–84.7, was isolated as an amorphous powder, and its elemental composition was determined to be C20H24O10 by high-resolution (HR) electrospray ionization (ESI) MS. The IR spectrum exhibited absorption bands ascribable to hydroxy groups (3308 cm−1), a carbonyl group (1734 cm−1), and an aromatic ring (1629 and 1573 cm−1). The presence of the aromatic ring was also supported by a UV absorption band at 320 nm. In the 1H-NMR spectrum, signals assignable to two singlet methyls, two aromatic protons, two olefinic protons in cis geometry [δH 6.23 (d, J = 9.5 Hz) and 7.88 (d, J = 9.5 Hz)], two oxymethines, and an anomeric proton [δH 4.73 (J = 7.8 Hz)] were observed (Table 1). The 13C-NMR spectrum displayed 20 signals (Table 2). Six of these were typical of a hexopyranose and this sugar was found to be D-glucose by HPLC analysis of the hydrolyzate of 1 using a chiral detector. The remaining 14 signals comprised two methyls, two oxymethines, one oxygenated aliphatic tertiary carbon, eight sp2 signals and a carbonyl carbon. The eight sp2 signals expected to form an aromatic ring, included four substituted ones and two with aromatic protons and a disubstituted double bond. One of the aromatic protons was coupled with an oxymethine proton, and the other appeared as a singlet. This evidence suggested that the benzene ring was substituted at the 1, 2, 4, and 5 positions, and the degrees of unsaturation acquired by MS analysis suggested two rings other than the aromatic one and a sugar moiety were present in the scaffold. The crucial heteronuclear multiple-bond correlations (HMBC) between one of the olefinic protons at H-4 (δH 7.88) and C-2 and C-5 (δH 163.3 and 129.4, respectively) and the ester absorption in the IR spectrum established the structure of 1 to be a coumarin derivative. Further HMBC correlation of H-5 (δH 7.70) and C-1′ (δC 69.3) suggested that the remaining five carbon unit was attached at the C-6 position, and this unit was also expected to have a cyclic system from the degree of unsaturation (Fig. 2). The 1H–1H correlation spectroscopy (COSY) correlation from H-1′ to H-2′, and HMBC correlations between H-1′ and C-3′ and H-2′ and C-4′ substantiated the planar structure of the aglycone of 1 to be decursidinol as shown Fig. 1. The position of the sugar linkage was determined to be on the hydroxy group at the 2′-position by HMBC correlation between H-1″ and C-2′, and the mode of linkage β from the coupling constant of the anomeric proton. Closely related compounds, 1′,2′-O-disenecioyloxy decursidinol (decursidin) and 1′,2′-O-diangeloyloxy decursidinol, were isolated from Angelica decursiva10,11) and Peucedanum decursivum,12) respectively, and their stereochemistry was intensively investigated.11) Kong et al. hydrolyzed 1′,2′-O-diangeloyloxy groups using KOH to give (+)-trans- and (−)-cis-decursidinols.12) The aglycone (1a) enzymatically derived from 1 showed an identical NMR spectra with those of (+)-trans-decursidinol including the trans coupling constant between H-1′ and H-2′ (J = 8.6 Hz, Ref. 10: J = 8.5 Hz); however, the sign of optical rotation value was opposite to that of (+)-trans-decursidinol. Therefore, the structure of 1 was elucidated to be (1′S,2′R)-(−)-trans-decursidinol 2′-O-β-D-glucopyranoside as shown in Fig. 1.
| 1 | 2 | 3 | 4 | |
|---|---|---|---|---|
| 2 | 7.33 d 1.8 | 7.02 d 1.8 | ||
| 3 | 6.23 d 9.5 | 6.57 s | ||
| 4 | 7.88 d 9.5 | |||
| 5 | 7.70 d 1.1 | 7.15 d 8.4 | 6.76 m | |
| 6 | 6.77 s | 7.39 dd 8.4, 1.9 | 6.76 m | |
| 7 | 2.85 2H t 8.0 | 7.56 d 15.8 | 2.64 2H t-like 6.9 | |
| 8 | 6.69 s | 2.57 2H t 8.0 | 6.31 d 15.8 | 3.64 2H t 6.9 |
| 1′ | 4.80 dd 8.2, 1.1 | 6.26 d 9.9 | 3.40 2H m | 4.75 d 7.5 |
| 2′ | 3.82 d 8.2 | 5.54 d 9.9 | 5.34 t 7.9 | 3.53 m |
| 3′ | 3.53 m | |||
| 4′ | 1.28 3H s | 1.36 3H s | 1.73 3H s | 3.44 dd 9.6, 8.8 |
| 5′ | 1.55 3H s | 1.37 3H s | 1.75 3H s | 3.71 ddd 9.6, 6.7, 2.2 |
| 6′ | 4.38 dd 11.9, 6.7 | |||
| 4.57 dd 11.9, 2.2 | ||||
| 1″ | 4.73 d 7.8 | 4.87 d 7.7 | 4.97 d 7.7 | |
| 2″ | 3.26 dd 9.1, 7.8 | 3.45 m | 3.51 dd 8.7, 7.7 | 6.92 s |
| 3″ | 3.35 m | 3.43 m | 3.45 m | |
| 4″ | 3.35 m | 3.43 m | 3.45 m | |
| 5″ | 3.35 m | 3.43 m | 3.45 m | |
| 6″ | 3.69 dd 11.8, 5.2 | 3.72 dd 12.0, 4.8 | 3.71 dd 12.1, 5.4 | 6.92 s |
| 3.88 dd 11.8. 2.1 | 3.89 dd 12.0, 2.0 | 3.89 dd 12.1, 2.0 | ||
| 7″ | 7.64 d 15.9 | |||
| 8″ | 6.43 d 15.9 | |||
| CH3O- | 3.63 3H s | 3.88 6H s |
m: multiplets or overlapped signals.
| 1 | 1a | 2 | 3 | 4 | |
|---|---|---|---|---|---|
| 1 | 123.6 | 129.9 | 132.2 | ||
| 2 | 163.3 | 163.3 | 157.4 | 130.2 | 119.6 |
| 3 | 113.6 | 113.6 | 104.8 | 132.9 | 146.5 |
| 4 | 145.8 | 145.8 | 153.9 | 158.5 | 146.8 |
| 5 | 129.4 | 129.6 | 117.2 | 116.1 | 117.0 |
| 6 | 123.8 | 124.4 | 128.5 | 128.5 | 125.4 |
| 7 | 157.4 | 157.7 | 26.4 | 145.8 | 39.6 |
| 8 | 105.3 | 104.6 | 35.8 | 117.8 | 64.3 |
| 9 | 156.2 | 156.2 | 177.8 | 171.1 | |
| 10 | 114.5 | 114.4 | |||
| 1′ | 69.3 | 69.5 | 122.9 | 29.3 | 104.4 |
| 2′ | 83.8 | 76.4 | 129.8 | 123.4 | 74.9 |
| 3′ | 80.8 | 81.5 | 77.4 | 133.8 | 77.5 |
| 4′ | 21.0 | 19.8 | 28.3 | 18.0 | 71.8 |
| 5′ | 27.3 | 27.1 | 28.2 | 25.9 | 75.8 |
| 6′ | 64.8 | ||||
| 1″ | 104.6 | 102.6 | 102.2 | 126.7 | |
| 2″ | 75.9 | 75.0 | 75.0 | 107.1 | |
| 3″ | 78.3 | 78.3 | 78.3 | 149.5 | |
| 4″ | 71.7 | 71.4 | 71.4 | 139.8 | |
| 5″ | 78.2 | 78.2 | 78.2 | 149.5 | |
| 6″ | 62.9 | 62.6 | 62.2 | 107.1 | |
| 7″ | 147.5 | ||||
| 8″ | 115.6 | ||||
| 9″ | 169.0 | ||||
| CH3O- | 52.1 | 57.0 |
Zanthoside B (2), [α]D23–53.5, was isolated as an amorphous powder, and its elemental composition was determined to be C21H28O9 by HR-ESI-MS. The IR and UV spectra were similar to those of 1. The 1H-NMR spectrum exhibited signals assignable to two singlet methyls, two olefinic protons in a cis geometry, two singlet aromatic protons, an anomeric proton, and methoxy functional group. The 13C-NMR spectrum displayed a typical methoxy along with 20 signals, six of which were assigned to those of glucopyranose. The remaining 15 signals comprised those of a 1,2,4,5-tetrasubstituted benzene ring, two methyls, two methylenes, one oxygenated tertiary carbon, a disubstituted double bond, and a carbonyl functional group. The HMBC cross peaks between H-6 and C-7 and C-1′, H2-7 and C-9, methoxy methyl and C-9 together with 1H–1H COSY correlations of H2-7 and H2-8, and H-1′ and H-2′ placed a propanoid unit at C-1 and an isoprene unit at C-5. Degrees of unsaturation required one more ring formation, and thus the structure of 2 was elucidated and is shown in Fig. 1.
Zanthoside C (3), [α]D25–43.8, was isolated as an amorphous powder, and its elemental composition was determined to be C20H26O8 by HR-ESI-MS. The IR spectrum exhibited absorption bands assignable to hydroxy groups, a ketone, and an aromatic ring. The 1H-NMR spectrum showed three aromatic signals coupled in an ABX system, two olefinic protons in a trans geometry, one triplet olefinic proton and an anomeric proton. In the 13C-NMR spectrum, 20 signals were observed, and six of them were assigned to glucopyranose. The remaining 14 signals comprised isoprene and propanoic acid units, and a core aromatic ring. The positions of substitution were substantiated by the HMBC spectrum in which correlations between H-2 [δH 7.33 (d, J = 1.8 Hz) and C-7 and C-1′, H-6 [δH 7.39 (dd, J = 8.4, 1.9)] and C-7, H-7 and C-9, and H2-1′ and C-4 and C-3′ were observed. The position and mode of sugar linkage were determined to be at the hydroxy group in the 4-position by the HMBC spectrum and also β by the coupling constant of the anomeric proton. The structure of 3 is shown in Fig. 1.
Zanthoside D (4), [α]D25–39.7, was isolated as an amorphous powder. Its elemental composition was determined to be C25H30O12 by HR-ESI-MS. The IR spectrum indicated the presence of a carbonyl and an aromatic ring similar to that of 3. In the 1H-NMR spectrum, there were two olefinic protons in a trans double bond, three aromatic protons, one singlet aromatic signal for two protons, four aliphatic protons for two continual methylenes, one methoxy signal for six protons, and anomeric proton. The 13C-NMR spectrum displayed 22 signals, 12 of these were assigned to hexose and a trisubstituted aromatic ring. The hexose was identified with D-glucose by HPLC analysis of its hydrolysate, and the remaining ten signals consisted of 13 carbons, namely the presence of a symmetrically substituted aromatic ring was suggested. The HMBC correlations from methylene protons (H2-7) to three aromatic carbon signals C-1, C-2 with δH 7.02 and C-6, and H-2 with C-4 suggested one of the aromatic rings carried an ethanol side chain and from those H-2 and C-6 and C-4, and H-5 and H-6 with C-3-and C-2, respectively, 1,3,4-substitution of this aromatic ring was established (Fig. 3). Further HMBC correlation between H-1′ and C-3 confirmed the position of the sugar linkage and the mode of the linkage was β. The symmetrical acyl group with two methoxy groups was expected to be sinapic acid from HMBC correlation between H-7″ (δH 7.64) and C-2″(6″) (δC 107.1) (Fig. 3), and the position of the ester linkage was at the hydroxyl group at C-6′ from HMBC correlation and downfield shifts of the H2-6′ proton in the 1H-NMR spectrum. Therefore, the structure of 4 was elucidated to be 3,4-dihydroxyphenethyl alcohol 3-O-β-D-glucopyranoside 6′-O-synapate, as shown in Fig. 1.
The structure of zanthoside A (1) was unambiguously determined to be a glucoside of prenylated coumarin, including the absolute stereochemistry of the aglycone. Zanthosides B and C (2, 3) were also prenylated phenylpropanoids, and the methyl ester in 2 may be formed during the extraction and isolation procedures.
Experimental section is provided as a supplementary material through an internet system. https://www.jstage.jst.go.jp/article/cpb/00/0/00_c00-01012/_article.
The authors are grateful for access to the superconducting NMR instrument (Brucker Avance III 600) at the Analytical Center of Molecular Medicine of the Hiroshima University Faculty of Medicine, and an Applied Biosystem QSTAR XL system ESI (Nano Spray)-MS at the Analysis Center of Life Science of the Graduate School of Biomedical Sciences, Hiroshima University. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Society for the Promotion of Science (Nos. 22590006, 23590130, 25860078, 15H04651 and 17K08336). Thanks are also due to the Research Foundation for Pharmaceutical Sciences and the Takeda Science Foundation for the financial support.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
