Article pubs.acs.org/jnp
Unexpected Secoiridoid Glucosides from Manulea corymbosa Chrysoula Gousiadou,† Tetsuo Kokubun,‡ Charlotte H. Gotfredsen,† and Søren R. Jensen*,† †
Department of Chemistry, Technical University of Denmark, DK-2800, Lyngby, Denmark Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, U.K.
‡
S Supporting Information *
ABSTRACT: From an extract of Manulea corymbosa were isolated four known secoiridoid glucosides (1−4), 10 new monoterpenoid esters of secologanol, namely, manuleosides A−I (5−11, 13, and 14) and dimethyl rhodanthoside A (12), and four new phenylpropanoid esters of carbocyclic iridoid glucosides, manucorymbosides I−IV (15−18). Also, the caffeoyl phenylethanoid glycoside verbascoside was isolated. The presence of secoiridoids apparently derived from loganic acid in the family Scrophulariaceae is unprecedented and greatly unexpected.
D
ethanoid glycoside verbascoside. Of these compounds, 5−18 are new naturally occurring iridoid glucosides.
■
Manuleoside A (5), the main iridoid constituent, was obtained as a syrup, [α]22D −92. For this and the following compounds the molecular formula was determined from the 13 C NMR and HRMS data. For 5, the formula was C27H40O12 with the quasimolecular ion obtained by LC-HRESIMS (observed m/z 557.2607 [M + H]+). The NMR data (Table 1) in methanol-d4 of this and the following compounds (6−18) were assigned using 2D techniques. The 1H NMR spectrum showed resonances corresponding to the presence of an iridoid
uring the last few decades accumulating evidence evolving from DNA sequence investigations has led to taxonomic rearrangements within the family Scrophulariaceae s.l. to an unprecedented extent.1−4 The distribution of specific types of iridoid constituents from different plant taxa has been shown to fit very well with their findings in different plant orders.5 Thus, it has been demonstrated that loganin-derived secoiridoids and the derived complex indole alkaloids are almost exclusively found in the order Gentianales, while the decarboxylated C9-iridoid compounds such as aucubin and catalpol arise from an alternative biosynthetic pathway and are mostly confined to Lamiales including the Scrophulariaceae.6 At lower ranks of classification, the patterns in the structural features of iridoids in families and genera fit remarkably well with the new DNA-based classification.7−9 So far, only a single report on the finding of loganin and biosynthetically derived secoiridoids in Lamiales has appeared, namely, from Lippia graveolens Kunth. (Verbenaceae).10 We can now report a second finding of secoiridoids that are most likely formed by biosynthesis from loganic acid, this time from Manulea corymbosa L. f., an annual herb from South Africa belonging to the Scrophulariaceae s.s. To our knowledge, this genus has never been investigated chemically before.
RESULTS AND DISCUSSION
Frozen plant material was extracted with EtOH, and the watersoluble part of the extract was subjected to a series of chromatographic procedures. Nineteen compounds were isolated and identified, namely, the secoiridoids sweroside (1),11 secologanol (2),12 secoxyloganin (3),13 secologanoside (4),13 manuleosides A−I (5−11, 13, 14), and dimethyl rhodanthoside (12) as well as the carbocyclic iridoid glucosides manucorymbosides I−IV (15−18) and the caffeoyl phenyl© 2013 American Chemical Society and American Society of Pharmacognosy
Special Issue: Special Issue in Honor of Otto Sticher Received: October 11, 2013 Published: December 12, 2013 589
dx.doi.org/10.1021/np400853f | J. Nat. Prod. 2014, 77, 589−595
Journal of Natural Products
Article
Table 1. 1H and 13C NMR Data of Manuleosides A−E (5−9) in Methanol-d4 5 position aglucone 1 3 4 5 6
13
97.7 153.6 111.5 31.6 30.2
6 1
C
H
5.55 d (6.2) 7.47 d (0.7) 2.90 q-like (6.3) 2.00 br sextet (7.0) 1.86 obsc.a 4.14 2H m 5.78 ddd (8.6, 10.4, 17.4) 2.64 td (6.4, 8.2) 5.30 br d (17.4) 5.25 br d (10.4)
C
97.7 153.6 111.7 31.3 30.1
7 8
64.3 135.7
9 10
45.4 119.2
11 OMe Glc 1′ 2′ 3′ 4′
169.1 51.8
3.66 3H s
169.3 51.6
100.2 74.7 78.0 71.6
4.69 3.18 3.36 3.24
100.1 74.7 77.9 71.4
5′ 6′
78.4 62.8
3.31 obsc. 3.65 dd (5.9, 11.9) 3.89 dd (2.1, 11.9)
d (7.9) dd (7.9, 8.9) t (8.9) dd (8.9, 9.3)
Glc 1″ 2″ 3″ 4″ 5″ 6″
a
13
64.2 135.8 45.3 119.5
78.3 62.7b
169.6 128.9 143.3 27.9
5‴
39.2
6‴ 7‴
138.4 125.7
8‴
59.4
9‴
12.5
10‴
16.2
6.74 qt (1.3, 7.5) 2.34 2H q-like (7.5) 2.16 2H t-like (7.5) 5.39 qt (1.3, 6.7) 4.08 2H br d (6.7)
1.82 3H br d (1.3) 1.69 3H br s
H
5.55 d (6.6) 7.47 d (0.6) 2.90 q-like (6.3) 2.02 br sextet (6.9) 1.85 obsc.a 4.15 2H m 5.78 ddd (8.6, 10.4, 17.3) 2.65 m 5.30 br d (17.3) 5.25 br d (10.4) 3.67 3H s 4.69 d (7.9) 3.17 dd (7.9, 8.9) 3.36 t (8.9) 3.25 dd (8.9, 9.3)b 3.31 obsc. 3.65 obsc.b
13
169.7 129.2 143.3 27.8
4.26 d (7.8) 3.18 dd (7.9, 8.9) 3.36 t (8.9) 3.27 obsc.b 3.23 obsc. 3.66 obsc.b 3.86 dd (2.1, 12.1)
8 1
C
97.7 153.4 111.3 31.4 30.0
64.2 135.5 45.2 119.4
H
5.55 d (6.6) 7.47 br s 2.90 q-like (6.3) 2.02 br sextet (7.1) 1.85 obsc.a 4.15 2H m 5.77 ddd (8.8, 10.5, 17.3) 2.65 m 5.30 br d (17.3) 5.26 br d (10.5)
13
97.7 153.6 111.5 31.6 30.2
64.2 135.8 45.4 119.5
63.9 135.5 45.1 119.3
5.55 d (6.5) 7.47 d (0.7) 2.90 q-like (6.6) 2.03 br sextet (6.9) 1.84 obsc.a 4.14 2H m 5.78 ddd (8.6, 10.4, 17.3) 2.64 m 5.30 br d (17.3) 5.25 br d (10.4) 3.67 3H s
100.0 74.5 77.8 71.4
4.70 3.18 3.36 3.25
100.2 74.7 78.0 71.6
4.69 3.18 3.36 3.24
99.9 74.5 77.8 71.5
4.69 3.18 3.36 3.27
78.2 62.6
3.32 obsc. 3.65 obsc.b
78.4 62.8
3.31 obsc. 3.65 dd (6.0, 12.0) 3.89 dd (2.0, 11.9)
77.9 62.6
3.30 obsc. 3.63 dd (5.6, 11.9) 3.89 dd (1.9, 11.9)
99.3 74.9 78.1 71.3 77.5 62.6
4.36 d (7.9) 3.16 obsc.b 3.32 t (8.9) 3.26 obsc.b 3.17 obsc. 3.66 obsc. 3.80 dd (2.4, 11.9)
d (7.9) obsc. t (8.9) obsc.b
3.90 dd (1.6, 11.9)
102.7 74.5 77.8 71.4 77.6 62.5
169.3 128.8 143.7 28.1
4.27 3.18 3.36 3.29 3.23 3.66 3.85
d (7.9) dd (7.9, 8.9) t (8.9) dd (8.9, 9.2)
d (7.8) obsc. t (8.9) obsc.b obsc. obsc.b dd (1.9, 12.0)
41.8
140.7 123.1
5.43 t-like (7.0)
73.6 145.9
65.8
4.33 dd (6.4, 12.0)
112.4
12.4
4.35 dd (6.1, 11.9) 4.25 dd (7.6, 11.9) 1.82 3H br d (1.0
12.4
1.82 3H br s
12.4
16.3
1.71 3H br s
23.3
1.78 3H br s
27.8
66.1
97.5 153.5 111.2 31.2 29.9
H
168.6 51.4
2.31, 2.25 ms
5.41 t-like (6.3)
2.90 q-like (6.3) 2.01 br sextet (7.0) 1.86 obsc.a 4.14 2H m 5.79 ddd (8.6, 10.4, 17.4) 2.64 td (6.4, 8.3) 5.29 br d (17.0) 5.25 br d (10.4)
1
C
3.66 3H s
31.6
136.9 122.4
5.55 d (6.6) 7.47 d (0.7)
13
169.1 51.8
6.74 br t (6.5) 2.32 2H m
39.1
H
3.67 3H s
169.7 128.6 144.1 24.5
6.74 qt (1.4, 7.3) 2.35 2H q-like (7.4) 2.19 2H t-like (7.4)
9 1
C
168.9 51.6
3.90 dd (2.1, 12.0) 102.7 74.7 77.9 71.4 77.9 62.6b
terp 1‴ 2‴ 3‴ 4‴
7 1
4.20 dd (7.8, 12.0)
d (7.9) obsc.b t (8.8) obsc.b
6.75 qt (1.4, 7.6) 2.23 2H m
169.1 128.3 144.1 24.2
6.77 qt (1.3, 7.3) 2.29 2H m
1.60 2H m
40.9
1.70 2H m
5.91 dd (10.8, 17.4) 5.22 dd (1.5, 17.4) 5.05 dd (1.5, 10.8) 1.80 3H br d (1.2) 1.27 3H s
80.9 144.0
12.1
5.95 dd (11.0, 17.7) 5.27 dd (1.2, 17.7) 5.05 dd (1.2, 11.0) 1.80 3H br s
23.1
1.40 3H s
116.0
Obsc.: signal obscured. bValues are pairwise interchangeable in the same column.
glucoside at δH 7.47 (d, H-3), 5.55 (d, H-1), and 4.69 (d, H-1′), as well as resonances from a vinyl group at δH 5.78 (ddd, H-8), 5.25 and 5.30 (d’s, CH2-10), suggesting that 5 is a secoiridoid glucoside. Furthermore, two three-proton resonances at δH 1.82 and 1.69 as well as signals at δH 6.74 and 5.39 suggested the presence of an additional monoterpenoid moiety. In the 13C
NMR spectrum, 27 resonances were observed. Six of these were consistent with the presence of a β-glucopyranosyl group. Of the remaining resonances in the spectrum, 11 (in addition to those from the sugar moiety) were similar to those seen for secologanol (2),12 a compound also found in the plant. The remaining 10 resonances fit well with the presence of a 590
dx.doi.org/10.1021/np400853f | J. Nat. Prod. 2014, 77, 589−595
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Table 2. 1H and 13C NMR Data of Manuleosides F−I (10, 11, 13, and 14) in Methanol-d4 10 position
13
aglucone 1 3 4 5 6
97.7 153.6 111.5 31.6 30.2
7 8 9 10
64.3 135.8 45.4 119.5
11 OMe Glc′ 1′ 2′ 3′ 4′ 5′ 6′
169.1 51.8 100.2 74.7 78.0 71.6 78.4 62.8
11 1
C
H
5.55 d (6.7) 7.47 d (0.7) 2.90 q-like (6.4) 2.00 br sextet (7.0) 1.85 obsc.a 4.15 2H m 5.79 ddd (8.5, 10.4, 17.4) 2.64 m 5.30 br d (17.4) 5.25 br d (10.4)
d (7.9) dd (7.9, t (8.9) dd (8.9, obsc. dd (5.9, dd (2.0,
8.9) 9.3) 11.9) 11.9)
Glc″ 1″ 2″ 3″ 4″ 5″ 6″
a
terp 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴
169.7 128.6 144.1 27.1 36.9 30.5 40.6 60.9
9‴ 10‴
12.4 19.8
6.76 qt (1.4, 7.5) 2.22 2H m 1.46, 1.29 m 1.60 m 1.36, 1.58 m 3.59 2H m 1.82 3H br d (1.1) 0.94 3H d (6.6)
13
C
1
97.7 153.6 111.3 31.6 30.1
5.56 d (6.7) 7.47 d (0.4)
64.2 135.7 45.4 119.6 169.1 51.8
3.66 3H s 4.69 3.18 3.36 3.25 3.31 3.65 3.89
13
13
H
2.90 q-like (6.3) 2.01 br sextet (7.0) 1.86 obsc.a 4.15 2H m 5.79 ddd (8.6, 10.4, 17.4) 2.64 m 5.30 br d (17.4) 5.25 br d (10.4)
4.69 3.18 3.36 3.26 3.33 3.65 3.90
d (7.9) dd (7.9, 8.9) t (8.9) obsc.b obsc. obsc.b dd (2.0, 12.0)
104.4 75.3 78.0b 71.8 78.0b 62.9
4.24 3.16 3.36 3.27 3.18 3.66 3.86
d (7.8) dd (7.9, 8.9) t (8.9) obsc.b obsc. obsc.b dd (1.6, 12.1)
12.4 20.0
64.3 135.8 45.4 119.6 169.2 51.9
3.67 3H s
100.2 74.7 78.0b 71.8 78.2b 63.1
169.6 128.6 144.1 27.1 37.1 30.6 37.6 69.1
97.7 153.6 111.5 31.7 30.3
100.2 74.8 77.9 71.7 78.3 62.9
169.5 129.4 142.4 27.6 40.2 158.8 118.1 170.7
6.76 qt (1.3, 7.5) 2.24 2H m 1.48, 1.29 m 1.65 m 1.70, 1.46 m 3.98 td (6.6, 9.5) 3.56 td (7.0, 9.5) 1.82 3H br s 0.94 3H d (6.5)
12.6 18.8
14 1
C
H
5.56 d (6.8) 7.47 d (0.5) 2.90 q-like (6.3) 1.99 br sextet (6.9) 1.87 obsc.a 4.15 2H m 5.79 ddd (8.5, 10.4, 17.4) 2.64 m 5.29 br d (17.4) 5.25 br d (10.4)
C
1
97.7 153.7 111.5 31.7 30.2
5.56 d (6.7) 7.47 d (0.7)
64.4 135.8 45.4 119.5 169.2 51.8
3.66 3H s 4.70 3.18 3.36 3.25 3.30 3.65 3.90
13
d (7.9) dd (7.9, 8.9) t (8.9) dd (8.9, 9.2) obsc. obsc. dd (2.0, 11.9)
6.72 dt (1.4, 7.2) 2.41 2H q-like (7.3) 2.31 2H t-like (7.3) 5.69 m
1.83 3H br d (1.0) 2.14 3H d (0.9)
H
2.90 q-like (6.3) 1.99 br sextet (6.9) 1.87 obsc.a 4.15 2H m 5.79 ddd (8.5, 10.4, 17.3) 2.64 td (6.7, 7.7) 5.30 br d (17.3) 5.25 br d (10.4) 3.66 3H s
100.2 74.7 78.0b 71.6 78.4 62.7
4.70 3.18 3.36 3.25 3.31 3.64 3.90
d (7.9) dd (7.9, 8.9) obsc. dd (8.9, 9.2) obsc. obsc.b dd (2.1, 11.9)
95.3 74.0 78.1b 71.0 78.8 62.4
5.48 3.34 3.42 3.34 3.31 3.67 3.83
d (8.0) obsc. t (8.9) obsc. obsc. obsc.b dd (2.0, 12.1)
169.4 129.7 142.1 27.5 40.4 163.0 116.4 166.4 12.6 19.2
6.71 dt (1.3, 7.1) 2.41 2H m 2.36 2H m 5.78 m
1.83 3H br d (1.2) 2.20 3H d (1.2)
Obsc.: signal obscured. bValues are pairwise interchangeable in the same column.
monoterpenoid acid moiety forming a carboxyl ester linkage (δC 169.6). Comparison with the spectra of known terpenoid esters of the iridoid glucoside catalpol, namely, nemoroside and the isomer 6″-(Z)-nemoroside,14 indicated that 5 is a foliamenthoyl ester of secologanol (2). The HMBC spectrum allowed the site of attachment between the iridoid moiety and the peripheral part of 5 to be discerned. Thus, the position of the ester group was confirmed by an HMBC correlation between the CH2-7 group (δH 4.14) and the carbonyl carbon of the foliamenthoyl moiety (δC 169.6). This was supported by the downfield shift (δC 64.3) of the CH2-7 group in 5 when compared to that of 2 (δC 61.1).12 Also, cross-peaks could be seen between H-3 (δH 7.47) and the high-field carbonyl carbon atom (δC 169.1) as well as between the methoxy group (δH
3.66) and C-1‴. Thus, manuleoside A (5) was assigned as the 7-O-foliamenthoyl ester of secologanol. Manuleoside B (6) was obtained as a syrup, [α]22D −90. The molecular formula was established as C33H50O17, as indicated by the LC-HRESIMS (observed m/z 719.3117 [M + H]+). The 1 H NMR spectrum (Table 1) was similar to that of 5, including a foliamenthoyl and a secologanyl moiety. However, additional resonances at δH 4.26 and between 3.1 and 3.9 ppm were present, suggesting the presence of an additional hexosyl group, and this was in keeping with the molecular formula. The 13C NMR data for 6 fit well with the presence of an additional βglucopyranosyl group, including an anomeric carbon atom (δC 102.7) and five carbinol atoms between δC 62.6 and 77.9. The remaining resonances were superimposable with those of 5, except for the resonances arising from C-6‴ to C-8‴ of the 591
dx.doi.org/10.1021/np400853f | J. Nat. Prod. 2014, 77, 589−595
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menthiafolic acid-6-O-β-D-glucosides15 as well as the -quinovosides and -xylosides16 have been repoted. In the R-forms the resonances of C-5 exhibit consistently greater upfield shifts by 1.7 to 2.3 ppm, whereas in the S-forms the corresponding shifts are between 0.5 and 0.7 ppm. These observations and the fact that in the case of the pair 8/9 the difference (Table 1) is 0.9 ppm, assuming that the sugar is D-glucose, suggest the configuration at 6‴ is in the S-form in both compounds. Manuleoside F (10) was obtained as a syrup, [α]21D −86. The molecular formula was C27H42O12, indicated by LCHRESIMS (observed m/z 559.2727 [M + H]+). The 1H NMR spectrum (Table 1) was in part similar to that of 5, but the molecular formula indicated that 10 is a dihydro form of 5. Thus, the H-7‴ resonance (δH 5.39) seen in 5 was absent in 10 and replaced with some additional resonances at high field. In keeping with this, the 10‴-methyl and the 8‴-methylene groups were also seen at relatively higher field (δH 0.94 and 3.59) than those in compound 5. The coupling pattern elucidated from the COSY spectrum confirmed the overall structure, except for the configuration at C-6‴. In the 13C NMR spectrum, most of the observed 27 resonances were superimposable with those in the spectrum of 5. Significant differences were that the signals arising from the C-6‴−C-7‴ double bond in 5 were replaced by two signals at high field. Therefore, manuleoside F (10) was established as C-6‴−C-7‴-dihydromanuleoside A. Manuleoside G (11), [α]21D −70, was obtained as a syrup with the molecular formula C33H52O17, as indicated by LCHRESIMS (observed m/z 721.3279 [M + H]+), suggesting that 11 is a glycosylated form of 10. A comparison of the NMR data (Table 2) with those of the pair 5/6 suggested that this is indeed the case. Thus, a significant difference was that the signal arising from the C-8‴-oxymethylene group (δC 69.1) was seen 8.2 ppm downfield when compared with that (δC 60.9) seen in 10, indicating that this is the point of glycosylation. In the HMBC spectrum, the anomeric proton at 4.24 ppm showed a correlation to C-8‴ (δC 69.1), confirming the position of the additional sugar moiety that was found to be a glucosyl group. In conclusion, manuleoside G (11) was proposed as 8‴-O-βglucopyranosyl manuleoside F. We have not been able to determine the absolute configurations of the terpenoid moiety in compounds 10 and 11. Compound 12 was obtained as a crystalline solid, mp 168− 170 °C; [α]23D −108. The molecular formula established was C44H62O22, as indicated by LC-HRESIMS (observed m/z 943.3827 [M + H]+). The NMR data showed that this compound contained two secologanyl and one monoterpenoid moiety, and these were superimposable with those reported for dimethyl rhodanthoside A,17−19 a compound prepared from the naturally occurring diacid rhodanthoside A, isolated from Gentiana rhodantha Franch. (Gentianaceae).17 The melting point was the same as that reported, but the optical rotation differed considerably from the literature value,17 namely, [α]25D −266.6. We are unable to explain this discrepancy other than speculating that the latter may be due to an error, considering also the reported value of the parent diacid is [α]25D −48.5.17 Methylation by CH2N2/Et2O is unlikely to have caused a molecular rearrangement, so a large change in conformation can be dismissed. Manuleoside H (13) was obtained as a syrup, [α]22D −91. The molecular formula was C27H38O13, as indicated by LCHRESIMS (observed m/z 571.2374 [M + H]+). The 1H and 13 C NMR data (Table 2) were similar to those seen for 12 but contained only resonances from a single secologanyl moiety.
terpenoid moiety. The point of attachment was indicated by the low-field shift of the C-8‴ carbon atom (δC 66.1) when compared to that of 5 (δC 59.4). Analysis of the HMBC spectrum confirmed this. Thus, a correlation was seen between the CH2-8‴ group (δH 4.35 and 4.25) and the anomeric C-1″ carbon atom (δC 102.7). Accordingly, manuleoside B was deduced as 8‴-O-β-glucopyranosyl manuleoside A. Manuleoside C (7) was isolated as a syrup that could not be purified completely, due to the small amount isolated. However, the structure could be established by spectroscopy, and the molecular formula was C33H50O17, as indicated by LCHRESIMS (observed m/z 719.3118 [M + H]+). The 1H NMR spectrum (Table 1) was very similar to that of 6, except for some differences at high field (1.8−2.4 ppm). Also, the 13C NMR data (Table 1) for 7 and 6 were similar, except for the resonances arising from C-5‴−C-8‴ and C-10‴, and this suggested that the two compounds are E/Z-isomers. Comparison with nemoroside and the isomer 6″-(Z)-nemoroside14 showed that 7 is the 6‴-Z-isomer of 6. Manuleoside D (8) was obtained as a syrup, [α]22D −91. The molecular formula was C27H40O12, as indicated by LCHRESIMS (observed m/z 557.2609 [M + H]+). The 1H NMR spectrum (Table 1) was reminiscent of that of 5, except for some differences in the terpenoid moiety. Thus, resonances from an additional vinyl group were present in the spectrum, indicating that in this case it was instead the menthiafoloyl ester of 2. This was in agreement with the low-field value (δH 1.27) seen for the 10‴-methyl group.14 In the 13C NMR spectrum, 27 resonances were observed. Of these, 17 were consistent with the presence of a secologanyl moiety. The remaining 10 resonances were almost identical to those reported for a menthiafoloyl ester.14 Again, the HMBC spectrum allowed the link between the two moieties to be determined, since a correlation was seen between the CH2-7 group (δH 4.14) and the carbonyl carbon of the menthiafoloyl moiety (δC 169.7). Thus, manuleoside D (8) was shown to be the 7-Omenthiafoloyl ester of secologanol. Manuleoside E (9), [α]22D −77, was purified as a syrup. The molecular formula was the same as those of 6 and 7, namely, C33H50O17, as indicated by LC-HRESIMS (observed m/z 719.3148 [M + H]+). The 1H NMR spectrum (Table 1) was in part similar to that of 6, again with some differences in the terpenoid moiety and by signals from an additional glucosyl group. Of the 33 resonances in the 13C NMR spectrum, 17 were identical to those of 5−8 and six could be affilliated to a βglucopyranosyl moiety. The remaining 10 resonances corresponded in part to those of the menthiafoloyl moiety in 8, but the shift of the C-6‴ carbon atom (δC 80.9) was found 7.3 ppm downfield when compared to that of 8. Also, the upfield shifts of C-7‴ and C-10‴ and the downfield shift of C-8‴ were consistent with the interpretation that the glucosyl moiety is attached to the tertiary C-6‴ oxygen. The data compared well with the 13 C NMR spectrum of 6β-O-glucopyranosyl menthiafolic acid methyl ester,15 allowing for the different solvents used. The HMBC spectrum confirmed the position of the glucosyl group. Thus, the anomeric proton H-1″ (δH 4.36) showed a correlation to C-6‴ (δC 80.9). Therefore, manuleoside E (9) was determined as 6‴-O-β-glucopyranosyl manuleoside D. The magnitude of glycosylation-induced shifts in the 13C NMR spectrum may allow the deduction of the stereostructure of the tertiary alcohol of the terpenoid moiety in the pair 8 and 9. Thus, the 13C chemical shifts of diastereomeric pairs of 592
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C26H32O11, as indicated by LC-HRESIMS (observed m/z 521.2011 [M + H]+). The 1H NMR spectrum revealed the presence of an iridoid glucoside moiety identical to cachineside I,22 an aldehyde first isolated from the leaves of Campsis chinensis (Lam.) Voss (Bignoniaceae). Characteristic signals were observed at δH 7.39 (d, H-3), 5.49 (d, H-1), 1.10 (d, H10), and 9.23 (s, H-11). The H-7 signal (δH 5.26) was deshielded significantly, implying substitution at this position as in 15 above. Additional resonances in the spectrum revealed the presence of an AA′BB′ aromatic system, a methoxy group (δH 3.83), and a trans double bond: Hα (δH 6.38, d, J = 16.0 Hz) and Hβ (δH 7.64, d, J = 16.0 Hz), typical of a 4-methoxy-(E)cinnamoyl moiety. In the HMBC spectrum, cross-peaks could be observed between the protons of the methoxy group and the high-field carbon atom C-4‴ (δC 163.2) as well as between H-3 and C-11 (δC 193.3). Thus, manucorymboside III (17) was assigned as the trans 4-methoxycinnamoyl ester of cachineside I. Manucorymboside IV (18) was observed to be the Z-isomer of 17 and was obtained as a syrup, [α]22D −56. The molecular formula was C26H32O11, as indicated by LC-HRESIMS (observed m/z 521.2009 [M + H]+). The 1H NMR spectrum revealed the presence of cachineside I esterified with a cis 4methoxycinnamoyl group. The structure was elucidated as above. Thus, resonances in the spectrum revealed the presence of an AA′BB′ aromatic system, a methoxy group (δH 3.81), and a cis double bond: Hα (δH 5.83, d, J = 12.7 Hz) and Hβ (δH 6.95, d, J = 12.7 Hz), typical of a 4-methoxy-(Z)-cinnamoyl moiety. Thus, manucorymboside IV (18) was assigned as the cis 4-methoxycinnamoyl ester of cachineside I. After standing for a week in the NMR tube in the laboratory daylight, the 1H NMR spectrum showed that ca. 15% of the E-isomer (17) was present in the sample. In contrast, no Z-isomer was formed in the sample of 17 under identical conditions. This demonstrated that manucorymboside IV (18) is a genuine constituent of the plant and not an extraction artifact. The finding of secoiridoids in the Scrophulariaceae is surprising, since iridoid glucosides so far reported from this family are all considered to be formed by a biosynthetic pathway involving 8-epi-deoxyloganic acid, a compound with the 8α-configuration.6 Neither loganin or loganic acid, the known precursors for secologanin and both with the 8βconfiguration, has so far been recorded from the Scrophulariaceae. The co-occurrence of the loganic acid ester (15) with manuleosides 5−14 indicates that the latter are formed by biosynthesis through loganic acid and secologanin, the pathway normally found in Gentianales, but different from that of the Oleaceae secoiridoids, which are derived from 7-epi-loganin or ketologanin.23
Except for differing intensities of the secologanyl signals, the spectra of the two compounds were superimposable. The link between the terpenoid and the secoiridoid moieties could be established by cross-peaks in the HMBC spectrum. Thus, both the CH2-7 group (δH 4.15) and the 9‴-methyl group (δH 1.83) shared correlations to the 1‴-carbonyl carbon atom (δC 169.5), and this was used to establish the structure given as 13. Manuleoside I (14) was obtained as a syrup, [α]22D −59. The molecular formula was C33H48O18, as indicated by LCHRESIMS (observed m/z 733.2905 [M + H]+). The 1H NMR data (Table 1) were very similar to those of 13, except for resonances from an additional sugar moiety in the spectrum of 14. Significantly, the anomeric proton signal from this moiety resonated at unusually low field (δH 5.48), suggesting that it is esterified at this position.20 The 13C NMR spectrum presented 33 resonances, of which 27 were superimposable with those of 13, except for those of C-6‴ to C-8‴, indicating that this end of the terpenoid part is involved in the linkage to the hexose moiety. The signal pattern revealed that it was again a β-glucopyranosyl moiety, except that the chemical shift of the anomeric carbon atom was at unusually high field (δC 95.3), corroborating the observation made in the 1 H NMR spectrum.20 The position of the link could be confirmed as above by correlations between the anomeric proton signal H-1″ (δH 5.48) as well as the 10‴-methyl group (δH 2.20) and the 8‴-carbonyl carbon atom (δC 166.4), establishing the structure proposed as 14. Manucorymboside I (15) was obtained as a syrup, [α]22D −34. The molecular formula was C27H34O13, as indicated by LC-HRESIMS (observed m/z 567.2067 [M + H]+). The 1H NMR spectrum showed resonances corresponding to the presence of an esterified loganic acid15,21 moiety as well as a trans 3,4-dimethoxycinnamoyl group. Signals corresponding to the iridoid glucoside were observed at δH 7.44 (d, H-3), 5.28 (d, H-1), and 1.10 (d, H-10). The pronounced downfield shift of the H-7 signal (δH 5.29) suggested the position of substitution. Additional resonances in the spectrum revealed the presence of an ABX aromatic system and two methoxy groups at δH 3.86 and 3.87, typical of a trans 3,4dimethoxycinnamoyl moiety. The position of the ester group was confirmed by an HMBC correlation between the CH-7 group (δH 5.29) and the carbonyl carbon of the trans 3,4dimethoxycinnamoyl moiety (δC 168.3). Cross-peaks could also be observed between the methoxy groups (δH 3.86, 3.87) and the carbon atoms C-3‴ (δC 150.3) and C-4‴ (δC 152.2) as well as C-2‴ (δC 111.1) of the aromatic ring. Finally, cross-peaks could be found between H-3 (δH 7.44) and the low-field carboxyl carbon atom (δC 170.4). Thus, manucorymboside I was found to be a trans 3,4-dimethoxycinnamoyl ester of loganic acid. Manucorymboside II (16) was obtained as a syrup, [α]22D −54. The molecular formula was C27H34O12, as indicated by LC-HRESIMS (observed m/z 551.2115 [M + H]+). The 1H NMR spectrum revealed that 16 has a structure identical to that of manucorymboside I, except for the presence of an aldehyde proton resonance at very low field (δH 9.23). On comparing the two 13C NMR spectra, a typical downfield shift of C-11 (δC 193.5) was observed, thus confirming that manucorymboside II is the 11-aldehyde corresponding to 15. Manucorymboside III (17) was obtained as a syrup that could not be purified completely, due to the small amount isolated. However, the structure was established using spectroscopic means, and the molecular formula was
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined on a Stuart smp30 apparatus. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. One-dimensional 1H, 13 C NMR and 2D DQF-COSY, gHSQC, gHMBC, and NOESY NMR spectra were recorded on a Varian Unity Inova 500 MHz spectrometer or a 400 MHz Bruker Avance III equipped with a BBO Prodigy probe in methanol-d4, and the chemical shifts are given as δ values with reference to the solvent peaks (δH 3.30 or δC 49.0). LC-HRESIMS was performed using a reversed-phase UHPLC-UV/vis-HRMS on a maXis G3 quadrupole time-of-flight (qTOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) connected to an Ultimate 3000 UHPLC system (Dionex, Sunnyvale, CA, USA) and equipped with a 10 cm Kinetex C18 column (Phenomenex Torrance, CA, USA), 593
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Table 3. 1H and 13C NMR Data of Manucorymbosides I−IV (15−18) in Methanol-d4 15 position aglucone 1 3 4 5 6a 6b 7 8 9 10 11 Glc 1′ 2′ 3′ 4′ 5′ 6′ aryl CO α β 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ OMe a
13
97.3 152.1 113.0 32.6 40.2 78.3 41.0 46.8 13.6 170.4 99.8 74.6 77.7 71.5 78.1 62.5
168.3 116.2 146.1 128.2 111.1 150.3 152.2 112.3 123.7 56.1 56.1
16 1
C
H
5.28 d (5.1) 7.44 d (1.0) 3.15 2.35 1.81 5.29 2.20 2.09 1.10
q-like (8) ddd (1.1, 7.6, 14.3) ddd (5.1, 8.0, 14.3) obsc.a m dt (5.2, 8.6) 3H d (6.9)
4.68 3.21 3.38 3.27 3.32 3.67 3.90
d (7.9) dd (7.9, 9.0) t (9.0) obsc. obsc. dd (5.9, 11.9) dd (1.9, 11.9)
6.42 d (15.9) 7.62 d (15.9) 7.23 d (1.9)
6.97 7.18 3.86 3.87
d (8.4) dd (1.9, 8.4) 3H s 3H s
13
98.1 163.2 126.1 30.0 39.9 78.3 40.5 46.7 13.1 193.5 99.9 74.5 77.6 71.3 78.1 62.4
168.5 116.2 146.5 128.7 112.3 150.8 152.9 111.4 124.0 56.2 56.2
17 1
C
13
H
5.49 d (4.1) 7.39 d (0.8) 3.13 2.33 1.83 5.27 2.12 2.16 1.10 9.23
q-like (7.7) ddd (1.6, 8.0, 14.5) ddd (5.4, 7.1, 14.5) t-like (4.1) obsc.a obsc. 3H d (6.4) s
4.70 3.20 3.37 3.27 3.32 3.67 3.91
d (7.9) dd (8.0, dd (8.9, obsc. obsc. dd (6.1, dd (2.1,
9.0) 9.0)
11.9) 11.9)
6.42 d (15.9) 7.63 d (15.9) 7.17 dd (1.9, 8.4)
6.97 7.23 3.86 3.86
d (8.4) d (1.9) 3H s 3H s
98.4 163.4 125.9 30.5 39.2 78.6 40.8 46.9 13.5 193.3 100.3 74.7 78.0 71.6 78.5 62.8
168.9 116.3 146.2 128.3 131.0 115.4 163.2 115.4 131.0 55.9
18 1
C
H
5.49 d (4.1) 7.39 d (0.9) 3.13 2.33 1.82 5.26 2.14 2.17 1.10 9.23
m ddd (1.7, 8.0, 14.6) m m obsc.a obsc. 3H d (6.5) s
4.70 3.20 3.37 3.26 3.28 3.66 3.91
d (7.9) dd (7.9, 9.0) dd (8..8, 9.0) obsc. obsc. obsc. dd (2.1, 11.9)
6.38 d (16.0) 7.64 d (16.0) 7.56 d (8.7) 6.95 d (8.7) 6.95 d (8.7) 7.56 d (8.7) 3.83 3H s
13
1
C
98.2 163.1 125.6 30.1 39.0 78.5 40.3 46.6 13.1 193.3 100.2 74.5 77.9 71.4 78.2 62.7
167.9 118.1 144.5 129.1 133.0 114.4 161.8 114.4 133.0 55.7
H
5.43 d (4.3) 7.36 d (0.9) 3.01 2.30 1.78 5.18 2.06 1.97 0.99 9.21
q-like (7.8) ddd (1.6, 8.0, 14.6) ddd (5.4, 7.2, 14.6) dt (1.3, 5.1) m ddd (4.3, 8.0, 9.3) 3H d (6.8) s
4.67 3.19 3.36 3.26 3.31 3.65 3.90
d (7.9) dd (7.9, dd (8.9, dd (8.9, obsc.a dd (6.0, dd (2.1,
9.0) 9.0) 9.1) 11.9) 11.9)
5.83 d (12.7) 6.95 d (12.7) 7.61 d (8.8) 6.90 d (8.8) 6.90 d (8.8) 7.61 d (8.8) 3.81 3H s
Obsc.: signal obscured.
running a 10−100% acetonitrile gradient system in 10 min at 40 °C. MS was operated in positive electrospray ionization and calibrated using sodium formate automatically infused before each analytical run.24 Initial LPLC separations were performed on a Merck Lobar RP18 column size B using MeOH−H2O mixtures. Further separations by HPLC were done on a Waters system (600 pump, 717-Plus autosampler, 996 photodiode array detector; Milford, MA, USA), with a Genesis C18 column (10 mm i.d. × 250 mm, 5 μm; Jones Chromatography, Mid Glamorgan, UK) at 30 °C, with either MeOH− H2O or MeCN−H2O mixtures as eluents at a flow rate of 4 mL/min. Known compounds were identified by 1H and 13C NMR spectra. Plant Material. Manulea corymbosa was grown from seeds obtained from Silverhill Seeds, Cape Town, SA. The plant was autheticated by Prof. Dirk Albach, University of Oldenburg, Germany, and a voucher specimen (IOK-20/2003) has been deposited at the Herbarium of Vienna. Extraction and Isolation. Frozen plant material (194 g) was homogenized with EtOH and left for 24 h. The extract was filtered, taken to dryness, and partitioned between H2O and Et2O. The aqueous phase was concentrated (6.58 g), and a portion (2.13 g) was redissolved in H2O and subjected to C18 reverse-phase chromatography (Lobar size B), eluting with MeOH−H 2 O mixtures. Compounds were obtained in order of elution: secologanoside (4; 38 mg); sweroside (1; 19 mg), and a fraction with 2 and 3 (26 mg) was further separated on a Merck LiChrosphere C18e column (4.0 mm i.d. × 250 mm, 5 μm)25 to give secologanol (2; 6 mg) and secoxyloganin (3; 8.1 mg). A subsequent fraction yielded verbascoside (75 mg). Two following Lobar fractions, A and B, eluted with
MeOH−H2O (1:1), were further purified with preparative HPLC with isocratic elution, obtaining from fraction A manuleoside I (14; 7.8 mg) and manucorymboside III (17; 3.7 mg). Fraction B gave manuleoside A (5; 24 mg) and manuleoside D (8; 7.7 mg). The remaining original extract (4 g) was prefractioned on the Lobar column as described above. Fraction C, eluted with MeOH−H2O (1:1) (56 mg), was separated further by repeated injections on preparative HPLC with 30% MeCN (isocratic to 14 min),26 giving manuleoside E (9; 3.4 mg), manuleoside B (6; 5.8 mg), manuleoside C (7; 2.6 mg), and manucorymboside I (15; 5.3 mg), and with 40% MeCN (isocratic from 14 min), giving manucorymboside II (16; 4.0 mg) and manucorymboside IV (18; 3.0 mg). The following fraction D, which was also eluted with MeOH−H2O (1:1) (174 mg) and subjected to preparative HPLC with 35% MeCN (isocratic), gave manuleoside G (11; 3.9 mg), manuleoside H (13; 3.6 mg) (followed by additional preparative HPLC runs with 60% MeOH, isocratic), manuleoside A (5; 77.6 mg), and manuleoside D (8; 20.9 mg). A subsequent fraction E was further purified by preparative HPLC with 63% MeOH and 68% MeOH, to give manuleoside F (10; 14.4 mg) and dimethylrhodanthoside A (12; 24.3 mg). Manuleoside A (5): colorless syrup; [α]22D −92 (c 0.3; MeOH); 1H and 13C NMR, Table 1; LC-HRESIMS m/z 557.2607 [M + H]+ (calcd for C27H41O12, 557.2595). Manuleoside B (6): colorless syrup; [α]22D −90 (c 0.3; MeOH); 1H and 13C NMR, Table 1; LC-HRESIMS m/z 719.3117 [M + H]+ (calcd for C33H51O17, 719.3121). Manuleoside C (7): impure syrup; 1H and 13C NMR, Table 1; LCHRESIMS m/z 719.3118 [M + H]+ (calcd for C33H51O17, 719.3121). 594
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Manuleoside D (8): colorless syrup; [α]21D −91 (c 0.5; MeOH); 1H and 13C NMR, Table 1; LC-HRESIMS m/z 557.2609 [M + H]+ (calcd for C27H41O12, 557.2593). Manuleoside E (9): colorless syrup; [α]22D −77 (c 0.3; MeOH); 1H and 13C NMR, Table 1; LC-HRESIMS m/z 719.3148 [M + H]+ (calcd for C33H51O17, 719.3121). Manuleoside F (10): colorless syrup; [α]21D −86 (c 0.4; MeOH); 1 H and 13C NMR, Table 2; LC-HRESIMS m/z 559.2727 [M + H]+ (calcd for C27H43O12, 559.2749). Manuleoside G (11): colorless syrup; [α]21D −70 (c 0.3; MeOH); 1 H and 13C NMR, Table 2; LC-HRESIMS m/z 721.3279 [M + H]+ (calcd for C33H53O17, 721.3277). Dimethyl rhodanthoside A (12): colorless, crystalline solid; mp 168−170 °C (dec); [α]23D −108 (c 0.2; MeOH); LC-HRESIMS m/z 943.3827 [M + H]+ (calcd for C44H63O22, 943.3806). Manuleoside H (13): colorless syrup; [α]22D −91 (c 0.2; MeOH); 1 H and 13C NMR, Table 2; LC-HRESIMS m/z 571.2374 [M + H]+ (calcd for C27H39O13, 571.2385). Manuleoside I (14): colorless syrup; [α]22D −59 (c 0.3; MeOH); 1H and 13C NMR, Table 2; LC-HRESIMS m/z 733.2905 [M + H]+ (calcd for C33H49O18, 733.2913). Manucorymboside I (15): colorless syrup; [α]22D −34 (c 0.4; MeOH); 1H and 13C NMR, Table 3; LC-HRESIMS m/z 567.2067 [M + H]+ (calcd for C27H35O13, 567.2072). Manucorymboside II (16): colorless syrup; [α]22D −54 (c 0.2; MeOH); 1H and 13C NMR, Table 3; LC-HRESIMS m/z 551.2115 [M + H]+ (calcd for C27H35O12, 551.2123). Manucorymboside III (17): impure syrup; 1H and 13C NMR, Table 3; LC-HRESIMS m/z 521.2011 [M + H]+ (calcd for C26H33O11, 521.2017). Manucorymboside IV (18): colorless syrup; [α]22D −56 (c 0.2; MeOH); 1H and 13C NMR, Table 3; LC-HRESIMS m/z 521.2009 [M + H]+ (calcd for C26H33O11, 521.2017).
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REFERENCES
(1) Olmstead, R. G.; Reeves, P. A. Ann. Mo. Bot. Gard. 1995, 82, 176−193. (2) Olmstead, R. G.; DePamphilis, C. W.; Wolfe, A. D.; Young, N. D.; Elisons, W. J.; Reeves, P. A. Am. J. Bot. 2001, 88, 348−361. (3) Oxelman, B.; Kornhall, P.; Olmstead, R. G.; Bremer, B. Taxon 2005, 54, 411−425. (4) Tank, D. C.; Beardsley, P. M.; Kelchner, S. A.; Olmstead, R. G. Aust. Syst. Bot. 2006, 19, 289−307. (5) Jensen, S. R. Ann. Mo. Bot. Gard. 1992, 79, 284−302. (6) Jensen, S. R. In Ecological Chemistry and Biochemistry of Terpenoids; Proceedings of the Phytochemical Society of Europe 31; Harborne, J. B.; Tomas-Barbaran, F. A., Eds.; Clarendon Press: Oxford, UK, 1991; Chapter 6, pp 133−158. (7) Rønsted, N.; Franzyk, H.; Mølgaard, P.; Jaroszewski, J. W.; Jensen, S. R. Plant Syst. Evol. 2003, 242, 63−82. (8) Taskova, R. M.; Gotfredsen, C. H.; Jensen, S. R. Phytochemistry 2006, 67, 286−301. (9) Jensen, S. R.; Li, H.-Q.; Albach, D. C.; Gotfredsen, C. H. Phytochemistry 2008, 69, 2162−2166. (10) Rastrelli, L.; Caceres, A.; Morales, C.; De Simone, F.; Aquino, R. Phytochemistry 1998, 49, 1829−1832. (11) Jensen, S. R.; Lyse-Petersen, S. E.; Nielsen, B. J. Phytochemistry 1979, 18, 273−277. (12) Damtoft, S.; Franzyk, H.; Jensen, S. R. Phytochemistry 1994, 35, 705−711. (13) Calis, I.; Sticher, O. Phytochemistry 1984, 23, 2539−2540. (14) Arslanian, R. L.; Anderson, T.; Stermitz, F. R. J. Nat. Prod. 1990, 53, 1485−1489. (15) Tanahashi, T.; Shimada, A.; Nakakura, N.; Inoue, K.; Ono, M.; Fujita, T.; Chen, C.-C. Chem. Pharm. Bull. 1995, 43, 729−733. (16) Kiuchi, F.; Gafur, M. A.; Obata, T.; Tachibana, A.; Tsuda, Y. Chem. Pharm. Bull. 1997, 45, 807−812. (17) Ma, W.-G.; Fuzzati, N.; Wolfender, J.-L.; Hostettmann, K.; Yang, C.-R. Helv. Chim. Acta 1994, 77, 1660−1671. (18) Ma, W.-G.; Fuzzati, N.; Wolfender, J.-L.; Yang, C.-R.; Hostettmann, K. Phytochemistry 1996, 43, 805−810. (19) Yu, G.-P.; Li, X.-C.; Liu, Y.-Q.; Yang, C.-R. Acta Bot. Yunnan. 1996, 18, 110−114. (20) Demuth, H.; Jensen, S. R.; Nielsen, B. J. Phytochemistry 1989, 28, 3361−3364. (21) Calis, I.; Sticher, O. J. Nat. Prod. 1985, 48, 108−110. (22) Imakura, Y.; Kobayashi, S.; Kida, K.; Kido, M. Phytochemistry 1984, 23, 2263−2269. (23) Jensen, S. R.; Franzyk, H.; Wallander, E. Phytochemistry 2002, 60, 213−231. (24) Andersen, M. R.; Nielsen, J. B.; Klitgaard, A.; Petersen, L. M.; Zachariasen, M.; Hansen, T. J.; Blicher, L. H.; Gotfredsen, C. H.; Larsen, T. O.; Nielsen, K. F.; Mortensen, U. H. Proc. Natl. Acad. Sci., U.S.A. 2013, 110, E99−E107 SE99/1-SE99/16. (25) Euerby, M. R.; Petersson, P. J. Chromatogr. A 2003, 994, 13−36. (26) Taskova, R.; Kokubun, T.; Alipieva, K. In High Performance Liquid Chromatography in Phytochemical Analysis; Chromatographic Science Series 102; Waksmundzka-Hajnos, M.; Sherma, J., Eds.; CRC Press: Boca Raton, FL, 2011; Chapter 28, pp 709−727.
ASSOCIATED CONTENT
S Supporting Information *
NMR spectra (1H and 13C or HSQC) of manuleosides A−I (5−14) and manucorymbosides I−IV (15−18) are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +45-45252103. Fax: +45-4593 3968. E-mail: srj@kemi. dtu.dk (S. R. Jensen). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research project is implemented within the framework of the Action “Supporting Postdoctoral Researchers” of the Operational Program “Education and Lifelong Learning” (Action’s Beneficiary: General Secretariat for Research and Technology) and is cofinanced by the European Social Fund (ESF) and the Greek State. We thank the staff of The Botanical Garden of Copenhagen for growing the plant material and Dr. A. Klitgård, BioCentrum, DTU, DK, for providing highresolution ESIMS data. We thank Prof. D. Albach, University of Oldenburg, Germany, for authenticating the plant and Ms. F. Ferris, South Africa, for permission to use her photograph of M. corymbosa.
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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry. 595
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