Chemotaxonomy of Pseudowintera: Sesquiterpene ...

ARTICLE IN PRESS Phytochemistry xxx (2010) xxx–xxx

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Chemotaxonomy of Pseudowintera: Sesquiterpene dialdehyde variants are species markers Kjirsten A. Wayman a, Peter J. de Lange b, Lesley Larsen c, Catherine E. Sansom c, Nigel B. Perry c,* a

Department of Chemistry, Humboldt State University, 1 Harpst Street, Arcata, CA 95521, USA Research, Development and Information, Department of Conservation, Box 68908, Newton, Auckland, New Zealand c New Zealand Institute for Plant and Food Research Ltd., Department of Chemistry, University of Otago, Box 56, Dunedin, New Zealand b

a r t i c l e

i n f o

Article history: Received 10 November 2009 Received in revised form 27 January 2010 Available online xxxx Keywords: Pseudowintera insperata Pseudowintera colorata Pseudowintera axillaris Winteraceae Chemotaxonomy Sesquiterpene dialdehydes Coumarates Cinnamates Drimanes Polygodial

a b s t r a c t Two sesquiterpene dialdehydes, the 1b-E-coumaroyl-5a-hydroxypolygodial plus the known 1b-E-cinnamoyl-5a-hydroxypolygodial, were isolated from the recently described species Pseudowintera insperata. This discovery is a further example of the rare sesquiterpene dialdehyde coumarate/cinnamate combination being found exclusively in the family Winteraceae. Another sesquiterpene dialdehyde, isopaxidal, with the rare rearranged drimane skeleton, was isolated from Pseudowintera axillaris. The sesquiterpene dialdehyde contents of leaves of 25 individual plants of the four Pseudowintera species, all endemic to New Zealand, were measured by HPLC. P. insperata individuals all had high levels (3.0– 6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2–6.9%) of paxidal, and Pseudowintera colorata from different areas of New Zealand contained varying levels of polygodial (1.4–2.9%) and 9deoxymuzigadial (0–2.9%). Therefore the sesquiterpene dialdehydes are good species markers. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Sesquiterpene dialdehydes occur in a variety of organisms, including fungi, marine sponges, liverworts and flowering plants (Jansen and de Groot, 2004). This is probably because the various biological activities of these compounds (Jansen and de Groot, 2004) protect these organisms. The most widespread sesquiterpene dialdehyde is polygodial (1) with a drimane skeleton. Dialdehydes with the rearranged drimane skeleton of 9-deoxymuzigadial (2) are much rarer, having only been reported from the plants Canella winterana (Al-Said et al., 1990; Ying et al., 1995), Warburgia salutaris (Clarkson et al., 2007), Warburgia ugandensis (Kioy et al., 1990; Kubo and Ganjian, 1981), Warburgia stuhlmannii (Kioy et al., 1990; Kubo and Ganjian, 1981; Wube et al., 2005), Pseudowintera colorata (Gerard et al., 1993) and Pseudowintera axillaris (Brennan et al., 2006). Canella and Warburgia are in the Canellaceae and Pseudowintera is in the Winteraceae, the two families in the

* Corresponding author. Tel.: +64 3 479 8354; fax: +64 3 479 7906. E-mail address: [email protected] (N.B. Perry).

order Cannelales (Takhatajan, 2009). Another variant on the sesquiterpene dialdehyde structure is substitution with cinnamate or coumarate at C1, as in paxidal (3), 1b-E-p-coumaroyl-6ahydroxypolygodial (8) and 1b-E-cinnamoyl-5a-hydroxypolygodial (9). Such compounds have only been found in the Winteraceae species: Drimys brasiliensis (Vichnewski et al., 1986), Drimys granadensis (Ferreto et al., 1988), Drimys winteri (Cechinel Filho et al., 1998; Malheiros et al., 2001), P. axillaris (Brennan et al., 2006), P. colorata (Larsen et al., 2007) and most recently in Zygogynum spp. (Allouche et al., 2009; Fotsop et al., 2008). In the light of these chemotaxonomic relationships, the discovery of a new species of Pseudowintera, Pseudowintera insperata (Heenan and de Lange, 2006), led us to expect that this plant might contain new sesquiterpene dialdehydes. Pseudowintera (horopito) is a New Zealand endemic genus of shrubs and small trees comprising four species: P. axillaris (J.R. Forst and G. Forst) Dandy, P. colorata (Raoul) Dandy, Pseudowintera traversii (Buchan.) Dandy (Allan, 1961) and the recently described P. insperata Heenan & de Lange (Heenan and de Lange, 2006). P. axillaris and P. colorata are wide ranging on both North and South Islands of New Zealand, though P. axillaris is mainly northerly. P. traversii is much less common, found only in the

0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.01.017

Please cite this article in press as: Wayman, K.A., et al. Chemotaxonomy of Pseudowintera: Sesquiterpene dialdehyde variants are species markers. Phytochemistry (2010), doi:10.1016/j.phytochem.2010.01.017

ARTICLE IN PRESS 2

K.A. Wayman et al. / Phytochemistry xxx (2010) xxx–xxx

Fig. 1. Pseudowintera collection sites and major dialdehydes; South Island data from Brennan et al. (2006) and Perry et al. (1996b).

north-west of the South Island. P. insperata is confined to the volcanic plugs above the Whangarei Harbour, north of Auckland on the Northland Peninsula (Fig. 1), and is a threatened species known only from 46 mature individuals (de Lange et al., 2009). In Northland P. axillaris and P. colorata are scarce, and there are plants with foliage, fruit and flower characteristics suggestive of hybridism between these species (Vink, 1970). Sampson (1980) argued against such hybrids, based on morphology and experimental attempts to generate them. While the naming of P. insperata resolved some of the uncertainty about Northland Pseudowintera, further morphological intermediates remain. In particular, Heenan and de Lange (2006) could not resolve the status of Northland plants from Logue’s Bush (unknown, Fig. 1), which were more similar to P. insperata than to any other Pseudowintera collection. Leaves of P. colorata contain polygodial (1) and 9-deoxymuzigadial (2) with anti-Candida, insect antifeedant and anthelmintic activities (Gerard et al., 1993; Lorimer et al., 1996; McCallion et al., 1982). Polygodial (1) is also intensely pungent to humans (Szallasi et al., 1996), so its presence at high levels in P. colorata leaves is the basis of the common name pepper tree (Riley, 1994). Individual P. colorata plants varied in their levels of 1 and 2, with plants from four populations on the South Island of New Zealand showing two chemotypes: a mixed chemotype with similar levels of polygodial and 9-deoxymuzigadial, and a polyg-

odial chemotype with very low levels of 9-deoxymuzigadial (Fig. 1) (Perry et al., 1996b). The leaves of P. axillaris contain sesquiterpene dialdehyde cinnamates, paxidal (3) and the 6-hydroxy derivatives 4 and 5, with activity against fungal plant pathogens (Brennan et al., 2006). P. axillaris leaves are much less pungent than those of P. colorata (Riley, 1994), despite paxidal (3) possessing the unsaturated 1,4-dialdehyde moiety associated with pungency (Szallasi et al., 1996). The only reported work on the rare P. traversii showed an unidentified dialdehyde (Larsen et al., 2007). Leaves of P. colorata also yielded the sesquiterpene cyclocolorenone (6) (Corbett and Speden, 1958), common flavonols and flavones (Williams and Harvey, 1982), and antioxidant anthocyanins and other flavonoid glycosides, which were at higher levels in the red portions of leaves around wounds (Gould et al., 2002). Fruits of P. colorata contained very high levels of 1 and 2, plus sesquiterpene dialdehyde cinnamate 7 and coumarate 8 (Larsen et al., 2007). We now report that leaves of P. insperata contain high levels of a new sesquiterpene dialdehyde, coumarate (10), plus the rare cinnamate (9). Another new sesquiterpene dialdehyde, isopaxidal (12), with the rare rearranged drimane skeleton, was isolated from P. axillaris. We also report HPLC analyses of the sesquiterpene dialdehyde contents of leaves of individual plants of the four Pseudowintera species, and discuss the chemistry of potential hybrids.


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O

O

R1

O

O

H

H 2

1 R1

12 9

4

6

O

H 13

6'

12 R1 = Cinnamate

12

1

9

O

4

6

Cinnamate =

14

11 O

1 14

11 O

O 15

O 15

H

R6

13

O

4'

O

1'

O

H 13

3 R1 = Cinnamate, R6 = H 4 R1 = Cinnamate, R6 = β-OH 5 R1 = Cinnamate, R6 = α-OH H O

6 R1

O 15

11 O

1 4

H 14

13

OH 12

9

O

6

Coumarate =

6' 4'

OH

7 R1 = Cinnamate 8 R1 = Coumarate

1'

O

OCH3 R1

O 15 1

11 O 9

4

O

4'

6

OH 14

6' 12

13

p-Methoxycinnamate = O 1'

9 R1 = Cinnamate 10 R1 = Coumarate 11 R1 = p-Methoxycinnamate

2. Results and discussion 2.1. Sesquiterpene dialdehydes 9 and 10 from P. insperata Leaves of P. insperata were much less pungent than those of P. colorata, so we did not expect high levels of polygodial (1). Ethanol extracts of P. insperata leaves showed the characteristic 1H NMR signals of unsaturated 1,4-dialdehydes, and dialdehydes 9 and 10 were isolated by silica gel column chromatography. The minor dialdehyde 9 showed 1H NMR signals characteristic of a polygodial derivative with cinnamate at C-1, similar to 7 from fruits of P. colorata (Larsen et al., 2007). The HREIMS of 9 was consistent with a molecular formula, C24H28O5, isomeric with 7. However, analysis of 2D NMR data (not shown) placed an a-OH at C-5 in 9, which was confirmed by our 1H and 13C NMR data matching those just reported for 1b-E-cinnamoyl-5a-hydroxypolygodial from Zygozynum spp. from New Caledonia (Allouche et al., 2009) (the only previous report of 9). The major dialdehyde 10 showed 1H NMR signals (Table 1) characteristic of a coumarate at C-1, similar to 8 from fruits of P.

colorata (Larsen et al., 2007). The molecular formula, C24H28O6, and the similarities of the 1H and 13C NMR signals of 10 to those of the sesquiterpene dialdehyde portion of 9 again suggested an a-OH at C-5. Full analysis of 2D NMR data (Supplementary Data) assigned the structure of 10 as 1b-E-p-coumaroyl-5a-hydroxypolygodial (insperadial), previously unreported. However, the pmethoxycinnamate derivative 11 (drimanial) has been reported from the bark of D. winteri (Malheiros et al., 2001). The 1H and 13 C NMR data for 10 and 11 were nearly identical with the exception of the signals around the p-OH and p-OMe groups. The absolute stereochemistry of 10 was assumed to be the same as that established for ()-polygodial (1) (Barnes and Loder, 1962). We found that the silica gel fraction containing 10 was unstable in CDCl3 solution. Pure 10 was precipitated, and the soluble portion showed increasing amounts of a dialdehyde that we identified as the cis-coumarate based on the 1H NMR signals assigned to H-30 at 6.86 ppm d (12 Hz) and H-20 at 5.75 ppm d (12 Hz) (Mahmood et al., 2003). The rate of isomerisation was much slower in acetone-d6, due to the stronger absorption of UV by acetone and/or the absence of DCl to catalyse the transformation. The propensity


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Table 1 1 H and 13C NMR dataa for compounds 10b and 12c. 10 Position

a b c

13

C

1 2

78.2 25.1

3

35.2

4 5 6

38.9 77.6 32.7

7 8 9 10 11 12 13 14 15 10 20 30 40 50 , 90 60 , 80 70 5-OH 70 -OH

150.6 141.5 56.4 47.5 201.3 193.2 27.8 25.6 13.7 166.6 116.0 145.6 127.0 131.0 116.7 160.6

Table 2 Levelsa of major sesquiterpene dialdehydes in leaves of individual Pseudowintera plants.

12 1

13

H

C

5.35 dd (11.5, 4.5) 1.84 ddd (12.5, 8.0, 4.0), 1.75 m 2.04 m, 1.28 ddd (13.5, 3.5, 3.5)

2.94 m 2.52 ddd (20, 5.5, 1.2) 7.03 m 3.74 m 9.78 9.36 1.06 1.23 1.17

d (3.5) s s s s

6.30 d (16) 7.57 d (16) 7.55 d (9.0) 6.90 d (8.5)

77.5 34.7

1

H

5.03 dd (10, 6) 2.12 br t (14), 2.47 dd (17, 6)

125.1 124.4 45.1 26.6 151.9 141.5 55.6 40.8 200.4 192.4 15.5 19.1 9.8 166.2 117.6 146.0 134.1 128.3 128.9 130.6

2.28 2.70 2.21 7.08

br d (14) dm (18) ddq (18, 12, 2) dt (6, 2)

3.42 dd (3, 2) 9.96 9.37 1.62 1.65 0.92

d (2.5) s d (1) s s

6.42 d (16) 7.69 d (16) 7.53 m 7.38 m 7.38 m

3.75 s 8.88 s

Chemical shifts in ppm multiplicity (coupling constants in Hz). In acetone-d6. In CDCl3.

for coumarates to undergo trans–cis photoisomerisation has been reported before (Kort et al., 1996). We could not purify the cis isomer, and only the trans isomer 10 was seen in 1H NMR spectra of P. insperata leaf extracts in acetone-d6. 2.2. Isopaxidal (12) from P. axillaris During re-isolation of paxidal (3) we noticed the presence of another dialdehyde at about one fifth the level of 3. This was very difficult to separate from paxidal (3) on silica gel or RP C18, but we eventually obtained a pure sample of 12 by preparative HPLC. The HREIMS of 12 was consistent with a molecular formula, C24H26O4, isomeric with 3. The 1H and 13C NMR spectra of 12 (Table 1) showed coumarate and dialdehyde signals similar to those of 3 (Brennan et al., 2006), but with two allylic methyl signals rather than the methyl doublet and exocyclic methylene signals of 3. Full analysis of 2D NMR data (Supplementary Data) suggested structure 12 with an endocyclic double bond. This is a new compound, for which we suggest the name isopaxidal. The only other sesquiterpene known with this 3,4 double bond in a rearranged drimane skeleton is 13, which has been reported only once, from C. winterana, in which it co-occurs with 9-deoxymuzigadial (2) at about one quarter the level of 2 (Al-Said et al., 1990). The 13C NMR signals of 12 around the 3,4 double bond (Table 1) are close to those reported for 13 (Al-Said et al., 1990), and the absolute stereochemistry is assumed to be the same as established for ()-polygodial (1) (Barnes and Loder, 1962). The co-occurrence of 12 and 3 strengthens the suggestion (Al-Said et al., 1990) that the 3,4 and 4(13) double bonds are alternative products of a carbocation intermediate in the biosynthesis of 2 and 3. 2.3. Variation of sesquiterpene dialdehydes in Pseudowintera In order to decide whether sesquiterpene dialdehydes are good markers for Pseudowintera species, we needed to know if there is

Species

Location

2

1

3

axillaris axillaris axillaris axillaris axillaris axillaris axillaris

Mt Bruce Mt Bruce Mt Bruce Mt Bruce Mt Bruce Waitakere Manawatu

10

9

0.1 0.2 0.3 0.1 0.1 0.1 0.1

0.1 0.1 0.2 0.1 0.1 0.1 0.0

3.3 3.2 2.2 2.9 5.1 6.9 4.9

colorata colorata colorata colorata colorata colorata colorata colorata colorata

Alpha Hut Mt Pureora Aorangi Mt Bruce Mt Bruce Mt Bruce Taihape Dunedin Dunedin

1.5

1.4 2.2 2.9 1.6 1.5 1.4 2.3 2.9 2.7

insperata insperata insperata insperata insperata axil col Unknown Unknown Unknown

Bream Head 06b Bream Head 08b Bream Head Bream Head Mt Manaia Waitakere Logue’s Bush Logue’s Bushc Logue’s Bushc

2.9 1.6 1.3 1.2 0.2 2.1 1.5 3.6 3.6 3.0 3.1 6.9

0.4 0.1 0.5 0.8

0.1

0.3 0.2

2.0 0.6 2.0 2.0 2.8

0.3 0.1 0.2 1.1 0.2

0.1

a % w/w of compound in dried leaves, mean of results on two leaves from a single plant unless indicated otherwise. b Leaves collected from the same plant in December 2006 and in March 2008. c Results from a single leaf.

much variation between individuals of the same species, especially as previous work on P. colorata showed two distinct chemotypes (Perry et al., 1996b). The previously reported reversed-phase HPLC method (Perry et al., 1996b) was adapted to separate the known Pseudowintera sesquiterpene dialdehydes, and calibrated with an internal standard to quantify the levels of polygodial (1), paxidal (3) and coumarate 10. Replicate analyses showed good precision, with relative standard deviations