Comparative Phloem Chemistry of Manchurian (Fraxinus mandshurica) and Two North American Ash Species (Fraxinus americana and Fraxinus pennsylvanica)

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J Chem Ecol (2007) 33:1430–1448 DOI 10.1007/s10886-007-9312-3

Comparative Phloem Chemistry of Manchurian (Fraxinus mandshurica) and Two North American Ash Species (Fraxinus americana and Fraxinus pennsylvanica) Alieta Eyles & William Jones & Ken Riedl & Don Cipollini & Steven Schwartz & Kenneth Chan & Daniel A. Herms & Pierluigi Bonello

Received: 12 March 2007 / Revised: 26 April 2007 / Accepted: 7 May 2007 / Published online: 24 May 2007 # Springer Science + Business Media, LLC 2007

Abstract Recent studies have investigated interspecific variation in resistance of ash (Fraxinus spp.) to the exotic wood-boring beetle, emerald ash borer (EAB, Agrilus planipennis). Manchurian ash (Fraxinus mandshurica) is an Asian species that has coevolved with EAB. It experiences little EAB-induced mortality compared to North American ashes. Host phloem chemistry, both constitutive and induced, might partly explain this interspecific variation in resistance. We analyzed the constitutive phloem chemistry of three ash species: Manchurian ash and North American white (Fraxinus americana) and green (Fraxinus pennsylvanica) ash. Analysis of the crude phloem extracts revealed the presence of an array of phenolic compounds including hydroxycoumarins, a A. Eyles : P. Bonello Department of Plant Pathology, The Ohio State University, 201 Kottman Hall, 2021 Coffey Road, Columbus, OH 43210, USA W. Jones : K. Chan Division Of Pharmaceutics, College Of Pharmacy, The Ohio State University, 308 Comprehensive Cancer Center, 410 W 12th Ave, Columbus, OH 43210, USA K. Riedl : S. Schwartz Department of Food Science and Technology, 110 Parker Food Science, The Ohio State University, 2015 Fyffe Court, Columbus 43210, USA D. Cipollini Department of Biological Sciences, Wright State University, 208 Biological Sciences Building, 3640 Colonel Glenn Hwy, Dayton, OH 45435, USA D. A. Herms Department Of Entomology, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Ave, Wooster, OH 44691, USA Present address: A. Eyles (*) Cooperative Research Centre for Forestry, Private Bag 12, Hobart 7001, Australia e-mail: [email protected], [email protected]

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monolignol, lignans, phenylethanoids, and secoiridoids. Both qualitative and quantitative differences were observed among the three ash species. Hydroxycoumarins and the phenylethanoids, calceloariosides A and B, were present only in the phloem of Manchurian ash and might represent a mechanism of resistance against EAB. Keywords Agrilus planipennis . LC-MS . Hydroxycoumarins . Phenolics . Coevolution . Host resistance . Invasive species

Introduction Since its accidental importation from Asia, emerald ash borer (EAB Agrilus planipennis Fairmaire (Col: Buprestidae), a phloem-feeding beetle, has infested and killed millions of ash (Fraxinus spp.) trees in Indiana, Ohio, Michigan, and Ontario (Herms et al. 2004; Cappaert et al. 2005; Poland and McCullough 2006). As EAB continues to spread, it has the potential to eliminate ash species from natural and urban forests throughout North America. In Ohio alone, there are 3.8 billion white ash trees (Griffith et al. 1991). Unlike congeneric woodboring beetles endemic to North America, including bronze birch borer (Agrilus anxius Gory) and two-lined chestnut borer (Agrilus bilineatus Weber) that colonize stressed trees (e.g., Anderson 1944; Dunn et al. 1990), EAB is more aggressive, killing even healthy trees on high quality sites (Haack et al. 2002; Herms et al. 2004; Poland and McCullough 2006). In a recent common garden study that investigated interspecific variation in ash resistance to EAB, Manchurian ash (Fraxinus mandshurica Rupr.) was shown to have low levels of EAB colonization and EAB-induced mortality, while North American ashes experienced high rates of colonization and severe mortality (Rebek et al. 2006). These observations are consistent with the hypothesis that Asian ashes have high resistance to EAB by virtue of their coevolutionary history. Reports indicate that EAB is rare in Asia, where Manchurian ash is a primary host (Haack et al. 2002; Liu et al. 2003; Schaefer 2005), and infestations appear restricted to stressed trees (Gould et al. 2005). This implies that Asian ashes may be generally resistant. Relatively little research has addressed mechanisms of resistance of angiosperm trees to wood-borers (e.g., Hanks et al. 1999). We propose that in the case of phloem-feeding buprestids, host resistance results from constitutive and induced physical and phytochemical defenses (Dunn et al. 1990). More specifically, defense may have three main components: (1) constitutive, or pre-attack, physical and chemical defenses, such as thick outer bark and preexisting pools of compounds in the phloem that are either toxic and/or serve as deterrents against the insect; (2) immediate, attack-induced de novo synthesis and accumulation of specific, low-molecular-weight secondary metabolites, phenolic polymers such as lignin and suberin, and defensive proteins (Kemp and Burden 1986; Bostock and Stermer 1989; Fernandes 1990; Pearce 1996; Kehr 2006), which may slow the growth of the invading organism enough to allow for (3) subsequent formation of wound (necrophylactic) periderms (i.e., callus tissue), which serve to isolate the wound, inhibit the spread of the colonizing organism (e.g., by encapsulation of insect larvae), and reestablish phellogen and cambial integrity (Mullick and Jensen 1973; Mullick 1977; Biggs et al. 1984; Bostock and Stermer 1989; Robinson et al. 2004). Allelochemicals are represented by a diverse array of plant secondary metabolites, including terpenes and phenolic derivatives such as tannins, flavonoids, and stilbenes. Whether constitutively expressed or induced in response to herbivory, they may serve a wide range of roles. The genus Fraxinus belongs to the family Oleaceae, which comprises around 600

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species in 25 genera (Wallander and Albert 2000; Perez et al. 2005). The olive (Olea europaea L.), cultivated for its fruit and oil, belongs to this family and has been studied extensively with regard to chemical constituents. Compound classes that have been reported to be commonly associated with olives include iridoid glucosides, phenylethanoids, coumarins, and lignan glucosides (Bianco et al. 2001; Jensen et al. 2002; Ryan et al. 2002; Cardoso et al. 2005). As part of a broader study investigating potential mechanisms of Manchurian ash resistance to EAB, we compared the constitutive composition of secondary metabolites in phloem from the main stem of white (Fraxinus americana L.), green (Fraxinus pennsylvanica Marsh.), and Manchurian ash. We hypothesized that: (a) Manchurian ash phloem contains concentrations of particular constitutive secondary metabolites different from white and green ash, and (b) that these compounds may be involved in resistance. To date, relatively few studies have investigated the phenolic chemistry of Fraxinus spp. phloem (Terazawa and Sasaya 1970; Tsukamoto et al. 1984; 1985; Nykolov et al. 1993; Kostova et al. 1995; Iossifova et al. 1998, 1999) with many focused on manna ash (Fraxinus ornus L.) (Steinegger and Brantschen 1959; Kostova 2001). The present paper addresses hypothesis (a), by providing chemical data that will be needed to investigate the role of putative defense compounds in resistance of Manchurian ash to EAB (hypothesis b).

Methods and Materials Plant Material Eight 4-yr-old saplings each of F. americana, F. mandshurica, and F. pennsylvannica were obtained from Bailey Nursery, Inc., St Paul, MN, USA. The ash saplings had a mean stem height of 181±0.03 (SEM), 216±0.06, and 149±0.03 cm, respectively, and a mean stem diameter of 15.8±0.5, 23.2±0.9, and 12.4±0.3 cm, respectively. The dormant bareroot saplings were stored in the dark at 4°C for 2 wk before sampling. Chemicals and Reagents Methanol and acetic acid (both HPLC grade) were purchased from Fisher (Pittsburg, PA, USA). Ultrapure water was prepared using a Milli-Q water system (Millipore Ltd, Bedford, MA, USA). Standards of caffeic acid, esculin, and vanillin were purchased from Sigma Aldrich (St. Louis, MO, USA). Chlorogenic acid, fraxin, oleuropein, and verbascoside, were purchased from Extrasynthese (Genay, France). Pinoresinol was purchased from Arbo Nova (Turku, Finland). Extraction One hundred milligrams of stem phloem were ground in liquid nitrogen and extracted twice with 500 μl of 100% methanol over 48 hr in the dark at 4°C. The pooled extract was transferred to a 1.5-ml microcentrifuge tube and centrifuged (12,000×g for 5 min) to remove solids. Samples were stored at −20°C and analyzed within 1 wk of extraction. Analysis of Phenolics with HPLC-UV, HPLC-ESI-MS, and 1H NMR and 13C NMR HPLC with UV detection and HPLC-ESI-MS were used to analyze phenolics. LC-MS analyses were conducted on a Hewlett Packard HPLC system (Palo Alto, CA, USA) model HP 1100 equipped with an HP DAD G1315A detector coupled to a QTof-1 mass spectrometer (Micromass, Manchester, UK). Chromatographic separation was carried out by using a Waters Xterra™ RP18, 5 μm, 4.6×150 mm column and a Waters Xterra ™ RP18, 3.9 μm, 3.0×20 mm guard column. The autosampler and column temperatures were set at 4 and 30°C, respectively.

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The binary mobile phase consisted of water/acetic acid (A) (99.9:0.1, v/v) and methanol/ acetic acid (B) (99.9:0.1, v/v), and the flow rate was set at 1 ml/min. The elution program was as follows (percentages refer to proportion of eluant B): 5–15% (0–15 min); 15–30% (15–35 min); 30–40% (35–40 min); 40–60% (40–50 min); 60–90% (50–55); 100% (55– 60 min). The injection volume for all samples was 5 μl. Compound separation was followed by post-column splitting (1:1), passing of the LC effluent through the Photodiode Array (PDA) Detector (scanning range, 200–400 nm) and an electrospray source (source block temperature, 120°C; desolvation temperature, 400°C). The MS detector was optimized to obtain maximum yields of [M-H]− ions of epicatechin/flavonoid standards. The optimized parameters in the negative ionization mode were as follows: capillary voltage, 3.2 kV; cone voltage, 32 V. Argon was used as the collision gas at 20 psi and nitrogen as the nebulizing gas at 15 l/hr. For full-scan MS analysis, the spectra were recorded in the range of m/z 100–900. We applied MS detection in survey scan mode to identify the major compounds in the HPLC eluate, and we compared these peaks with the PDA trace. In this MS method, full MS scans (survey) were analyzed on-the-fly by the instrument to identify the most abundant parent ions. The mode temporarily switched to MS/MS mode to perform daughter scans of the presumptive parent ions. Data acquisition and processing were performed by using a MassLynx version 3.5 data system (Micromass). The identifications of esculin, fraxin, verbascoside, oleuropein, and apigenin were unequivocally confirmed by spiking samples with the respective standards and comparing retention times and ultraviolet (UV) spectra. Additional mass data for syringin was obtained with a LCQ Classic (Finnigan) by using a slightly modified version of the above LC-MS method. Calceolariosides A and B, oleuropein, and pinoresinol glucoside were isolated by semipreparative HPLC and further purified by normal-phase preparative TLC (1,000 μm thickness silica gel plates) with chloroform:methanol:toluene:acetic acid (80:30:8:2). These compounds were analyzed by 1H NMR and 13C NMR. Spectra were obtained in methanol-d4 by using a Bruker Avance DRX-400 (400 MHz) spectrometer. 1H NMR and 13C NMR spectra were calibrated to the residual solvent peaks at δH 3.31 and δC 49.0, respectively. Quantification of Selected Phenolics After identification, the compounds (Table 4) were quantified against external standards as mg g−1 FW. Lack of suitable external standards made it impossible to quantify all compounds. Contents were expressed as verbascoside equivalents for calceolariosides A and B, fraxin equivalents for fraxetin and mandshurin, esculin equivalents for esculetin and methylesculin, oleuropein equivalents for ligustroside and oleuropein hexoside, pinoresinol equivalents for pinoresinol dihexoside, pinoresinol glucoside, and syringaresinol. Quantitative HPLC analyses were performed on an Alliance 2690 separation module (Waters, Milford, MA, USA) equipped with an autosampler and a 996 Photodiode Array Detector (Waters). The same column and gradient program as described for the LC-MS system were used. The mobile phase consisted of water/acetic acid (A) (98:2, v/v) and methanol/acetic acid (B) (98:2, v/v). Monitoring and processing of data were performed at 280 nm. Statistical Analysis The contents of selected phenolic compounds that were present in two or more ash species were examined by univariate analysis of variance (ANOVA) using SPSS 14.0 for Windows (SPSS Inc., Chicago). Levene’s test was used to examine variance equality. Data that did not have equal variances were log-transformed to satisfy this assumption.

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Results Characterization of Phenolic Compounds Among the three ash species, a total of 38 individual compounds were analyzed in detail. The characterization of unknown phenolic compounds was based on comparison of UV, mass spectrometry (MS), MS fragmentation behavior, and published NMR data. Liquid chromatography/tandem mass spectrometry (LC-TMS) with electrospray ionization has proven to be a powerful tool for the characterization of nonvolatile, polar, and thermally labile phenolic compounds obtained from plant extracts (Eyles et al. 2003a, b; Cardoso et al. 2005; March et al. 2006). Comparison of the ESI-MS/MS data with published data and standards revealed a diverse range of phenolic compounds in the crude phloem extracts including hydroxycoumarins, lignans, phenylethanoids, and secoiridoids. Tables 1, 2, 3 summarize the tentative identification of the chromatographic peaks on the basis of retention times, molecular weights, and the main ions obtained by TMS for compounds in each species. Given that the majority of the compounds detected are known and have been characterized in previous studies (Tables 1, 2 and 3), only brief explanations of the structural analyses of the major compounds are presented (Fig. 1). Hydroxycoumarins Seven compounds were identified as hydroxycoumarins (5, 7, 8, 12, 14, 15, and 16). Most were identified as a glycoside containing one sugar moiety, usually a hexoside as characterized by a neutral loss of 162 Da (Fig. 2). Esculin (5) and fraxin (14) were identified by comparing with standards. Compound 8 exhibited a [M-H]− ion at m/z 353 and UV spectrum maxima at 290 and 335 nm. Its MS/MS spectrum included ions at m/z 191, which corresponds to a loss of hexose (162 Da) and at m/z 176, which corresponds to a loss of a methoxy group (15 Da) from m/z 191. These findings are consistent with those reported for methylesculin (Steinegger and Brantschen 1959; Vereecke et al. 1997). Compounds 12 and 15 were tentatively identified as fraxidin hexoside and mandshurin, respectively (Table 2, Fig. 3). Despite both compounds exhibiting the same [M-H]− ion at m/z 383 and MS/MS spectrum (m/z 221, 206), it was possible to identify each compound by comparing their UV spectra to those previously reported (Terazawa and Sasaya 1970; Jensen and Nielsen 1976). Lignans Compounds 19, 20, 26, and 28 showed maxima of absorption bands at around 281 nm, characteristic of furofuran lignans (Tsukamoto et al. 1984) and similar to the pinoresinol standard. Compounds 19 and 26 were identified as pinoresinol dihexoside and pinoresinol glucoside, respectively. Pinoresinol dihexoside (19) displayed a [M-H]− ion at m/z 681, and its MS/MS spectrum gave ions at m/z 519 and 357, originating from successive losses of 162 Da, suggesting the presence of two hexosyl residues. By contrast, pinoresinol glucoside (26) yielded only one neutral loss of 162 Da. Furthermore, the 1H NMR and 13C NMR data of this compound were consistent with the literature values (Bodesheim and Holzl 1997). Compound 22 exhibited a [M-H]− ion at m/z 523, corresponding to a neutral loss of a hexose moiety (162 Da) and the MS/MS spectrum (m/z 329, 359) was consistent with those reported for lariciresinol (Smeds et al. 2006). Thus, this compound was tentatively characterized as lariciresinol hexoside. Monolignol Compound 10 exhibited an [M-H]− ion at m/z 371 and its MS/MS gave a [MH-162]− ion at m/z 209, corresponding to the loss of a hexose. Furthermore, the product ion spectrum of m/z 371 yielded fragments at m/z 194 and 179, indicating the successive losses of 15 Da and the presence of two methoxyl groups (loss of methyl radicals) (Ye et al. 2005).

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Table 1 Characterization of phenolic compounds from the phloem of green ash, Fraxinus pennsylvanica No. Rt [M-H]− Fragments m/z (Ordered (min) by Decreasing Intensity)

UV Maxima Tentative Identificationa

References

1

5.78

315

153,135, 119, 123

278

Cardoso et al. 2005

3

7.88

299

4

11.10 431

6

12.67 601

119, 113, 137, 131, 149, 276 179, 161 149, 299, 179, 191, 161, 276 131, 251, 119 403, 223, 179, 161 sh 240

10 13

13.96 371 16.60 377

209, 194, 179, 151 197, 153

265 260

18

22.17 353

191

325

19

27.42 681

519, 357, 161

274

20

29.16 535

21 24

31.50 523 33.10 391

26

35.39 519

373, 211, 343, 181, 193, 278 179, 151 361 278 179, 161, 135, 311 sh 290, 331 357, 151 278

28

37.16 417

181, 402, 387, 166

277

29

37.94 701

539, 377, 275, 307, 437

sh 280

31

39.64 685

32

40.06 623

33

41.37 539

36 37

44.62 523 46.37 609

38

54.44 269

523, 453, 421, 299, 223, 330 179 461, 161 sh 285, 331 377, 275, 307, 223, 345, sh 281 149, 179 291, 361, 259, 223, 179 sh 281 301, 271 255, 353 149, 151, 225 267, 339

Hydroxytyrosol hexoside Tyrosol hexoside

Kammerer et al. 2005

Unknown Elenolic acid derivative Syringin Oleuropein aglycone Caffeoyl-quinic acid Pinoresinol dihexoside Hydroxypinoresinol hexoside Unknown Unknown Pinoresinol glucosideb Syringaresinol Oleuropein hexoside Nuzhenide Verbascosidec Oleuropeinb,c Ligustroside Quercetin diglycoside Apigeninc

Wang and Sun 2005 Cardoso et al. 2005 Cardoso et al. 2005 Boyer et al. 2005 Iossifova et al. 1998

Ye et al. 2005 Evtuguin and Amado 2003 Bianco et al. 2001 Ryan et al. 2002; Mulinacci et al. 2005 Cardoso et al. 2005 Tanahashi et al. 1998; Bianco et al. 2001 Ryan et al. 2002 Cardoso et al. 2005 Fabre et al. 2001

sh Shoulder a

Tentative identification based on MS data and UV spectrum consistent with literature

b

Identification confirmed by NMR

c

Identification based on retention time, mass spectral and UV spectrum consistent with those of standard

Based on these observations and a characteristic UV spectrum with a peak of 265 nm, 10 was tentatively identified as syringin (Wang and Sun 2005). Secoiridoids Oleuropein (33) was identified by comparing with the standard. The 1H NMR and 13C NMR data for this compound were consistent with those found in the literature

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Table 2 Characterization of phenolic compounds from the phloem of Manchurian ash, Fraxinus Mandshurica No.

Rt (min)

[M-H]−

Fragments m/z (Ordered by Decreasing Intensity)

UV Maxima

Tentative Identificationa

References

1

5.69

315

135, 119, 153, 113, 123

280

Cardoso et al. 2005

2

7.03

167

152, 122, 121

276

3

7.77

299

276

5

11.82

339

119, 113, 137, 131, 149, 179, 161 177

Hydroxytyrosol hexoside Homovanillic alcohol Tyrosol hexoside Esculinb

Parejo et al. 2004

7

12.74

177

133, 149, 105

Esculetin

Concannon et al. 2000

8

13.13

353

191, 176

Methylesculin

Vereecke et al. 1997

9

13.36

389

Oleoside

Cardoso et al. 2005

11

13.89

353

165, 183, 121, 209, 345, 119 191, 179, 135, 173

Cardoso et al. 2005

12

15.57

383

221, 191, 163, 135, 206

14

17.01

369

207, 191, 354

15 16 19

17.37 20.49 26.89

383 207 681

221, 206, 191, 161 193, 165, 109 519, 357, 161

292, sh 340 sh 300, 341 329 335 276

Caffeoyl-quinic acid Fraxidin hexoside Fraxinb

21 22

30.78 32.26

523 521

361 329, 359

278 278

25 26

33.18 35.01

335 519

179, 135, 161 357, 151, 342, 136

276 278

27

36.77

477

161, 179, 135, 315

290, 328

29

37.10

701

539, 377, 275, 307, 437

sh 278

31

39.11

685

32

39.38

623

523, 453, 421, 299, 223, 179 461, 161

33

41.14

539

not detected sh 290, 331 sh 281

35

42.07

477

36 37

44.50 46.01

523 609

377, 275, 307, 223, 345, 149, 179 161, 315, 135, 179, 251, 281, 221 361, 291, 259, 223, 127 301, 271

290, 336 sh 290, 335 sh 290, 335 Not detected 287, 335

285, 328 sh 280 256, 353

Mandshurin Fraxetin Pinoresinol dihexoside Unknown Lariciresinol hexoside Unknown Pinoresinol glucoside b Calceolarioside Ac Oleuropein hexoside Nuzhenide Verbascosideb Oleuropeinb,c Calceolarioside Bc Ligustroside Quercetin diglycoside

Kammerer et al. 2005

Jensen and Nielsen 1976 Godecke et al. 2005 Terazawa and Sasaya 1970 Liu et al. 2005 Boyer et al. 2005

Smeds et al. 2006

Ye et al. 2005 Nicoletti et al. 1986 Bianco et al. 2001 Ryan et al. 2002; Mulinacci et al. 2005 Cardoso et al. 2005 Tanahashi et al. 1998; Bianco et al. 2001 Nicoletti et al. 1986; Ersöz et al. 2002 Ryan et al. 2002 Cardoso et al. 2005

sh Shoulder a

Tentative identification based on MS data and UV spectrum consistent with literature

b

Identification based on retention time, mass spectral and UV spectrum consistent with those of standard

c

Identification confirmed by NMR

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Table 3 Characterization of phenolic compounds from the phloem of white ash, Fraxinus americana No. Rt [M-H]− Fragments m/z (Ordered (min) by Decreasing intensity)

UV Maxima Tentative Identificationa

References

1

5.82

315

153, 123, 135

278

Cardoso et al. 2005

3

7.77

299

6 8

10.98 431 12.27 565

119, 113, 137, 131, 149, 179, 276 161 149, 299, 179, 191, 221, 251 275 403, 179, 223, 161, 119, 143 239

9 10 13

13.36 389 13.36 371 16.47 377

165, 183, 121, 209, 345, 119 209, 194, 179, 151 197, 153

266 265 262

17

21.60 537

375, 327, 195, 179

19

27.02 681

519, 357, 161

270, sh 305 276

20

28.70 535

373, 343, 211, 181, 193

278

21 22

30.96 523 32.10 521

361 329, 341, 359

278 278

23

32.58 555

26

35.00 519

273, 393, 307, 361, 179, 181, sh 290, 375, 419 332 357, 151, 136, 342 278

28

37.16 417

181, 402, 387, 166

277

29

37.18 701

539, 377, 275, 307, 437

sh 278

30

38.40 621

179, 161, 459, 487

32

39.11 623

33

40.98 539

34

42.00 685

286, 331 461, 161 sh 285, 331 377, 275, 307, 223, 345, 149, sh 281 179 361, 291, 259, 179 277

36 37

44.37 523 45.98 609

291, 361, 223, 179, 127 301, 271

38

54.45 269

149, 151, 225, 201

280 255, 353 267, 337

Hydroxytyrosol hexoside Tyrosol hexoside Unknown Elenolic acid derivative Oleosideb Syringinb Oleuropein aglycone Unknown

Kammerer et al. 2005

Cardoso et al. 2005 Wang and Sun 2005 Cardoso et al. 2005

Pinoresinol Boyer et al. 2005 dihexoside Hydroxypinoresinol Iossifova et al. 1998 hexoside Unknown Lariciresinol Smeds et al. 2006 hexoside Unknown Pinoresinol glucosidec Syringaresinol Oleuropein hexoside Unknown

Ye et al. 2005 Evtuguin and Amado 2003 Bianco et al. 2001

Verbascosided

Cardoso et al. 2005

Oleuropeinc,d

Tanahashi et al. 1998; Bianco et al. 2001

Ligustroside derivative Ligustroside Quercetin diglycoside Apigenind

Ryan et al. 2002 Cardoso et al. 2005 Fabre et al. 2001

sh Shoulder a

Tentative identification based on MS data and UV spectrum consistent with literature

b

Peaks co-eluted

c

Identification confirmed by NMR

d

Identification based on retention time, mass spectral and UV spectrum consistent with those of standard

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Hydroxycoumarins: R3 R1

R2

O

O

R4

Esculin: Fraxin: Fraxidin: Mandshurin:

R1

R2

R3

R4

Glc OMe OMe Glc

OH OH OMe OMe

H H H OMe

H Glc OH H

Lignans: OH

R

O H O

O H

O HO

Pinoresinol: Syringaresinol:

R

R=H R = OMe

Fig. 1 Chemical structure of major compounds in crude ash extracts

(Tanahashi et al. 1998). Compound 29 exhibited the same characteristic UV spectrum as oleuropein but gave an [M-H]− ion at m/z 701. Its MS/MS spectrum yielded an intense ion at 539, consistent with a neutral loss of a hexose (162 Da) plus other ions (m/z 377, 275, 307) identical to those for oleuropein. Therefore, this compound was tentatively identified as oleuropein hexoside. The MS/MS spectrum of compound 36 yielded ions at m/z 291, 361, 223, 179, and 127. These findings are consistent with ligustroside (Ryan et al. 2002). Compound 34 exhibited a similar MS/MS spectrum as ligustroside (m/z 361, 291, 259, 159) but with a [M-H]− ion at m/z 685, which corresponds to an addition of 162 Da. Therefore, it was tentatively identified as a ligustroside derivative. Phenylethanoids Compound 32 was characterized by the [M-H]− ion at m/z 623 and CID gave two major ions at m/z 461 and 161 that are in accordance with the fragmentation of

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Monolignol: O OH

O O O HO

OH HO OH

Syringin

Secoiridoid glycosides: O

O

R

O O O

HO O

O

HO

HO

OH OH

Ligustroside: Oleuropein:

R=H R = OH

Fig. 1 (continued)

verbascoside (Cardoso et al. 2005). Compounds 27 and 35 exhibited [M-H]− ion at m/z 477 with almost identical UV and mass spectra suggesting them to be isomers of the same compound (Fig. 4). In both cases, their MS/MS spectra gave an [M-H-162]− ion at m/z 315,

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Phenylethanoids:

OR 2 O

R1 O

O

OH

OH

OH

OH

Calceolarioside A: R1 = Caffeoyl Calceolarioside B: R1 = H

R2 = H R2 = Caffeoyl

Fig. 1 (continued)

indicating the loss of a caffeic acid moiety. In addition, caffeic acid (m/z 179) and its decarboxylated product (m/z 135) generated signals of low intensity (Fig. 4). The intense fragment ion at m/z 161 is most likely due to the fragmentation of the hexose moiety by the loss of water (Tolonen et al. 2004). Thus, compounds 27 and 35 were assigned as calceolariosides. The 1H NMR and 13C NMR data showed them to be identical with those reported for calceolariosides A (Nicoletti et al. 1986) and B (Ersöz et al. 2002), respectively. Comparison of the Three Ash Species Both qualitative and quantitative differences were observed among the three species (Fig. 2, Tables 1, 2, 3 and 4). Comparisons of 17 individual phenolic compounds revealed a number of differences in composition as listed in Table 4. In particular, Manchurian ash was distinguished from the North American ash species by the presence of hydroxycoumarins, particularly mandshurin (15), fraxin (14), and fraxidin hexoside (12), in decreasing order of abundance. Other minor hydroxycoumarins that were not detected in either green or white ash included esculin (5), esculetin (7), methylesculin (8), and fraxetin (16). The phenylethanoids, calceolariosides A (27) and B (35), also were only found in Manchurian ash. The main lignan in green and white ash was syringaresinol (28) followed by smaller amounts of pinoresinol glucoside (26) and pinoresinol dihexoside (19). Manchurian ash contained amounts of pinoresinol dihexoside (19) that were 11-fold higher than in green and 10-fold higher than in white ash (F2,23 =238, P
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