fireweed toxicity facts and perspectives€¦ · fireweed toxicity facts and perspectives page 5 of...

Fireweed Toxicity

Facts and

Perspectives

Steven M Colegate BSc(Hons), PhD

October 2008

Photos: Forest&Kim Starr

Fireweed Toxicity Facts and Perspectives

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Executive Summary

• HPLC-MS analysis of fireweed collected in the Bega Valley (NSW) in the

spring of 2006 and 2008 showed the presence of dehydropyrrolizidine alkaloid

esters at levels up to about 220 milligrams per kilogram of plant.

• Due to the adverse effects of dehydropyrrolizidine alkaloid esters, a

comprehensive assessment of livestock health, welfare and productivity in the

infested areas should be undertaken.

• Animal-derived food for humans, especially bee-products and honey produced

by bees foraging on fireweed, require monitoring for the presence of fireweed

pyrrolizidine alkaloids.

• All possible measures should be undertaken to manage and control the

infestation and potential spread of fireweed in Australia.


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Summary

• Senecio madagascariensis (fireweed) is a non-indigenous invasive weed that

is expanding its range in Australia.

• It is closely related to the indigenous Senecio lautus in both morphology and

its distribution.

• The dehydropyrrolizidine alkaloid esters have been associated with acute and

chronic liver toxicity, pneumotoxicity, carcinogenicity and genotoxicity.

• Toxic dehydropyrrolizidine alkaloids have been detected in samples of

fireweed collected from the Bega Valley in the spring of 2006 and 2008.

• The alkaloid profiles of young plant and mature green-stemmed plant and

mature red-stemmed plant are very similar.

• In this limited survey, young plants contain higher levels (up to about 220

milligrams per kilogram of plant) of the toxic pyrrolizidine alkaloids than the

mature plant (up to about 110 milligrams per kilogram of plant).

• The presence of alkaloids was confirmed using ion trap mass spectrometry and

time of flight mass spectrometry. Additionally a sample was analyzed by

another laboratory experienced in Senecio madagascariensis.

• The suspected presence of a low level of a toxic pyrrolizidine alkaloid,

associated with fireweed, in a single, blended wild honey sample of unclear

heritage remains unconfirmed.


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CONTENTS

Executive Summary ....................................................................................................... 1 Executive Summary ....................................................................................................... 2 Summary ........................................................................................................................ 3 List of Figures ................................................................................................................ 5 Introduction .................................................................................................................... 7 Potential Toxicity Issues ................................................................................................ 8

Toxicology of Dehydropyrrolizidine Alkaloids (DHPAs) ........................................ 8 Potential Toxicity of DHPAs for Livestock ......................................................... 11 Potential Toxicity of DHPAs for Humans ........................................................... 12

Relevance of DHPA Content and Toxicity to Fireweed .......................................... 13 General Approach and Examples ............................................................................. 16 Analysis of Fireweed ............................................................................................... 19

Ion trap mass spectrometry .................................................................................. 19 Time-of-flight mass spectrometry ........................................................................ 24

Analysis of Wild Honey from the Bega Valley ....................................................... 27 Conclusions and Recommendations ............................................................................ 28 References .................................................................................................................... 30


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List of Figures

Figure 1: Common structural cores of dehydropyrrolizidine alkaloid esters

Figure 2: Structures of pyrrolizidine alkaloids isolated from Senecio

madagascariensis sourced from Hawaii and Australia (Gardner et al.,

2006). The bold number is the mass of the molecular ion adduct

(MH+) for each alkaloid.

Figure 3: HPLCesiMS ion chromatograms showing alkaloidal profiles for

Echium plantagineum flowers (A) and leaves (B). Each of the peaks

represents a different pyrrolizidine alkaloid. In this case the alkaloids

are in their N-oxide form.

Figure 4: Base ion (m/z 200 – 500) HPLC-ESI-MS chromatograms for (A)

pollen loads from bees foraging on Echium vulgare; (B) pollen loads

from bees foraging on Eupatorium cannabinum; (C) pollen, hexane-

washed from anthers of Senecio jacobaea; and (D) pollen loads from

bees foraging on Senecio ovatus. The internal standard (IS) is used to

standardize the chromatograms. The presence of different suites of

alkaloids from each sample is evident. The alkaloids are in their N-

oxide forms.

Figure 5: The HPLC-MS ion chromatogram of an extract of honey produced by

bees that foraged in the vicinity of Echium vulgare. Each of the six

“arrowed” peaks is a hepatotoxic pyrrolizidine alkaloid that is also

found in the plant itself.

Figure 6: HPLC-esiMS base ion (m/z 200-500) chromatograms of the alkaloidal

components of Senecio madagascariensis (fireweed): A. fresh young

plant; B. fresh mature plant, and C. dried and milled mature plant.

Figure 7: HPLC-esi-ion trap MS base ion (m/z 200-500) chromatograms of: A.

fresh young fireweed; B. fresh mature fireweed; and C. dried and

milled mature fireweed. Also shown are example mass spectra for

selected peaks: a. Unidentified alkaloid, m/z 424 (1); b. N-oxide of

senecionine type alkaloid, m/z 352 (2); c. N-oxide of a retrorsine type

alkaloid, m/z 368 (3).


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Figure 8: HPLC-esi-ion trap MS base ion chromatograms of: A. an extract of

young Senecio madagascariensis spiked with; B. integerrimine-N-

oxide, C. usaramine, D. retrorsine, and E. senecionine-N-oxide.

Figure 9: An HPLC-MS comparison of a zinc/acid-reduced extract of fireweed

collected in the Bega Valley, New South Wales in about October 2006

with that collected in northern New South Wales (Gardner et al.,

2006). The bold numbers are the MH+ values for each peak (see

Figure 2). The analyses were done by Dr Dale Gardner of the USDA

Poisonous Plants Research Laboratory in Logan, Utah, USA

Figure 10: Direct infusion, ESI-TOF MS analysis of fresh Senecio

madagascariensis collected in the Bega Valley, October 2008. A.

young plants; B. mature, red-stemmed plants; and C. mature, green-

stemmed plants.

Figure 11. HPLC-TOF mass spectrometric analysis of fresh, green-stemmed

mature samples (green trace) and fresh, red-stemmed mature samples

(red trace) of Senecio madagascariensis. The numbers shown are the

masses of the ions observed.


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Introduction

In North America, “fireweed” is a trivial name used for the purple-flowered

Epilobium angustifolium and Erechtites hieracifolia. In Australia, in addition to

“fireweed” being used to describe a toxic blue-green algae (Lyngbya majuscule),

“fireweed” can be a generic, trivial name applied to a number of weed species in the

Senecio genus of the Asteraceae (daisy/thistle) Family. In the context of this report

“fireweed” refers to Senecio madagascariensis Poiret that is spreading prolifically

along the east coast of Australia and specifically, in the particular context of this

report, the Bega Valley in New South Wales where it is a declared plant under the

Noxious Weeds Act 1993 (www.dpi.nsw.gov.au/agriculture/ pests-

weeds/weeds/profiles/fireweed).

Senecio madagascariensis, a native of South Africa, is closely related to S. lautus

which is native to Australia

(www.esc.nsw.gov.au/weeds/Sheets/herbs/H%20Fireweed.htm) and is one of over

1200 Senecio species world-wide. It was first observed in the Hunter Valley region of

Australia in about 1918. Apparently, it is not a declared noxious plant in this region

(www.lhccrems.nsw.gov.au/weeds_cd/extras/fireweed.pdf).

Since Senecio spp. produce alkaloids that are toxic to livestock and humans, and since

previous phytochemical analyses of S. madagascariensis in particular have shown the

presence of hepatotoxic (liver damaging) alkaloids, it was deemed essential to

complete a phytochemical assessment of the fireweed collected in the Bega Valley of

New South Wales.

This report, and the research described within, have been completed at the request of

the New South Wales, Bega Valley Fireweed Committee and is intended to be

complementary to related reports (also commissioned by the Bega Valley Fireweed

Committee) that describe other aspects of this plant such as its identification, growth

habits, dispersal rates and distribution, and methods of control (if not eradication)

including biological options for control.


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Herein, potential problems associated with the expected presence of

dehydropyrrolizidine alkaloid (DHPA) esters in the fireweed are discussed. The

results of a limited phytochemical research project aimed at identifying the

complement of toxic DHPAs in the fireweed and in wild honey gathered in the

vicinity of fireweed are presented and discussed.

Potential Toxicity Issues

The potential toxicity issues for livestock and humans associated with fireweed are

due to the likely presence of DHPA esters. This diverse class of natural chemicals

comprises over 400 different entities occurring in over 6000 species of plants world-

wide.

The basic structural variations of the alkaloids include monoesters, open chain

diesters and macrocyclic diesters of the free base and corresponding N-oxide forms of

dehydropyrrolizidine-based and the otonecine-based alkaloids (Figure 1).

There exists a high level of variation in the suite of DHPAs found in different plant

genera and different species. There can even be variation in the DHPA complement

between different populations of the same species.

Toxicology of Dehydropyrrolizidine Alkaloids (DHPAs)

Following absorption into the blood stream, the DHPA esters are rapidly metabolized

by the liver to form the didehydropyrrolizidine alkaloid form commonly referred to as

the “pyrrolic” form. It is this pyrrolic form that then reacts with macromolecules

within the liver (eg., proteins and DNA) to initiate the chain of events culminating in

clinical disease (Stegelmeier et al., 1999).

The DHPAs are primarily liver toxins (hepatotoxic) but can also “spill out” of the

liver to damage the lungs (pnuemotoxic). In some animals, some DHPAs have been

shown to cause cancers (carcinogenic) and damage genes (mutagenic).


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Not all DHPAs are as toxic as each other. Not all animal species are equally

susceptible to the DHPAs. This is due, in the main, to the relative ease (or difficulty)

with which the DHPAs are metabolized to the reactive pyrrolic forms. For example,

sheep are relatively resistant to the effects of DHPAs and, as such, have been

recommended as grazing control options (Schmidl, 2006; www.dpi.nsw.gov.au

/__data/assets/pdf_file/0007/49840 /fireweed _-_primefact_126-final.pdf). On the

other hand, cattle and horses are relatively susceptible to the toxic effects of ingested

DHPAs.

Even without knowing the reasons why, the toxicity of Senecio spp. to livestock was

first recognized in the early 20th century. Similarly, the danger to humans was first

recognized in 1920 when cirrhosis of the liver was attributed to senecio poisoning

(Willmot and Robertson, 1920).

Subsequently there has been extensive research into the detection of DHPAs and the

determination of their mechanism of action and their toxicities. A relatively recent

review states: "Pyrrolizidine alkaloids are the leading plant toxins associated with

disease in humans and animals. The PAs present a serious risk to human populations

.... Some PA adducts are persistent in animal tissues and the metabolites may be

released and cause damage long after the initial period of ingestion. ..." (Prakash et

al.,1999).


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Figure 1: Common structural cores of dehydropyrrolizidine alkaloid esters

C

Otonecine diester

N

HOH H C H 2

O CO

CHC C

O H

CH3 H3 C OCH3 CH3 H

monoester

N

OH H CH2

O C O

CC C

OH

CH3 OCH3 CH3 H

CO

C C

H

CH3 CH3

CH3 HO

Diester

N

O O

CH3

O

otonecine base necine base

N

OH C H 2

O CO

C CH 3 O H

CO

CC

H

CH3

CC H H HCH3

O

C H3

N

HO O

N

OH H CH2

O C O

C CH3 OH

CO

CC

CCH H HCH3

CH3

H

O

N

OH H CH2

O CO

C C H 3OH

CO

C C

CCH H HCH3

H 3

H

Macrocyclic diester

N-oxide

N

HO O

N-oxideO


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Potential Toxicity of DHPAs for Livestock

The acute and chronic effects of exposure of livestock to dietary DHPAs have been

well-documented (for example: Stegelmeier et al., 1999). The major effect is on the

liver and many clinical signs are related to this initial insult. For example, chronic

poisoning of horses by dietary DHPAs causes a neurological syndrome that is related

to increased levels of systemic ammonia (particularly in the brain) as a result of the

damaged liver’s decreased capacity to process ammonia to urea.

While cattle, horses, pigs and chickens are quite susceptible to the poisonous effects

of the DHPAs, sheep and goats are fairly resistant. However, sheep are very

susceptible to copper poisoning and exposure to DHPAs can exacerbate the

accumulation of copper in the liver of the sheep. Eventually an acute, copper-induced

haemolytic crisis occurs, as the red blood cells are disrupted, causing the death of the

animal. Clinical signs of chronic exposure to DHPAs vary depending upon the animal

exposed but can be attributed to the loss of liver function. Signs of exposure can

include a rough, unkempt appearance, oedema of gastro-intestinal tract, ascites (fluid-

filled body cavities eg the peritoneal cavity), diarrhea, prolapsed rectum, dullness and

lethargy, photosensitization and general abnormal behaviour. As the liver disease

progresses then jaundice will be observed. A summary of clinical signs resulting from

exposure to DHPAs is presented in the Merck Veterinary Manual

(www.merckvetmanual.com/mvm/index.jsp?cfile =htm/bc/212800.htm).

On post-mortem examination, the livers in most species are shrunken, hard to the

touch and fibrotic in nature following chronic exposure to the DHPAs. In sheep,

however, the livers can be soft and mushy rather than hard. Microscopic indicators of

exposure to the DHPAs include the observation of enlarged liver cells (hepatocytes)

referred to as megalocytosis.

The long term prognosis for affected livestock is not good due to the primary effect on

the liver. It has also been shown that the fatal effects can be delayed with no

immediate signs of liver damage following the initial exposure. For example, steers

given a single sub-lethal dose of Senecio riddellii, S. jacobaea or S. longilobus died

from a progressive liver failure several months later (Molyneux et al., 1988).


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Using sheep as grazing control agents for the DHPA-producing plants is not without

its risks to the sheep since, although relatively resistant to the effects of the DHPAs,

they are not totally resistant. As a result, the use of sheep as biocontrol agents may be

unacceptable on animal welfare grounds (www.bbc.co.uk/dna/h2g2/A22548774). It

has not been positively confirmed that such “biocontrol” animals do not accumulate

toxic metabolites of the DHPAs in meat destined for the human food supply.

The lethal effects (and economic losses associated with them) of acute or chronic

exposure of livestock to the DHPAs are not expected to be the major source of loss to

producers. Low-level, sub-clinical exposures can lead to severe losses in productivity

that may include poor feed utilization and reduced weight gains, reduced milk yields

and potential reproductive/breeding adverse effects.

Potential Toxicity of DHPAs for Humans

A review published in 1989 clearly demonstrated the exposure of humans to the

DHPAs and the adverse consequences (Huxtable, 1989). Since then, there have been

continuing reports of humans being poisoned by dietary DHPAs, including exposure

via medicinal herbs preparations, cooking spices and flour made from grain

contaminated with seeds from DHPA-producing plants.

The major, reported clinical symptoms of pyrrolizidine alkaloid intoxication in

humans result from a veno-occlusive disease of the liver (Prakash et al., 1999). In

addition to outbreaks of “bread poisoning”, when the seeds of pyrrolizidine alkaloid-

producing plants were co-harvested with milling grains (Ahmad, 2001), pyrrolizidine

alkaloids in herbs and spices have been associated with many instances of fatal

hepatic disease in adult, neonatal and prenatal humans. Whilst the effects of acute

exposure to pyrrolizidine alkaloids on humans and animals have been well

documented, the effects of long term, low level exposure to DHPAs via the diet

(grain, milk, meat, honey, and related products) (Colegate et al., 1998) are a relatively

unknown factor. In addition, and for many reasons, adverse clinical effects can be

very difficult to unambiguously associate with the sources of dietary DHPAs.

After a comprehensive risk assessment of toxic pyrrolizidine alkaloids, the German

Federal Health Bureau established regulations that restrict oral exposure to


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pyrrolizidine alkaloids or their N-oxides in herbal preparations to 0.1 microgram per

day with the exclusion of pregnant and lactating women for which zero exposure is

recommended (German Federal Health Bureau, 1992). In the Netherlands, levels of

pyrrolizidine alkaloids and their N-oxides are restricted to 0.1 microgram per 100 g of

food (H van Egmond, The Netherlands National Institute for Public Health and the

Environment, personal communication). In Australia, the Food Standards Australia

New Zealand (FSANZ, 2001) has set a limit of exposure to 1 microgram per kilogram

bodyweight per day. For example, a 30 kg child would be allowed 30 micrograms of

DHPAs (or their N-oxides) per day. In contrast to the European deliberations, the

FSANZ did not consider DHPA-induced carcinogenicity or genotoxicity as a major

factor with humans and therefore developed their recommendations on the occurrence

(or prevention of the occurrence) of hepatic veno-occlusive disease.

In addition to the clearly proven contamination of grains, and the use of cooking

spices and medicinal herbs either prepared or contaminated with DHPA-producing

plants, the potential exists for humans to be exposed to DHPAs (and their N-oxides)

via honey and bee products (Edgar et al., 2001; Beales et al., 2004; Betteridge et al.,

2005; Boppré et al., 2005), milk (James et al., 1994), eggs (Edgar and Smith, 2000)

and meat (Seawright, 1994).

Relevance of DHPA Content and Toxicity to Fireweed

In Australia, grazing of fireweed (Senecio madagascariensis)-contaminated pasture

has previously been associated, but not proven, with livestock poisonings in New

South Wales (Seaman and Walker, 1985). Another report describes pyrrolizidine

alkaloidosis in a 2 month-old foal and associated it with in utero exposure of the

foetus to DHPAs ingested by the mare grazing a pasture heavily infested with

fireweed (Small et al., 1993). An intoxication of cattle was associated with S. lautus

(Noble et al., 1994) but, apparently, subsequent re-examination of the herbarium

specimens identified the plant as S. madagascariensis (referred to in Gardner et al.,

2006).

Despite the previous associations of fireweed with overt disease of livestock, it was

only recently that the DHPAs, in fireweed collected in Hawaii and in Australia, were


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identified and compared (Gardner et al., 2006). This report, that used gas

chromatography-mass spectrometry (GCMS) to identify 12 macrocyclic DHPAs

(Figure 2) at levels that were potentially toxic to livestock (217 – 1990 micrograms

per gram of plant material). The DHPA profile for the fireweed collected in Hawaii

was essentially identical to the profile determined for a composite sample of fireweed

collected from northern New South Wales. Citing this latter observation the authors

supported the contention that fireweed in Hawaii arrived via Australia.

The research by Gardner et al (2006) on the fireweed in Hawaii highlighted the high

variation in DHPA content and profile from plants collected at different locations.

Therefore, to help complete the understanding of the fireweed population, and

problems posed by it, in the Bega Valley area of New South Wales, a limited

phytochemical analysis of the plants collected in the area was conducted.


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Figure 2: Structures of pyrrolizidine alkaloids isolated from Senecio

madagascariensis sourced from Hawaii and Australia (Gardner et al., 2006). The

bold number is the mass of the molecular ion adduct (MH+) for each alkaloid.

H O O

OO

O

N

H

H

Senecivernine (336)

HO O

O O

O

N

HO

O

OO

O

N

Senecionine (336)

Integerrimine (336)

O

OO

O

N

O

H O

Senkirkine (366)

C H2OHHO

O

O O

O

N

Mucronatinine (352)

C H 2O HH OO

O O

O

N

Retrorsine (352)

CH 2O H H OO

O O

O

N

Usaramine (352)

H OO

OO

O

O

N

O

Otosenine (382)

O

OO

O

N

O

O

O

Acetylsenkirkine (424)

O

OO

O

O H

N

O

H OC l

Desacetyldoronine (418)

O

O O

O

O

N

O

O

O

Florosenine (424)

O

OO

O

OH

N

O

O

O

Cl

Doronine (460)


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Analysis for Dehydropyrrolizidine Alkaloids

In mid to late spring of 2006, samples of mature, flowering plants and juvenile plants

were collected in the Bega Valley and immediately sent to Dr Steven Colegate,

Leader of the CSIRO Plant-associated Toxins Research Laboratory in Geelong,

Victoria, for processing and analysis. In mid spring of 2008 other samples of plants

and samples of wild honey collected in the same area were sent to Dr Steven Colegate

for analysis at Deakin University once the CSIRO had closed down its plant toxins

research group. The plants included mature flowering plants, some with a red hue to

the stems and some just green, and juvenile plants. There is no botanical separation at

this stage of the green-stemmed fireweed and the red-stemmed fireweed.

All samples were received in a fresh condition and were immediately processed for

analysis.

The earlier samples processed at CSIRO were analysed using high pressure liquid

chromatography (HPLC)-electrospray ionization (ESI) ion trap mass spectrometry

(MS) while the samples analyzed at Deakin University were processed using HPLC-

ESI-time of flight (TOF) MS.

General Approach and Examples

The plant and honey samples were analyzed for the presence of DHPAs using a

sequential combination of solvent extraction, solid phase concentration and

subsequent high pressure liquid chromatography-mass spectrometry (HPLC-MS) in a

manner previously described for:

• plants eg., Echium plantagineum (Paterson’s Curse, Salvation Jane) (Colegate

et al., 2005) (Figure 3), Echium vulgare, Senecio jacobaea (ragwort) and

Senecio ovatus (Boppré et al., 2008)

• pollen collected from plants or bees eg., Echium vulgare (Boppré et al.,

2005), Eupatorium cannabinum, Senecio jacobaea and Senecio ovatus

(Boppré et al., 2008) (Figure 4)

• honey (Betteridge et al., 2005) (Figure 5)


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In all examples shown, and in the analyses of fireweed in the following section, the

individual DHPAs are represented by peaks in an ion chromatogram. Identities are

rationalized using the mass spectrometric data collected for each peak and subsequent

comparison with authentic standards of those alkaloids or with mass data reported in

the scientific literature. In general, because the alkaloids within a species of plant are

structurally similar, the size of the peak reflects the relative abundances of the

individual pyrrolizidine alkaloids in the sample.

Figure 3: HPLCesiMS ion chromatograms showing alkaloidal profiles for Echium plantagineum flowers (A) and leaves (B). Each of the peaks represents a different pyrrolizidine alkaloid. In this case the alkaloids are in their N-oxide form.

In contrast to the GC-MS method employed by Gardner et al (2006), this HPLC-MS

method of analysis allows the simultaneous extraction, recovery and analysis of the

pyrrolizidine alkaloids and their N-oxides. Since the N-oxides are also toxic (Chou et

al., 2003) it is important that they are fully accounted.

The ion chromatograms shown in Figure 3 demonstrate how different parts of the

same plant can have slightly different alkaloid profiles. In this case the flowers

produce acetylated derivatives of the alkaloid-N-oxides detected in the leaves

(Colegate et al., 2005). However, Figure 4 clearly shows the different profiles of

B

A

RT: 1.49 - 12.06

2 3 4 5 6 7 8 9 10 11 12Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

8.81414.2

10.26398.2

9.83456.2

11.20440.2

6.54316.2

7.82358.2

6.39316.2

2.13332.1

3.42332.1

8.25254.1

7.08254.1

5.84374.2

9.31416.2

10.82592.2

8.82414.2

10.28398.2

6.56316.2

8.68398.2

6.42316.2

2.13332.1

8.25254.1

7.82358.1

7.10254.1

9.83456.2

3.47332.1

9.29416.2

NL:1.27E8

Base Peak m/z= 200.0-600.0 MS smc070104lcq21

NL:4.05E7

Base Peak m/z= 200.0-600.0 MS SMC070104LCQ17


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Figure 4: Base ion (m/z 200 – 500) HPLC-ESI-MS chromatograms for (A) pollen loads from bees foraging on Echium vulgare; (B) pollen loads from bees foraging on Eupatorium cannabinum; (C) pollen, hexane-washed from anthers of Senecio jacobaea; and (D) pollen loads from bees foraging on Senecio ovatus. The internal standard (IS) is used to standardize the chromatograms. The presence of different suites of alkaloids from each sample is evident. The alkaloids are in their N-oxide forms.

alkaloids that might be expected from different plant genera i.e., Echium versus

Senecio versus Eupatorium, and even from different species of the same genus i.e.,

Senecio jacobaea versus Senecio ovatus (Boppré et al., 2008).

As a final example of the application of this methodology, Figure 5 shows the

detection of hepatotoxic pyrrolizidine alkaloids detected in honey produced by bees

that foraged in the vicinity of the DHPA-producing plant Echium vulgare (Betteridge

et al., 2005). The levels detected in this commercial honey would clearly exceed

European and Australian regulations.

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.50

20 40 60 80

1000

20 40 60 80

1000

20 40 60 80

1000

20 40 60 80

100

A

B

C

D

IS

IS

IS

IS

Time (min)

Rel

ativ

e A

bund

ance

(%

)


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Figure 5: The HPLC-MS ion chromatogram of an extract of honey produced by bees that foraged in the vicinity of Echium vulgare. Each of the six “arrowed” peaks is a hepatotoxic pyrrolizidine alkaloid that is also found in the plant itself. Analysis of Fireweed

Samples of fireweed were processed and analysed using two different mass

spectrometry approaches: 1. ion trap mass spectrometry, and 2. time of flight (TOF)

mass spectrometry.

Ion trap mass spectrometry

Qualitatively, there were no major differences observed in the alkaloid profiles of the

mature plant and the juvenile plants collected in 2006 and analyzed using HPLC-ESI-

ion trap MS i.e., the chromatograms looked very similar.

Within the profiles, several, potentially toxic DHPAs were identified (Figure 6) in

extracts of the Bega Valley fireweed. The structures of the alkaloids were indicated by

comparison of their mass spectrum characteristics (for example, Figure 7) with the

major

D:\YU210504CQ39 22/05/2004 12:04:15 Honey Nov.2002-5(Arataki clover blend) elute 1 (1Eho2)

RT: 0.00 - 19.99

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Tim e (m in)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bund

ance

496.2

310.1414.2

310.1

310.1

310.1

278.0 310.1480.2

380.2414.2310.1

326.1

309.1 398.2310.1326.1

310.1 456.2

336.3310.1

217.0310.1

326.1 396.2

269.1396.2276.1251.1 310.1 391.3310.1

491.3485.3374.2383.1 324.1 370.2346.3 232.3346.2 470.4318.8 336.3 278.8 339.8

NL:3.79E7

Base Peak m /z= 200.0-500.0 MS YU210504CQ39


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Figure 6: HPLC-esi-ion trap MS base ion (m/z 200-500) chromatograms of the alkaloidal components of Senecio madagascariensis (fireweed): A. fresh young plant; B. fresh mature plant, and C. dried and milled mature plant.

literature report (Gardner et al., 2006) and using authentic standards (Figure 8) from

the collection of the CSIRO Plant Toxins Research Group.

The major peaks with MH+ ions at m/z 368 and 352 all showed a significant dimer ion

signal at m/z 735 and 703 respectively. This is good evidence for the N-oxide

character (Figure 1) of the alkaloids (Colegate et al., 2005), in this case the N-oxides

of the usaramine-type alkaloids (MH+ 352) and senecionine-type alkaloids (MH+ 336)

respectively (see Figure 2). On the contrary, the major peaks with MH+ ions at m/z

382 and 424 did not show the presence of dimeric ions and this is supportive of their

structures being of the otonecine type (Figure 1) such as otosenine and florosenine

respectively (see Figure 2).

RT: 0.00 - 19.99

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Time (min)

0

10

20

30

40

50

60

70

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90

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10

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0

10

20

30

40

50

60

70

80

90

100352.2

424.2

368.2

352.2

336.3 482.3352.1 352.2 392.1426.1 423.3 257.0

424.2

352.2

368.2

336.2314.2

382.2352.2316.2 287.1300.2366.2

352.2

368.2

424.2352.2

336.2

205.0 382.2366.2 352.2 394.2

NL:1.38E7

Base Peak m/z= 200.0-500.0 MS yu260906lcq69

NL:1.30E8


NL:2.12E8


A

B

C


of 33

Figure 7: HPLC-esi-ion trap MS base ion (m/z 200-500) chromatograms of: A. fresh young fireweed; B. fresh mature fireweed; and C. dried and milled mature fireweed. Also shown are example mass spectra for selected peaks: a. Unidentified alkaloid, m/z 424 (1); b. N-oxide of senecionine type alkaloid, m/z 352 (2); c. N-oxide of a retrorsine type alkaloid, m/z 368 (3).

RT: 5.30 - 13.20

6 7 8 9 10 11 12 13Time (min)

10

20

30

40

50

60

70

80

90

100

10

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100

Rel

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10

20

30

40

50

60

70

80

90

100352.2

424.2

368.2

352.2

336.3 482.3

352.1 352.2332.1 426.1

424.2

352.2424.2

368.2

336.2314.2

382.2352.2472.1366.2

352.2

368.2

368.2

424.2

352.2

336.2

205.0382.2

366.2 352.2 394.2

NL:1.38E7


NL:1.30E8


NL:2.12E8


yu260906lcq69 #735-743 RT: 10.12-10.22 AV: 9 NL: 8.33E6T: + c ESI sid=35.00 Full ms [ 100.00-1200.00]

100 150 200 250 300 350 400 450 500m/z

0

20

40

60

80

100

Re

lativ

e A

bu

nda

nce

424.1

122.1150.1

168.1

425.2380.2364.2

320.2 396.1336.2169.1 460.1210.1 292.2250.1 507.8

yu260906lcq30 #641-651 RT: 9.05-9.16 AV: 11 NL: 5.77E7T: + c ESI Full ms [ 100.00-1200.00]

100 200 300 400 500 600 700m/z

0

20

40

60

80

100

Rel

ativ

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bu

nda

nce

352.2

703.1

704.1705.1354.2 392.3 486.1336.3120.2 687.3220.1 560.1 594.3246.0 772.8176.1


100 200 300 400 500 600 700 800m/z

0

20

40

60

80

100

Re

lativ

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bun

dan

ce

368.2

735.0

369.2314.2 736.0

680.9 737.1138.1 470.1408.3156.1 502.1 626.8 790.9313.3 576.3

A

B

C c

b

a

RT: 5.30 - 13.20

6 7 8 9 10 11 12 13Time (min)

10

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100

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Rel

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30

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50

60

70

80

90

100352.2

424.2

368.2

352.2

336.3 482.3

352.1 352.2332.1 426.1

424.2

352.2424.2

368.2

336.2314.2

382.2352.2472.1366.2

352.2

368.2

368.2

424.2

352.2

336.2

205.0382.2

366.2 352.2 394.2

NL:1.38E7


NL:1.30E8


NL:2.12E8


yu260906lcq69 #735-743 RT: 10.12-10.22 AV: 9 NL: 8.33E6T: + c ESI sid=35.00 Full ms [ 100.00-1200.00]

100 150 200 250 300 350 400 450 500m/z

0

20

40

60

80

100

Re

lativ

e A

bu

nda

nce

424.1

122.1150.1

168.1

425.2380.2364.2

320.2 396.1336.2169.1 460.1210.1 292.2250.1 507.8


100 200 300 400 500 600 700m/z

0

20

40

60

80

100

Rel

ativ

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bu

nda

nce

352.2

703.1

704.1705.1354.2 392.3 486.1336.3120.2 687.3220.1 560.1 594.3246.0 772.8176.1


100 200 300 400 500 600 700 800m/z

0

20

40

60

80

100

Re

lativ

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bun

dan

ce

368.2

735.0

369.2314.2 736.0

680.9 737.1138.1 470.1408.3156.1 502.1 626.8 790.9313.3 576.3

A

B

C c

b

a 1

1

1

2

2

2 3

3

3


of 33

Figure 8: HPLC-esi-ion trap MS base ion chromatograms of: A. an extract of young Senecio madagascariensis spiked with; B. integerrimine-N-oxide, C. usaramine, D. retrorsine, and E. senecionine-N-oxide.

The alkaloid profile observed for the Bega Valley fireweed samples was similar but

seemed sufficiently different from the report on the profiles of both the Hawaiian and

Australian (northern NSW) samples previously analyzed (Gardner et al., 2006). It was

the apparent close similarity in these latter samples that prompted the conclusion that

the Hawaiian fireweed was originally from Australia. Therefore, a sample of the

extract of the Bega Valley plants was sent to Dr Dale Gardner of the USDA

Poisonous Plants Laboratory in Logan, Utah, USA for comparative analysis with their

sample of Australian fireweed collected in northern New South Wales (Figure 9).

RT: 0.00 - 20.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)

0

10000000

20000000

30000000

Inte

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424.1

352.2368.2

442.2 456.2352.2

352.2

368.2 352.2424.1

461.3 368.2461.4461.3352.2 482.2 460.4

352.2

368.1352.1 424.1

352.1 482.3456.2

352.2

424.1368.2 352.2

456.1382.1 482.2461.4

352.1

424.1368.2

352.1 461.3442.2 461.4

NL:1.85E8


NL:3.21E7


NL:1.82E7


NL:2.85E7


NL:3.52E7


A

B

C

D

E


of 33

Figure 9: An HPLC-MS comparison of a zinc/acid-reduced extract of fireweed collected in the Bega Valley, New South Wales in about October 2006 with that collected in northern New South Wales (Gardner et al., 2006). The bold numbers are the MH+ values for each peak (see Figure 2). The analyses were done by Dr Dale Gardner of the USDA Poisonous Plants Research Laboratory in Logan, Utah, USA.

The major differences evident between the two Australian samples include the greater

levels of the senecionine-type alkaloids (MH+ 336, Figure 2), the relative imbalance

of the retrorsine-type alkaloids (MH+ 352, Figure 2) and the apparent appearance of

dehydroretrorsine-type alkaloids (MH+ 350) in the Bega Valley sample. The latter are

possibly related to seneciphylline and have not been previously reported in this plant.

RT: 0.00 - 20.00 SM: 3B

0 2 4 6 8 10 12 14 16 18Time (min)

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13.13

12.19

10.169.818.26

6.89 14.805.65 9.30 17.0415.6811.97 17.28 19.692.43 4.791.73 2.870.40

14.72

14.77

12.25

9.78

14.07

10.078.26 8.80

15.187.76 11.49 18.685.55 18.3715.766.971.25 5.444.653.08

NL:1.96E7

Base Peak MS PA121106_01

NL:6.04E6

Base Peak MS PA121106_02

Northern NSW

Bega Valley

336

336

336

460

460

424

424

424

352

352 352

350

442

442 350

418

382

382

418

352


of 33

In a similar way to the quantitation of alkaloids in honey and pollen (Betteridge et al.,

2005; Boppré et al., 2005; Boppré et al., 2008), the approximate concentrations of the

DHPAs and their N-oxides were estimated by comparison to calibration curves

generated using the authenticated DHPA lasiocarpine and lasiocarpine-N-oxide

respectively. Thus, fresh young plant was estimated to contain between 100 and 220

ppm (milligrams of alkaloids per kilogram of plant) and mature fresh plant between

70 and 110 ppm. Another sample of mature plant that was dried and milled to a fine

powder was estimated to contain between 140 and 170 ppm of pyrrolizidine alkaloids.

In this very limited survey, it appears that young fresh plant contains more

pyrrolizidine alkaloids than the fresh mature plant. The higher levels estimated in the

dried and milled mature plant reflects the increased efficiency of extraction.

Significantly, there was no apparent selective degradation of most of the alkaloids

during the drying process however, significant loss of the alkaloid with MH+ 424 did

seem to occur. This observation would require further checking for confirmation.

The levels of pyrrolizidine alkaloids detected in the Bega Valley samples was at the

lower end of the scale (217 – 1990 ppm) for the levels detected in samples collected

from various sites in Hawaii (Gardner et al., 2006). It is unlikely that this results from

differences in extraction efficiency but may reflect the relatively limited sampling

procedure employed for the Bega Valley samples, combined with inter-plant

differences in levels.

Time-of-flight mass spectrometry

Samples of fresh fireweed (entire plant) were extracted in the usual way and analyzed

using TOF mass spectrometry to complement the results obtained using ion trap mass

spectrometry. Three plant samples, collected in spring of 2008, were analyzed: 1.

mature fireweed plants with green stems; 2. mature fireweed plants with red stems;

and 3. small (juvenile) fireweed plants.


of 33

Figure 10: Direct infusion, ESI-TOF MS analysis of fresh Senecio madagascariensis collected in the Bega Valley, October 2008. A. young plants; B. mature, red-stemmed plants; and C. mature, green-stemmed plants

The direct infusion TOF mass spectrometric analysis (Figure 10) of the three 2008

samples showed the presence of ions compatible with the major pyrrolizidine

alkaloids reported for S. madagascariensis (Gardner et al., 2006) and that were

observed using the ion trap mass spectrometric analysis on plants collected two

seasons earlier in 2006. In particular the ion profiles of the juvenile and mature-red-

stemmed plants were very similar. The ion profile for the mature, green-stemmed

plants was also qualitatively similar to the other samples but it showed a relative

increase in the presence of the ions at 352 and 368. This apparent relative increase

could be a result of the sampling technique i.e., accidentally choosing plants with

slightly different alkaloid profiles, or it could reflect the presence of other alkaloids

with the same molecular weights. The first explanation can be addressed by collecting

more samples and completing seasonal alkaloid profiles of plant populations. The

5x10

0

1

2

3

4

+ Scan (0.194 min) RMFW100.d

424.22699

382.21328368.19640352.20047

Counts vs. Mass-to-Charge (m/z)

320 325 330 335 340 345 350 355 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 445

5x10

0

2

4

6

8

+ Scan (0.182 min) JFW100.d

424.22976

382.21541352.20260

368.19856

Counts vs. Mass-to-Charge (m/z)

320 325 330 335 340 345 350 355 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 445

A

B

C


of 33

Figure 11. HPLC-TOF mass spectrometric analysis of fresh, green-stemmed mature samples (green trace) and fresh, red-stemmed mature samples (red trace) of Senecio madagascariensis. The numbers shown are the masses of the ions observed.

second explanation was addressed by examining the high pressure liquid

chromatographic (HPLC) separation of the alkaloids (Figure 11) as with the ion trap

mass spectrometry analysis described for the 2006 samples.

The HPLC-TOF mass spectrometric analysis showed that the alkaloid with a

molecular ion adduct (MH+) of 424 is predominant. In the samples analyzed, the red-

stemmed variation of the plant shows an additional peak with an MH+ of 382 that is

not observed in the green-stemmed variation. The red-stemmed variation also shows

an additional 2 peaks with an MH+ of 368. The green-stemmed variation shows a

larger relative amount of the free alkaloid at 336 (derived from the N-oxide at 352).

Despite these minor differences, the two variations are very similar in the profile of

alkaloids.

424

352

336

382

368


of 33

Analysis of Wild Honey from the Bega Valley

Three small samples of honey-in-the-comb, taken from a wild hive in the vicinity of

previous fireweed flowerings in the Bega Valley, were each extracted with dilute acid.

The three separate extracts were then combined to improve the sensitivity of the

detection method.

The dilute acid extract was processed using solid phase extraction in a slight

modification of the method used to process the plants. The resultant sample was

analyzed using HPLC-TOF-MS.

There was no unambiguous detection of fireweed pyrrolizidine alkaloids in this honey

sample. Nonetheless, the very low level presence in the honey of toxic pyrrolizidine

alkaloids derived from Senecio spp. was indicated by the observation of a peak with

an MH+ of 352 suggestive of senecionine-N-oxide.

The lack of convincing evidence for toxic pyrrolizidine alkaloids in this sample

should not be taken as an indication that fireweed honey generally does not contain

such alkaloids. The actual history of the honey sample presented is unknown and it

may well be that fireweed contributed very little to the foraging bees.


of 33

Conclusions and Recommendations The results of this very limited phytochemical survey of plants collected in the Bega

Valley of New South Wales, clearly demonstrate the presence of hepatotoxic

dehydropyrrolizidine alkaloids. There is increasing international concern about these

alkaloids in animal feed and in the human food supply (European Food safety

Authority, 2007).

On the basis of potential toxicity to livestock and to humans it would seem prudent to

control the spread of fireweed. In the first instance this should involve the rigorous

containment of current populations thereby preventing incursions into new areas.

Where small, invading populations are discovered, they should be immediately

removed. The success of this approach has been reported on Hawaii where, in some

areas, regular monitoring and hand-pulling of fireweed plants has resulted in a

dramatic decrease in the plants observed

(www.hawaiiinvasivespecies.org/iscs/kisc/pdfs/wow3text.pdf). The example set, and

the concern shown, by the Hawaiian Invasive Species Council and the Kauai Invasive

Species Committee (www.hawaiiinvasivespecies.org/iscs/kisc/wow.html) should be

examined and emulated in Australia. Thorne et al (2005) describe an adaptive

management approach to the control of fireweed. The essential component is an

integrated weed management plan comprising three levels of activity i.e., prevention,

control and immediate response. In concert with controlling fireweed where it is

established, the fireweed should be prevented from invading new areas and, when it

does, immediate action should be taken to eradicate it from the new area. Thorne et al

(2005) describe six steps to adaptive management control of fireweed i.e., establish

goals; set management priorities; identify appropriate methods; develop and

implement an integrated weed management plan; monitor results; and adaptive

modification to improve the plan.

Livestock health and productivity should be carefully monitored (taking into

consideration the delayed onset of some clinical signs) in fireweed-endemic areas.

Animal-derived food products, including meat, milk and bee products such as honey

and pollen, should be checked for the presence of hepatotoxic pyrrolizidine alkaloids.


of 33

Because of the potential for higher exposures, locally-produced and consumed honey

and pollen products should be specifically monitored for hepatotoxic pyrrolizidine

alkaloid content.


of 33

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