Synthesis of citrophilus mealybug sex pheromone using chrysanthemol extracted from Pyrethrum (Tanacetum cinerariifolium)

Article abstract of DOI:10.1080/14786419.2018.1446136

A commercial pyrethrum extract was used as a source of chrysanthemol for the synthesis of the citrophilus mealybug (Pseudococcus calceolariae) sex pheromone. The chrysanthemic acid esters (pyrethrins I) were isolated and subsequently reduced to obtain chrysanthemol, which was used for ester pheromone synthesis. Field tests showed that the pheromone synthesized using plant-derived chrysanthemol was as attractive to male P. calceolariae as the pheromone obtained using a commercial isomeric chrysanthemol mixture.

Full text of DOI:10.1080/14786419.2018.1446136

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Synthesis of citrophilus mealybug sex pheromone
using chrysanthemol extracted from Pyrethrum
(Tanacetum cinerariifolium)
Jan Bergmann, Jaime Tapia, Manuel Bravo, Tania Zaviezo & M. Fernanda
Flores
To cite this article: Jan Bergmann, Jaime Tapia, Manuel Bravo, Tania Zaviezo & M. Fernanda
Flores (2018): Synthesis of citrophilus mealybug sex pheromone using chrysanthemol
extracted from Pyrethrum (Tanacetum cinerariifolium), Natural Product Research, DOI:
10.1080/14786419.2018.1446136
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Natural Product research, 2018
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2018.1446136
ꢀ
Synthesis of citrophilus mealybug sex pheromone using
chrysanthemol extracted from Pyrethrum (Tanacetum
cinerariifolium)
Jan Bergmanna , Jaime Tapia , Manuel Bravo , Tania Zaviezo and
a
a
b
M. Fernanda Floresa
ꢆ
b
Inꢂꢁiꢁꢇꢁꢄ ꢃꢈ Qꢇímiꢉꢆ, Pꢄnꢁifiꢉiꢆ univꢈꢅꢂiꢃꢆꢃ cꢆꢁóꢊiꢉꢆ ꢃꢈ Vꢆꢊpꢆꢅꢆíꢂꢄ, Vꢆꢊpꢆꢅꢆíꢂꢄ, cꢀiꢊꢈ; Fꢆꢉꢇꢊꢁꢆꢃ ꢃꢈ agꢅꢄnꢄmíꢆ
ꢈ Ingꢈniꢈꢅíꢆ Fꢄꢅꢈꢂꢁꢆꢊ, Pꢄnꢁifiꢉiꢆ univꢈꢅꢂiꢃꢆꢃ cꢆꢁóꢊiꢉꢆ ꢃꢈ cꢀiꢊꢈ, sꢆnꢁiꢆgꢄ, cꢀiꢊꢈ
ABSTRACT
ARTICLE HISTORY
A commercial pyrethrum extract was used as a source of chrysanthemol
for the synthesis of the citrophilus mealybug (Pseudococcus
calceolariae) sex pheromone.The chrysanthemic acid esters (pyrethrins
I) were isolated and subsequently reduced to obtain chrysanthemol,
which was used for ester pheromone synthesis. Field tests showed
that the pheromone synthesized using plant-derived chrysanthemol
was as attractive to male P. calceolariae as the pheromone obtained
using a commercial isomeric chrysanthemol mixture.
rꢈꢉꢈivꢈꢃ 13 Nꢄvꢈmbꢈꢅ 2017
aꢉꢉꢈpꢁꢈꢃ 24 Fꢈbꢅꢇꢆꢅy 2018
KEYWORDS
cꢀꢅyꢂꢆnꢁꢀꢈmꢄꢊ; pyꢅꢈꢁꢀꢅinꢂ;
mꢈꢆꢊybꢇg pꢀꢈꢅꢄmꢄnꢈ
1
. Introduction
The citrophilus mealybug, Pseudococcus calceolariae (Maskell) (Hemiptera: Pseudococcidae)
is a cosmopolitan pest insect found in many regions of the world, attacking various plant
species, and causing economic losses in fruit crops (García Morales et al. 2016). Control is
usually based on conventional chemical control methods, which are not always efficient due
CONTACT Jꢆn Bꢈꢅgmꢆnn
jꢆn.bꢈꢅgmꢆnn@pꢇꢉv.ꢉꢊ
sꢇppꢊꢈmꢈnꢁꢆꢊ ꢃꢆꢁꢆ fꢄꢅ ꢁꢀiꢂ ꢆꢅꢁiꢉꢊꢈ ꢉꢆn bꢈ ꢆꢉꢉꢈꢂꢂꢈꢃ ꢆꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2018.1446136.
©
2018 Infꢄꢅmꢆ uK limiꢁꢈꢃ, ꢁꢅꢆꢃing ꢆꢂ tꢆyꢊꢄꢅ & Fꢅꢆnꢉiꢂ Gꢅꢄꢇp

2
J. BERGMANN ET AL.
to the cryptic habits of the mealybugs. On the other hand, monitoring mealybug populations
is done by visual inspections, which is a very time-intensive labour and requires trained
personnel. The P. calceolariae sex pheromone produced by the females has been identified
as (1R,3R)-chrysanthemyl (R)-2-acetoxy-3-methylbutanoate (El-Sayed et al. 2010; Unelius
et al. 2011). The potential use of the pheromone for detection, monitoring, and control has
been studied in New Zealand and Chile with promising results (Flores et al. 2015; Suckling
et al. 2015), so it is desirable to explore possible sources of chrysanthemol for synthesis of
the pheromone to be used in field applications.
Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) (Asteraceae) is a perennial herb
that has been used for a long time as a natural insecticide. The active ingredients are six
compounds, which are usually grouped according to their chemical structure into chrysan-
themic acid esters (pyrethrins I) and pyrethric acid esters (pyrethrins II). The typical pyrethrin
I + II content of dried T. cinerariifolium flowers is about 1–2%, and in view of the demand for
this botanical insecticide, breeding methods for the improvement of pyrethrin content (Li
et al. 2014), and extraction methods for the isolation of pyrethrins have been studied exten-
sively. For the latter, extraction using different solvents such as hexane, acetone, and ethanol
for example (Nazari and Kambarani 2008; Ban et al. 2010; Nagar et al. 2015), ultrasound-
assisted extraction (Babić et al. 2013), and extraction with supercritical CO (Kiriamiti et al.
2
2003; Martín et al. 2012), among other methods, have been studied regarding their efficiency.
Furthermore, numerous methods have been developed for the quantification of pyrethrins
(
Essig and Zhao 2001; Henry et al. 2001; Ban et al. 2010).
Chrysanthemol can be obtained from pyrethrins I by chemical reduction. Furthermore,
pyrethrum extract is commercially available and should yield pure (1R,3R)-chrysanthemol,
as opposed to commercially available chrysanthemol, which is usually sold as a stereoiso-
meric mixture. Considering the possible advantages (price and stereoisomeric purity), the
present work studies if pyrethrum extract can be used as a source of chrysanthemol for
citrophilus mealybug sex pheromone synthesis.
2
. Results and discussion
2
.1. Pyrethrin extraction, purification, and reduction and synthesis of sex
pheromone
Ten grams T. cinerariifolium extract was treated with methanol at low temperature (experi-
mental details provided in supplementary material). This procedure was performed because
the pyrethrins are soluble in this solvent, while other plant extract components, such as
waxes and pigments, are not extracted at low temperatures, thus obtaining a refined extract
which is more suitable for flash chromatography. Three variables which were considered to
most likely have an impact on extraction yield were selected for a simple optimization pro-
cedure. Their contribution to the mass obtained from the initial extract was evaluated using
a two-level full factorial design in a reduced number of runs. Variables examined, levels
considered, and results obtained are presented in Table 1. The normal probability plot
obtained for these experiments revealed a significant positive effect for the solvent volume
(
Figure 1), while the factors time and temperature presented non-significant effects. This
means that for the latter two factors, the most convenient experimental values (i.e. shorter
times, temperatures closer to room temperature) can be chosen. The mean mass obtained

NATURAL PRODUCT RESEARCH
3
Table 1. expꢈꢅimꢈnꢁꢆꢊ vꢆꢅiꢆbꢊꢈꢂ ꢆnꢃ ꢅꢈꢂꢇꢊꢁꢂ ꢄbꢁꢆinꢈꢃ fꢄꢅ ꢁꢀꢈ ꢄpꢁimizꢆꢁiꢄn ꢄf ꢁꢀꢈ ꢊꢄw-ꢁꢈmpꢈꢅꢆꢁꢇꢅꢈ ꢈx-
ꢅꢆꢉꢁiꢄn ꢄf pyꢅꢈꢁꢀꢅꢇm ꢈxꢁꢅꢆꢉꢁ.
ꢁ
Experiment
Volume (mL)
Time (min)
Temperature (ºC)
Mass (g)
1
2
3
4
5
6
7
8
9
75
75
75
30
30
60
60
30
30
60
60
45
45
45
−10
−70
−10
−70
−10
−70
−10
−70
−40
−40
−40
6.73
6.65
6.78
7.32
8.22
8.10
7.96
7.46
8.76
8.33
8.47
75
150
150
150
150
112
112
112
10
1
1
Figure 1. Nꢄꢅmꢆꢊ pꢅꢄbꢆbiꢊiꢁy pꢊꢄꢁ fꢄꢅ ꢂꢁꢆnꢃꢆꢅꢃizꢈꢃ ꢈffꢈꢉꢁꢂ ꢄbꢁꢆinꢈꢃ fꢄꢅ ꢁꢀꢈ ꢄpꢁimizꢆꢁiꢄn ꢄf ꢁꢀꢈ ꢈxꢁꢅꢆꢉꢁiꢄn
pꢅꢄꢉꢈꢂꢂ.
in three experiments at the intermediate‘0’levels was 8.52 g, while the predicted value was
7.70 g. This suggests a response curvature due to a maximum in the experimental domain.
Generally, this situation is resolved conducting additional experiments and modelling the
results with a second experimental design order. However, in order to decrease the number
of experiments, the experiment which presented the maximum response in the factorial
design, in this case the ‘0’level, was chosen as the optimum extraction condition.
Pyrethrins I were isolated from the refined extract in a 13–18% yield based on the mass
of the refined extract submitted to chromatography (Table S1). The identity of pyrethrins I
was confirmed by comparing gas chromatographic retention times and mass spectra of
isolated compounds with those of an authentic standard. The commercially available plant
extract contained 25% pyrethrins. According to GC analysis, it was estimated that approxi-
mately two thirds of the total pyrethrin content of the plant extract corresponded to pyre-
thrins I and one third to pyrethrins II, i.e. 17% and 8% ca., respectively. Considering the
13–18% yield of pyrethrins I from the refined extract, and the 85% yield of refined extract,
this translates to an approximate 66–92% recovery of pyrethrins I from the commercial
extract. This simple calculation, however, does not take into account a possible degradation

4
J. BERGMANN ET AL.
which pyrethrins might suffer over time and which may account for substantial losses
(Atkinson et al. 2004).
The conversion of pyrethrins I to the mealybug sex pheromone was achieved by reduction
and subsequent esterification of the resulting chrysanthemol with 2-acetoxy-3-methylbu-
tanoic acid (Figure 2). Treatment of purified pyrethrins I with lithium aluminium hydride
resulted in the formation of chrysanthemol, which was isolated and purified by column
chromatography in 80% yields. The relative configuration of chrysanthemol was determined
to be trans by comparing gas chromatographic retention times and NMR spectra with those
of an isomeric mixture (Figure S1) (Unelius et al. 2011). Furthermore, upon gas chromatog-
raphy using a chiral stationary phase, the chrysanthemol obtained presented the same
retention time as synthetic (1R,3R)-chrysanthemol (Figure S2). Gas chromatographic analysis
of an isomeric mixture of chrysanthemol on a chiral stationary phase showed three peaks
with ratios of 50:32:18 ca. (Figure S2(a)). Considering the cis-trans ratio of the chrysanthemol
isomeric mixture (ca. 35:65), the first peak must correspond to a trans-isomer, co-eluting
with a cis-isomer, the second peak to a trans-isomer, and the third peak to a cis-isomer.
Furthermore, the second peak showed the same retention time as the pure trans-(1R,3R)-
isomer (Figure S2(b)), as well as the chrysanthemol obtained from the Tanacetum extract
(
Figure S2(c)). In conclusion, although a complete resolution of all four stereoisomers was
not achieved, the absolute configuration of the natural chrysanthemol could be assigned,
as expected, as (1R,3R), matching the configuration of P. calceolariae pheromone. Finally, the
conversion of (R)-2-acetoxy-3-methylbutanoic acid into the corresponding acid chloride by
treatment with oxalyl chloride, and subsequent esterification with the plant-derived chry-
santhemol yielded (1R,3R)-chrysanthemyl (R)-2-acetoxy-3-methylbutanoate in 60–70%
overall yield (Figures 2, S3, S4) (El-Sayed et al. 2010).
We have shown that by using a simple optimization procedure, a recovery of up to 90%
of pyrethrins I from the plant extract can be achieved, while the reduction of pyrethrins I to
chrysanthemol proceeded with a yield of 80%, without any optimization. These are promising
results in view of a possible scale-up of the methodology, particularly considering that there
should be room for improvement, for example by further optimizing the amounts of solvents
and reagents. In a similar approach, Tabata et al. (2015) reported the use of lavender essential
oils in the synthesis of isolavandulyl butyrate, the sex pheromone of the mealybug
Planococcus kraunhiae, without the need of isolating or purifying lavandulol, and showed
LiAlH4
O
R
OH
O
O
O
O
OH
1
. (COCl)2
Ac2O
O
O
HO
2. (1R,3R)-chrysanthemol
HO
O
O
O
O
Figure 2. tꢅꢆnꢂfꢄꢅmꢆꢁiꢄn ꢄf pyꢅꢈꢁꢀꢅinꢂ I ꢁꢄ ꢉꢀꢅyꢂꢆnꢁꢀꢈmꢄꢊ ꢆnꢃ ꢂynꢁꢀꢈꢂiꢂ ꢄf ꢉꢀꢅyꢂꢆnꢁꢀꢈmyꢊ 2-ꢆꢉꢈꢁꢄxy-3-
mꢈꢁꢀyꢊbꢇꢁꢆnꢄꢆꢁꢈ.

NATURAL PRODUCT RESEARCH
5
the potential for the use of the lavender oil derived pheromone in mating disruption. In our
case, an additional reduction step is required to obtain the monoterpene alcohol needed
for the sex pheromone synthesis. Considering the (1R,3R)-stereochemistry of the chrysan-
themolobtainedfrompyrethrum, itcanalsobeusedforthesynthesisof(1R,3R)-chrysanthemyl
(R)-2-methylbutanoate, the sex pheromone of the Madeira mealybug, Phenacoccus madeiren-
sis (Ho et al. 2009), but not for the synthesis of the sex pheromone of the striped mealybug,
Ferrisia virgata, (1S,3R)-chrysanthemyl tiglate (Tabata and Ichiki 2017).
2
.2. Field tests
Traps baited with pheromone prepared from chrysanthemol obtained from the T. cinerarii-
folium extract (‘extract pheromone’) and those prepared with commercially available chry-
santhemol (‘synthetic pheromone’) captured the same amount of males, 347 ± 122 vs.
347 ± 94 males (mean per trap in 7 days ± standard deviation), respectively, while no males
were captured in traps treated with solvent only. Pheromone molecules have three chiral
centres and females produce the (R,R,R)-isomer exclusively (Unelius et al. 2011). Furthermore,
stereoisomers with (S)-configuration in the acid part are not attractive to males (Unelius
et al. 2011), and the unnatural isomers do not interfere with attracting males to the traps in
the field (Flores et al. 2015). The ‘synthetic pheromone’ used in the field test is a mixture of
8
stereoisomers, containing ca. 16% active (R,R,R)-isomer (i.e. ca. 5 μg in 30 μg), while the
‘extract pheromone’corresponded to the pure (R,R,R)-isomer.
The advantage of using the pheromone obtained by the method described here is the pos-
sibility to use much lower doses as compared to the isomeric mixture without any loss of activity
(Flores et al. 2015), and this advantage may outweigh a possible higher cost for its production.
3
. Conclusions
Pyrethrum can be used as an alternative source for chrysanthemol in the synthesis of the
citrophilus mealybug sex pheromone. The economic feasibility of our method remains to
be analysed in more detail. Future studies should be directed at simplifying the procedure
presented in this work in order to facilitate a scale-up.
Acknowledgements
This work was supported by the Fondo de Fomento al Desarrollo Científico y Tecnológico (FONDEF)
under grant D10i1208, the Fondo de Equipamiento Científico y Tecnológico – Conicyt (Fondequip)
under grant EQM130154, Deutscher Akademischer Austauschdienst (via material resources program
to JB), and the Dirección de Investigación-PUCV.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Fondo de Fomento al Desarrollo Científico y Tecnológico (FONDEF)
[
[
grant number D10I1208]; the Fondo de Equipamiento Científico y Tecnológico – Conicyt (Fondequip)
grant number EQM130154].

6
J. BERGMANN ET AL.
ORCID
Jan Bergmann
http://orcid.org/0000-0003-3566-1979
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