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Comparison of pyrethrins extraction methods efficiencies

Authors:
  • Institut za poljoprivredu i turizam Porec
  • Institute of Agriculture and Tourism, Poreč, Croatia

Abstract and Figures

Extraction efficiency of insecticidal active compounds from dried Dalmatian pyrethrum flowers (Chrysanthemum cinerariaefolium /Trevir/Vis) was tested using different techniques and solvents. The research included six treatments which are the combination of two techniques (soxtec and ultrasound) and three different solvents (hexane, ethanol and petroleum ether). Dalmatian pyrethrum is a perennial herb native to Croatia. Its powder prepared from dried flower heads has been used as natural insecticide for centuries in traditional Croatian farming systems. It has no toxicity to man and animals but possesses ecological benefits that have led to increasing worldwide production of this natural insecticide. Nowadays, it is cultivated mainly at higher altitudes in tropical countries such as Kenya, Tanzania and Rwanda. The present investigation was directed in identifying a simple and reliable extraction treatment using solvents with lower cost and toxicity and an adequate method for the identification and separation of active compounds (pyrethrins) with possible application in enterprises or industry. Best developed method was used for determination of pyrethrin content in three different natural populations of Chrysanthemum. The results revealed high content of total pyrethrins in populations grown in Croatia. Developed method and good quality product give a possibility for this culture to become again an exporting and economically valid product for Croatia.
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African Journal of Biotechnology Vol. 9 (18), pp. 2702-2708, 3 May, 2010
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2010 Academic Journals
Full Length Research Paper
Comparison of pyrethrins extraction methods
efficiencies
Dean Ban1, Barbara Sladonja1*, Marina Luki1, Igor Luki1, Viviane Lušeti1, Karin Kovaevi
Gani2 and Dragan Žnidari3
1Institute of Agriculture and Tourism Pore, K. Hugues 8, p.p. 31, 52 440 Pore, Croatia.
2Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia.
3Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI - 1111, Ljubljana, Slovenija.
Accepted 5 March, 2010
Extraction efficiency of insecticidal active compounds from dried Dalmatian pyrethrum flowers
(Chrysanthemum cinerariaefolium /Trevir/Vis) was tested using different techniques and solvents. The
research included six treatments which are the combination of two techniques (soxtec and ultrasound)
and three different solvents (hexane, ethanol and petroleum ether). Dalmatian pyrethrum is a perennial
herb native to Croatia. Its powder prepared from dried flower heads has been used as natural
insecticide for centuries in traditional Croatian farming systems. It has no toxicity to man and animals
but possesses ecological benefits that have led to increasing worldwide production of this natural
insecticide. Nowadays, it is cultivated mainly at higher altitudes in tropical countries such as Kenya,
Tanzania and Rwanda. The present investigation was directed in identifying a simple and reliable
extraction treatment using solvents with lower cost and toxicity and an adequate method for the
identification and separation of active compounds (pyrethrins) with possible application in enterprises
or industry. Best developed method was used for determination of pyrethrin content in three different
natural populations of Chrysanthemum. The results revealed high content of total pyrethrins in
populations grown in Croatia. Developed method and good quality product give a possibility for this
culture to become again an exporting and economically valid product for Croatia.
Key words: Chrysanthemum cinerariaefolium, extraction, pyrethrum, reversed phase-high performance liquid
chromatography, soxtec, ultrasound.
INTRODUCTION
Pyrethrum is a natural insecticide produced from
Dalmatian Chrysanthemum (Chrysanthemum cinerariae-
folium) flowers. There are many areas of application of
pyrethrum. The most important use is probably in
mosquito control for both rural and urban areas where
*Corresponding author. E-mail: barbara@iptpo.hr. Tel: +385 52
408 300. Fax: +385 52 431 659.
Abbreviations: SFE, Super critical fluid extraction; RP-HPLC,
reversed phase-high performance liquid chromatographic; NP-
HPLC, normal phase- high performance liquid chromatography;
GC, gas-liquid chromatography; CEC, capillary electro
chromatography; HPLC, high performance liquid
chromatography; RSD, relative standard deviation.
human safety is of prime considerations (US
Environmental Protection Agency Office of Pesticide
Programs, 2009). It is applied as an insecticide in home
gardens and in organic farming. Pyrethrum is already
included in most lists of approved insecticides for organic
production throughout the world and has become the
dominant insecticide (Glynne-Jones, 2001). The term
“pyrethrum” referres to the plant, flower or flower extract,
with the active insecticidal components of pyrethrum
known as “pyrethrins” (Morris et al., 2006). Pyrethrins are
esters of chrysanthemic (pyrethrins I, PI) and pyrethric
(pyrethrins II, PII) acid. Thus, “total pyrethrins” refers to
sum of pyrethrins I and pyrethrins II esters (Casida and
Quistad, 1995). Pyrethrins I group include pyrethrin I,
cinerin I, and jasmolin I, while pyrethrins II group consists
of pyrethrin II, cinerin II and jasmolin II (Essig and
Zhao, 2001a). Among these compounds, pyrethrin I and
pyrethrin II are the most predominant and active (Casida
and Quistad, 1995). Pyrethrins content can vary from 0.9
to 1.3% by weight of dried flowers in native populations
(Kolak et al., 1999; Casida and Quistad, 1995). Tas-
manian commercial varieties contain 1.8 - 2.5% (Morris et
al., 2006), while 3.0% of pyrethrin was reported in flowers
of clones and breeding lines from breeding programs in
Australia, Kenya, and USA (Casida and Quistad, 1995).
The extraction yields of each pyrethrin ester depend on
extraction conditions (temperature, solvent) but their
relative proportions do not vary significantly (EU project,
2002). Because of separation difficulties and lack of
absolute standards for each compound, the content of
pyrethrum extract is not usually reported nor analysed for
the individual pyrethrins, but for total pyrethrins or total
pyrethrins I and total pyrethrins II.
There are many organic solvents and extraction meth-
ods which are not sufficiently effective or are too expen-
sive for large scale profitable pyrethrum production.
Among methods of pyrethrin extraction, the classic
organic solvent extraction methods are still the most
commonly used in industry and laboratories. Ultrasound
(Kasaj et al., 1999), soxtec (Otterbach and Wenclawiack,
1999) and recently the super critical fluid extraction (SFE)
methods (Pan et al., 1995; Wynn et al., 1995; Otterbach
and Wenclawiack, 1999; Della Porta and Reverchon,
2002; EU project, 2002; Pol and Wenclawiack, 2003;
Reverchon and De Marco, 2006) have been widely
investigated. Different solvents have been tested for their
efficiency in pyrethrins extraction. These include n-
hexane (Pan et al., 1995; Kasaj et al., 1999; EU project,
2002), methanol, ethanol, propanol (EU project, 2002),
dichloromethane (Kasaj et al., 1999; EU project, 2002),
and petroleum ether (Della Porta and Reverchon, 2002).
Among the separation techniques, many have been
reported and compared: reversed phase-high perfor-
mance liquid chromatographic (RP-HPLC) (Pan et al.,
1995; Wang et al., 1997; Kasaj et al., 1999; EU project,
2002), normal phase-high performance liquid chroma-
tography (NP-HPLC) (Essig and Zhao, 2001a; Essig and
Zhao, 2001b), gas-liquid chromatography (GC) (Nguyen
et al., 1998; Della Porta and Reverchon, 2002), and
some others, e.g. high-performance capillary electro-
phoresis (HPCE) (Henry III et al., 1999) and capillary
electro chromatography (CEC) (Henry III et al., 2001)
with the official AOAC titration method used as a referent
method (Casida and Quistad, 1995). The pyrethrins are
light (especially UV), oxygen, water, and elevated temp-
erature sensitive (Casida and Quistad, 1995). Termol-
abile pyrethrins could be extracted without decomposition
in the temperature range of 20 to 40°C (Della and
Reverchon, 2002) and therefore the HPLC to the GC
technique for analysing pyrethrins was preferred. The
advantage of reversed-phase HPLC over normal-phase
HPLC methods is the very low level of interferences in
the chromatography (Wang et al., 1997). Chrysanthemum is
a native plant in Croatia (Kolak et al., 1999) and the world
Ban et al. 2703
need of natural insecticides has increased considerably
recently. A study on methods of extraction, sep-
eration and identification of pyrethrum active compounds
is of particular interest for potential industrial or home
production. Extraction efficiency of six different treat-
ments (combinations of two extraction techniques and
three solvents) for isolation of natural pyrethrins was
investigated. The extraction recovery and repeatability,
cost for eventual commercial extraction and the lowest
toxicity of solvents used were considered. Developed
method was used for determination of pyrethrin content in
three native populations of C. cinerariaefolium.
MATERIALS AND METHODS
Chemicals and reagents
Pyrethrum extract containing 25% pyrethrin I + pyrethrin II,
4’methoxyflavanone and acetonitrile HPLC grade were obtained
from Fluka (Buchs, Switzerland). Ethanol, methanol, hexane and
petroleum ether were p.a. grade, purchased from Kemika (Croatia).
Pure water was obtained from an Ellix 3 purification system
(Millipore, USA).
Plant materials
The seed of native pyrethrum plants C. cinerariaefolium /Trevir./Vis
was collected in their natural habitat near Split, Croatia. Seed was
sown and one month old transplants were planted at experimental
field of the Institute of Agriculture and Tourism-Pore (Croatia).
During the growing season, common cultural practices was applied.
Flowers were hand harvested in 2004 at optimum maturity and the
flowers were spread in a thin layer on wooden pallets. Prior
analysis, flowers were dried for two months under dry, cool and
dark conditions. Dry flowers (91.4% dry matter) were pulverised
(with an electric mixer) and stored at C in a dark well tapped
glass. Dry samples were dusted in a dark room and extracted at the
lowest possible temperature. They were preserved at -18°C in well
closed flasks protected with parafilm and Al-folium and the time
between extraction and analysis was as short as possible.
Treatments
The research included six treatments which were the combination
of two techniques (soxtec and ultrasound) and four solvents
(ethanol, hexane, methanol and petroleum ether).
Instrumentation
The extractions were made on a soxtec avanti 2055 manual system
(Foss, Sweden) and in the ultrasound bath (Branson, The
Netherlands). To achieve better and uniform elution of pyrethrins
from crude oleoresin, a laboratory stirrer 3005 (GFL, Germany) was
used. Solvent was evaporated on a rotary evaporator, Laborota
4000, comprising a Rotavac vacuum pump (Heildorph, Germany).
HPLC analyses were performed on a Varian Pro Star HPLC
system comprising a Pro Star 230 solvent delivery module, Pro Star
UV-Vis detector and manual 7725I Rheodine injector with a 20 µl
sample loop. Separation of compounds was achieved using a
Chrompack Omnisphere C18 column (250 × 4.6 mm, 5 µm particle
size). Monitoring, pump control and data processing were
performed by means of Star LC Workstation Version 5.5 software.
2704 Afr. J. Biotechnol.
The spectra of individual esters were obtained using a Varian Pro
Star HPLC system including: Pro Star 230 pump, Pro Star 330 UV-
Vis Photodiode Array Detector, Pinaclle C18 column (250 × 4.6
mm, 5 µm particle size) and LC Workstation Version 6.20 software.
Soxtec extraction
Pulverised material (1 g) was extracted in soxtec apparatus with
each solvent at a recommended temperature and duration (155°C/
85 min for hexane, 200°C/ 110 min for ethanol, 135°C/ 80 min for
petroleum ether). The evaporation of the solvent and its recovery
was automated. Dried pyrethrum extract was collected in Al-
vessels. The extraction was repeated on the already extracted
sample of the pulverised material. The obtained crude extract was
then eluted with acetonitrile (25 ml in two portions) using an electric
laboratory stirrer for 10 min at 200 RPM. The elutes were collected
with Pasteur pipettes in 25 ml graduated flasks and kept at - 18°C
prior to analysis. The elution of the same crude extract was made
five times in the preliminary studies, and two times for the analysed
samples.
Ultrasound extraction
Pulverised material (1 g) and 15 ml of solvent were transferred into
25 ml flasks and sonicated for 1 h. The extraction of the same
sample was repeated 5 times in the preliminary studies, and four
times for the analysed samples. After filtration, the combined
extracts of the first three extractions were collected into 25 ml
graduated flasks and then the solvent was evaporated on a
Rotavac (30°C, vacuum, 150 RPM). Preliminary studies included
the fourth and fifth extraction (collected and analysed separately).
To get a purified extract of the residue, the elution of pyrethrins was
performed the same way as described in the soxtec extraction
method. Results are expressed as g/100 g (or %) of dried flowers
for each ester and calculated as a mean value of three replicates.
Chromatographic conditions (RP-HPLC)
RP-HPLC method proved to be in good correlation with the
standard AOAC method and has been successfully used to
separate the pyrethrins from the pyrethrum extract (Kasaj et al.,
1999). By modifying the HPLC conditions (Table 1), excellent
separation and resolution of all six compounds and the internal
standard was achieved (Figure 1). The mobile phase components
used were acetonitrile (solvent A) and water (solvent B). The flow
rate was 1 ml/ min. The pyrethrins were detected at 230 nm. The
same gradient program was used to obtain spectra for each ester
with the use of (diode array detection) DAD scanning over a
wavelength range from 200 to 400 nm (Table 1).
Standard solutions
Stock solutions containing pyrethrin I and pyrethrin II (3 mg/ml) and
4’-methoxyflavanone (internal standard; 2.417 mg/ml) in acetonitrile
were used to prepare 9 standard pyrethrin mixtures containing
0.147 - 4.705 ml of the stock solution and 1 ml of the 4’-
methoxyflavanone solution in 25 ml graduated flasks.
Data analysis
For comparing the efficiency of each combination of techniques
(ultrasound, soxtec) and solvents (ethanol, hexane, and petroleum
ether), two-way ANOVA was used. We used t-test to determine the
difference between populations of C. cinerariaefolium.
Table 1. HPLC condition as listed in literature (Kasaj et al.,
1999) and modified in our study.
Time (min) Acetonitrile
(Solvent A, %) Kasaj et al. Modification
58 0 - 5 0 - 5
58 - 75 5 - 35 5 - 50
75 - 100 35 - 36 50 - 51
Table 2. Separation factor [ = tR(B) / tR(A); tR = tR-tR(i.st.)] of
individual successive pyrethrin esters (A, B) for validated (Kasaj
et al., 1999) and our modified method.
*
B/A Kasaj et al. (1999) Modified
Pyrethrin II/ CinerinII 1.127 1.112
Jasmoline I/ Pyrethrin II 1.437 1.442
Cinerin I/ Jasmoline II 1.565 1.645
Pyrethrin I/ Cinerin I 1.044 1.036
Jasmoline I/ Pyrethrin I 1.250 1.152
Cinerin II/ Jasmoline I 0.302 0.317
* = Separation factor, = tR(B) / tR(A); tR = tR-tR(i.st.); A, B = individual
successive pyrethrin esters.
RESULTS AND DISCUSSION
Identification and quantification
Individual esters were identified by matching the
separation factors () from our analysed samples to
those calculated from the relative retention times of the
esters reported in the work of Kasaj et al. (1999) as
shown in Table 2. The identification was confirmed
comparing the UV spectra of each ester from the
analysed sample with those from the standard solution.
The found UV maximum matched those reported in the
literature as listed in Table 3. The individual pyrethrum
esters are unavailable, thus most HPLC quantification
methods use a commercial pyrethrum mixture with an
estimated amount of 25% of total pyrethrins as a stan-
dard solution. The amount of total pyrethrins in the
assayed sample was estimated by calculating the sum of
measured peak areas of individual pyrethrins. Figure 1
shows the chromatogram overlaying for a prepared
standard mixture and for an analysed sample. The
calibrating curves for total pyrethrins and for each
pyrethrin were obtained from the prepared standard
mixtures. The calibrating intervals covered the range of
occurrence of all six compounds in the analysed sample.
These calibrating curves were used to determine the
amounts of total pyrethrins, pyrethrins I, pyrethrins II, as
well as the amounts of each pyrethrin ester in the assay
and their percent in dried flowers. The same detector
response for all six esters based on their very similar
chemical structure was assumed (Figure 2).
Ban et al. 2705
Table 3. Absorbance maximum wavelengths (max, nm) of the
pyrethrin compounds as referred in the literature (Casida and
Quistad, 1995) and found in our study.
Pyrethrin
compounds
max, nm
(literature)
max, nm
(found)
Pyrethrin I 226 224.31
Cinerin I 226 225.87
Jasmoline I 226 225.89
Pyrethrin II 229 228.13
Cinerin II 234 233.70
Jasmoline II 234 233.49
O
H
O
CH3
O
H
HH
H
CH3
CH3
CH3
O
H
O
CH3
O
H
HH
H
CH3
CH3
CH3
O
H
O
CH3
O
H
H
H
H
CH3
CH3
CH2
Pyrethrin I
Cinerin I
Jasmolin I
O
H
O
CH3
O
H
HH
H
CH3
H3CO2CCH3
O
H
O
CH3
O
H
HH
H
CH3
H3CO2C
CH2
O
H
O
CH3
O
H
HH
H
CH3
H3CO2CCH3
Pyrethrin II
Cinerin II
Jasmolin II
Figure 1. Chemical structure of individual pyrethrins.
Linearity of acetonitril standard solutions and
detection limits
For all assays, the relationship between the signal (peak
area normalised by the internal standard) and the
concentration was linear, and the regression coefficient
was higher than 0.999. Detection limits were estimated
by the analysis of standard solutions and real samples.
The obtained figures corresponded to the concentration
at which the signal-to-noise ratio became 3. The
estimated detection limits for pyrethrins I ranged from
0.025 to 0.028 mg/L and for pyrethrins II from 0.034 to
0.038 mg/L.
Repeatability of results
Table 4 shows the relative standard deviation (RSD) for
all treatments. Comparing the extraction techniques,
soxtec extraction showed lower repeatability than
ultrasound extraction, probably because of the low
content of the fatty material, and for only one gram of
dried flowers taken into extraction. For the soxtec system
used, the more sample taken into extraction, the more
the reliabliability of the results (Table 4).
Extraction recovery
Both techniques of extraction (ultrasound and soxtec)
and the elution of obtained crude extract with acetonitrile
were repeated several times to achieve exhaustive
extraction of the plant material. According to HPLC
analysis, the second soxtec and the forth and fifth
ultrasound extraction did not yield pyrethrins. The first
acetonitrile elute contained more than 99% of pyrethrins
2706 Afr. J. Biotechnol.
Retention time (min)
Figure 2. Reversed-phase HPLC chromatogram of standard (blue line) and sample (red line) of pyrethrum extract.
(Mobile phase flow rate 1 mL min-1, injection volume 20 µL, UV detector at 230 nm)
Table 4. Relative standard deviation (RSD) for each pyrethrin compounds obtained with ultrasound or soxtec extraction using
ethanol, petroleum ether or hexane as a solvent.
RSD
Ultrasound Soxtec
Pyrethrin
compounds Ethanol Petroleum ether Hexane Ethanol Petroleum ether Hexane
Pyrethrin I 0.78 11.30 7.57 51.64 15.55 13.72
Cinerin I 3.40 10.59 5.54 42.50 12.25 2.85
Jasmoline I 1.54 9.21 1.49 9.68 8.61 11.95
Pyrethrin II 1.14 10.59 5.17 21.21 17.50 16.20
Cinerin II 0.81 9.64 5.37 10.49 12.37 2.74
Jasmoline II 1.69 10.58 5.12 6.25 12.58 3.53
and the second one contained the rest, whereas the third,
forth and fifth elution did not contain pyrethrins. There-
fore, the results for each pyrethrin were calculated summ-
ing the peak areas obtained, analysing the first and
second elution.
Methanol was included at the beginning of the study
but soon rejected because of low extraction efficiency
even though it was reported to be the most effective
among tested solvents: ethanol, methanol, propanol and
acetonitrile (EU project, 2002). Moreover, methanol is
very toxic and as such not suitable for commercial
extraction.
The influence of the extraction technique and the
solvent on the extraction efficiency
The extraction efficiency for these treatments on the
amounts of pyrethrins I and II, and total pyrethrins was
observed since their content depends on the amount of
predominant singular compounds of pyrethrin 1 and
pyrethrin 2 (Table 5), the ones being the most active too
(Casida and Quistad, 1995; EU project, 2002). Guided by
that fact, and in order to clarify the differences between
treatments results in Table 6 that shows the percent of
TP extracted with each treatment were summarized. For
the pyrethrins (p1 and p2), as well as for PI, PII and TP,
the ultrasound-ethanol treatment showed the best
extraction efficiency. Next treatments, in decreasing order
of efficiency that also showed good results are the
soxtec-petroleum ether, ultrasound-petroleum ether, and
ultrasound-hexane, but soxtec-petroleum ether showed
the worse repeatability. All three ultrasound treatments
and the soxtec-petroleum ether treatment did not show
significant difference in extracting pyrethrins (P > 0.01).
Ethanol as a solvent was less hazardous than the other
Ban et al. 2707
Table 5. Treatments with related order based on extraction efficiency of pyrethrin compounds (p1, c1, j1, p2, c2, j2), the
groups of pyrethrins I (PI) and II (PII), and for total pyrethrins (TP).
Ultrasound Soxtec
Pyrethrin
compounds Ethanol Hexane Petroleum ether Ethanol Hexane Petroleum ether
TP 1 4 3 6 5 2
PI 1 4 3 6 5 2
PII 1 4 3 6 5 2
p1 1 3 2 5 4 2
c1 3 4 5 6 1 2.
j1 4 4 2 5 3 1
p2 1 4 2 6 5 3
c2 3 4 4 5 1 2
j2 3 4 5 4 1 2
Table 6. Percentage of total pyrethrins extracted by two
techniques and tree solvents.
Technique Solvent
Total pyrethrins*
(%, flower dry
weight)
Ethanol 1.207 a
Hexane 0.967 b
Ultrasound
Petroleum ether 1.020 ab
Ethanol 0.530 c
Hexane 0.743 c
Soxtec
Petroleum ether 1.100 ab
*The results are expressed as a mean of three measurements.
Means within a column followed by the same letter are not
significantly different at P 0.01 by LSD test.
compared solvents (OJEC, 2001), has lower cost than
hexane, the most frequently used in laboratories and
referred as the most effective (Casida and Quistad, 1995;
Kasaj et al., 1999; EU project, 2002). These assumptions
make it a preferable solvent for potential commercial use.
The treatments soxtec-hexane, and soxtec-ethanol gave
the worse results, without significant difference between
them (P > 0.01).
It is clear that the extraction efficiency does not depend
on the use of a particular solvent, nor a particular method
of extraction, but it depends on the use of a proper
solvent for a particular method, for example, ethanol used
with ultrasound gave the best results, while used with
soxtec, the worse, or the most suitable solvent for
ultrasound is ethanol, and for soxtec is petroleum ether
(Table 5). We cannot say that the polarity of the solvents
affects the extraction efficiency, probably due to bipolar
character of pyrethrin compounds. Further investigations
should be carried out on the combination of solvents.
Even though it has been reported that the degradation of
pyrethrins starts from 40°C (Della Porta and Reverchon,
2002), the soxtec treatment soxtec-petroleum ether, gave
as good results as the best treatment ultrasound-ethanol.
Table 7. Content of total extracted pyrethrins for three
populations of Chrysanthemum cineraraefolium originating
from Dalmatia, Croatia.
Population Total pyrethrins
(g/100 g dried
flowers)
RSD (%)
I 1.25 ab* 5.32
II 1.16 b 1.08
III 1.30 a 1.99
*Means within a column followed by the same letter are not
significantly different at P 0.01 by LSD test.
The conditions were 13C/80 min. Among all other
soxtec treatments, this was the mildest.
Considering the results so far, and after the method
was validated, the ultrasound-ethanol treatment which
showed the best efficiency and repeatability, as well as
the lowest cost and toxicity, was chosen for isolation of
active ingredients in three Chrysanthemum populations
(Table 7). The average pyrethrins content in wild
populations collected from Dalmatia (Croatia) and planted
in Kenya was 0.89% ranging from 0.75 to 1.04% (Casida
and Quistad, 1995). The present results with values
higher than these, confirm that the Croatian costal area is
very suitable for pyrethrum growing, not surprising since
this plant is native to Dalmatia (Croatia). In further
researches, population III should be included due to
significantly (P 0.01) highest amount of total pyrethrins.
New investigations embracing the production technology
development, clonal selection and pyrethrum product
development are in course. Selection and introduction of
commercial clones in local plantings would make pyre-
thrum crops grow on economically profitable agricultural
activity.
Conclusion
A method that is efficient, reliable and simple, with low
2708 Afr. J. Biotechnol.
toxicity and cost for routine analyses of pyrethrins was
described. Two extraction techniques (ultrasound and
soxtec) were tested with no difference in efficiency. How-
ever, soxtec was less reliable, even though it requires
less sample manipulation. To our knowledge this is the
first report on using soxtec for pyrethrin extraction.
For the first time, the results demostrated that ethanol
could be more effective in extraction of pyrethrins than
hexane or methanol. Considering its lower cost and
toxicity, it is being recommended as the optimal solvent
for laboratory and industrial scale purposes. The content
of total pyrethrins in three different natural populations of
C. cinerariaefolium grown in Croatia demonstrates that
Croatian coast is a very suitable place for growing and
commercialising of this culture.
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... The extraction method also has a certain influence on the total pyrethrin content in the pyrethrum extract (Pan et al. 1995;Kiriamiti et al. 2003;Ban et al. 2010;Nagar et al. 2015;Gallo et al. 2017); including the solvent used for the extraction (Ban et al. 2010;Gallo et al. 2017) (see further). ...
... The extraction method also has a certain influence on the total pyrethrin content in the pyrethrum extract (Pan et al. 1995;Kiriamiti et al. 2003;Ban et al. 2010;Nagar et al. 2015;Gallo et al. 2017); including the solvent used for the extraction (Ban et al. 2010;Gallo et al. 2017) (see further). ...
... These values are accordant to the few previous reports on pyrethrin content in the dry flower weight of natural Dalmatian pyrethrum populations: 1.2% (Ambrožič Dolinšek et al. 2007), 1.1-1.3% (Ban et al. 2010), and 1-1.2% (Babić et al. 2012). Content in samples from Ugljan island was 1.58% for wild populations and 2.09% for cultivated plants (Rončević et al. 2014). ...
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Pyrethrin is a potent biopesticide, a natural mixture of six compounds (pyrethrin I and II, cinerin I and II, and jasmolin I and II), biosynthesized in plants of Dalmatian pyrethrum (Tanacetum cinerariifolium/Trevir./Sch. Bip.), a species endemic to the eastern Adriatic coast, but grown worldwide. Not many known natural insecticides encompass such superior qualities as pyrethrin—very high efficacy against a broad spectrum of pests in combination with minor adverse effects on human health and the environment. In previous decades, pyrethrin was largely replaced by its synthetic derivatives, pyrethroids. However, due to their harmful effects on various species and ecosystems, the use of pyrethrin should again take the lead. This review summarizes one century (1920–2020) of research on the properties and use of pyrethrum as a source of pyrethrins. The primary focus is on presenting its current advantages and disadvantages, toxicity on target and non-target species, biosynthesis, factors that influence the pyrethrins content in pyrethrum, comparison of different methods of their extraction and determination; as well as its production potential and development of new products. The final goal is to present possible approaches to improve and enhance the use of this highly effective but still underused phytochemical insecticide with unique properties.
... 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 extensively. 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), ultrasoundassisted extraction (Babić et al. 2013), and extraction with supercritical CO 2 (Kiriamiti et al. 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). ...
... 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), ultrasoundassisted extraction (Babić et al. 2013), and extraction with supercritical CO 2 (Kiriamiti et al. 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). ...
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Full-text available
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.
... 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 extensively. 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), ultrasoundassisted extraction (Babić et al. 2013), and extraction with supercritical CO 2 (Kiriamiti et al. 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). ...
... 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), ultrasoundassisted extraction (Babić et al. 2013), and extraction with supercritical CO 2 (Kiriamiti et al. 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). ...
Article
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.
... Previous studies on natural populations found similar values for the total pyrethrin content: 0.36 to 1.30% (Grdisǎ et al., 2022), 1.1-1.3% (Ban et al., 2010), and 1-1.2% (Babićet al., 2012). In the more recent study by (Varga et al., 2021), a lower average pyrethrin content of 0.22 to 0.87% was reported, which could be due to the different extraction method used. ...
Article
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The insecticidal compound pyrethrin is synthesized in Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch.Bip.; Asteraceae), a plant species endemic to the eastern Mediterranean. Pyrethrin is a mixture of six compounds, pyrethrin I and II, cinerin I and II, and jasmolin I and II. For this study we sampled 15 natural Dalmatian pyrethrum populations covering the entire natural distribution range of the species; Croatian coastal regions and the islands, inland Bosnia and Herzegovina and Montenegro. The plants were grown in a field experiment under uniform growing conditions to exclude a short-term response to environmental factors and instead observe variation in pyrethrin content and composition among and within populations due to genetic adaptation to the native environment. The drivers of local adaptation were explored by examining the role of bioclimatic factors as a cause of population differentiation. Pyrethrins were extracted by ultrasound-assisted extraction, and the extracts were analyzed by HPLC-UV-DAD. The populations differed significantly in the content and composition of pyrethrins. The highest levels of total pyrethrins (1.27% flower DW), were found in population P14 Budva and the significantly highest levels of pyrethrin I in population P14 Vranjske Njive, Podgorica (66.47% of total pyrethrin). Based on bioclimatic conditions of the sampling sites, populations were grouped into five bioclimatic groups (A, B, C, D, and E), which showed qualitative and quantitative variability in pyrethrin content. The most abundant bioclimatic group was bioclimatic group E, which was characterized by the highest average values for pyrethrin I (53.87% of total pyrethrin), total pyrethrin content (1.06% flower DW) and the ratio of pyrethrin I and II (1.85). The correlation analysis between the pyrethrin compounds and some of the bioclimatic variables (e. g., BIO03 Isothermality and BIO04 Temperature seasonality) showed their significant contribution in explaining the variation of pyrethrins in T. cinerariifolium. The differences in pyrethrin content and composition may be partly due to genetic adaptation to the ecological conditions of the native environment. The obtained data would enable the selection of source populations for breeding programs aimed at producing cultivars with desirable biochemical properties and adaptation to different bioclimatic conditions.
... The biosynthesis and accumulation of pyrethrin is highly dependent on a variety of factors such as genotype [15,18], morphological traits mainly related to floral characteristics [17,[19][20][21][22], as well as environmental factors and climatic conditions [23,24]. Its content in the dry flower weight also depends on the extraction methods [25][26][27][28][29], storage conditions [16], drying methods [16,30,31], and agrotechnical methods applied [32] including the harvest time [33][34][35]. ...
Article
Full-text available
Pyrethrin is a specialized metabolite of Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), Asteraceae, known worldwide as an effective bioinsecticide. It consists of six active compounds: Pyrethrin I and II, cinerin I and II, and jasmolin I and II. Pyrethrin accumulates mainly in the flower heads and its content depends on numerous factors, such as the flower developmental stage. This study aims to investigate the accumulation patterns of six pyrethrin compounds in the flower heads of Dalmatian pyrethrum over the eight developmental stages (FS1 to FS8), and to make a comparison in six natural populations. Ultrasound assisted extraction was used to extract the pyrethrin, while qualitative and quantitative analysis was performed by High performance liquid chromatography. The accumulation patterns of different pyrethrin compounds were generally similar and also synchronous between different populations, while the pyrethrin I/pyrethrin II ratio showed irregular patterns. In all populations studied, the highest increase of all compounds was observed from FS1 to FS2. Their concentration continuously increased, reaching the highest values at FS4 stage (2–5 rows of open disc flowers), and generally decreased gradually towards FS6 or FS7 and then stagnated until the FS8 stage. Despite the very similar accumulation pattern of pyrethrin compounds in the different populations, the significant differences in their content suggest a different genetic background. Knowledge of the dynamics of pyrethrin compounds accumulation across flower development stages is valuable for determining the optimal harvest time of pyrethrum flower heads.
... Previous studies analysing pyrethrin composition in natural pyrethrum were conducted on very small sample sizes (Ambrožič Dolinšek et al., 2007;Ban et al., 2010;Babić et al., 2012;Rončević et al., 2014). The only comprehensive study of pyrethrin content and composition of natural Dalmatian pyrethrum from Croatia to date (Grdiša et al., 2013), was conducted by analysing combined population samples and the study showed high diversity at the population level. ...
Article
Seeds collected from 10 Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip. Asteraceae) populations naturally occurring along the Adriatic coast and islands, were used to establish a common garden with the aim of estimating the variation of pyrethrin compounds between and within populations. A recently optimized matrix solid phase dispersion (MSPD) method was used for the extraction of six pyrethrin compounds from dry flower heads of 200 Dalmatian pyrethrum individuals. Separation and quantification of pyrethrin compounds were performed by high performance liquid chromatography with diode array detector (HPLC-DAD). High variability in pyrethrin content and composition was observed within and between populations. Total pyrethrin content in individual samples varied from 0.10% to 1.35% of flower dry weight. On average, the lowest total pyrethrin content was observed in the population of Pelješac (0.22%) and the highest in the island populations of Mali Lošinj and Zlarin (0.87%). The population of Mali Lošinj had the most favourable pyrethrin profile, including the lowest variability in total pyrethrin content, the highest pyrethrin I (PI) content (up to 60.47%) and PI/PII ratio (up to 5.88), as well as the lowest pyrethrin II (PII) content. In contrast, the populations from Biokovo and Pelješac represented pyrethrin profiles with the lowest content of total pyrethrin, PI and PI/PII ratio. The Biokovo population also had the highest PII content (43.18% on average), while the Pelješac population had the highest jasmolin I (JI) and jasmolin II (JII) content. Four different chemotypes were determined by cluster analysis. Chemotype P1 is the most promising for future breeding programs and is characterized by the highest total pyrethrin content, PI content and PI/PII ratio, as important measures of insecticidal activity. Correlation analysis of each pyrethrin compound with 25 environmental variables and Principal Component Analysis (PCA) were carried out and the importance of optimum temperature and precipitation on pyrethrin quantity and quality was suggested. Total pyrethrin content was significantly correlated with six temperature and five precipitation parameters. Pyrethrin I, PII and cinerin II (CII) were significantly correlated mainly with temperature parameters; JII and cinerin I (CI) with precipitation parameters, while JI was correlated with temperature range and precipitation parameters. The mountainous Biokovo population was an outlier in the PCA analysis, mainly due to the extremely low values of temperature parameters at a higher altitude. Precipitation variables separated the central Adriatic islands inhabiting drier habitats from populations inhabiting habitats with greater precipitation. The high-resolution data from this study provide the opportunity for individual-based selection for breeding plants with the best pyrethrin profiles, as well as for increased adaptation to broader climatic conditions.
... Pyrethrins (six related insecticidal compounds of which the most abundant are pyrethrins I and II) are found predominantly in the ovaries of the flower (El-Wakeil, 2013) and its content can vary from 0.9 to 1.3% by weight of dried flowers in native populations (Casida & Quistad, 1995;Kolak et al., 1999). Ban et al. (2010) showed that content of pyrethrins from coast of Croatia reaches more than 1.2%. Natural pyrethrins act mainly by contact or ingestion on both central and peripheral nervous system, like neurotoxic action, blocking voltage-gated sodium channels in axons, thus causing immediate insect 'knockdown' paralysis (El-Wakeil, 2013). ...
Article
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The presence of olive moth (Prays oleae Bern.) and effectiveness of different control treatment against it has been conducted on 160 olive trees of Leccino variety in Istria, Croatia. The treatments were: dimethoate, Bacillus thuringiensis var. kurstaki (Bt), pyrethrins, consociation of olive and planted pyrethrum and control. Olive moth has been monitored weekly using pheromone-baited delta traps and different coloured sticky plates in 12 and eight monitoring. Moth's life stages (non-destroyed, destroyed eggs and larvae) has been counted in seven monitoring. The predator activity has been determined as the ratio of destroyed eggs in the total number of observed eggs on 50 randomly chosen samples of flowers and fruits. On pheromone traps, the maximum catch has been determined on May 15th on control, when maximum of moth's flight on control, dimethoate and pyrethrins treatments coincided in time. There were more adults detected on pheromone traps then on sticky traps (16.8 times over). With time, moth's first preference was red and then blue sticky plates. The highest number of eggs (non-destroyed and destroyed) has been found on dimethoate on May 15th (2.8 ± 1.1 and 4.5 ± 1.6) and June 18th (0.8 ± 0.5) as a consequence of accidental high oviposition rate at dimethoate plots before treatment applications. The highest number of larvae were detected on control on July 2nd (2.0 ± 0.7).
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Article
The mixture of six components (pyrethrin I and II, cinerin I and II, and jasmolin I and II) constituting natural insecticide pyrethrin is responsible for the insecticidal activity of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.). In order to obtain deeper knowledge of its insecticidal potential, the extraction of mentioned components from plant samples was carried out for the first time, by matrix solid-phase dispersion. The influence of different factors such as the type of sorbent, sorbent-sample ratio, the elution solvent and its volume, addition of co-sorbent and drying agent, affecting extraction yield was evaluated. The pyrethrin components were extracted under optimized conditions: florisil (0.5 g) as the sorbent, mixed with 0.25 g of pyrethrum flower heads, 5 mL of acetone-ethyl acetate (1:1, v/v) as the elution solvent and 0.4 g of Na2SO4 as the drying agent. All six components in the extracts were qualitatively and quantitatively determined by HPLC-DAD. Analytical method provides high degree of linearity with r2≻0.9974, except for pyrethrin II (0.9961). Also, the method showed good precision (intra- and inter-day) with a relative standard deviation below 14 %, and recoveries mainly above 80 %. In order to demonstrate the applicability of the developed method, three Dalmatian pyrethrum natural populations from Croatia were analyzed.
Article
The authors separated and collected milligram quantities of six pyrethrin esters in pyrethrum extract by semipreparative, normal-phase high performance liquid chromatography (HPLC). They structurally confirmed each ester by Fourier transform-infrared spectroscopy and gas chromatography-mass spectrometry. They also checked retention times based upon previously developed HPLC methodology. They determined the purity of each ester based upon the chromatographic area percentage. The authors then used the esters as individual standards to restandardize the content of pyrethrin esters in two selected pyrethrum extracts, which they previously had assayed using the Association of Official Analytical Chemists International titration method. They will submit one of the selected pyrethrum extracts to the U.S. Pharmacopoeia as a potential reference standard for additional applications.
Article
Nineteen collaborating laboratories (including the authors') analyzed 6 blind, duplicate pairs of various technical materials, pyrethrum extracts, concentrates, and finished products by split injection capillary gas chromatography (GC) with flame ionization detection. This procedure simultaneously quantitates with speed, ease, accuracy, and precision all 6 insecticidal compounds in pyrethrum: pyrethrin I, jasmolin I, cinerin I, pyrethrin II, jasmolin II, and cinerin II, as well as butylcarbityl 6-propylpiperonyl ether (BPE, the predominant compound in technical piperonyl butoxide, also commonly known as piperonyl butoxide) and both the endo and exo isomers of N-octyl bicycloheptene dicarboximide (MGK 264). Repeatability ranged from 4.28 to 7.22% for total pyrethrins, from 2.41 to 7.04% for BPE, and from 2.20 to 4.91% for total MGK 264. Reproducibility ranged from 5.22 to 9.71% for total pyrethrins, from 4.37 to 7.04% for BPE, and from 2.66 to 6.01% for total MGK 264. The capillary GC method for these insecticidal compounds in technical materials, concentrates, and finished products has been adopted first action by AOAC INTERNATIONAL.
Article
Pyrethrum is the most important natural botanical pesticide. Pyrethrins are effective against virtually all insect pests, yet are of very low toxicity to mammals, and break down quickly on exposure to light and air. This study of the place of pyrethrum in ecology and pest management includes discussion of the new synthetic pyrethroids that have been developed, in addition to continuing work on natural product pyrethrins.
Article
Three different extraction methods (ultrasonic extraction (USE), Soxhlet extraction (SOX) and supercritical fluid extraction (SFE)) were compared for the extraction of pyrethrins from chrysanthemic flowers and commercial insecticide powder. Allethrin was extracted from paper strips. All extracts and the kinetics were analyzed by supercritical fluid chromatography and flame ionization detector.
Article
The pyrethrins, six insecticidally active esters fromChrysanthemum cinerariaefolium have been separated by high-performance liquid chromatography (HPLC). A method for the separation and quantitative determination of these compounds in pyrethrum extracts by RPHPLC has been developed and validated. Quantification of the pyrethrins was performed by internal and external standardization. Normal-phase HPLC was used to isolate the individual pyrethrins. Different commercial pyrethrum extracts and samples of Flos Pyrethri of different origin were investigated by use of the new method. There was good correlation between our results and those obtained by quantification of the pyrethrins in some of the samples by the AOAC method.
Article
A simple, rapid method for the direct determination of six pyrethrin esters in pyrethrum extracts by reversed-phase high-performance liquid chromatography with diode-array detection has been described. The separation of the six esters was based on a binary mobile phase optimization, temperature control and the use of a C8 octyl column with 5-μm particles. Diode array detection and quantitation were selectively performed at 230 and 240 nm. The method demonstrated acceptable linearity, specificity, limit of sensitivity for the determination of six pyrethrin esters in pyrethrum extracts.
Article
Supercritical extraction and fractionation of natural matter is one of the early and most studied applications in the field of supercritical fluids. In the last 10 years, studies on the extraction of classical compounds like essential and seed oils from various sources: seeds, fruits, leaves, flowers, rhizomes, etc., with or without the addition of a co-solvent have been published. Supercritical extraction of antioxidants, pharmaceuticals, colouring matters, and pesticides has also been studied. The separation of liquid mixtures and the antisolvent extraction are other processes that can perform very interesting separations. Mathematical modelling has also been developed and refined for some of these processes.The objective of this review is to critically analyze traditional and new directions in the research on natural matter separation by supercritical fluids extraction and fractionation.
Article
Windrowed pyrethrum stems were air dried under a range of storage conditions to examine whether the current commercial practice of drying crop material is conducive to pyrethrins' degradation. Crop material was stored for up to 12 days in a commercial windrow, a shed receiving indirect light or a dark, 5 degrees C cool-room. Analysis of pyrethrins extracted from flowers of all treatments demonstrated that pyrethrins were not degrading in windrowed crops, plant material stored in the shed or in the 5 degrees C cool-room. The small differences obtained in pyrethrins content among the treatments can be explained by the natural variation in pyrethrins content of pyrethrum crops. The observation that the achenes were unchanged during this drying period supported the pyrethrins analysis. These results demonstrate that pyrethrins in planta do not degrade as rapidly as extracted pyrethrins. (C) 2005 Elsevier B.V. All rights reserved.
Article
A preparative supercritical fluid extraction system is described and was used with supercritical carbon dioxide to extract the active insecticide components pyrethrin I (PI) and pyrethrin II (PII) successfully from pyrethrum flower. A high-performance liquid chromatography method was developed and was used to separate and analyze the supercritical carbon dioxide extracts. Extraction efficiencies under several different extraction conditions were examined. Under the conditions examined, the most effective extractions of PI and PII (140 +/- 18 mg and 55 +/- 9 mg per 100 g of dry pyrethrum flower powder) were performed at 40 degrees C and 1200 psi. The results showed that extraction efficiencies of supercritical carbon dioxide are much better than those of n-hexane for pyrethrins I and II. During the extraction process, the most efficient extraction period was the first 3 h of the experiment.