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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 4°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 135°C/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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