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Journal of Chromatography A, 1125 (2006) 172176

Extraction of rotenone from Derris elliptica and Derris malaccensis


by pressurized liquid extraction compared with maceration
Attawadee Sae-Yun a , Chitchamai Ovatlarnporn b , Arunporn Itharat c ,
Ruedeekorn Wiwattanapatapee a,
a Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
b Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
c Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University,

Hat Yai, Songkhla 90112, Thailand


Received 21 February 2006; received in revised form 19 May 2006; accepted 19 May 2006
Available online 19 June 2006

Abstract
The extraction of active compounds from plants is one of the most critical steps in the commercial development of natural products for medicinal,
herbicidal or pesticidal use. The focus of this study was to compare conventional maceration and pressurized liquid extraction (PLE) techniques
for the efficient extraction of rotenone from the stem and root of Derris elliptica Benth and Derris malaccensis Prain. The effects of experimental
variables, such as solvent, temperature and pressure, on PLE efficiency have been studied. Chloroform was determined to be a good extraction solvent
(rotenone content 40.6%, w/w) compared to commonly used solvent, 95% ethanol (rotenone content 15.0%, w/w). The optimal conditions for
PLE were 50 C and 2000 psi. PLE showed higher extraction efficiency (rotenone content 46.1%, w/w) as compared with conventional maceration
method (rotenone content 40.6%, w/w). The order of rotenone content found in crude extract obtained by optimized method from the highest
to the lowest was root (46.1%, w/w) and stem (9.4%, w/w) of D. elliptica and stem of D. malaccensis (5.2%, w/w), respectively. Moreover, the
results from this study indicated that PLE was considerably less time and solvent consuming (30 min, 3 ml/g of dried sample) than the conventional
maceration techniques (72 h, 10 ml/g of dried sample).
2006 Elsevier B.V. All rights reserved.

Keywords: Pressurized liquid extraction; Rotenone; Derris elliptica; Derris malaccensis

1. Introduction properties are due to the presence of rotenone (Fig. 1). Rotenone
is known to be safe to the farmers, since it known to be toxic only
Pest management is a major problem in almost all agricultural to cold-blooded animals and less toxic to warm-blooded crea-
countries. Many types of insecticides have been used to control tures. Rotenone is not stable in air, light and alkaline conditions.
insect pests. However, with the development of resistant insects, It is rapidly broken down in soil and water. Therefore, almost
the threat of pesticide contamination of food, high production all toxicity may be lost after 23 days of summer exposure, so it
and purchase costs and environmental pollution problems, plant is good for the environment and safe for agriculturists and other
extracts are increasingly of interest as pest control alternatives users [27].
[1]. Derris elliptica Benth and Derris malaccensis Prain (Legu- Extraction methods, such as soxhlet extraction, stirring soak-
minosae) have previously been known as important sources for ing and solvent extraction, are commonly used to extract
compounds with pesticidal properties. They grow widely in rotenone from derris plants [1,8]. However, these methods are
Southeast Asian countries, and their extracts have been used over time consuming, involve high solvent consumption and may
centuries as fish poison and as insecticidal compounds. These have lower extraction efficiencies. In order to reduce the use
of organic solvents and improve the extraction processes, pres-
surized liquid extraction (PLE; Dionex trade name ASE for
Corresponding author. Tel.: +66 74 288820; fax: +66 74 218503. accelerated solvent extraction) has been used since 1995 as an
E-mail address: wruedeek@pharmacy.psu.ac.th (R. Wiwattanapatapee). alternate technique. PLE is an extraction procedure under ele-

0021-9673/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2006.05.075
A. Sae-Yun et al. / J. Chromatogr. A 1125 (2006) 172176 173

stock solution (0.10 mg/ml) was prepared by dissolving 10.0 mg


of rotenone in 100 ml methanol. The working solutions were
freshly prepared by suitable dilution of the stock solution with
methanol. Since rotenone is known to decompose when exposed
to light, the stock solution was kept in the dark at 4 C and used
within 1 month. This solution was stable over this period as
determined by an HPLC assay (data not shown).

2.2. Plant samples

The stems and roots of D. elliptica were collected from the


botanical garden of the Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Thailand, in December 2004. The
stems of D. malaccensis were collected from Chana District,
Fig. 1. Chemical structure of rotenone.
Songkhla Province, Thailand, in August 2004. Authentication
of plant materials was carried out at the herbarium center of the
Department of Forestry, Bangkok, Thailand, where herbarium
vated temperature and pressure. It allows the universal use of vouchers have been kept identifying the plant species. Voucher
solvents or solvent mixtures with different polarities and indi- specimens of these plants were also kept in the Herbarium of
vidually variable pressure of 5003000 psi in order to maintain Southern Center of Thai Medicinal Plants at Faculty of Phar-
the extraction solvent in a liquid state, and temperature ranging maceutical Sciences, Prince of Songkla University, Songkhla,
from room temperature up to 200 C for accelerate extraction Thailand. Only stems and roots with a diameter size less than
[9,10]. 1.5 cm were used. They were washed with tap water to remove
Several researchers have successfully used PLE to extract the remaining soil and other unwanted materials, cut into small
active components from natural products, such as polyphenols pieces (5 mm cubes) and dried in air overnight. The dried plants
from apple [11], dianthrons from St. Johns wort, escin from were stored in plastic bags and kept in a dark at room temperature
horse chestnut seed, silybin from milk thistle fruit, curcuminoid (2830 C).
from turmeric rhizome, the essential oil from thyme [12], gin-
senosides from the roots of American ginseng [13], flavonones 2.3. Extraction of rotenone by maceration using organic
and xanthones from the root bark of the osage orange tree solvents
[14], carotenoids from Haematococcus pluvialis and Dunaliella
salina, kavalactones from Piper methysticum [15], antioxidants The dried plant sample (50 g) was placed in a stopped conical
from rosemary leaves [16], antioxidants from Spirulina platensis flask and macerated with 500 ml of chloroform at room temper-
[17], berberine from Coptidis rhizoma, aristolochic acids from ature (2830 C) for 3 days with occasional stirring. The solvent
Radix aristolochiae [18], proanthocyanidins from malt [19], cat- was then filtered and evaporated in a rotary evaporator (Eyela,
echin and epicatechin from grape seeds [20], isoflavones from Tokyo, Japan) under vacuum at 45 C. The residue was placed
soybeans [21] and sesquiterpenes from Curcuma rhizomes [22]. in a vacuum oven (Napco, Chicago, USA) at room temperature
Until now, the use of PLE to extract rotenone from derris plants until dry. The crude extract was stored in a well-closed container
has not been reported. The objective of this study was there- and protected from light and kept in a desiccator at 20 C. The
fore to examine the extraction process of rotenone from the extraction was carried out on three separate occasions (n = 3).
dried stems and roots of D. elliptica and the dried stems of D. The above procedure was repeated using 95% ethanol as sol-
malaccensis by pressurized liquid extraction, and compare this vent instead of chloroform, again replicated on three occasions
technique with maceration of the dried plants using the same (n = 3).
solvents.
2.4. Extraction of rotenone by PLE
2. Experimental
Extractions were performed on a Dionex ASE 200 Accel-
2.1. Chemicals erated solvent extractor (Archemica, Bangkok, Thailand). The
dried plant sample (10 g) was packed in a 22-ml stainless steel
HPLC grade acetonitrile and analytical grade methanol were extraction cell. A cellulose filter was placed at the bottom of
purchased from Labscan Asia (Bangkok, Thailand). Commer- the extraction cell. The PLE was carried out with static time
cial grade chloroform and 95% ethanol were obtained from High of 6 min per 1 cycle. Solvent, temperature and pressure were
Science distributor (Bangkok, Thailand). All other solvents were varied to optimize the extraction efficiency. The experimental
AR grade purity. Nitrogen gas (99.9% purity) was purchased conditions are shown in Table 1. Three replicate extractions for
from TIGT (Songkhla, Thailand). Water was purified by pass- each experimental condition were performed (n = 3). The extrac-
ing it through a Milli-Q purification system. Standard rotenone tion procedures were as follows: (i) extraction cell and collection
was purchased from SigmaAldrich (Steinheim, Germany). The vial were loaded onto the automated carousel, (ii) cell was filled
174 A. Sae-Yun et al. / J. Chromatogr. A 1125 (2006) 172176

Table 1 3. Results and discussion


The absolute yield of rotenone from dried stem of D. malaccensis using PLE as
extraction method in different experimental conditions
3.1. Analytical characteristics
Condition Solvent Temperature Pressure Absolute yield of
( C) (psi) rotenone (%, w/w) Analytical determinations of rotenone are commonly carried
(n = 3)
out by HPLC [2327]. The method described by Cabizza et
1 95% EtOH 50 1500 0.046 0.015 al. [28] afforded a good resolution of compounds with a gra-
2 CHCl3 50 1500 0.054 0.007 dient of acetonitrile/water. An acetonitrile/water gradient with
3 CHCl3 60 1500 0.037 0.004
4 CHCl3 70 1500 0.041 0.006
an initial concentration of 50:50 (%, v/v) and a final concen-
5 CHCl3 50 1000 0.047 0.006 tration of 70:30 (%, v/v) in 30 min was used in the present
6 CHCl3 50 1750 0.044 0.011 study. Standard calibration curves of rotenone were constructed
7 CHCl3 50 2000 0.038 0.012 by plotting concentrations against peak areas. A good linearity
8 CHCl3 30 (RT) 2000 0.038 0.004 was achieved with a correlation coefficient of 0.9999 over the
concentration range 020 g/ml. The limit of detection (LOD)
value for rotenone was 0.24 g/ml and the limit of quantita-
with solvent, (iii) heat-up time was applied, (iv) static extraction tion (LOQ) value was 0.71 g/ml, respectively, as calculated
was undertaken, in which all system valves were closed, (v) cell by signal to noise ratio with the help of VP software program
was rinsed with 60% of cell volume with extraction solvent, (vi) Version 6.1 by Shimadzu. The reproducibility of the method
solvent was purged from cell with gaseous N2 for 60 s and (vii) was demonstrated by repeated injections of rotenone standards.
depressurization took place. About 30 ml of solvent was used Five daily injections over a 5-day period gave intraday relative
in each of extraction process. The extracts were collected into standard deviation (RSD) ranging from 0.59 to 1.18% while
glass collection vials. The solvent was evaporated to dryness by inter-day relative standard deviation ranged from 2.18 to 3.22%,
rotary evaporator under vacuum at 45 C and the residue was respectively. The retention time of rotenone was 16.6 min, and
placed in a vacuum oven at room temperature (2830 C). The the peak was separated from others as shown by HPLC chro-
crude extract was stored in a well-closed container and protected matogram given in Fig. 2, peak 1 corresponds to rotenone. The
from light and kept in a desiccator at 20 C. peak purity was investigated using photodiode array detector
and it was found that no other compounds were co-eluted with
2.5. HPLC analysis the peak of rotenone (data not shown).

The chromatographic system (Shimadzu, Kyoto, Japan)


3.2. Effect of solvent types on the yields of rotenone from
consisted of SCL-10A VP system controller fitted with LC-
dried plant
10AD VP pump, DGU-14A degasser, SPD-10A VP UVVisible
detector, SIL-10AD VP auto-injector and CTO-10AS VP col-
The yields of rotenone from the stems of D. malaccensis by
umn oven. The column was a Waters Spherisorb S5 ODS2
PLE and maceration when using different type of solvents were
(250 mm 4.6 mm, 5 m). The gradient profile for the separa-
calculated as absolute yields of rotenone (g) in 100 g of dried
tion of rotenone was as follows: initial mobile phase acetoni-
plant (%, w/w) and shown in Fig. 3. Chloroform extracts from
trile/water (50:50, %, v/v) reaching 70:30 (%, v/v) in 30 min.
maceration were found to contain significantly higher absolute
Before each injection, the LC system had to be stabilized for
yields of rotenone than ethanolic extracts, whereas the absolute
10 min with an acetonitrile/water mobile phase (50:50, %, v/v).
yields of rotenone of chloroform extracts and ethanolic extracts
Standard solutions of rotenone (020 g/ml) were prepared
from PLE at 50 C, 1500 psi were not significantly different
in methanol, and 20 l of each was injected onto the HPLC col-
umn via the auto-injector three separate times (n = 3). The mean
peak areas for each concentration were calculated, and standard
calibration curves were constructed by plotting concentrations
against peak areas.
The rotenone contents in the extracted samples were deter-
mined as above. Before analysis, the portions of crude extracts
obtained from PLE and maceration were accurately weighed and
dissolved in 25 ml of methanol and sonicated until complete sol-
ubilization. The injection volume was 20 l, and the flow rate
was 1 ml/min. The analysis was performed at a wavelength of
294 nm.

2.6. Statistical data analysis

Statistical data analysis was performed using the t-test with Fig. 2. HPLC chromatograms of rotenone (1) in standard solution at 12 g/ml
p < 0.05 as the minimal level of significance. (A) and of crude extract solution (B).
A. Sae-Yun et al. / J. Chromatogr. A 1125 (2006) 172176 175

Fig. 5. Rotenone contents of crude extracts from the stems and the roots of D.
elliptica and the stems of D. malaccensis. Extractions were performed by using
ASE compared with maceration (n = 3).

that the solvent remains in intimate contact with the sample


Fig. 3. Absolute yields of rotenone from dried stem of D. malaccensis using
95% ethanol as extraction solvent compared with chloroform (n = 3). [10,11,21]. It was also reported that the pressure does not signif-
icantly affect extraction [13,19,21]. In this study, pressure was
(p > 0.05). This may be due to the PLE condition assisting the observed to have no effect on the extraction of rotenone. The
solubility of rotenone both in chloroform and in 95% ethanol. increase in pressure from 1000 to 2000 psi using chloroform
as the extraction solvent (at 50 C) led to no significant differ-
3.3. Effect of temperature and pressure on PLE procedures ence in the rotenone content of the extracts (p > 0.05) (Fig. 4B).
However, the pressure of 2000 psi was chosen to ensure that the
The effects of other factors such as temperature and pressure solvent remained in the liquid phase.
on PLE procedures on rotenone content of crude extract were
also investigated and the results are displayed in Fig. 4. By main- 3.4. Comparison of the rotenone content of the extracts
taining the pressure at 1500 psi, the highest rotenone content was from the different parts of the plants and the different
obtained when using chloroform as extraction solvent at 50 C extraction methods
(Fig. 4A). At higher temperatures (60 and 70 C), rotenone con-
tents of the extracts decreased. This decrease in the rotenone The extracts from the stems of D. elliptica and D. malac-
content could be due to a possible degradation of rotenone at censis contained small amounts of rotenone (mg) in 100 mg of
temperatures above 50 C. The main reasons for the enhanced crude extract (0.379.37%, w/w), whereas the extracts from the
performance when using PLE over the other conventional meth- roots of D. elliptica contained much higher rotenone contents
ods of extraction are the higher solubility of analytes in solvent (15.0346.08%, w/w) (Fig. 5). Chloroform displayed better sol-
and higher diffusion rate as a result of higher temperature. At vent properties for rotenone than 95% ethanol, since the crude
higher temperature, the strong solutematrix interaction caused extract using chloroform contains significantly higher amount
by van der Waals forces, hydrogen bonding and dipole attrac- of rotenone compared to the crude extract using 95% ethanol
tions between solute molecules and active sites on the matrix are (p < 0.05). It was observed that the rotenone contents from the
disrupted [10]. However, high temperature may promote decom- extraction by PLE using chloroform at room temperature (30 C)
position of compounds. Pressure is used to maintain the solvent were comparable to maceration. The extraction performed by
in the liquid phase during the extraction process and to ensure PLE at 50 C also showed significantly higher rotenone con-

Fig. 4. Rotenone contents of chloroform crude extracts from the stems of D. malaccensis by using ASE with variation of temperature at 1500 psi (A) and pressure
at 50 C (B) (n = 3).
176 A. Sae-Yun et al. / J. Chromatogr. A 1125 (2006) 172176

Table 2
The comparison of the extraction method
Maceration PLE (at 50 C, 2000 psi)

Volume of solvent/weight of dried plants 10 ml/g 3 ml/g


Extraction time 72 h 30 min
Rotenone content of extract from root of D. elliptica (n = 3) 40.57% (w/w) (1.73); RSD 4.26% 46.08% (w/w) (0.20); RSD 0.43%

tents than at 30 C (p < 0.05). However, the chloroform extracts References


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