Solvent Extraction Research and Development, Japan, Vol. 19, 137 – 145 (2012) – Technical Reports – Extraction of Limonin from Orange (Citrus reticulata Blanco) Seeds by the Flash Extraction Method Jing LIU, 1 Can LIU, 1 Yonghai RONG, 1 Guolun HUANG 2 and Long RONG 1* 1 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, P R China 2 Jiangmen Haolun Company, Jiangmen 529100, P R China (Received January 10, 2012; Accepted February 14, 2012) A flash extraction method was investigated for mass production of limonin from orange (Citrus reticulata Blanco) seeds. The limonin was extracted by using flash extraction for 2 min. The extraction conditions optimized by response surface methodology were as follows: ethanol concentration, 72% (v/v); solvent/solid ratio, 29:1 mL/g; rotational speed, 4000 r/min. The limonin was crystallized from the mixed solution of dichloromethane and isopropanol (1:3) at 4 o C for 1 h. The limonin crystals were identified by high performance liquid chromatography (HPLC) and from their infrared spectrum (IR). The purity of limonin was 95%, the yield of limonin was 6.8 mg/g and the recovery yield of limonin was 97.1%. Thus, flash extraction is an efficient method for the mass production of limonin. 1. Introduction Limonin, primarily isolated from Navel and Valencia oranges in 1938, is a highly oxygenated triterpene derivative of limonoids found to be rich in citrus seeds from the Rutaceae families [1, 2]. The studies in vitro and in vivo indicated that limonin is a potential bioactive compound which has many properties for promoting human health, such as lowering cholesterol, anticancer, antiviral and a number of other pharmacological activities [3-7]. The identification of limonin has also been reported [4, 8-11]. With respect to methods for the extraction of limonin, Soxhlet extraction is the main method using organic solvents such as ethyl acetate, acetone and dichloromethane [7, 9, 12, 13]. Recently, limonin was extracted from sour orange seeds by using aqueous hydrotropic solutions (Na-Sal or Na-CuS) [14]. Another method for the extraction of limonin from grapefruit seeds is the supercritical carbon dioxide (SC-CO 2 ) extraction technique [15]. Although hydrotropes were used instead of organic solvents in the extraction of limonin, the purification of limonin from the solutions with high concentration hydrotropes (2 M) would cause some problems. On the other hand, while the SC-CO 2 technique also reduced the use of organic - 137 -
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Extraction of Limonin from Orange Citrus reticulata … from Orange (Citrus reticulata Blanco) Seeds by the Flash Extraction Method Jing L IU, 1 Can LIU,1 Yonghai RONG,1 Guolun HUANG2
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Solvent Extraction Research and Development, Japan, Vol. 19, 137 – 145 (2012)
– Technical Reports –
Extraction of Limonin from Orange (Citrus reticulata Blanco) Seeds by the Flash
Extraction Method
Jing LIU,1 Can LIU,
1 Yonghai RONG,
1 Guolun HUANG
2 and Long RONG
1*
1 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of
Biological Science and Medical Engineering, Beihang University, Beijing 100191, P R China 2 Jiangmen Haolun Company, Jiangmen 529100, P R China
(Received January 10, 2012; Accepted February 14, 2012)
A flash extraction method was investigated for mass production of limonin from
orange (Citrus reticulata Blanco) seeds. The limonin was extracted by using flash
extraction for 2 min. The extraction conditions optimized by response surface
methodology were as follows: ethanol concentration, 72% (v/v); solvent/solid ratio,
29:1 mL/g; rotational speed, 4000 r/min. The limonin was crystallized from the mixed
solution of dichloromethane and isopropanol (1:3) at 4 oC for 1 h. The limonin crystals
were identified by high performance liquid chromatography (HPLC) and from their
infrared spectrum (IR). The purity of limonin was 95%, the yield of limonin was 6.8
mg/g and the recovery yield of limonin was 97.1%. Thus, flash extraction is an
efficient method for the mass production of limonin.
1. Introduction
Limonin, primarily isolated from Navel and Valencia oranges in 1938, is a highly oxygenated triterpene
derivative of limonoids found to be rich in citrus seeds from the Rutaceae families [1, 2]. The studies in
vitro and in vivo indicated that limonin is a potential bioactive compound which has many properties for
promoting human health, such as lowering cholesterol, anticancer, antiviral and a number of other
pharmacological activities [3-7]. The identification of limonin has also been reported [4, 8-11].
With respect to methods for the extraction of limonin, Soxhlet extraction is the main method using
organic solvents such as ethyl acetate, acetone and dichloromethane [7, 9, 12, 13]. Recently, limonin was
extracted from sour orange seeds by using aqueous hydrotropic solutions (Na-Sal or Na-CuS) [14]. Another
method for the extraction of limonin from grapefruit seeds is the supercritical carbon dioxide (SC-CO2)
extraction technique [15]. Although hydrotropes were used instead of organic solvents in the extraction of
limonin, the purification of limonin from the solutions with high concentration hydrotropes (2 M) would
cause some problems. On the other hand, while the SC-CO2 technique also reduced the use of organic
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solvents for the extraction of limonin, the high pressure conditions and high costs would limit its
application for scaling up. In conclusion, there is no appropriate method to extract limonin for mass
production.
Although considerable research of limonin has been devoted to discovering its diverse physiologically
activities, investigating the mechanism of pharmacology and establishing novel methods for determination
[4, 8-11], rather less attention has been paid to improving the extraction efficiency for mass production of
limonin. Therefore, in this work, a flash extraction method was investigated to improve extraction
efficiency for mass production of limonin. It is reported that flash extraction is an efficient method [16, 17].
At room temperature and pressure, the bioactivity can be preserved primarily within a few minutes and the
extraction yield can also be considerably increased. In our previous studies, the mogrosides and other active
constituents from Siraitia grosvenorii have been successfully extracted by flash extraction [18]. To the best
of our knowledge, it is the first time that a Herbal Blitzkrieg Extractor (HBE) has been used for the
extraction of limonin.
2. Materials and Methods
2.1 Materials and chemicals
Defatted orange (Citrus reticulata Blanco) seeds were obtained from Jiangmen Haolun Co. A standard
sample of limonin was purchased from Sigma Chemical Co. Acetonitrile, obtained from J&K Chemical Co.,
was of HPLC grade, and all other chemicals were analytical reagent grade.
2.2 Instrumentation
2.2.1 Flash extraction apparatus
In this work, a HBE with a
volume of 2L (JHBE-50S,
Henan Jinnai Sci-tech
Development Ltd., China) was
used. A larger type with a
volume of 2500L (JHBE-05T)
for mass production is also
available. The HBE was
originally developed by Liu et
al. [19] and a detailed diagram
is shown in Figure 1a. The raw
materials were placed in the
stainless steel container and
thoroughly macerated in the
extraction solvent. Then the
high-speed rotating cutter head rapidly pulverized the materials, thus allowing the active constituents to
dissolve in the solvent. Moreover, the two-layer cutter head (with a gap of 300 μm) could allow granules of
the materials into its inner volume and the shear force effectively ground the granules. Meanwhile,
vibration of the cutter head could cause ultrasonic cavitation which would contribute to the extraction. In
brief, this method combined the effects of soaking, pulverising, stirring, vibrating to extract the active
Figure 1. Schematic drawings of a Herbal Blitzkrieg Extractor (1
motor; 2 main shaft; 3 container; 4 cutter head; 5 controller) (a) and
the recovery scheme for limonin from defatted orange (Citrus
reticulata Blanco) seeds (b).
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constituents from natural products [20].
2.2.2 The HPLC and the IR systems
In this work, high performance liquid chromatography (HPLC) was used to determine limonin. The
Waters HPLC system was equipped with a pump, a controller (Waters 600) and a dual λ (210 nm was used)
absorbance detector (Waters 2487). The chromatographic separation was carried out using a reversed-phase
C18 5 μm (4.6 × 150 mm) HPLC column (Waters SunFire). 45% (v/v) acetonitrile was used as the mobile
phase and the flow rate was controlled at 1 mL/min. The injection volume was 20 μL. All standards and
samples were filtered through a 0.45 μm millipore filter.
In order to further identify the extracted limonin, the infrared spectrum (IR) of the limonin sample was
compared with the IR of the limonin standard. The IR spectrum in the range of 4000-400 cm−1
was
obtained from a Nicolet spectrometer using KBr wafers (sample 1 wt%).
2.3 Experiment design
The experimental conditions for the flash extraction of limonin from orange seeds were optimized by
the Box-Behnken design of response surface methodology (RSM) [21] . Three parameters, A (ethanol
concentration, %), B (solvent/solid ratio, mL/g) and C (rotational speed, r/min), were selected for the
extraction experiments at three variation levels, as shown in Table 1.
2.4 Extraction procedure for limonin
Figure 1b showed the recovery scheme for limonin from defatted orange (Citrus reticulata Blanco)
seeds and the method mainly included the following steps:
a) To extract limonin: flash extraction was conducted at room temperature and pressure with HBE at an
ethanol concentration of 72%, i.e. a mixed solution of ethanol and water (72:28), a solvent/solid ratio of
29:1 mL/g, a rotational speed of 4000 r/min, and the extraction time was 2 min.
b) To obtain crude limonin: the extracted solution was evaporated under reduced pressure (rotary
evaporation) at 55 oC and the extract (crude limonin) was obtained.
c) Crystallization: the obtained crude limonin was crystallized from a mixed solution of
dichloromethane and isopropanol (1:3) [22] at 4 oC for 1 h.
d) Purification: the obtained crystals of limonin were washed with isopropanol and sodium hydroxide
solution (20 mM) in turn. This is because some pigments and impurities are very likely to dissolve in the
water containing a little sodium hydroxide, while the limonin is insoluble. Finally, the white limonin
crystals were dried at 50 oC under vacuum for 1 h.
2.5 Data processing
The data of RSM was analyzed by Design Expert software (Version 7.1.6).
3. Results and Discussion
3.1 Determination of extraction time
For the HBE apparatus, the maximum extraction time should not exceed 4 min. Thus, the effect of
extraction time on limonin yield by flash extraction was investigated for an ethanol concentration of 70%, a
solvent/solid ratio of 30:1 mL/g and a rotational speed of 4000 r/min, and the results were shown in Figure
2. It can be seen that the limonin yield increases with increasing extraction time, with the highest limonin
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yield being obtained at 2 min. There was no
significant change when the extraction time
exceeded 2 min. This is probably due to the fact that
the orange seeds are completely pulverized in two
minutes and limonin then is easily dissolved by the
ethanol solvent. This result suggested that the
method of flash extraction was rapid and efficient.
Therefore, two minutes were chose as the extraction
time in the subsequent experiments. All the
experiments were done in triplicate and yields were
averaged.
3.2 Optimization of extraction conditions
The selected experimental parameters for the
extraction of limonin by flash extraction, which were
ethanol concentration, solvent/solid ratio and
rotational speed, were optimized using the
Box-Behnken design of the RSM. The conventional multifactor experiment is time-consuming and ignores
the combined interactions between physicochemical parameters, while the RSM can be employed as a
useful approach to implement optimal process conditions by performing a minimum number of
experiments. The Box-Behnken design (BBD) is an independent quadratic design that is an efficient and
creative three-level composite design for fitting second-order response surfaces [23, 24]. The mathematical
relationship between the three independent variables and the response surface is described by the following