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

second-order polynominal equation:

Y = β0 + β1A + β2B + β3C + β12AB + β13AC + β23BC + β11A2 + β22B

2 + β33C

2 (1)

where Y is the yield of limonin, A, B and C are the independent variables for the ethanol concentration, the

solvent/solid ratio and the rotational speed, respectively. In Eq. (1), β0 is the regression coefficient for the

intercept, β1, β2, and β3 are the linear coefficients, β12, β13, and β23 are the interactive coefficients and β11, β22,

and β33 are the quadratic coefficients. Table 1 showed the experimental design and the limonin yield (Y)

obtained under 15 test conditions. The extraction yields of limonin, which varied significantly depending

on the different extraction conditions, were from 4.1 to 6.6 mg limonin/g dry seeds.

The analysis of variance (ANOVA) obtained by the software is presented in Table 2. The ANOVA for

the regression model demonstrated that the model was highly significant, as was evident from the fact that

the F-value (Fmodel = 45.83) for the regression was greater than the tabulated Ftab value (F0.05, 9, 5 = 4.77).

Since Fmodel > Ftab, the Fisher F-test had a 95% confidence [25]. The lack-of-fit measured the failure of the

model to represent data in the experimental domain at points which were not included in the regression.

The F value of 1.87 for lack of fit implied that it was not significant compared to the pure error. The p-value

was used as a tool to check the significance of each coefficient and to indicate the interaction strength

between each independent variable. The p-value of the model was significant (P < 0.05) while the lack of fit

value of the model was 0.3664 (P > 0.05, not significant). Therefore, these values indicated that the

regression model explained a significant amount of the variation in the response values. The goodness of fit

of the model can be checked by the correlation coefficient (R2). The R

2 value of 0.9880 indicated that a

Figure 2. Effect of extraction time on the

limonin yield by the method of flash extraction

at an ethanol concentration of 70%, a

solvent/solid ratio of 30:1 ml/g and a rotational

speed of 4000 r/min. The number of

independent experiments n=3.

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good relation between the experimental and predicted values of the response. In conclusion, ANOVA of the

response surface quadratic model for the limonin yield showed that the model is highly significant and that

the experiments are accurate and reliable.

The software also generated the following quadratic equation which demonstrated the relationship

between the three parameters and their response Y in terms of coded units:

Y = 6.37 + 0.23A + 0.68B + 0.24C + 0.13AB - 0.14AC - 0.27BC - 1.05A2 - 0.40B

2 - 0.17C

2 (2)

where Y is the limonin yield, A, B, and C are the ethanol concentration, the solvent/solid ratio and the

rotational speed, respectively. Eq. (2) demonstrated that the limonin yield depended more on the

solvent/solid ratio followed by the ethanol concentration and rotational speed.

Table 1. Box-Behnken design of three variables with limonin yields

Run

Parameters and levels Limonin

yields

(mg/g)

A: Ethanol

concentration (%)

B: Solvent/solid ratio

(mL/g)

C: Rotational speed

(r/min)

1 70(0) 10(-1) 5000(+1) 5.7

2 60(-1) 20(0) 5000(+1) 5.3

3 70(0) 20(0) 4000(0) 6.2

4 80(+1) 20(0) 3000(-1) 5.3

5 60(-1) 10(-1) 4000(0) 4.1

6 70(0) 30(+1) 5000(+1) 6.6

7 60(-1) 20(0) 3000(-1) 4.7

8 70(0) 10(-1) 3000(-1) 4.5

9 70(0) 20(0) 4000(0) 6.4

10 80(+1) 30(+1) 4000(0) 6.0

11 70(0) 30(+1) 3000(-1) 6.4

12 70(0) 20(0) 4000(0) 6.5

13 80(+1) 20(0) 5000(+1) 5.3

14 80(+1) 10(-1) 4000(0) 4.4

15 60(-1) 30(+1) 4000(0) 5.2

Table 2. ANOVA of the quadratic model for the limonin yield

Source Sum of

squares

Degrees of

freedom Mean square F value

p-value

Prob > F

Model 9.47 9 1.05 45.83 0.0003

Residual 0.11 5 0.023

Lack of fit 0.085 3 0.028 1.87 0.3664

Pure error 0.030 2 0.015

Total 9.58 14

The three-dimension (3D) response surface showed the effects of the ethanol concentration, the

solvent/solid ratio and the rotational speed on the limonin yield. Figure 3a shows the effects of ethanol

concentration and solvent/solid ratio on the limonin yield at a fixed rotational speed of 4000 r/min. The

interaction between the ethanol concentration and rotational speed is shown in Figure 3b at a fixed

solvent/solid ratio of 20:1 mL/g. Figure 3c shows the effects of the solvent/solid ratio and rotational speed

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on the limonin yield at a fixed ethanol concentration of 70%.

The ethanol concentration showed a parabolic effect on the limonin yield, and the maximum limonin

yield was obtained at 72%, followed by a decline with a further increase in the ethanol concentration. Such

a decrease may be due to protein denaturation in the seeds which would influence the extraction yield; the

limonin yield increased significantly in the solvent/solid ratio range from 10:1 to 29:1 mL/g but there was

no significant change when the solvent/solid ratio was larger than 29:1 mL/g. In addition, it was found that

a higher limonin yield was obtained at a rotational speed of 4000-4500 r/min. When considering the fact

that a lower rotational speed would save energy and cost, 4000 r/min is the preferred rotational speed for

the extraction of limonin.

Figure 3. Response surface for the effect of ethanol concentration and solvent/sample ratio at a fixed

rotational speed of 4000 r/min (a), effect of ethanol concentration and rotational speed at a fixed

solvent/sample ratio of 20:1 mL/g (b), effect of solvent/sample ratio and rotational speed at a fixed ethanol

concentration of 70% (c) on the limonin yield.

The maximum predicted yield of limonin obtained from the RSM was 6.7 mg/g and the extraction

conditions could be optimized at an ethanol concentration of 72%, a solvent/solid ratio of 29:1 mL/g and a

rotational speed of 4000 r/min. The validation experiments were repeated three times under these optimized

extraction conditions and the average yield of limonin was 6.8 ± 0.1 mg/g. The above results indicated that

the Box-Behnken design was suitable for the extraction of limonin in this work. From the comparison of

extraction time and limonin yields with different extraction methods, as shown in Table 3, the maximum

yield of limonin could be obtained by flash extraction.

Table 3. Comparison of extraction time and limonin yields with different reported extraction methods

Extraction methods Extraction time (min) Limonin yields (mg/g)

Soxhlet extraction [9] 480 3.9

Hydrotropic extraction [14] 360 0.65

SC-CO2 extraction [15] 60 6.3

Flash extraction 2 6.8 ± 0.1 (6.7*)

* Predicted value from the response surface methodology

In this work, a higher yield of limonin (6.8 mg/g) can be obtained with a shorter extraction time (2 min)

by using flash extraction. To date, the highest yield of limonin (6.3 mg/g) was obtained by the SC-CO2

extraction method [15], but the high pressure conditions and high costs would limit its application for

scaling up. Although the limonin yield from flash extraction is approximately the same level as that from

SC-CO2 extraction, the extraction time and the cost of flash extraction are dramatically reduced.

Additionally, instead of the organic solvents normally used such as acetone and dichloromethane [7, 9, 13],

ethanol, as the extraction solvent used in this work, is more environmental friendly.

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3.3 HPLC and IR analysis

The purity of limonin was determined by

HPLC. As shown in Figure 4a-b, the retention

time for limonin sample was 7.7 min, which was

the same as for the standard sample. The

calibration curve was expressed by the following

linear equation:

Y = 16671 X + 35617 (r = 0.9986) (3)

where Y is the peak area, X is the concentration

of limonin (175-300 μg/mL). The purity of

limonin was calculated according to the

following equation:

P = (C × V / M) × 100% (4)

where P (%) is the purity of limonin, C (g/mL) is

the concentration of limonin, V (mL) is the total

volume of limonin and M (g) is the weight of

sample. The purity of limonin calculated by Eq.

(4) was 95%. The equation of recovery yield

was:

R = (W1 / W2) × 100% (5)

where R (%) is the recovery yield, W1 (g) is the

weight of obtained limonin and W2 (g) is the

weight of limonin in the extract solution. The

limonin content (7.0 mg/g) of the extract

solution (see Figure 4c) and the limonin yield

(6.8 ± 0.1 mg/g) in this work were calculated by

Eq. (3) respectively, and the recovery yield (97.1 ± 1.4%) was calculated by Eq. (5).

Figure 5 shows the IR spectra of the standard and limonin sample and the chemical structure of limonin.

Both IR spectra (Figure 5a-b) exhibited the bands associated with C=O vibrational modes at 1711, 1755

and 2071 cm−1

, along with C=C stretching at 1632 cm−1

. A broad absorption envelope at 3441 cm−1

was

attributed to hydrogen-bonded O–H stretching vibration of physically bound water while the C–O bond

stretching was located at 1130 cm−1

. The peak at 1385 cm−1

was attributed to flexible vibrations of the

methyl groups. The similarities of the two spectra indicated that the limonin structure did not change after

the process of flash extraction. It is well known that limonin is a highly oxygenated triterpene derivative of

limonoids in Meliaceae and Rutaceae [26, 27]. The IR spectrum showed the presence of lactone (1755

cm-1

), a furanyl ring (1504, 876 cm-1

), a ketone (1711 cm-1

) and methyl groups (1385 cm−1

). It can be

concluded that the results of the IR analysis were consistent with the chemical structure of limonin (see

Figure 5c).

Figure 4. HPLC of limonin and the sample of

extract solution from orange seeds (a the limonin

standard; b the limonin sample; c the extract

solution sample) using a reversed-phase C18

column at 210 nm, injection volume = 20 μL,

mobile phase of acetonitrile in water (45:55), flow

rate = 1 mL/min.

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Figure 5. IR of limonin from orange seeds (a the limonin standard; b the limonin sample) and the chemical

structure of limonin (c)

4. Conclusion

A simple and rapid extraction method was investigated for the mass production of limonin from orange

(Citrus reticulata Blanco) seeds. The extraction conditions were optimized by response surface

methodology at an ethanol concentration of 72% (v/v), a solvent/solid ratio of 29:1 mL/g and a rotational

speed of 4000 r/min. The purity of limonin was 95% and the recovery yield of limonin was 97.1%. At room

temperature and pressure, a higher yield of limonin (6.8 mg/g) can be obtained with a shorter extraction

time (2 min) by using this method. Thus, flash extraction is more efficient than other methods for the mass

production of limonin.

Acknowledgements

This work was supported by Guangdong government fund (No. 2009B090300323).

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