Solvent Extraction Research and Development, Japan, Vol. 22, No 2, 187 – 199 (2015)

Liquid Carbon Dioxide Extraction of Xanthones from the Pericarps of Garcinia Mangostana Linn.

Enhanced by Ultrasonic Irradiation

Kenji MISHIMA,1* Hirofumi KAWAMURA,

2 Shota ITO,

1 Yuuta INOUE,

1 Tetsuya HIROTA,

1

Takafumi KATO,1 Tanjina SHARMIN,

1 Ryo KAWAKAMI,

1 Keiichi IRIE,

3 Kenichi MISHIMA,

3

Takunori HARADA,4 Salim MUSTOFA,

5 Fauziyah HASANAH,

6 Yusraini Dian Inayati SIREGAR,

6

Lily Surayya Eka PUTRI6 and Agus SALIM

6

1Department of Chemical Engineering, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma

Jonanku, Fukuoka 814-0180, Japan 2Department of Seasoning & Foods Division, San-Ei Gen F.F.I., Inc., 1-1-11, Sanwa-cho, Toyonaka, Osaka

561-8588, Japan 3Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1,

Nanakuma Jonanku, Fukuoka 814-0180, Japan 4Department of Chemistry, Faculty of Engineering, Oita University,

5Research Center for Technology of Nuclear Industrial Material, Indonesia Nuclear Energy Agency,

Gedung 42, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten 15419, Indonesia

6Faculty of Science and Technology, Syarif Hidayatullah State Islamic University (UIN) Jakarta,

JL.Ir.H.Juanda Ciputat, Tangerang, Indonesia

(Received December 23, 2014; Accepted March 12, 2015)

The liquid carbon dioxide (CO2) extraction of xanthones, such as α-mangostin from the pericarp of

mangosteen (Garcinia mangostana Linn.), was enhanced using ultrasound at 5, 20 and 25 ºC. Mixtures of

carbon dioxide and ethanol were used as solvents. The extraction pressure was from 8 to 14 MPa. To

enhance the extraction yield, the solvent was irradiated with ultrasound. The yields of xanthones, such as

α-mangostin, in the extraction were significantly improved without any change in the selectivity of the

extract. We also qualitatively and quantitatively analyzed the xanthones in the extract using HPLC

(high-performance liquid chromatography) as well as the extraction behavior. The effect of four operating

parameters, temperature, pressure, ultrasound irradiation time and the composition of ethanol in the liquid

CO2 solution, on the extraction yield was investigated using single-factor experiments. The improved ratio

in yields of each xanthone between the ultrasonic-enhanced case and unenhanced case was calculated. The

yields of xanthones in the extract were significantly improved by adjusting the operating temperature,

ultrasound irradiation time and the composition of ethanol in the liquid CO2 solution, whereas no change in

the selectivity of the extract was observed.

1. Introduction

Xanthones, such as α-mangostin from the pericarp of mangosteen (Garcinia mangostana Linn.), have

recently attracted much attention because of their pharmacological activity, such as their anti-allergy [1],

anti-bacterial [2,3], anti-tumoral [4,5], anti-inflammatory [6], anti-oxidant [7-9] and anti-hypertensive

properties [10]. Mangosteen is a tropical tree that grows naturally in Southeast Asia. It has been used as a

traditional curative agent for various diseases and ailments, including cholera, diarrhea, dysentery, skin rash

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and inflammation [11].

Conventionally, xanthones are extracted from natural matrices using organic solvents including ethanol,

methanol, acetone and toluene, which is time-consuming and leaves solvent residues. The interest in

alternatives to conventional solvent extraction, such as supercritical carbon dioxide (supercritical CO2)

extraction, is increasing because CO2 is nonflammable, nontoxic, inexpensive and environmentally benign,

and its critical condition is easily accessible [12-19]. Thus, supercritical CO2 has provoked considerable

interest as a separation technique in many fields. Previous literature has explored the possibility of

extracting xanthones with supercritical CO2. For example, Zarena et al. described the xanthone extraction

process from mangosteen pericarps using supercritical CO2 with and without alcohol as the cosolvent at 30

MPa and 50 ºC [20,21]. In our previous study, the feasibility of supercritical CO2 extraction of xanthones

from mangosteen pericarps was demonstrated for several mild conditions. Huang et al. reported that the

solubility of xanthone and xanthene in supercritical CO2 was very low [22]. Improvement in the solubility

of xanthones in supercritical CO2 is required. Recently, supercritical-fluid technology enhanced by

ultrasound has been proposed for several applications.

Here, we describe the possibility of increasing the yield in the xanthone extraction from mangosteen

pericarp using liquid CO2. We also determined the extraction conditions when liquid CO2 was used and

enhanced by ultrasound.

2. Experimental

2.1 Reagents

Peel of mangosteen and mangosteen powder were kindly supplied by University Islam Malaysia.

α-Mangostin, 8-desoxygartanin and 3-isomangostin were purchased from Wako Co. Ltd. The chemical

structures of 8 major xanthones (α-mangostin, 3-isomangostin, mangostanol, gartanin, garcinone E,

9-hydroxycalabaxanthone, β-mangostin, 8-desoxygartanin) in mangosteen pericarp have been reported

elsewhere. Methanol and acetic acid were purchased from Wako Co. Ltd. Analytical-grade ethanol was used

as a cosolvent in the supercritical CO2 extraction and as a solvent in the HPLC analysis. High-purity CO2

(more than 99 %, Fukuoka Sanso Co. Ltd.) was used as received.

2.2 Liquid CO2 Extraction Equipment and Procedures

Mangosteen pericarps were cut into an exocarp and a mesocarp-endocarp composite (hereinafter

called mesocarp+endocarp), which was dried at room temperature, and then ground and powdered using a

freezer mill 6750 (SPEX CentriPrep Co. Ltd.). The mesocarp+endocarp powder was used for the extraction.

The average particle diameter was less than 1 mm. The samples were stored in a cold and dark place because

of xanthone photosensitivity [23]. The mangosteen pericarp powders were placed in an extraction cell of a

liquid CO2 extraction apparatus.

A schematic diagram of the experimental apparatus used for liquid CO2 extractions with and without

ultrasonic irradiation is shown in Figure 1. The extraction cell was approximately 150 cm3 in volume (34

mm i.d. × 165 mm long, Toyo Koatsu Co. Ltd.). A titanium ultrasound horn is equipped in the upper part of

the extraction cell, and driven by electrical signals from an ultrasonic processor (VC-750, Sonic and

Materials Inc.). The ultrasonic processor produced ultrasonic waves at a frequency of 20 kHz with a

maximum power capability of 750 W. During ultrasonic irradiation, waves of constant amplitude were

delivered from the ultrasonic processor by automatically adjusting the power supply. The maximum

amplitude was 61 μm, and the amplitude control was set at 100%. The extracting experiments were

performed at a constant amplitude of 15.3 μm (the amplitude control was set at 25%). To prevent a rapid

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increase in system temperature from the ultrasonic irradiation, the ultrasonic irradiation was performed

intermittently (on time: 5 sec; off time: 2 sec), and the ultrasonic horn was cooled using a cooling jacket.

Prior to the extraction, the mangosteen sample (0.3 g) was directly loaded into the high-pressure cell.

Liquid CO2 was pumped first from a gas cylinder at a constant flow rate (2 cm3/min) through the dryer, and

cooling unit, then into the extraction cell by a HPLC pump (SCF-get, JASCO), up to the desired pressure.

The cooling unit is used to cool the pump-head which keeps the CO2 liquid. A back pressure regulator

(880-81, JASCO), located at the outlet of the cell, is adjusted to maintain the desired pressure within an

accuracy of ±0.3 MPa and monitored by a digital pressure gauge (Shinwa Electronics Co., model DD-501,

accuracy ± 0.3%). The high-pressure cell is immersed in a water bath to monitor and control the

temperature. The temperature of the water bath was maintained at 5, 20 and 25 ºC. The temperature inside

the high-pressure cell was controlled to an accuracy of ±0.1 ºC. Amounts of ethanol were injected through

a co-solvent feeder. The solvent composition x EtOH was defined by the mole fraction of ethanol in the

solution of CO2 and ethanol. The mole fraction of ethanol, xEtOH, in the solvent composition was

determined by adjusting the amount of ethanol. After the pressure and temperature of the high-pressure cell

reached the desired values, the ultrasonic irradiation was performed using the ultrasonic processor. After

each experiment, the cell was slowly depressurized to atmospheric pressure. The extract was collected from

the high-pressure cell, an expansion valve, all tubes, and U-tube cold traps at atmospheric pressure. The

amount of CO2 used was measured by a gas flow meter.

1.Gas cylinder 2. Dryer 3. Cooling unit 4. Filter

5. Pump 6. Pressure gauge 7. Safety valve 8. Pre-heater

9. Check valve 10. High-pressure cell 11. Ultrasonic processor

12. Water bath 13. Pressure gauge 14. Safety valve 15. Heater

16. Thermometer 17. Ultrasonic horn 18. Quartz glass windows

19. Co-solvent feeder 20. Cold traps 21. Gas flow meter

V-1,V-7. Back pressure regulators V-2-V-6. Stop valves

Figure 1. Schematic diagram of the experimental apparatus for the liquid CO2 extraction using ultrasonic

irradiation.

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The extraction process was essentially affected by the solute solubility. The solute solubility was

sensitive to the temperature, solvent composition and density of CO2 and ethanol cosolvent. In our study, the

effects of the temperature, liquid CO2 pressure and mole fraction ratio of ethanol to liquid CO2 in the

extraction cell were studied to optimize the extraction yield. The time for extraction was set at 60 min. The

extraction was performed at the temperatures of 5, 20 and 25 ºC and a pressure of 8~14 MPa and the mole

fractions of ethanol in liquid CO2 were 0, 0.06, 0.131, 0.262, 0.393 and 1. The compositions of the collected

samples were analyzed using HPLC.

2.3 HPLC Analysis

The HPLC system consisted of a Tosoh LC-8010 system, which was equipped with a UV detector. The

detection wavelength was set at 340 nm. The xanthones were separated using a TSK-GEL (Tosoh Co.)

ODs-80Ts column (1504.6 mm), which was set at 40°C. The injection volume was 10 or 50 μL, which

was selected based on the extract concentration to avoid detector saturation. Two mobile phases were used

during the separations: (A) methanol and (B) 0.1 % acetic acid in water. The methanol gradient profiles

were as follows: 75 % over 0-10 min; 75-90 % over 10-50 min; 90 % over 50-60 min; 75 % over 60-70 min.

The flow rate of the solvent was set at 0.8 mL/min.

3. Results and discussion

Previously, many researchers proposed various HPLC profiles to more accurately separate the

components in the extraction. We applied the modified method that was originally developed by Zarena et

al [21]. To estimate the validity of our elute composition, the liquid CO2 extract and standard samples, such

as α-mangostin, 8-desoxygartanin and 3-isomangostin were analyzed. The liquid CO2 extraction was

performed at 25 ºC and 10 MPa. Ethanol was added to the high-pressure cell as a cosolvent. The results of

the HPLC analysis is shown in Figure 2 and table 1. Through an analysis of the standard xanthone samples

and a literature survey [21,24], we confirmed that the main peaks that appeared after the extraction were

3-isomangostin, mangostanol, 8-desoxygartanin, gartanin, α-mangostin, garcinone E,

9-hydroxycalabaxanthone and β-mangostin.

Figure 2. HPLC chromatogram of xanthones extracted from mangosteen pericarp powder using

supercritical CO2 with ethanol as the cosolvent at 25 ºC and 10 MPa.

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Table 1. List of extracted xanthones using liquid CO2 extraction with ethanol (xEtOH=0.131) as the cosolvent

at 25 ºC and 10 MPa.

No. Retention Time (min) Peak Name Area (%) Height

1 22.05 3-Isomangostin 1.4401 33170

2 28.98 Mangostanol 0.4297 13969

3 31.70 8-Desoxygartanin 21.9276 322501

4 35.84 Gartanin 1.8747 52124

5 40.95 α-Mangostin 67.9567 1214088

6 46.06 Garcinone E 1.9446 30122

7 52.34 9-Hydroxycalabaxanthone 0.0149 1873

8 55.48 β-Mangostin 1.6449 67973

In our previous work, mesocarp and endocarp yielded more xanthones than exocarp [25]. Therefore,

we used mesocarp and endocarp as the extraction material for the following experiments. Additionally, the

most appropriate extraction time for ethanol extraction at constant temperature (25 ºC) with or without

ultrasound was investigated. As previously described, the extraction was performed using a heating and

shaking apparatus, and sampling was performed at several time intervals. Qualitative and quantitative

information was obtained using HPLC. The α-mangostin yield significantly increased over 0-60 min, and

the saturation of the extract concentration was subsequently observed. It was concluded that the appropriate

time of treatment was circa 60 min.

To determine the enhancing effect of ultrasound irradiation on yield a time-dependent assay of the

extraction was performed for the mangosteen pericarp powders with and without ultrasound under

atmospheric pressure. Methanol and ethanol were used as the solvents and the temperature was controlled

at 25ºC. The total time of ultrasonic irradiation was 200 sec. The experimental extraction curves for

α-mangostin with and without ultrasound are presented in Figure 3. It shows that in every case, either

methanol or ethanol, the yield of -mangostin gradually increased over time till 60 min at which point

saturation occurred. Therefore, it is reasonable to decide that the optimum time for extraction is around 60

min. Although the extract yield was greater with methanol, it is apparent that, for both methanol and

ethanol extraction, the total yield extracted with ultrasound was higher than that without ultrasound at each

identical time. The yields of -mangostin with or without ultrasound were found to be 16.3 mg/g and 14.4

mg/g with methanol and 15.06 mg/g and 14.1 mg/g with ethanol respectively. Therefore, these results

imply that ultrasonic irradiation clearly improves the yield of -mangostin extractions.

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Figure 3. Rate of extraction of -mangostin from mangosteen mesocarp+endocarp powder using methanol

or ethanol as the solvent at 25 ºC, with and without ultrasound under atmospheric pressure. The total time

of ultrasonic irradiation was 200 sec. Symbols ▲ and △ show the mass extracted with and without

ultrasound using ethanol, whereas ■ and □ show the mass extracted with and without ultrasound using

methanol.

The effect of temperature on the total amount of extracted xanthones using liquid CO2 extraction with

ethanol (xEtOH=0.131) was studied at 10 MPa with or without ultrasonic irradiation. The total time of

ultrasonic irradiation was 200 sec. Previously, we studied the efficiency of supercritical CO2 extraction

with ethanol as the cosolvent and without ultrasonic irradiation. To compare the efficiency between the

extractions with and without ultrasonic irradiation, the yields of each xanthone are showed in Figure 4. The

temperature of the air bath was set at 5, 20 and 25 ºC. Thus, changing the temperature significantly affects

the density of liquid CO2, which changes the mole fraction ratio of ethanol to liquid CO2 (xEtOH) in the cell;

thus, the added mass of ethanol was varied to balance the mole fraction ratio. The pressure was set at 10

MPa, and the extraction was performed for 60 min. As a cosolvent, ethanol was added to the extraction cell

before the extraction. As previously observed, the α-mangostin yield, in this work was by far the greatest

among the xanthones with or without ultrasound (26.8 mg/g and 19.6 mg/g respectively). This result shows

that the eluent obtained at 25 ºC had the highest concentration, which implies that lower or higher

temperatures were not suitable for xanthone extraction from mangosteen peel. In addition, the tendency for

elution at the extraction temperature hardly differed between α-mangostin and other xanthones.

Furthermore, these results implied that ultrasonic irradiation improves the yield of xanthone extractions.

This tendency was the same for almost all of the xanthones. However, the yields obtained at 5 ºC and 20 ºC

were clearly lowest for all xanthones.

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Figure 4. Effect of temperature on the extraction yields of xanthones obtained by liquid CO2 extraction with

ethanol (xEtOH=0.131) at 10 MPa with or without ultrasonic irradiation.

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The improvement in extraction yield, which was attributed to the ultrasonic irradiation, was evaluated

based on the ratio of the yield obtained with ultrasound to the yield obtained without ultrasound. The

results are shown in table 2. The liquid CO2 extraction with ultrasonic irradiation improved the xanthone

yields for each condition. In particular, at 25 ºC, the xanthone yields obtained with ultrasonic irradiation

were approximately two times higher than those without ultrasonic irradiation.

Table 2. Improvement ratio of the yield obtained with ultrasound to the yield obtained without ultrasound.

Compounds 5 ºC 20 ºC 25 ºC

3-isomangostin 1.972 1.309 1.184

mangostanol 5.382 2.805 1.199

8-desoxygartanin 1.287 1.316 1.416

Gartanin 1.194 1.122 1.101

α-mangostin 1.141 1.312 1.367

garcinone E 3.772 4.629 4.324

9-hydroxycalabaxanthone 1.934 3.394 2.578

β-mangostin 1.126 1.552 1.975

Furthermore, we examined the effect of the extraction pressure on the yields of each xanthone with and

without ultrasonic irradiation using liquid CO2 extraction with ethanol (xEtOH=0.131) at 25 ºC. The pressure

was maintained at 8, 10 and 14 MPa. The temperature was set at 25 ºC as the highest yield was obtained at

this temperature and also to avoid unstable phases of gas and liquid which exist near the critical

temperature of CO2, such as 32 ºC. The total time of ultrasonic irradiation was 200 sec. The densities of

liquid CO2 at 8, 10, and 14MPa at 25 ºC are 766, 812, and 871kg/m3, respectively. As shown in Figure 5,

the highest yields of α-mangostin (26.8 mg/g) plus almost all the xanthones in ethanol were obtained at

10MPa which were further successfully enhanced by ultrasonic irradiation. This result shows that the eluent

obtained at these pressures gave similar concentrations, implying that the yields of xanthones were

insensitive to the operating pressure of liquid CO2 and the extraction rate clearly was enhanced by

ultrasonic irradiation. Although the yield of xanthones was expected to be higher at 14MPa, which gave a

higher density of CO2, than that at 10MPa, the yield of xanthones at 14MPa was just a little lower than that

at 10MPa. It is considered that the amount of ethanol, as the cosolvent, decreases in the liquid CO2 phase

because the ethanol distributed more to the vapor-phase at 14MPa. The effects of operating temperature and

pressure on the extraction yield are similar to the results of other liquid CO2 extractions [16, 25-28]. Under

these extraction conditions (temperature = 25 ºC, pressure = 8, 10, and 14 MPa), gas and liquid phases

coexist in the extraction vessel. The change in the density of liquid CO2 is very small with the change in the

operating pressure. So the solvent power of liquid CO2 is considered to be almost constant under these

conditions.

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Figure 5. Effect of pressure on the extraction yields of xanthones obtained by liquid CO2 extraction with

ethanol (xEtOH=0.131) at 25 ºC with or without ultrasonic irradiation.

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Figure 6. Effect of time of ultrasonic irradiation on the amount of extract using liquid CO2 extraction with

ethanol (xEtOH=0.131) at 25 ºC and 10 MPa from mangosteen mesocarp+endocarp powder. Symbol ■,

□, and ▲ show the yield of extract of α-mangostin, 8-desoxygartanin and gartanin, respectively.

We also examined the effect of ultrasonic irradiation time on the yields of each xanthone using

liquid CO2 extraction with ethanol (xEtOH=0.131) at 25 ºC and 10 MPa. The total time of ultrasonic

irradiation was set at 75, 200 and 250 sec. The yield of each of the xanthones increased with an

increase in the total time of ultrasonic irradiation. These values were almost saturated at 200 sec. It is

considered that ultrasonic irradiation can improve the extraction yield and 200 sec of ultrasonic

irradiation time is sufficient.

To elucidate the effect of changing the mole fraction (xEtOH) of ethanol in the liquid CO2 on the yield

of -mangostin, we performed the extraction using ethanol and liquid CO2 alone and in combination; xEtOH

=0, 1 denotes pure CO2 and ethanol, respectively. The pressure was set at 10 MPa, the temperature was set

at 25 ºC and extraction was performed for 60 min. The total time of ultrasonic irradiation was 200 sec. In

order to change the mole fraction of ethanol in the mixtures of liquid CO2 and ethanol through the range of

0 to 1, we used three experimental schemes; for xEtOH = 0, this means no ethanol added, so the extraction

was performed with only liquid CO2; for xEtOH = 1, this method is the traditional extraction with ethanol

without liquid CO2. For other ranges of xEtOH, the extraction was performed with mixtures of liquid CO2

and ethanol. That is, the mole fraction of ethanol was set at 0.131, 0.26 and 0.39 so as to reveal the effects

of the mole fraction of ethanol more precisely. The yield of each xanthone is showed in Figure 7. The

maximum yield of 26.8 mg/g and 19.6 mg/g were obtained with and without ultrasound respectively at

xEtOH = 0.131. This result implies that lower or higher mole fractions of ethanol reduce the yield of

-mangostin under our extraction conditions. This result is consistent with the results of our previous CO2

extraction results for resveratrol [29].

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Figure 7. Effect of the mole fraction of ethanol in the liquid CO2 and ethanol solvent on the yield of

-mangostin extracted at 25 ºC and 10 MPa with or without ultrasound.

Comparison of the -mangostin yields with or without ultrasound with ethanol or methanol or

mixtures of CO2 and ethanol (mole fraction of ethanol, xEtOH = 0.131) at 25ºC and 10MPa are shown in

Figure 8. The extractions were performed for 60 min in each case. This figure clearly shows that the

highest amount of yield of -mangostin was obtained with the mixtures of liquid CO2 and ethanol (mole

fraction of ethanol, xEtOH = 0.131) and which was further enhanced by ultrasonic irradiation.

Figure 8. Comparison of -mangostin yields extracted from mangosteen mesocarp+endocarp powder at 25

ºC and 10 MPa with (■) or without ultrasound (□).

4. Conclusions

Xanthones, such as α-mangostin, were successfully extracted from mangosteen pericarp (Garcinia

mangostana Linn.) using a liquid fluid extraction with ultrasonic irradiation. Carbon dioxide and ethanol

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were used as the liquid solvent and cosolvent, respectively. Ultrasonic irradiation enhanced the xanthone

extraction yields by approximately two times. The yields of xanthones in the liquid CO2 extraction were

significantly improved by adjusting the operating temperature and pressure (25 ºC, 10MPa), ultrasound

irradiation time (total time= 200 sec, amplitude= 15.3 μm, frequency= 20 kHz) and mole fraction of ethanol

in the liquid CO2 solution (xEtOH = 0.131).

Acknowledgments

This work was partially supported by a Grant-in-Aid for Scientific Research (Grant Nos. 20560707

and 23560913).

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