Process Biochemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Supercritical carbon dioxide extraction of α-mangostin from mangosteen

pericarp with virgin coconut oil as co-extractant and in-vitro bio-accessibility
measurement
Wan Jun Leea, Chea Chi Nga, Jin Shuen Nga, Richard Lee Smith Jr.c, Siew Lee Koka, Yen Yi Heea,
Sin Yee Leed, Wei Kiat Tana, Nur Hanani Zainal Abidina, Sarina Abdul Halim Lima,
Gun Hean Chonga,b,
⁎

a
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
b
Supercritical Fluid Centre (SFC), Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
c
Research Center of Supercritical Fluid Technology, Tohoku University, Aramaki Aza Aoba, 6-6-11-403, Aoba-ku, Sendai, 980-8579, Japan
d
School of Science, Monash University Malaysia, Bandar Sunway, 47500, Subang Jaya, Selangor, Malaysia

ARTICLE INFO ABSTRACT

Keywords: This work aims to develop a method for producing α-mangostin that has high bio-accessibility through the use of
Supercritical fluid extraction supercritical carbon dioxide (scCO2) extraction of mangosteen pericarp extract (MPE) with virgin coconut oil
Xanthone (VCO) as co-extractant. Extractions with scCO2 were carried out at 30 MPa to 40 MPa, 60 °C, CO2 flow rate of
Garcinia mangostana Linn 1.7 g/min and co-extractant VCO (0% to 20%). MPE yield and extraction yield increased with the amount of co-
Virgin coconut oil
extractant added. The highest yield of MPE obtained was 17% at conditions of 35 MPa with 20% of VCO. The
Co-extractant
Bio-accessibility
scCO2 MPE had higher bio-accessibility for α-mangostin (91%) than α-mangostin obtained by extraction with
ethanol (30%) or hexane (50%). Thus, supercritical CO2 extraction used with virgin coconut oil co-extractant
provides a method for producing high bio-accessible α-mangostin from mangosteen pericarp extract.

1. Introduction ethanol with scCO2 is generally considered as necessary for increasing

Mangosteen fruit (Garcinia mangostana Linn) consists of edible white
segments and an outer layer of purplish pericarp that is approximately
three quarters of the fruit’s weight and is often discarded as agricultural
waste. There is interest in using mangosteen pericarp (MP) as an in-
gredient in functional products or as a dietary supplement due to its
high concentration of xanthone [1]. The medicinal properties of xan-
thone, include treatment for diabetes, obesity, pain and inflammatory,
neurotoxicity, Alzheimer's disease and cancer and has been reviewed by
Ovalle-Magallanes et al. [2]. More than 50 types of xanthones have
been isolated from MP [3] and the most examined compound is the α-
mangostin, which is reported to have powerful antioxidant with anti-
inflammatory, anti-cancer, anti-bacterial and anti-fungal properties [4].
Presently used xanthone separation techniques include solvent ex-
traction [5], hot water extraction [6,7], deep eutectic solvent extraction
[8], subcritical ethanol extraction [9], liquefied dimethyl ether ex-
traction [10], and supercritical carbon dioxide (scCO2) extraction aided
with organic co-solvents [3,11,12]. The use of a co-solvent such as
extraction yield, because the solubility of xanthone in scCO2 is rela-
tively low (ca. yxanthone = 10−5) [13]. On the other hand, vegetable oil
can be introduced as a co-extractant and as a substitute for organic co-
solvents [14]. Unlike a co-solvent, vegetable oil used as a co-extractant
enhances mass transfer of the targeted compounds within the solid
matrix and it can have desirable properties in the end product [15]. For
instance, separation of vegetable oil in the end product obtained after
scCO2 extraction is not required, because vegetable oils are safe for
consumption and they may contain natural bioactive compounds that
are deemed beneficial to human health. In the scCO2 extraction of ly-
copene from tomato, hazelnut oil, tomato seed oil, and canola oil have
been used [16–18]; for extraction of carotenoids from carrot and
pumpkin, canola oil and olive oil have been used [19,20]; for extraction
of lutein from marigold, soybean oil has been used [21]; for extraction
of astaxanthin from microalgae (Haematococcus pluvialis), soybean oil,
olive oil and sunflower oil have been used [22,23]. The co-extractant
oils used in the aforementioned studies were chosen for the purpose of
enhancing the stability and to avoid degradation of the targeted

⁎
Corresponding author at: Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, Serdang, Selangor,
Malaysia.
E-mail address: gunhean@upm.edu.my (G.H. Chong).

https://doi.org/10.1016/j.procbio.2019.09.009
Received 19 June 2019; Received in revised form 23 August 2019; Accepted 7 September 2019
1359-5113/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Wan Jun Lee, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.09.009
W.J. Lee, et al.
Fig. 1. Overview of experimental design. Assessment of vegetable oil for extraction of mangosteen pericarp extract, extraction steps and bio-accessibility tests.
bioactive compounds and to increase their extraction yield.
It has been reported that α-mangostin has restricted bioavailability
[24,25]. Presence of oil as co-extractant potentially enhances the bio-
accessibility of α-mangostin from the MPE since the oil could act as a
carrier lipid for lipophilic bioactive components [26]. The role of oil co-
extractant in both influencing the scCO2 extraction yield and bio-ac-
cessibility of α-mangostin has not been reported.
In this work, virgin coconut oil was used as co-extractant to increase
the extraction yield of mangosteen pericarp extract (MPE) and α-man-
gostin. The objectives of this study were to determine effective con-
centrations of co-extractant and extraction conditions for separating
MPE and α-mangostin and to investigate the effect of co-extractant on
the in-vitro bio-accessibility of α-mangostin.
2. Materials and method
2.1. Materials
Dried MP and virgin coconut oil (VCO) were supplied by
Adirondack (M) Sdn. Bhd (Selangor, Malaysia), and liquid CO2 (purity:
99.99%) was purchased from NIG Gases (M) Sdn Bhd. (Selangor,
Malaysia). Rice bran oil (RBO), palm kernel oil (PKO), grape seed oil
(GSO), canola oil (CO), sunflower oil (SO) and olive oil (OO) were
purchased from AEON CO. (M) BHD (Selangor, Malaysia). HPLC grade
solvents (ethanol, methanol, acetonitrile, orthophosphoric acid), hy-
drochloric acid, sodium chloride, sodium hydroxide, ethyl acetate were
purchased from Thermo Fisher Scientific (New Hampshire, United
States). Enzymes pepsin (P-7000), pancreatin (P-1750) and bile extract
(B-8631) from porcine source and α-mangostin analytical standard
(purity ≥98%) were purchased from Wuhan ChemFaces Biochemical
Co. (Hubei, China).
2.2. Sample preparation
Prior to extraction, the dried MP (moisture content of 8%) was
2
Process Biochemistry xxx (xxxx) xxx–xxx
ground into powder form (RT-CR30S 3HP, Rong Tsong precision
technology CO, Taiwan) with measured particle size of < 2 mm, cor-
responding to 18 mesh.
2.3. Soxhlet extraction
MP powder (20 g) was extracted with hexane (ca. 68 °C), ethanol
(ca. 78 °C) and ethyl acetate (ca. 77 °C) for 6 h using a Soxhlet appa-
ratus. The solvent was evaporated at 40 °C and the extract was flushed
with nitrogen and sealed before being kept in the dark at −18 °C. The
results from solvent extraction are used as controls in the assessment of
the experiments.
2.4. Dissolution of MPE in solvents and vegetable oils
2.4.1. Solubility of MPE in different solvents
Dissolution experiments of MPE in ethanol, ethyl acetate and
hexane were performed as follows. MPE (0.05 g to 0.2 g) was mixed
with 4 mL of solvent in a test tube. The dissolution of MPE was visually
observed under different temperatures (30 to 70) °C with continuous
stirring.
2.4.2. Suitability of different vegetable oils as co-extractant
To determine a suitable type of vegetable oil as co-extractant for
scCO2 extraction, the ability of the MPE to dissolve in the oil was de-
termined. The dissolution of MPE in VCO, RBO, PKO, GSO, CO, SO and
OO were studied. MPE (0.025 g to 0.2 g) was mixed with oil (4 g) in a
test tube before subjecting the mixture to different temperatures (ca.
50 °C, 60 °C, 70 °C, 80 °C, 90 °C and 98 °C) using a water bath with
continuous stirring for 5 min. The oils that allowed for the highest MPE
dissolution by visual observation at the lowest possible temperature
were selected as the co-extractant for the subsequent scCO2 extraction.
Extractions with scCO2 were carried out at moderate temperatures (ca.
60 °C) and 35 MPa to select the most suitable type of vegetable oil based
on the extraction yield. Experiments were conducted in triplicate.
W.J. Lee, et al. Process Biochemistry xxx (xxxx) xxx–xxx

An overview on the experimental design for this work is shown in
Fig. 1.
2.5. scCO2 extraction
A laboratory scale system as described by Lee et al. [27] was used
for the extraction of MPE with scCO2. The system consisted of an ex-
traction vessel (110 mL), an oven (UFE 600, Memmert, Germany), a
CO2 pump (P-2024SFC, ChromTech, USA) and a CO2 measuring flow
metre (#20136, Restek, USA). VCO was selected as the co-extractant for
the scCO2 extraction (discussed in Section 3.1). MP powder (5 g) was
mixed with different amounts of VCO (5, 10, and 20% w/w) before
being loaded into the extraction vessel in which it was preliminary
determined that the addition of VCO above 20% caused powder ag-
glomeration. Hence the maximum co-extractant amount was set to
20%. A control sample without the addition of VCO was also prepared
for the scCO2 extraction. Glass wool (#20411, Supelco, USA) was
placed at both ends of the extraction vessel to hold the MP powder in
place. The scCO2 extractions were conducted at 30 MPa, 35 MPa and
40 MPa, ca. 60 °C and CO2 flow rate of 1.7 g/min for 4 h. The weight of
collected MPE was recorded and the samples were flushed with ni-
trogen and sealed before being kept in the dark at −18 °C. The ex-
periments were conducted in triplicate.
The extraction yield was calculated from Eq. (1)
Extraction yield = mMPE /(mmp + moil) × 100%
where, mMPE is the mass of mangosteen pericarp extract collected from
scCO2 extraction and mmp is the total mass of mangosteen pericarp
powder and moil is the amount of VCO loaded into the extraction vessel.
2.6. α-mangostin with HPLC
and small intestinal digestion model for the extracted MPE were carried
out using the methods adapted from Bumrungpert et al. [29] and Goh
et al. [30]. The MPE solution was prepared by mixing 32 mL of 0.9%
NaCl to 0.2 g of MPE, followed by homogenisation and acidification to
pH 2.0 with HCl. Simulation of the gastric digestion was carried out by
adding pepsin into the MPE solution followed by incubation for 1 h at
37 °C. Simulation of intestinal digestion was done by adding pan-
creatin/bile extract solution and bile extract to the MPE solution (ad-
justed to pH 5.3). The pH of the mixture was further adjusted to 7.5 and
incubated for 2 h at 37 °C. Micellar fraction was obtained from the su-
pernatant after the digesta was centrifuged (×5000g, 4 °C, and 45 min)
and filtered (0.22 μm). Extraction of α-mangostin from the micellar
fraction was done by adding in 1.5 mL of ethyl acetate followed by
centrifugation at ×2000g for 5 min. The organic layer was collected
and the extraction step with ethyl acetate was repeated for another
time. The solvent was then evaporated under reduced pressure at 40 °C
to obtain α-mangostin. Concentration of α-mangostin was then quan-
tified with HPLC as in Section 2.6.
The bio-accessibility of α-mangostin was calculated from Eq. (5):
Bio-accessibility = (BCdialysed/ BCnon-digested) × 100% (5)
where, BCdialysed and BCnon-digested correspond to the α-mangostin con-
centration (mg/g) in the micellar fraction and non-digested MPE, re-
spectively.
(1)
2.8. Statistical analysis
Analysis of variance (ANOVA) was performed followed by Tukey
HSD post hoc test using Minitab 16 statistical software (Minitab Inc.,
Pennsylvania). The limit of probability of significance was fixed at
p < 0.05.

The α-mangostin was quantified with an HPLC system (Agilent
Technologies 1200). The HPLC system was equipped with two precision
pumps and the chromatographic separations were performed using a
SGE RP-18 (146 × 4.6 mm, 5 μm) with UV–vis detector. Samples
(20 μL) of α-mangostin were analysed at wavelengths of 245 nm and
319 nm, with mobile phase of (A) 0.03% orthophosphoric acid in water
and (B) acetonitrile/methanol (75:25 v/v), being delivered at 0.8 mL/
min with gradient elution [28]. The gradient elution was as follows:
0 min, 25% A and 75% B; 0–15 min, 10% A and 90% B; (15 to 30) min,
100% B; (30 to 35) min, 25% A and 75% B. The α-mangostin present in
samples was calculated on the basis of a calibration curve using α-
mangostin standard that had a correlation coefficient of R2 = 0.9994
(Supplementary Materials, Fig. S1).
The α-mangostin yield and selectivity were calculated from the
following equations:
3. Results and discussion
3.1. Dissolution tests with solvents and oils
Table 1 shows results of the dissolution tests of MPE for ethyl
acetate, ethanol and n-hexane. MPE was highly-soluble in both ethyl
acetate (instantaneous dissolution) and in ethanol at 30 °C whereas the
MPE had very low solubility in n-hexane at 30 °C and only slightly
dissolved in n-hexane at higher temperatures of 60 °C and 70 °C.
Kamlet-Taft parameters can be used to explain the mechanism of dis-
solution of MPE in different solvents, expressed as dipolarity/polariz-
ability (π*), hydrogen-bond acidity (α) and hydrogen-bond basicity (β)
as presented in Table 1. It can be suggested that the hydrogen-bond
basicity (β) and dipolarity/polarizability (π*) are able to be used to

α-mangostin yield = mα-mangostin (g)/ mmp (g) × 100% (2) Table 1

Dissolution of mangosteen pericarp extract (MPE) in different solvents.
α-mangostin selectivity = mα-mangostin (g)/ (mMPE–mα-mangostin) (g) × 100%
Solvent Kamlet-Taft parameters Solubility characteristics
(3)
α β π*
where, mα-mangostin is the mass of α-mangostin, mmp is the mass of
mangostineen pericarp used for extraction, and mMPE is the amount of Ethyl acetate 0.00 0.45 0.55 a
Full dissolution at 30 °C
MPE collected from the scCO2 extraction. (instantaneous)
a
The co-extractant effectiveness was calculated from Eq. (4): Ethanol 0.86 0.75 0.54 Full dissolution at 30 °C
a
Hexane 0.00 0.00 0.04 No noticeable dissolution at 30 °C
b
Co-extractant effectiveness = mα-mangostin (g)/ mVCO (g) × 100% (4) Slight dissolution at 60 °C and
70 °C
where, mα-mangostin is the mass of α-mangostin and mVCO is the amount c
Slight dissolution at 60 °C and
of VCO used for the extraction. 70 °C

α: hydrogen bond donor; β: hydrogen bond acceptor; π*: dipolarity/polariz-

2.7. In-vitro gastrointestinal digestion ability.
Kamlet-Taft parameter was adapted from Weerachanchai et al. 2014 [49].
The simulation of an oral phase was neglected because samples are a
MPE:solvent ratio of 0.2:4.
in liquid state, thus their residence time in the mouth is negligible. b
MPE: solvent ratio of 0.1:4.0.
Simulations on the two phases in-vitro approach of the human stomach c
MPE:solvent ratio 0.05:4.0.

3
W.J. Lee, et al. Process Biochemistry xxx (xxxx) xxx–xxx

Table 2 probably due to their high concentration of medium-chain fatty acids;

Assessment of vegetable oils to dissolve mangosteen pericarp extract (MPE). i.e. 45% to 53% in VCO and 45% to 55% in PKO [31]. Oils that are rich
Dissolution characteristics of MPE for various MPE:oil ratios at 60 °C to 98 °C. in medium chain fatty acids are more polar and hydrophilic than oils
Vegetable oil Ratio of MPE: vegetable oil Temperature (°C) high in long chain fatty acids [32] such as the SO, RBO, GSO and CO.
The MPE which contains high concentration of xanthones (slight polar
60 70 80 90 98 structure) therefore, was able to readily dissolve in VCO or PKO. An-
other possible reason affecting the dissolution of MPE is oil viscosity.
Rice bran oil 0.2:4 – – – – √
0.1:4 – – – √ √ For high oil viscosity, the mass transfer coefficient of the target com-
Sunflower oil 0.2:4 – – – – √ pound into the oil is low. For a given temperature, the viscosities of
0.1:4 – – – √ √ VCO and PKO were lower than other vegetable oils. For instance, at
Olive oil 0.2:4 – – – √ √
50 °C, VCO had a viscosity of 19 mPa⋅s, followed by PKO (20 mPa⋅s),
0.1:4 – – – – √
Red palm oil 0.2:4 – – – – √
sunflower oil (26 mPa⋅s) and olive oil (30 mPa⋅s) [33]. The mass
0.1:4 – – – – √ transfer coefficient is influenced by the viscosity; oils with low viscosity
0.1:4 – – – √ √ (VCO and PKO) and similar chemical interactions with the solute will
Grape seed oil 0.2:4 – – – – √ generally have high dissolution rates.
0.1:4 – – – – √
Canola oil 0.2:4 – – – – √
0.1:4 – – – – √ 3.2. Extraction tests with co-extractant
Virgin coconut oil 0.2:4 – √ √ √ √
0.1:4 √ √ √ √ √ Extraction yield of MPE in the presence of VCO and PKO as co-
0.05:4 √ √ √ √ √
extractant (P = 35 MPa, T = 60 °C, co-extractant = 20% w/w) was
Palm kernel oil 0.2:4 – – √ √ √
0.1:4 – √ √ √ √
17% and 13%, respectively. One of the reasons for the higher extraction
0.05:4 √ √ √ √ √ yield for VCO was suggested to be due to the favorable dissolution
characteristics of MPE (0.1 g) in VCO, showing complete dissolution at
MPE: mangosteen pericarp extract (extracted with ethanol as in Section 2.3); √: 60 °C, whereas complete dissolution was only observed at 70 °C with
MPE dissolved completely; –: dissolved partially or did not dissolve. PKO (Table 1). Moreover, VCO has higher solubility in scCO2, 0.0309 g/
Solvent amount of 4 mL and MPE amount of 0.05 to 0.2 g; stirring time: 5 min. g CO2 at 34.5 MPa, 40 °C [34] compared to solubility of PKO, 0.0285 g/
g CO2 [35] at similar operating conditions, contributing to the higher
distinguish between the solubility characteristics of MPE and as a cri- extraction yield. Thus, the VCO was selected for the subsequent ex-
tical key to determine appropriate solvents for MPE extraction. Full traction processes as the most suitable co-extractant for MPE and α-
MPE dissolution was attained with solvents having β values in the range mangostin.
of 0.45 to 0.75 and π* values in the range of 0.54 to 0.55. The MPE was
not able to dissolve in hexane due to the low β and π* values of hexane. 3.3. Extraction of MPE and α-mangostin
The dissolution of MPE in vegetable oils at different temperature is
shown in Table 2. The effect of oil to MPE ratio on the dissolution was Table 3 shows the extraction yield of MPE and α-mangostin ob-
tested and it was found that at higher ratios of MPE to vegetable oil, tained from scCO2 and solvent extraction. The scCO2 extraction yield
higher temperatures were needed for complete dissolution for all types ranged from 1% to 17%, α-mangostin yield of 0.6% to 9%, α-mangostin
of vegetable oils. The viscosity of vegetable oil decreases with in- selectivity of 2% to 9% and, co-extractant effectiveness of 1% to 9%.
creasing temperature and promotes mass transfer and hence, high dis- The highest yield and α-mangostin concentration were obtained at
solution rates of MPE in the vegetable oil occurs at higher temperatures. 35 MPa, 20% VCO (Run 8), that contained 92 mg/g mangosteen peri-
For comparison, the dissolution of MPE in different vegetable oils was carp. The concentration of α-mangostin was higher than the results
examined whereas MPE showed complete dissolution in both VCO and obtained from the literature reported as xanthone [6,9,36]. The results
PKO at lower temperatures (ca. 70 °C) while higher temperatures (ca. obtained from this work confirmed the ability of VCO as a co-extractant
90 °C and 98 °C) were required for other type of oils to dissolve MPE to enhance the extraction of α-mangostin. However, the α-mangostin
completely. The ability of MPE to dissolve in both VCO and PKO was concentration was still lower in this work in comparison to the work

Table 3
Yields of α-mangostin obtained from mangosteen pericarp extract (MPE) using supercritical carbon dioxide extraction (60 °C, 1.7 g/min flow rate) and virgin coconut
oil (VCO) co-extractant. Results obtained from MPE for organic solvent extraction are shown for comparison.
Runa Pressure VCO (%, MPE (g) Extraction α-mangostin α-mangostin α-mangostin
(MPa) w/w) yield (%) (mg/g pericarp) yield (%) selectivity (%)

1 30 0 – – – – –
2 5 0.29 ± 0.03 5.56 ± 0.50 6.12 ± 0.14 0.61 ± 0.01 2.14 ± 0.15
3 10 0.34 ± 0.06 6.12 ± 1.01 9.41 ± 0.22 0.94 ± 0.02 2.87 ± 0.43
4 20 0.69 ± 0.03 11.45 ± 0.51 15.60 ± 0.87 1.56 ± 0.09 2.32 ± 0.18
5 35 0 – – – – –
6 5 0.27 ± 0.01 5.16 ± 0.22 20.86 ± 0.94 2.09 ± 0.09 8.35 ± 0.22
7 10 0.63 ± 0.14 11.49 ± 2.47 35.16 ± 0.75 3.52 ± 0.08 5.89 ± 1.10
8 20 1.05 ± 0.15 17.55 ± 2.55 92.49 ± 2.09 9.25 ± 0.21 9.63 ± 1.39
9 40 0 0.05 ± 0.01 1.09 ± 0.20 – – –
10 5 0.23 ± 0.01 4.30 ± 0.11 16.11 ± 0.81 1.61 ± 0.08 7.69 ± 0.36
11 10 0.38 ± 0.02 6.84 ± 0.31 22.83 ± 0.20 2.28 ± 0.02 6.46 ± 0.38
12 20 0.88 ± 0.11 14.63 ± 1.84 34.02 ± 1.07 3.40 ± 0.11 4.03 ± 0.68
Hexane extraction (at 68 °C) 0.15 ± 0.04 3.05 ± 0.72 11.53 ± 0.75 1.15 ± 0.08 8.18 ± 1.97
Ethanol extraction (at 78 °C) 1.90 ± 0.12 38.09 ± 2.30 21.75 ± 0.40 2.18 ± 0.04 1.16 ± 0.05
Ethyl acetate extraction (at 77 °C) 2.02 ± 0.08 40.36 ± 1.67 36.65 ± 0.86 3.66 ± 0.09 1.85 ± 0.04

a
Total extractable α-mangostin was taken to be 121 mg/g pericarp (Source: Ghasemzadeh et al. [50]).

4
W.J. Lee, et al.
done by Zarena et al. [3] which employed ethanol (5% w/w) as co-
solvent in scCO2 extraction, recording xanthone yields of 14.8%. De-
spite the lower extraction yields, VCO as co-extractant is still a great
alternative to using ethanol from the point of food processing, since it
avoids contamination issues. VCO can have desirable properties to the
end product such as enhancing the bio-accessibility of α-mangostin
(Section 3.4) and does not have to be removed from the extract.
Ethanol, when used as co-solvent, must be removed from the final
product, which increases production cost.
3.3.1. Co-extractant
Without co-extractant (VCO = 0%), the MPE yield was below the
weighable limit (Run 1 and 5) and the concentration of α-mangostin
was below detection limit (Run 1, 5 and 9). Table 2 shows by increasing
the amount of VCO from 0% to 20%, the extraction yield of MPE in-
creased for the pressures of 30 MPa, 35 MPa and 40 MPa. The increment
in the yield was contributed by the higher available amount of VCO to
be co-extracted during the extraction process. The extraction yield of α-
mangostin also increased with high amount of VCO. Similar observa-
tions were made by Shi et al. [20] whereby the extraction yield of
carotenoid increased from 4.2 mmol/kg to 4.73 mmol/kg (∼10% in-
crement) with increasing olive oil concentration of 5% to 15%.
3.3.2. Effect of pressure
The concentration of α-mangostin increased when the operating
pressure was raised from 30 MPa to 35 MPa, however, at 40 MPa, the
concentration decreased. The increased in solvation power when the
density of scCO2 increased at high pressures allowed for high solubility
of solute. For the case of α-mangostin, it was postulated that the re-
pulsive solute–solvent interactions became large which lowered the
solubility in scCO2 [21] and thus, lower concentration was obtained at
40 MPa. This observation is consistent to the reported findings by Ma
et al. [21], whereby the extraction yield of lutein esters started to de-
cline beyond pressure of 30 MPa.
3.3.3. Comparison with solvent extraction
Table 2 shows a comparison between the extraction yield obtained
from scCO2 extraction (Table 2, Run 8) (scCO2 extracted MPE) and from
solvent extraction; ethanol-extracted MPE, hexane-extracted MPE and
ethyl acetate-extracted MPE. The yield of ethyl acetate-extracted MPE
was the highest (40.4%), followed by ethanol-extracted MPE (38%),
scCO2-extracted MPE (17%) and lastly, hexane-extracted MPE (3%). It
was postulated that one of the main reasons that affect extraction yield
of MPE was solvent polarity according to the β and π* Kamlet-Taft
parameters as discussed in Section 3.1. Ethyl acetate had the highest β
and π* values followed by ethanol, hence, high extraction yields were
recorded for those two solvents. As for the scCO2, it was reported in the
literature that it had α value of 0, β value of 0.01, and π* of −0.03
(100 MPa, 60 °C) [37]. With the aid of VCO as co-extractant, it was
postulated that the β and π* of scCO2 was modified and recorded a
higher extraction yield compared with that of hexane extraction.
Therefore, the importance of addition of VCO as co-extractant was
demonstrated. Although both ethyl acetate and ethanol showed higher
MPE yield than scCO2, the selectivity of α-mangostin was lower for li-
quid solvents compared with that of scCO2-MPE. Solvent extracts often
contain large quantities of matrix organics that act as product im-
purities that may or may not affect bioactivity.
Fig. 2 shows HPLC chromatograms of the extracts obtained from
scCO2 extraction and solvent extractions. Original HPLC chromato-
grams are provided in supplementary materials (Fig. S2). As shown
(Fig. 2), α-mangostin was the main peak in all the MPE analyses, ap-
pearing at a retention time of around 2.9 min. A comparison of the
chromatogram obtained in this work with the work by Zarena et al. [28]
was carried out to define the unidentified peaks in the chromatograms.
Other than α-mangostin, the scCO2-extracted MPE potentially contains
8-deoxygartanin and gartanin; ethanol-extracted MPE could contain 3-
5
Process Biochemistry xxx (xxxx) xxx–xxx
isomangostin hydrate, γ-mangostin, 8-deoxygartanin, gartanin, and α-
mangostin; and hexane-extracted MPE could contain γ-mangostin, 8-
deoxygartanin, gartanin and α-mangostin. Ethyl acetate was not con-
sidered since MPE had high solubility in the solvent and many peaks
would appear. Further work could be carried out to identify the pre-
sence of bioactive components in each of the extracts.
3.4. In-vitro bio-accessibility of α-mangostin
Although ethyl acetate had favorable extraction characteristics for
MPE, the in-vitro bio-accessibility study using ethyl acetate extracted
MPE was not performed due to consideration of the Globally
Harmonized System (GHS) and its compatibility with functional food
products.
The in-vitro bio-accessibility of α-mangostin obtained from scCO2
extraction (Run 8) and the solvent extraction are shown in Fig. 3. The α-
mangostin from scCO2 extracted MPE showed the highest bio-accessi-
bility (91%) followed by α-mangostin from ethanol-extracted MPE
(47%) and α-mangostin from hexane-extracted MPE (5%). The α-man-
gostin in scCO2 extract could be considered almost fully digested and
reached a complete bio-accessibility through in-vitro gastrointestinal
digestion as it reached more than 90% bio-accessibility of the amount
ingested. Hexane-extracted MPE mainly contained non-polar hydro-
phobic compounds that were lipophilic, thus it had low solubility in the
aqueous solution and remained intact that caused low bio-accessibility.
As for scCO2 extracted MPE, the high bio-accessibility could be due to
the scCO2 affecting the molecular properties of compounds and directly
affecting the release of bioactive compounds and its bio-accessibility
through gastrointestinal digestion. A recent study disclosed that lipo-
philic compounds (terpenes) in marigold supercritical extracts showed
high bio-accessibility (> 75%) due to the significant correlation be-
tween molecular properties of compounds, such as molecular flexibility
and lipophilicity with their bio-accessibility [38].
To demonstrate the importance of VCO in enhancing the bio-ac-
cessibility of α-mangostin, the bio-accessibilities of α-mangostin from
ethanol-extracted MPE and hexane-extracted MPE in the presence of
20% VCO were tested, recording values of 67% and 11% (Fig. 2), re-
spectively. It was evident that with the addition of VCO, the bio-ac-
cessibilities of α-mangostin from ethanol-extracted MPE and hexane-
extracted MPE increased significantly. The capability of α-mangostin to
be taken up during digestion is hindered due to the lipophilic molecular
structure which has limited solubility in an aqueous micelle [39,40].
The VCO acts as a key positive effector by promoting the formation and
incorporation of the α-mangostin into micelles before absorption
[41,42]. Similar observations on enhanced bio-accessibility and bioa-
vailability of α-mangostin or xanthone in the presence of lipid have
been reported [29,43,44].
3.4.1. Effect of consumption quantities
The influence of consumption quantities of the scCO2-extracted MPE
in the gastrointestinal digestion was investigated. From previous
Section 3.4, the consumption of 0.2 g of scCO2-extracted MPE showed
high bio-accessibility of α-mangostin (91%). As the consumption
quantity increased to 0.5 g, 1.0 g and 3.0 g, the bio-accessibility of α-
mangostin decreased to 66%, 27% and 18%, respectively. Beyond
consumption of 0.2 g of extracts, the α-mangostin was postulated to
remain intact and dissolve only within the undigested oil (upper phase)
after the in-vitro gastrointestinal digestion steps. The absorption of
scCO2-extracted MPE was substantially related to the lipid digestion
mechanism, whereby the rate and extent of digestion were affected by
the decrease in the (1) ratio of lipase: oil in the digesta and (2) the ratio
of bile salt: oil. The decrease in lipase and bile salt or the increment of
oil content slows down the dissolution of fatty acid released during
digestion which inhibits the process of lipid lipolysis [45,46]. Results
were supported by previous studies on the bioactive components re-
lease and lipid hydrolysis of the lipophilic bioactive compounds such as
W.J. Lee, et al. Process Biochemistry xxx (xxxx) xxx–xxx

Fig. 2. Analyses of co-extractant, raw materials, and extracts made with high performance liquid chromatography (HPLC). Panels (A): virgin coconut oil (VCO), (B):
α-mangostin standard (retention time at 2.9 min), (C): mangosteen pericarp extract (MPE) extracted using supercritical carbon dioxide with VCO as co-extractant,
(D): ethanol-extracted MPE and (E): hexane-extracted MPE.

β-carotene, phytosterols, vitamin D3, curcumin and coenzyme Q10
[47,48].
4. Conclusions
The extraction yield of α-mangostin from mangosteen pericarp ex-
tract (MPE) with supercritical CO2 was enhanced with the addition of
virgin coconut oil (VCO) as co-extractant to MPE. At extraction con-
ditions of 35 MPa, 60 °C, and VCO concentration of 20% (w/w), high
yields of α-mangostin were obtained and the supercritical CO2 extracted
α-mangostin had high bio-accessibility (91%). The presence of VCO
enhanced the bio-accessibility of α-mangostin by 30% in ethanolic MPE
and by 50% in hexane-extracted MPE. The use of VCO has the potential
to replace organic solvents (i.e. ethanol) in extracting xanthones or
specifically α-mangostin from mangosteen pericarp. Future work in
determining the application of VCO in SFE to other types of xanthones
is of interest, because many of these compounds have valuable phar-
macological properties.

6
W.J. Lee, et al.
Acknowledgment
This work was supported by Geran Putra (GP-PI/2016/9481200),
Universiti Putra Malaysia. Gratitude is expressed to Adirondack (M)
Sdn. Bhd (Selangor, Malaysia) for the free supply of dried mangosteen
pericarp and virgin coconut oil that were used in this work. The authors
acknowledge collaborative effort on the project through the MoU be-
tween Department of Food Technology, Supercritical Fluid Centre
(Universiti Putra Malaysia) and Research Center of Supercritical Fluid
Technology (Tohoku University).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.procbio.2019.09.009.
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