Food Chemistry 130 (2012) 203–208

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Optimisation of ethanol modified supercritical carbon dioxide on the extract

yield and antioxidant activity from Garcinia mangostana L.
A.S. Zarena a, N.M. Sachindra b, K. Udaya Sankar a,⇑
a
Food Engineering Department, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570020, India
b
Meat Fish and Poultry Technology, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570020, India

a r t i c l e i n f o a b s t r a c t

Article history: Xanthones are an important class of secondary metabolites present in mangosteen fruit pericarp. Herein
Received 2 March 2011 we have used supercritical fluid technology to extract the active constituents from mangosteen pericarp.
Received in revised form 10 June 2011 Ethanol was added (5% w/w) as a co-solvent to increase the polarity of the CO2, thus favouring the extrac-
Accepted 5 July 2011
tion of xanthones. The maximum extraction yield of 15 wt.% was achieved at a pressure of 280 bar, tem-
Available online 14 July 2011
perature of 50 °C and a time of 8 h, while without co-solvent the yield was 7.5 wt.%. Conditions for
antioxidant activity as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay
Keywords:
was 280 bar at 50 °C and 5 h. Box–Behnken design was used to study the efficiency of extraction pressure
Supercritical extraction
Mangosteen
(180, 280, and 380 bar), temperature (40, 50, and 60 °C) and time (2–8 h) on the total extraction yields
Antioxidant activity and their radical scavenging activity. Experimental results of the total extract yield and radical scaveng-
Response surface methodology ing activity were close to the predicted values calculated from the polynomial response surface models
Xanthones equations (R2 = 0.99 and 0.98).
UV–Vis spectra Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction possess excellent extractive properties such as high compressibil-

The consumption of mangosteen (G. mangostana L.) products
has increased as dietary supplement because of their potential
pharmaceutical property. To date there are more than 50 xanthon-
es isolated from mangosteen pericarp (Pedraza-Chaverri,
Rodríguez, Orozco-Ibarra, & Pérez-Rojas, 2008). Xanthones being
relatively polar compounds; a higher concentration of organic sol-
vent and higher temperature are needed to guarantee a better
extraction performance in Soxtec™ method (Zarena & Udaya
Sankar, 2009a). Classical solvent extraction techniques are well-
known. However, these methods are time-consuming and tedious.
Also large amounts of high purity solvents, which are expensive
and toxic, are inevitable to be used adding to extra cost.
There are various opinions on the type of solvents to be used for
better extraction. These differences may be due to the types of phe-
nolic compounds in the plant materials. In general, a good balance
in polarity is needed in extracting phenolics from plant sources
(Horax, Hettiarachchy, & Chen, 2010). However; the use of organic
solvent is quite harmful to human health and environment, so its
use in food and pharmaceutical industries is restricted. Supercriti-
cal carbon dioxide (SC-CO2) is becoming popular as a cost effective
and environmentally friendly method for extracting useful compo-
nents (Mitra, Ramaswamy, & Chang, 2009). Regarding the extrac-
tion of antioxidants, SC-CO2 has received considerable attention
(Abbas, Mohamed, Abdulamir, & Abbas, 2008). Supercritical fluids
⇑ Corresponding author. Tel.: +91 821 2514874; fax: +91 821 2517233.
E-mail address: udaya@cftri.res.in (K. Udaya Sankar).
ity, liquid-like density, low viscosity and high diffusivity. CO2 is
an abundant, inert, non-toxic solvent and is readily acceptable in
food industry.
In our earlier study we reported that ethanol modified extract
gave a higher yield (Zarena & Udaya Sankar, 2009b). Herein we have
used different temperature and pressure for the extraction of active
components in the presence of co-solvent. The composition of the
extracts was determined by HPLC. Optimisation of the operating
conditions represents a critical step in the development of SC-CO2
extraction method. Box–Behnken design was selected as response
surface designs, as it provides the minimum number of experiments
(Lahlali, Massart, Serrhini, & Jijakli, 2008), a shorter time of operation
and feasibility in providing valuable information on the interactions
among experimental parametres (Gong, Zhang, Xu, Wei, & Lee,
2007). In the present study, three operating parametres pressure,
temperature and time were investigated on the total extraction
yields and radical scavenging activity. Furthermore, the antioxidant
activity of extracts, obtained under optimised SC-CO2 conditions
was determined by means of the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical-scavenging assay.
2. Materials and method
2.1. Materials
Carbon dioxide (purity 99.9%, M/S Kiran Corporation, India), eth-
anol (double distiled), acetonitrile, methanol, orthophosphoric acid
was from Merck (Mumbai, India). 2,2-diphenyl-1-picrylhydrazyl

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.07.007
204 A.S. Zarena et al. / Food Chemistry 130 (2012) 203–208

(DPPH) were purchased from Sigma–Aldrich (Steinheim, Germany). 2.4. HPLC–ESI/MS

All other reagents were of analytical grade.
HPLC analysis for the identification of xanthones was performed
on SGE RP-18 (146  4.6 mm, 5 lm particle sizes) column using
2.2. Extraction by supercritical carbon dioxide
Shimadzu LC 10A (Japan) with PDA detector (SPD–M 10 Avp,
Shimadzu, Japan). Class 10 software was utilised for instrument
Extractions were carried out in NOVA Swiss equipment (WERKE
control, data collection and data processing. UV–visible spectra
AG, EX 1000, 1.4–1.2 V type, Switzerland) using a mixture of
were simultaneously recorded from 200 to 600 nm during sample
CO2 + 5% ethanol in a stainless cylindrical extractor provided with
running. Reference samples of 8-deoxygartanin, gartanin, a-
a pump (Milton Roy, USA). The percentage of modifier was
mangostin, b-mangostin, 9-hydroxycalabaxanthone were isolated
adjusted by controlling the ethanol flow rate. In the present work,
and confirmed by UV, MS, 1H NMR and 13C NMR data. Garcinone
the modifier concentration was kept constant at 5%, since preli-
C; 3-isomangostin hydrate; c-mangostin, garcinone E, tovophyllin
minary trials (not reported here) showed 5% ethanol to be most
A were separated and purified in the same key laboratory and con-
suitable. A detailed description of the instrument and extraction
firmed by UV, MS and with the data from the literatures.
process is given extensively elsewhere (Zarena, Manohar, & Udaya
Six experimental points were employed for establishing a cali-
Sankar, 2010). Hundred grams of dried powdered mangosteen
bration curve for the different xanthones presented in the extract.
pericarp was weighed and loaded into the extraction vessel. Car-
The curves were linear with correlation coefficients (R2) varying
bon dioxide from a CO2 liquid cylinder was pressurised and
from 0.981 to 0.994, respectively. For quantitative determination,
pumped into the system. A flow metre was used for continuous
peak areas in the sample chromatograms were correlated with the
measurement and each extraction run lasted for 8 h. The flow rate
concentrations according to the calibration curve. Peaks without
of CO2 varied between 1.2–2.6 kg/h. Depending on the suction
standards were tentatively identified by ESI-MS. LC analysis was
pressures the flow rates tended to be higher. Ethanol was removed
performed using Waters Alliance system 2695 separation module
from the extracts using a rotavapor (RE111, Buchi, Switzerland).
with auto sampler and Waters 2996 Photo diode array detector
Samples were collected at specific intervals of time (2, 5, and
(USA). The ESI-MS of the extracts were obtained with a negative
8 h) and the extracts obtained at the different conditions were
operating mode. A detailed description of the gradient mobile phase
tested for the antioxidant activity.
and methodology for the analysis is reported by Zarena et al. (2010).

2.3. DPPH free radical scavenging assay

The antioxidant activity of the extracts was measured on the
basis of the scavenging activity of the stable radical DPPH accord-
ing to the method of Wang, Yen, Ling, and Wu (2003). The extracts
at different concentration range (20–100 lg) were mixed in the
freshly prepared 0.5 mM DPPH in ethanol and 0.1 M acetate buffer
(pH 5). Absorbance at 517 nm was determined after 30 min. The
scavenging activity was calculated using the following equation.
 
ðA517 of control  A517
%DPPH scavenging activity ¼
A517 of control
 100
The percentage of scavenging activity was plotted against the
sample concentration to obtain EC50 (effective concentration) de-
fined as the concentration of sample necessary to scavenge 50%
of the DPPH radicals. BHT and a-tocopherol were used as reference
antioxidants. The EC50 values of BHA and a-tocopherol were 20.2
and 24.0 lg/mL, respectively.
2.5. Experimental design and statistical analysis
A Box–Behnken (1960) design with three variables was used to
analyse the response pattern and to establish a model. The three
independent variables used in the study were pressure: 180, 280,
and 380 bar (X1); temperature: 40, 50, and 60 °C (X2) and time: 2,
5, and 8 h (X3), with three levels for each variable, while the depen-
dent variable was the total extraction yield and DPPH radical scav-
enging activity. A total of 15 experiments were conducted to
of sample Þ optimise the extraction conditions. The extractions were carried
in the presence of co-solvent (5% w/w EtOH). The factors, experi-
mental and predicted data obtained are shown in Table 1.
The results of each design were analysed using the software
Statistica™ version 5 (Statsoft, city, USA). Both linear and quadratic
effects of each of the variables (factors) under study, as well as
their interactions and their significance were evaluated by analysis
of variance.
In developing the regression equation, the test variables were
coded according to the equation:

Table 1
Box–Behnken design matrix along with the experimental and predicted values of total extraction yield and DPPH radical scavenging activity.

Run No. X1 X2 X3 Observed yield% (w/w) Predicted yield% (w/w) Observed DPPH EC50 (lg/mL) Predicted DPPH EC50 (lg/mL)
1 180 (1) 40 (1) 5 (0) 7.41 7.37 47.2 49.0
2 380 (1) 40 (1) 5 (0) 9.98 9.98 37.1 37.9
3 180 (1) 60 (1) 5 (0) 5.88 5.56 46.2 46.4
4 380 (1) 60 (1) 5 (0) 9.89 9.93 34.8 33.0
5 180 (1) 50 (0) 2 (1) 5.88 5.56 46.2 46.4
6 380 (1) 50 (0) 2 (1) 6.28 5.82 38.6 39.8
7 180 (1) 50 (0) 8 (1) 11.48 11.94 43.9 42.7
8 380 (1) 50 (0) 8 (1) 14.82 15.14 26.9 26.7
9 280 (0) 40 (1) 2 (1) 5.01 5.37 39.1 37.1
10 280 (0) 60 (1) 2 (1) 5.09 5.51 27.8 28.4
11 280 (0) 40 (1) 8 (1) 12.95 12.53 25.1 24.6
12 280 (0) 60 (1) 8 (1) 14.41 14.05 21.9 23.9
13 280 (0) 50 (0) 5 (0) 13.32 13.21 14.0 15.2
14 280 (0) 50 (0) 5 (0) 13.10 13.21 15.3 15.2
15 280 (0) 50 (0) 5 (0) 13.22 13.21 16.4 15.2

X1, pressure (bar); X2, temperature (°C); X3, time (hrs).

The coded values are indicated in the parenthesis.
 A.S. Zarena et al. / Food Chemistry 130 (2012) 203–208 205

X i  X CP Table 3
Xi ¼ Analysis of variance (ANOVA) of the regression parametres.
DX i
Factors/interactions SS df MS F
where xi is the independent variable coded value. Xi is the indepen-
dent variable real value. Xcp is the independent variable real value at Total extract yield
**
Pressure (X1) 5.96 1 5.96 23.06
the centre point and DXi is the step change of the real value of the ***
Pressure ðX 21 Þ 13.83 1 13.83 53.47
variable ‘i’ corresponding to a variation of a unit for the dimension-
Temperature (X2) 1.38 1 1.383 5.34 NS
less value of the variable ‘i’. Temperature ðX 22 Þ 17.62 1 17.62 68.13 ***

A statistically significant multiple regression relationship Time (X3) 123.28 1 123.28 476.52 ***

between the independent variables (X1, X2 and X3) and the re- Time ðX 23 Þ 10.20 1 10.20 39.44 **

sponse variable (Y) was established. A second order polynomial X1X2 0.78 1 0.78 3.02 NS
*
model of the design was fitted to evaluate the yield (response var- X1X3 2.16 1 2.16 8.35
X2X3 0.47 1 0.47 1.82
iable, Y) as a function of independent variables (X) and their
Error 1.29 5 0.25
interactions. Total SS 171.57 14
R2 0.99
Y ¼ B0 þ B1 X 1 þ B2 X 2 þ B3 X 3 þ B11 X 21 þ B22 X 22 þ B33 X 23 þ B12 X 1 X 2 Adjusted R2 0.97
MS residual 0.25
þ B23 X 2 X 3 þ B13 X 1 X 3 þ e
DPPH activity
B0 is the constant coefficient, B1, B2, B3 are the linear coefficients, Pressure ðX 1 Þ  ðX 21 Þ 1471.15 2 735.57 163.46 ***

B11, B22, B33 are the quadratic coefficients, B12, B13 and B23 are the Temperature ðX 2 Þ  ðX 22 Þ 263.81 2 131.90 29.31 **

**
interaction coefficient, respectively of the model and X1, X2 and X3 Time ðX 3 Þ  ðX 23 Þ 256.19 2 128.09 28.46
were the independent variables and e is the random error. X1X2 0.04 1 0.04 0.008 NS
X1X3 22.09 1 22.09 4.90 NS
X2X3 16.40 1 16.40 3.64 NS
3. Results and discussion Error 22.49 5 4.49
Total SS 1918.50 14
Response surface methodology was used to study the effect of R2 0.98
Adjusted R2 0.96
SC-CO2 extraction on total extract yield and radical scavenging
MS residual 4.49
activity of mangosteen pericarp. Table 2 shows the polynomial
functions of total extract yield and antioxidant activity. The devel- SS, sum of square; df, degree of freedom; MS, mean square; F, F ratio; NS, non-
significant.
oped regression model for the relationship between dependent *
Significant at 0.05.
variable and the coded values of independent variables of pressure **
Significant at 0.01.
(X1), temperature (X2) and time (X3) and their interaction is shown ***
Significant at 0.001.
in the following equation Eq. (1) for total extraction yield & Eq. (2)
for radical scavenging activity.
the behaviour of variations. All the quadratics of the process vari-
Y ¼ 72:1485 þ 0:1269X 1 þ 1:8968X3  0:0001X 21  0:0218X 22 ables namely pressure, temperature (p 6 0.001) and time
(p 6 0.01) showed significant fit. Pressure (p 6 0.01) and time
 0:1847X 23 þ 0:0024X 13 ð1Þ
(p 6 0.001) significantly affected the extraction yield in a linear
Y ¼ 404:9315  1:0285X 1  8:2640X 2  8:7284X 3  0:0018X 21 manner. The interaction between pressure and time (X1X3) was po-
þ 0:0772X 22 þ 0:6134X 23 ð2Þ sitive and significant at p 6 0.01 indicating a synergetic effect ex-
isted between pressure and time on the extraction yield.
The adequacy of the models was evaluated by coefficient of The 2D contour plots were obtained using Eq. (1) by presenting
determination R2 and adjusted R2 values. The analysis of variance the response as a function of two factors and keeping the third one
(ANOVA) shown in Table 3 indicates a good model fitting with an at the constant mid-level. Fig. 1A–C shows the contour plots of the
R2 value of 0.99 and adjusted R2 of 0.97 among linear, quadratic, total extract yield. The elliptical contours are obtained when there
cross-product of the total model. The adjusted R2 is a measure of is a perfect interaction between the independent variables
the amount of variation about the mean explained by the model (Muralidhar, Chirumamila, Marchant, & Nigam, 2001). The total
and R2 is defined as the ratio of the explained variations to the total yield varied markedly from 5.0% to 14.8 wt.% with the different
variation and is a measure of degree of fitness. Guan and Yao (2008) levels of the factors.
reported that R2 should be at least 0.80 for the good fit of a model. On From Fig. 1A it is evident that increase in extraction time from 5
the other hand the smaller the value of R2, the lesser will be the to 8 h and pressure from 250 to 380 bar within the studied range
relevance the dependent variables in the model have in explaining yielded a maximum yield of 13–15 wt.% indicating higher extrac-
tion time and elevated pressures facilitate solvent penetration
Table 2
Regression coefficient of polynomial functions of response surfaces of total extract through the interior of the sample matrix to the outer solvent sur-
yield and antioxidant activity. face thereby increasing the mass transfer rate (Zhang et al., 2009).
Similarly in Fig. 1B at pressure range from 230 to 370 bar and tem-
Factors/interactions Term Co-efficient (total Co-efficient
extract yield) (DPPH EC50) perature range of 45–58 °C the extraction yield increased to
12 wt.% but below or above this range the extract yield decreased.
Intercept B0 72.1485 404.9315
X1 B1 0.1269 1.0285 The possible reason may be that the solubility of the compounds
X2 B2 0.0001 0.0018 depends on the balance between SC-CO2 density and solute vapour
X3 B3 2.2932 8.2640 pressure which are controlled by fluid pressure and temperature.
X 21 B11 0.0218 0.0772 The increase in pressure from 180 to 380 bar results in an increase
X 22 B22 1.8968 8.7284
in CO2 density, increasing the solvating power of SCF. Thus higher
X 23 B33 0.1847 0.6134
pressure is responsible for quantitative yields and stronger interac-
X1X2 B12 0.0004 1E04
tion between the fluid and the matrix.
X1X3 B13 0.0024 0.0078
X2X3 B23 0.0114 0.0675 One of the important effects on extraction yield was exhibited
by the time of extraction or the amount of SC-CO2 passed through
206 A.S. Zarena et al. / Food Chemistry 130 (2012) 203–208
Fig. 1. Contour plot showing the effect of time and pressure (A), temperature and
pressure (B), time and temperature (C) on total extraction yield.
the extractor. Fig. 1C demonstrates the effect of temperature and
time on the total extract yield. The yield increased with increasing
time, and reached a maximum at 8 h within the tested range irre-
spective of the applied temperature indicating a sufficient contact
time is given for the active components in the solvent to reach
equilibrium thereby more xanthones were recovered (Bulley,
Fattori, Meisen, & Moyls, 1984). Similar results are also reported
for extraction of oil from kalahari melon seeds (Nyam, Tan, Lai,
Long, & Che Man, 2009).
In the present study 5% (w/w) ethanol was used in the extraction
and higher yields were obtained within the studied range of pressure
and temperature (280 bar and 50 °C). Even at high densities, CO2 has
a limited ability to dissolve high-polarity compounds therefore addi-
tion of modifiers to CO2 can improve the extraction efficiency by
increasing the solubility of the solute. There was a definite increase
in the yield of the extracts compared to our previous study (Zarena
et al., 2010) in which no modifier was used and temperature had
the predominate effect whereas in the present study we found den-
sity to be the more prominent factor. Considering the interactions
possible, the effects of co-solvents could be the result of several
mechanisms such as, increase in the mixture density, which
enhances the solubility as well as physical interactions like dipole–
dipole, dipole-induced dipole and induced dipole interactions (Ting,
Macnaughton, Tomasko, & Foster, 1993; Zarena & Udaya Sankar,
2009b). However, too high concentration of modifier may under-
mine the advantages of supercritical fluid extraction (Seo, Burri,
Quan, & Neidlinger, 2005) and may lead to ethanol saturation.
Search for optimum yield within the range of variables showed
higher yield above 15 wt.% could be obtained at temperatures of
50 °C and pressures of 280 bar and a time period of 8 h by solving
Eq. (1). The statistical analysis of the experiment indicated that
pressure (X1), extraction time (X3) and the interaction between
pressure and time (X1X3) had significant effect on the yields.
Table 3 also shows the analysis of variance (ANOVA) of the model
for DPPH activity. The high values of R2 = 0.98 and R2 adjusted = 0.96
of the model showed a close agreement between the experimental
results and the theoretical values. From the corresponding contour
plots (Fig. 2A–C), it is evident that the interactions among the vari-
ables were not significant. The linear and quadratic terms of pres-
sure were very significant at p 6 0.001 and temperature and time
showed significance at p 6 0.01 as evaluated by statistical analysis.
However according to Table 3 all the linear terms of the variable had
antagonistic effect on radical scavenging activity as the values were
negative but the quadratic terms were positive.
For the effect of experimental conditions on antioxidant activity
EC50 was used as an index, as it expresses the amount of antioxi-
dant needed to decrease the radical concentration by 50%. The low-
er the EC50 value, higher is the antioxidant activity of the tested
sample. Since EC50 value is negatively related to the antioxidant
activity, therefore the 2D plots were concave in surface. Fig. 2A
and B shows effect of operating time, temperature and pressure
for the extracts radical scavenging activity. The results indicate
as the extraction time increases from 4 to 8 h and at pressures from
250 to 340 bar and temperatures from 45 to 58 °C the EC50 value of
the extract was 18 lg/mL. In the predicted model, the temperature
did not have an important effect on the antioxidant activity as in
the case of total extract yield, indicating a predominant influence
of the solvent density over the effect of solute vapour pressure.
Fig. 2C depicts the effect of operating temperature and time for
the extract radical scavenging activity. Similar to the above para-
metres it indicates with an increase in temperature from 45 to
56 °C along with the extraction time from 4 to 8 h that the radical
scavenging activity showed an EC50 of 17 lg/mL. The results indi-
cate that solubility of active group is higher at the above tested
range of pressure and time which affects the extraction yield and
the radical scavenging activity of the extracts.
From the plots, it is evident that the radical scavenging activity is
sensitive to the quadratics of extraction pressure, temperature and
time. By solving the matrix with software design expert, the statis-
tical model gave the minimum point for antioxidant activity at the
optimum levels of 50 °C, 280 bar and 5 h. Under these conditions,
the maximum EC50 of the extracts was about 15 lg/mL. Beyond
6 h radical scavenging activity was less effective. The possible
UV–Vis spectra ranging from 200 to 350 nm.
retention time of the peak profile in comparison to ESI-MS and

all the three variables used within the tested range.

of antioxidant activity were obtained at the intermediate value of
ing activity are less extracted. The most desirable results in terms
reason may be that the components attributing to radical scaveng-

pressure (B), time and temperature (C) on DPPH scavenging activity.

Fig. 2. Contour plot showing the effect of time and pressure (A), temperature and
Individual xanthones were identified by HPLC based on the

A.S. Zarena et al. / Food Chemistry 130 (2012) 203–208

Table 4
Identification and quantification of the xanthones extracted by SC-CO2 + 5% w/w ethanol under different operational conditions.

Amount (% w/w)
Pressure (bar) Temperature (° C) 1 2 3 4 5 6 7 8 9 10 Total xanthone content
180 40 0.15 ± 0.01 0.80 ± 0.07 0.61 ± 0.03 0.71 ± 0.30 0.66 ± 0.04 40.23 ± 7.31 0.21 ± 0.01 0.92 ± 0.03 1.15 ± 0.09 0.66 ± 0.02 46.10 ± 7.91
280 40 0.33 ± 0.02 0.89 ± 0.09 3.48 ± 1.02 1.91 ± 0.92 1.57 ± 0.76 46.27 ± 11.21 0.43 ± 0.03 2..30 ± 0.42 2.91 ± 0.09 0..30 ± 0.04 60.39 ± 14.60
380 40 0.28 ± 0.02 0.72 ± 0.06 3.62 ± 0.92 1.01 ± 0.41 0.69 ± 0.09 41.01 ± 8.62 1.68 ± 0.14 1.14 ± 0.21 1.22 ± 0.04 0.31 ± 0.07 51.68 ± 10.58
180 50 0.31 ± 0.02 0.69 ± 0.04 3.33 ± 0.85 0.73 ± 0.03 0.96 ± 0.04 43.23 ± 9.21 1.86 ± 0.15 1.82 ± 0.18 1.29 ± 0.09 0.41 ± 0.12 54.63 ± 10.73
280 50 0.5 ± 0.04 0.76 ± 0.07 5.89 ± 1.51 3.24 ± 0.69 3.26 ± 0.88 51.09 ± 13.26 3.36 ± 1.69 4.85 ± 1.12 2.96 ± 0.98 0.37 ± 0.02 75.29 ± 20.26
380 50 0.44 ± 0.03 0.89 ± 0.08 5.45 ± 0.75 1.64 ± 0.35 2.87 ± 0.14 49.39 ± 12.47 3.53 ± 0.76 4.82 ± 0.96 2.26 ± 0.35 0.67 ± 0.04 71.96 ± 15.93
180 60 0.22 ± 0.03 0.65 ± 0.05 4.28 ± 0.97 0.79 ± 0.04 0.68 ± 0.06 40.83 ± 7.36 1.03 ± 0.51 1.02 ± 0.32 1.20 ± 0.14 0.26 ± 0.01 50.96 ± 9.49
280 60 0.36 ± 0.02 0.62 ± 0.05 6.25 ± 0.88 1.38 ± 0.34 2.10 ± 0.26 48.81 ± 11.32 2.76 ± 0.63 4.18 ± 1.03 1.68 ± 0.24 0.36 ± 0.02 68.51 ± 14.79
380 60 0.34 ± 0.04 0.54 ± 0.04 6.07 ± 1.05 0.75 ± 0.05 1.24 ± 0.61 45.84 ± 8.31 2.13 ± 0.08 1.94 ± 0.01 1.65 ± 0.20 0.28 ± 0.04 60.78 ± 10.43

(1), Garcinone C; (2), 3-isomangostin hydrate; (3), c-mangostin; (4), deoxy gartanin; (5), gartanin; (6), a-mangostin; (7), garcinone E; (8), 9-hydroxycalaba xanthone; (9), b-mangostin; (10), tovophyllin A.

207
208 A.S. Zarena et al. / Food Chemistry 130 (2012) 203–208
Due to the presence of benzene ring, xanthones have a maxi-
mum absorbance at 254 nm. Major xanthones were eluted be-
tween 5 and 25 min with satisfactory resolution. At the lowest
level of extraction (180 bar, 40 °C) xanthone composition (46% w/
w) and antioxidant activity (46 lg/mL) were comparatively lower.
At optimum level of extraction (280 bar, 50 °C) the antioxidant
activity (14 lg/mL) was found to increase with an increase in com-
position (75% w/w). The results from the test systems suggested
that the phytochemicals extracted had significant antioxidant
activity and the effect increased with increase in xanthone compo-
sition. The positions of hydroxyl and prenyl groups on the xan-
thone nucleus, affects the antioxidant activity of the compounds.
The mangosteen extracts were further analysed by ESI mass
spectrometry coupled to liquid chromatography on reversed-phase
columns to characterise the xanthonic compounds. The m/z values
observed on the ESI-MS spectrum for these phenolic compounds
were in accordance with those previously described (Zarena &
Udaya Sankar, 2009b). In the negative ion mode, signals at m/z
413 (garcinone C), 427 (3-isomangostin hydrate), 395 (c-mango-
stin), 379 (8-deoxygartanin), 395 (gartanin), 409 (a-mangostin),
463 (garcinone E), 407 (9-hydroxy calabaxanthone), 423
(b-mangostin), 461 (tovophyllin A) were respectively attributed
to xanthones.
Table 4 shows the compositions of the extracts at various pres-
sure and temperatures. When ethanol was used, the extraction
yield was 4.0–15.4 wt.% and the xanthone composition was
46–75% w/w, while the extraction yield without co-solvent as
reported previously by the same authors (Zarena & Udaya Sankar,
2011) was 2.6–7.5 wt.% and the xanthone composition in the total
extract was 29–65% w/w. The individual xanthone content and the
total extract yield varied at different extraction conditions; higher
xanthone recovery was obtained in presence of co-solvent than
without co-solvents. This difference is influenced by the type of
xanthones extracted at that particular condition and their interac-
tion with SC-CO2 and the co-solvent if used. Solvent density deter-
mines the number of interactions between CO2 and the molecule. If
sufficient interactions occur, the cohesive forces between individ-
ual molecules of the organic compound are broken and solubilisa-
tion will occur (Baysal & Starmans, 1999). Therefore, addition of a
small amount of polar co-solvent can enhance significantly the
extraction efficiency and the active components.
4. Conclusion
In the present study, the SC-CO2 operating conditions were
optimised to achieve an efficient extraction of the extract yield
from mangosteen pericarp, and their antioxidant activity. The high
correlation of the mathematical model indicated that a quadratic
polynomial model could be employed to optimise the xanthone
yield and its scavenging activity by supercritical carbon dioxide
extraction. From response plots, three factors (pressure, tempera-
ture and time) significantly influenced the radical scavenging
activity independently and rather than interactively. Studies on
SC-CO2 in presence of ethanol indicate that the overall extraction
yield obtained was higher and superior in recovery of xanthones.
Acknowledgement
The first author A.S. Zarena acknowledges the Council of Scien-
tific and Industrial Research, India for providing Senior Research
Fellowship.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.foodchem.2011.07.007.
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