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Supercritical Carbon Dioxide Extraction of Marigold


Lutein Fatty Acid Esters: Effects of Cosolvents and
Saponification Conditions
a

Wisinee Palumpitag , Phattanon Prasitchoke , Motonobu Goto & Artiwan Shotipruk

Faculty of Engineering, Department of Chemical Engineering , Chulalongkorn University ,


Bangkok, Thailand
b

PTT Chemical Public Company Limited , Rayong, Thailand

Department of Applied Chemistry and Biochemistry , Kumamoto University , Kumamoto,


Japan
Published online: 09 Mar 2011.

To cite this article: Wisinee Palumpitag , Phattanon Prasitchoke , Motonobu Goto & Artiwan Shotipruk (2011) Supercritical
Carbon Dioxide Extraction of Marigold Lutein Fatty Acid Esters: Effects of Cosolvents and Saponification Conditions,
Separation Science and Technology, 46:4, 605-610, DOI: 10.1080/01496395.2010.533739
To link to this article: http://dx.doi.org/10.1080/01496395.2010.533739

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Separation Science and Technology, 46: 605610, 2011


Copyright # Taylor & Francis Group, LLC
ISSN: 0149-6395 print=1520-5754 online
DOI: 10.1080/01496395.2010.533739

Supercritical Carbon Dioxide Extraction of Marigold Lutein


Fatty Acid Esters: Effects of Cosolvents and
Saponification Conditions
Wisinee Palumpitag,1 Phattanon Prasitchoke,2 Motonobu Goto,3 and
Artiwan Shotipruk1
1

Downloaded by [Christian Chandra] at 22:22 08 January 2015

Faculty of Engineering, Department of Chemical Engineering, Chulalongkorn University, Bangkok,


Thailand
2
PTT Chemical Public Company Limited, Rayong, Thailand
3
Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto, Japan

Extraction of lutein fatty acid esters from marigold ower using


supercritical carbon dioxide (SC-CO2) with cosolvent was investigated. Without the cosolvent, the total xanthophylls yield increased
with increasing temperature and pressure of SC-CO2, and the
optimal condition was found to be at 60 C and 40 MPa. At this
condition, the highest total xanthophylls percent recovery was
74.4  0.9%. Palm oil was found to be a more efcient cosolvent
than soybean oil, olive oil, and ethanol, resulting in a 16% increase
in the total xanthophylls recovery to 87.2  4.4% when 10% (w/w)
of palm oil was used. Furthermore, saponication of the oleoresin
for 3 h at 75 C with 40% w/v KOH solution at the oleoresin to
solution ratio of 1 g to 2 ml was found to suitably convert lutein
fatty acid esters into free lutein.
Keywords cosolvent;
extraction;
xanthophylls

Lutein;

saponication;

INTRODUCTION
Marigolds are grown all over the world for ornamental
purposes. In eastern countries, marigold owers are used in
garlands for social and religious purposes, but nowadays,
extraction of marigold owers for carotenoids is becoming
of importance due to its vital role in the enhancement of
immune systems and ghting off tissue damage. The major
component of marigold ower extract is a xanthophyll
called lutein, which offers many health benecial effects
such as reducing failure of eyesight due to age-related
macular degeneration (AMD), and preventing coronary
heart disease and cancer (1,2).
In marigold owers, lutein generally exists in the form of
lutein fatty acid esters; however, since lutein can be taken
Received 16 June 2010; accepted 18 October 2010.
Address correspondence to Artiwan Shotipruk, Faculty of
Engineering, Department of Chemical Engineering, Phayathai
Rd, Patumwan, Bangkok 10330, Thailand. Tel.: 662-218-6868;
Fax: 662-218-6877. E-mail: artiwan.sh@chula.ac.th

up by the human body in its free form only (3,4), marigold


oleoresin must therefore be saponied with alkali solution,
i.e., KOH solution, to obtain free lutein (5). Conventional
solvent extraction (generally using hexane) suffers several
drawbacks such as requiring long extraction time and leaving toxic residues (6,7). Supercritical carbon dioxide
(SC-CO2) extraction has been gaining considerable interest
(814). The process can be applied for extraction of plant
compounds, particularly for those that are easily degraded
by light, oxygen, and high temperatures. In addition,
SC-CO2 extraction offers other advantages such as low
toxicity, low critical point temperature, and easy product
isolation, simply by depressurization. Examples of previous
work on the extraction of carotenoids with SC-CO2 include
extraction of pitanga fruits (15) and rosehip fruits (16).
These studies demonstrated strong dependence of carotenoids (lycopene, b-carotene, and lutein) yields on temperature and pressure. In SC-CO2 extractions of carotenoids
from natural sources, the use of cosolvents such as ethanol
and vegetable oils has been demonstrated to improve the
extraction efciency (1720). The enhanced recovery was
possibly due to the improved interactions between cosolvents and solute as well as cosolvents and the sample
matrix, leading to higher solubility of lutein fatty acid
esters. Ma et al. (21) investigated the use of soybean oil
as cosolvents, which were added at the beginning of the
process for SC-CO2 extraction of the solute from marigolds. A maximum amount of lutein fatty acid esters of
approximately 1040 mg=100 g marigold was obtained with
use of 6.9% (w=w) soybean oil.
In this work, SC-CO2 extraction of lutein fatty acid
esters was carried out to determine the suitable extraction
conditions and the effects of palm oil, as a colsovent on
extraction efciency, compared with soybean oil, olive
oil, and ethanol. In addition, saponication of the extract

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was carried out to determine the suitable condition to


obtain free lutein.
MATERIALS AND METHODS
Materials and Chemicals
The dried marigold owers were obtained from PTT
Chemical (Rayong, Thailand). The marigold owers were
dried and then ground to small particle ranging from
0.190.22 mm in size. The samples were stored in the dark
at room temperature until use. Extractions were carried out
with high purity carbon dioxide obtained from Thonburiwattana Co. (Bangkok, Thailand). Hexane, ethanol, potassium hydroxide, and hydrochlolic acid were purchased
from Sigma-Aldrich, Germany. Palm oil was obtained
from P.S Pacic Company Limited (Petchaburi, Thailand).
Soybean oil was obtained from Thai Vegetable Oil Public
Company Limited (Nakornpathom, Thailand) and olive
oil (Bertolli, Italy) was purchased from a local supermarket. Nitrogen used for evaporating the extraction solvents
from the oleoresin was obtained from Thai Industrial
Gas Co. Ltd. (Bangkok, Thailand). Lutein standard (99%
purity) was purchased from Sigma-Aldrich, Germany.
SC-CO2 Extraction
To conduct an extraction experiment, one gram of
ground sample with the corresponding palm oil concentration (w=w) was loaded into a 10 ml SC-CO2 extractor
(model SFX-220, ISCO). The extraction was carried out
at a desired temperature and pressure for up to 4 h. The
extract was then collected in a side-armed glass tube
(wrapped with aluminum foil), where it was trapped with
hexane. The marigold extract was stored at 4 C in the dark
until analysis. In this experiment, the effect of extraction
conditions (pressure, temperature, type, and amount of
cosolvents) were investigated on the extraction efciency
of lutein fatty acid esters, assayed spectroscopically as the
amount of total xanthophylls. The ranges of the operating
variables are summarized in Table 1. The extraction
efciency was dened on the basis of the total amounts
of xanthophylls from marigold owers, determined by
repeated extractions of marigold powder with hexane at a

volume ratio of sample 1:5 at 40 C. The initial extraction


was carried out for 4 h. The extract was ltered and the
residue was re-extracted repeatedly with equal volume
of hexane at 30 C 24 h, 72 h, and 240 h after the initial
extraction. Each of these extracts was collected and was
determined for the amount of total xanthophylls by a
spectrophotometer. The total amount of xanthophylls
was the summation of the amount in each extract. For subsequent saponcation experiments, the resulting SC-CO2
extracts obtained at the most suitable conditions were
purged with nitrogen to evaporate off the hexane to obtain
marigold oleoresin. All extraction experiments were carried
out in duplicate.
Saponification of Lutien Fatty Acid Esters
Saponication was carried out to convert lutein fatty
acid esters to free lutein to determine the appropriate
process conditions such as concentration of KOH solution,
the volume to weight ratio of KOH solution to marigold
oleoresin, and saponication time. Initially, the suitable
concentration of KOH solution was determined, in which
marigold oleoresin was dissolved with 40% KOH solution
in ethanol at 75 C at the ratio of KOH solution to oleoresin of 2 ml to 1 g. The mixture was then vortexed for
1 min, and the reaction was allowed to take place for 4 h
during which time the mixture was vortexed every hour.
The reaction was then stopped by cooling the saponied
solution to 65 C and adjusting the pH to about 7.0 with
25 vol% of HCL aqueous solution. For the analysis of
saponied solution, 50 ml ethanol was added to the sapnonied product and the mixture was then sonicated at
30  5 C for 15 min. The mixture was ltered to separate
insoluble impurities and the resulting clear solution was
then analyzed for the amount of free lutein by HPLC. With
the suitable concentration of KOH solution, the desirable
volume to weight ratio of KOH solution to marigold oleoresin was then determined. At such concentration and ratio
of KOH solution to marigold oleoresin, the suitable saponifcation time was determined. The ranges of all variables
for saponication study are summarized in Table 2. All
saponication experiments were carried out in duplicate.

TABLE 1
Ranges of variables for SC-CO2 extraction
Variable
Temperature of extraction ( C)
Pressure (MPa)
Concentration of palm oil
% (w=w)
Type of vegetable oil as
cosolvent

TABLE 2
Ranges of variables saponication

Ranges
30, 45, 60, 75
30, 35, 40
0, 5, 10, 15, 20
palm oil, soybean oil,
olive oil

Variable
Concentration of KOH
solution % (W=V)
Ratio of KOH solution to
oleoresin (ml=g)
Saponication time (h)

Ranges
30, 35, 40, 45
1:1, 2:1, 4:1, 6:1, 8:1
0, 2, 3, 4, 5

SUPERCRITICAL CARBON DIOXIDE EXTRACTION OF MARIGOLD LUTEIN FATTY ACID ESTERS

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Analysis of Total Xanthophylls


The total xanthophylls content in the extract of
marigold ower was determined using a spectrophotometer
following the AOAC method, in which Sudan was used as
a standard. The absorbance of the mixture was then
measured by a spectrophotometer at 474 nm (22).
HPLC Analysis of Free Lutein
The analyses of free lutein in the extracts were carried
out by HPLC, using the method reported by Roberta
et al. (6), with modications. The reversed-phase HPLC
analysis was carried out using Inertsil-ODS-3. The gradient
mode of the mobile phase was employed with acetonitrile:methanol (9:1, v:v) (Solvent A) and ethyl acetate (Solvent
B), using a linear gradient at a ow rate of 1 ml=min running from 0% to 100% of B in 30 min. The sample injection
volume was 20 ml and the detection wavelength was
at 450 nm.
RESULTS AND DISCUSSION
Total Xanthophylls in Marigold Flower Sample
The amount of total xanthophylls in the dried marigold
sample was determined from repeated extraction. Table 3
summarizes the amounts of total xanthophylls obtained
after each extraction cycle. Approximately 75.2  0.6%
(20.5 mg=g) were extracted in the rst extraction (extraction at 40 C for 4 h), and only small amounts, 17.6 
0.7%, 5.2  1.0%, 1.6  0.2%, and 0.4  0.1% were extracted in subsequent extractions, with hexane at 30 C for 1,
2, 7, and 10 days, respectively. This therefore sums up to
the total amount of total xanthophylls of approximately
27.9  2.0 mg=g dry marigold owers.
Determination of Suitable SC-CO2 Extraction Conditions
Effects of Temperature of SC-CO2 Extraction
Initially, SC-CO2 extraction was performed with 1 g of
dry marigold sample without the cosolvent for 4 h. The
pressure was controlled at 35 MPa and the temperature
was varied: 30 C, 45 C, 60 C, and 75 C. The results showing the amounts of lutein fatty acid esters in terms of total

FIG. 1.
esters.

Effect of temperature on SC-CO2 extraction of lutein fatty acid

xanthophylls recoveries are depicted in Fig. 1. The plots in


Fig. 1 show an increase in the amount of total xanthophylls
with increasing temperature from 37.5  1.3% at 30 C to
71.1  0.7% at 60 C. For SC-CO2 extraction, the maximum
amount of total xanthophylls (71.1  0.7%) was obtained
at 60 C. At 75 C however, the extraction yield decreased
since lutein fatty acid esters are known to be heat sensitive, and thus could possibly be degraded at such high
temperature.
Effects of Pressure of SC-CO2 Extraction
The effects of pressure of SC-CO2 extraction on the
yield of total xanthophylls recovery were investigated at
various pressures: 30, 35, and 40 MPa. These sets of experiments were operated at 60 C, the SC-CO2 extraction temperature determined previously. The results shown in Fig. 2
revealed that the amount of total xanthophylls recovery
increased with the increasing pressure from 58.6  3.4%
to the maximum yield of 74.4  0.9% at 30 MPa to
40 MPa. This is due to the fact that the SC-CO2 density
increased with increasing pressure, allowing solutesolvent
interactions to be enhanced. This inuenced the increase in

TABLE 3
Total xanthophylls content in marigold ower
Temperature=time
( C)
40 C=4 h
30 C=1 day
30 C=2 day
30 C=7 day
30 C=10 day
Sum

Total xanthophylls
(mg=g sample)

Recovery
(%)

20.49  1.66
4.80  0.55
1.41  0.17
0.43  0.03
0.12  0.03
27.26  1.99

75.18
17.63
5.17
1.58
0.43
100.00

FIG. 2. Effect of pressure on SC-CO2 extraction of lutein fatty acid


esters.

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solute solubility. These results agreed with the previous


study by Danielski et al. (23) which reported that the
increase in SC-CO2 density could strongly inuence the solvent power, the signicant factor that enhanced the overall
efciency of SC-CO2 extraction.
Comparison of Vegetable Oils as Cosolvents
The comparison of the efciency of cosolvents: palm oil,
soybean oil, olive oil, and ethanol, was made for the extraction of lutein fatty acid esters with SC-CO2. Each extraction experiment was carried out for 4 h at the suitable
pressure and temperature determined previously (at 40 MPa
and 60 C). Prior to the extraction, the cosolvent was added
into the sample at 10 wt%. The results shown in Fig. 3
revealed that palm oil could improve the extraction yield
of lutein fatty acid esters more efciently compared with
other cosolvents. Lutein fatty acid esters recovery was
increased more than 16% using palm oil. Although ethanol
has been shown to be an effective cosolvent for SC-CO2
extraction of various lipids, due to the effect of intermolecular interactions between the solute and ethanol cosolvent
such as H-bonding interaction (24), in this case it did not
enhance the extraction efciency of lutein fatty acid esters.
This is possibly due to the large differences in the molecular
sizes and structures between the solute and the colsolvent.
The high extraction efciency resulting from the palm oil
co-solvent was possibly due to the high content of
shorter-chain saturated fatty acids in the palm oil (45%
palmitic acid) compared with other oils. This has been
reported to have higher solubility in SC-CO2 (25).
Effect of Concentration of Palm Oil Cosolvent
To investigate the effect of the concentration of palm oil
used as a cosolvent on the amount of the total xanthophylls
recovery, the most suitable SC-CO2 extraction condition
determined previously (60 C and 40 MPa) was applied.

FIG. 3. Comparison of extraction efciency of co-solvents: ethanol,


palm oil, soybean oil, and olive oil.

FIG. 4. Effect of concentration of palm oil as a co-solvent on SC-CO2


extraction.

The experiments were carried out at various concentrations


of palm oil: 0, 5, 10, 15, and 20% (w=w). The results are
shown in Fig. 4. The results indicated that the extraction
efciency of lutein fatty acid esters increased with increasing amount of added palm oil up to 10% w=w. As palm oil
permeated into the samples, it made the tissue swell, and
thus increased the contacting surface area of the sample
with SC-CO2. In addition, the palm oil that permeated into
the cell structure of the marigold sample caused SC-CO2 to
diffuse through the matrix more easily since the solubility
of lutein fatty acid esters in the mixture of SC-CO2 and
palm oil was higher than that of pure SC-CO2 (19). The
highest lutein fatty acid esters recovery of 87.2  4.4%
was obtained when using 10% w=w of palm oil as a cosolvent. However, further addition palm oil decreased the
extraction yield, possibly as a result of the decrease in the
homogeneity of the extraction mixture.
Determination of Suitable Saponification Conditions
Effect of KOH Concentration
The effect of KOH concentration for saponication of
lutein fatty acid esters on the yield of free lutien was
determined. The results shown in Fig. 5 indicate that up
to 40% KOH solution, the concentration of free lutein

FIG. 5.

Effect of concentration of KOH solution on free lutein yield.

SUPERCRITICAL CARBON DIOXIDE EXTRACTION OF MARIGOLD LUTEIN FATTY ACID ESTERS

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increased with increasing KOH concentration. However, at


a higher concentration of KOH, the conversion to free
lutein decreased. This was possibly due to the degradation
of lutein fatty acids at high pH conditions. This result
agreed with that of Sowbhagya et al. (1) which reported
that lutein was sensitive to extreme pHs. The highest
amount of free lutein was obtained at 40% KOH solution
(151.9  6.4 mg=g oleoresin), thus this concentration was
used in the subsequent experiment to determine the suitable
ratio (v=wt) of KOH to oleoresin and the suitable saponication time.
Effect of Volume to Weight Ratio of KOH Solution
to Marigold Oleoresin
The effect of the ratio of 40% KOH solution to marigold
oleoresin was investigated at 75 C for 4 h in the range of
1:1, 2:1, 4:1, 6:1, and 8:1 v=wt. The results shown in Fig. 6
revealed that the highest amount of free lutein
(151.9  6.4 mg=g oleoresin) was obtained with the ratio
of 2 ml of 40% KOH solution to 1 g oleoresin. The amount
of free lutein decreased when the ratio of KOH solution
volume to oleoresin was higher than 2:1 possibly due to
the degradation caused by the large volume of the KOH
solution.
Effects of Saponication Time
The effect of saponication time shown in Fig. 7
revealed the amount of the free lutein obtained at different
saponication times. The results indicated that the amount
of free lutein increased with increasing saponication time
up to 3 h, after which the concentration of free lutein
decreased possibly due to long exposure with high temperature and alkali condition (5). The highest amount of free
lutein was achieved in 3 h (157.2  4.4 mg=g oleoresin).

FIG. 7. Effect of saponication time on free lutein yield.

CONCLUSIONS
The use of palm oil as cosolvent for SC-CO2 extraction
of marigold ower could enhance the yield of lutein fatty
acid esters by approximately 16%, with the most suitable
concentration of palm being 10% w=v palm oil. At this condition, approximately 87.2  4.4% recovery of lutein fatty
acid esters was achieved after 4 h extraction at 60 C and
40 MPa. To further obtain free lutein, saponication of
the marigold oleoresin for 3 h with 2 ml of 40% (w=v)
KOH solution per 1 g of oleoresin resulted in the maximum
conversion of lutein esters, giving approximately 157.2 
4.4 mg of free lutein=g oleorsin.
ACKNOWLEDGEMENTS
Financial support from Thailand Research Fund is
greatly appreciated.
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FIG. 6. Effect of volume to weight ratio of KOH solution to marigold


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