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Procedia Food Science 1 (2011) 1953 – 1959

11th International Congress on Engineering and Food (ICEF11)

Separation and fractionation of Aquilaria Malaccensis oil

using supercritical fluid extraction and the cytotoxic
properties of the extracted oil
A. H. Ibrahima, S. S. Al-Rawib, A. M. S. Abdul Majida*, N. N. Ab. Rahmanc, K.
M. Abo- Salahd, M. O. Ab Kadirb
a
EMAN Testing & Research Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, 11800, Malaysia
b
School of Industrial Technology, Universiti Sains Malaysia, Penang 11800, Malaysia
c
School of Distance Education, Universiti Sains Malaysia, Penang 11800, Malaysia
d
King Abdulla Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia

Abstract

Most cancer chemotherapy procedure employs cytotoxic drugs that target tumor cell. Some natural product contains
cytotoxic compound but in low concentration. However, fractionation method can significantly increase the
concentration of the cytotoxic compound present, resulting in more effectiveness, which can easily achieve using
Supercritical extraction. Therefore, this study aims to extract and fraction Aquilaria Malaccensis oil using
supercritical fluid extraction, and investigate the cytotoxic properties of the extracted and fractioned oil. Aquilaria
Malaccensis oil was extracted using supercritical extractor at temperature of 40-50°C, pressure of 20.7, 27.6 and 34.5
MPa and extraction dynamic time 30 min. The extract with the highest extraction yield was then fractionated using
the best obtained operating condition to extract the most active fraction. Both samples and fractions were tested for
anticancer activity by employing MTT assay on human colon (HCT116) cancer cell line. The result of this study
shows that the highest amount of extracted oil was obtained at 50 °C, applied pressure of 34.5 MPa within 30 min
extraction time, using CO2 flow rate of 1 ml/min. The most cytotoxic fraction was obtained at the first ten minutes at
operating temperature of 50°C, pressure 34.5 MPa. The cytotoxicity result of the tested cell showed a significant cell
growth inhibition of 99% for using the whole sample and 94% for using the fraction and IC50 values against the tested
cell was 4 μg/ml. These finding reveals that the supercritical extraction oil of Aquilaria Malaccensis has strong
anticancer activity towards human colon cancer cells and hence can be a good candidate for treating cancer.

© 2011 Published by Elsevier B.V. Open access under CC BY-NC-ND license.

Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive
Committee.

Keywords: Supercritical; separation; fractionation; oil; Aquilaria; Malaccensis;

* Corresponding author. Tel.: +6046534582; fax: +6046534582.

E-mail address: aminmalikshah@gmail.com.

2211–601X © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress on Engineering
and Food (ICEF 11) Executive Committee. Open access under CC BY-NC-ND license.
doi:10.1016/j.profoo.2011.09.287
1954 A. H. Ibrahim et al. / Procedia Food Science 1 (2011) 1953 – 1959

1. Introduction

Many studies have shown that extracts from higher plant species contain compounds that have useful
medicinal properties, which have led to the development of current drugs in the market. The unique
characteristic of these medicinal plants is their ability to generate a large number of secondary
metabolites, aromatic substances, offshoot plants and microorganisms. The important role of these
compounds for a number of diseases has highlighted their usefulness to prevent and treat variety of
human illness such as cancer, cardiovascular disease and many other ailments.
Aquilaria species is an aromatic plant that is commonly known as “Gaharu wood” in South East Asia
[1]. It is found mostly in Malaysia, Indonesia, India, Iran, Singapore, Bangladesh, Myanmar, Philippine,
and Thailand [2]. A. malaccensis produces valuable resin marinate in the heartwood. This resin originates
as a consequence of natural immune response towards fungal attack. Endophyte is a fungus that lives
inside healthy plant tissues, some of these endophytes have been found to have anticancer and anti-
diabetic properties [2,3]. It is well known that the essential oil of A. malaccensis is safe, simple to
produce and commonly used in traditional medicine to relive pain, fever, rheumatism and asthma. A
number of compounds have been extracted from A. malaccensis such as alkaloids, tannins, phenols,
terpenoids, Quinones and Àavonoids [4]. Generally most cancer chemotherapy approach employs
cytotoxic drugs to target the actively proliferating cancer cell populations [5]. However, in
pharmaceutical industries the requirement for new, fast, powerful, cleaner and cheaper techniques are
required to meet these demands. Moreover, limitations are currently being proposed to eliminate solvent
residues in the traditional method used in food, pharmaceutical and cosmetic industries. Supercritical
fluid extraction (SFE) is an alternative method to the traditional solvent extraction technique. The unique
characteristic of this technique is it’s a rapid, selective, cheap and convenient method to separate and
fractionate the active compound [6,7]. Moreover, SFE was found to be a ground breaking extraction
method that can give the highest number of compounds [8] Carbon dioxide is the most preferable
supercritical solvent in the extraction of fragrance compounds, since it is nontoxic with low critical
pressure and it enables supercritical operation at low pressures and low temperature [9] which is
nonflammable and chemically stable [10] In addition, the short extraction time in SFE compared to other
extraction method such as water distillation provides additional benefit [11]. All these factors make the
SFE a good extraction method for pharmaceutical production.
To date, no information regarding anticancer activity of the supercritical extract of A. malaccensis oil
are exist and only a limited number of reports concerning A. malaccensis oil extracted using supercritical
extraction. Thus, the aim of this study is to extract and fraction Aquilaria Malaccensis oil using
supercritical fluid extraction and to investigate the anticancer activity of both the extracted and fractioned
oil towards human colon cancer cell lines.

2. Materials & Methods

2.1. Chemicals and Materials

Commercial liquid carbon dioxide gas with purity of 99 g kg-1 was purchased locally from Malaysian
Oxygen, Penang, Malaysia in a gas cylinder at temperature below -5ºC. Aquilaria Malaccensis stem bark
samples were sourced commercially from local supplier in Malaysia. Human cancer cell line HCT116
was obtained from ATCC, USA. RPMI 1640 cell culture media, foetal bovine serum (FBS), Penicillin
and Streptomycin solutions were from Gibco, USA. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, a yellow tetrazolium compound, and Dimethyl sulfoxide (DMSO),
Suramine was acquired from Sigma Aldrich, Germany. HCT-116 cell line were maintained in RPMI 1640
cell culture medium supplemented with 10% FBS and 1% P/S. The cells were cultured in class II
biosafety cabinet (ESCO, USA) under sterile conditions.
 A. H. Ibrahim et al. / Procedia Food Science 1 (2011) 1953 – 1959 1955

2.2. Sample preparation & supercritical extraction

Samples were prepared prior to extraction as the fresh stem bark (clean and infected part with
endophyte) was cut and chopped into small pieces. The small pieces were dried in oven at 50ºC
overnight. The samples were grinded using analytical mill (IKA (R) Retsch). The grounded powder was
then passed through an aperture sieve to get particle size of ” 500 μm. Supercritical CO2 extractions were
performed using an SFE extraction system (ISCO, Inc., Lincoln, NE. USA model SFX 220). About 2 g of
the sample was placed in the vessel and was extracted at 40°C and 50°C with pressure ranged from 20.7
27.6 and 34.5 MPa, using CO2 flow rate ” 1 ml/min for 60 min. The oil was collected in a pre-weighted
collection glass vial. The oil yield was calculated by the weight increment at the end of the extraction
process and kept at -20°C. The A. Malaccensis oil was fractioned using supercritical extraction at 50°C,
pressure 34.5 MPa, CO2 flow rate of ” 1ml/min and the particle size ” 500 μm into 3 fractions using
dynamic time of 10 min for each fraction.

2.3. Cytotoxicity assay

Cytotoxicity of A. Malaccensis oil on tumor cells was measured by microculture tetrazolium (MTT)
assay [12]. A 96-well microplate were seeded with 300 μl of medium containing 1 x 10-3 cells of HCT
116 in suspension and incubated for at least 24 h prior to treatment. The cells were treated with the
supercritical extracted oil sample at concentration of 25μg/ml. DMSO was used as a negative control and
suramin was as a positive control. All tested samples were performed in triplicate and repeated three
times. After 48 h incubation in 5% CO at 37ºC, (Sigma) tetrazolim salt was added as cytotoxicity
indicator for cell viability. The absorbance reading was recorded at 590 nm and at reference wavelength
of 650 nm with a scanning Multiskan Ascent micro plate reader (Thermolab system 354, Finland). Values
are expressed as the percentage of mean cell viability relative to the untreated cultures. The same
experiment was repeated using the fractions oil at the same concentration of 25 μg /ml and at 2.5, 5, 10,
15, 20 and 25 μg/ml and incubated for 48 h in 5% CO2 at 37ºC, in order determine it respective IC50
values.

3. Results & Discussion

The effect of temperature and pressure on the extraction yield of A. Malaccensis by supercritical CO2
was examined in this study as well as the cytotoxic property of the extracted samples. The supercritical
fluid extraction was chosen in this study due to its advantage in producing solvent free samples that
impose measurable level of cytotoxic activity. The extraction parameters were studied to investigate the
extracting condition that gives the highest oil yield from A. Malaccensis. These parameters are;
temperature (40 and 50ºC) pressure (20.7, 27.6 and 34.5 MPa). A fixed matrix particle size (” 500μm)
and CO2 flow rate (” 1ml/ min). The sample size was fixed to ” 500 μg/ml based on preliminary study in
our laboratory as well as from previous studies. It has been known that matrix particle size reduction
increases the extraction yield. Moreover, decreasing particle size in SFE creates more surface area leading
to increase extraction yield [13]. Flow rate was found to have strong influence on the extraction
efficiencies in supercritical extraction. The slower the fluid velocity, the deeper it penetrates the matrix,
which enhances the extraction yield positively [13]. In this study, the supercritical extraction result
showed that extraction yield increases with increasing both pressure and temperature. Other studies also
reported that increasing temperature and pressure will increase extraction yield in supercritical extraction
[14]. However, the temperature increment enhanced the extraction significantly. The highest extracted oil
was 3.66 g/100g sample, which is obtained at 50ºC, 34.5 MPa with dynamic time of 30 min. Increasing
temperature from 40 to 50ºC resulted to increase the extraction yield. However, the extracted yield was
higher at all pressure at temperature of 50ºC, as shown in Fig.1. While lowering the temperature requires
1956 A. H. Ibrahim et al. / Procedia Food Science 1 (2011) 1953 – 1959

the need of higher extraction pressure in order to extract more yields. In our study it was found that at
40ºC the highest extracted yield was 2.3 g oil/100g sample at the pressure of 34.5 MPa, whereas the
lowest extracted oil yield was at 1.7g/ 100g sample at 40 ºC with pressure of 20.7 MPa. This is due to the
decrease of the CO2 density with increasing temperature at constant pressure, which leads to increase in
solute vapour and improvement in the extraction quality. This data is consistent with other studies
reported [15], which showed that increasing temperature increased the extraction yield at constant
pressure.

Fig. 1.The effect of operating temperature on the extraction Fig. 2. The effect of operating pressure on the extraction
yield at 20.7, 27.6 and 34.5 MPa, CO2 flow rate İ1ml/min, yield at 40 and 50ºC, CO2 flow rate ” 1ml/min, particle size
particle size İ 500 μm for 30 min ” 500 μm for 30 min

The extraction yield increased significantly with increasing pressure at higher temperature as shown in
Figure 2. The highest extracted yield was 3.66 g oil/100g sample at pressure of 34.5 MPa; at 50ºC with
CO2 flow rate ” 1ml/min, particle size ” 500 μm for 30 min. It was found that increasing pressure at
constant temperature will increase CO2 density, which results in an increase in the solubility of the oil
components and enhancement of the extraction yield [16]. Cytotoxic property of the extracted oil of A.
Malaccensis using supercritical was investigated by employing human colon cancer cell HCT-116. The
result of the experiment shows that all the supercritical extracted samples of A. Malaccensis are cytotoxic
towards the colon cancer cell HCT 116 using the concentration of 25 μg/ml of extracted oil. However,
temperature showed significant effect on the extracted sample and increased it cytotoxic property. It was
found that all the extracted samples at temperature of 50ºC have potent cytoxicity than the extracted
samples at 40ºC at constant pressure. The most potent cytotoxic sample was the extracted sample at
temperature of 50ºC, pressure 20.7 MPa, which inhibited 99% of the colon cancer cell growth at the
concentration of 25 μg/ml as shown in figure 3. This difference in activity is contributed to increasing
temperature at constant pressure will decrease the CO2 density, which leads to increase the solute vapor
and increasing the solubility and increase the extracted compounds selectivity. Nevertheless, the
supercritical pressure has a diverse effect on the cytotoxicity properties of the extracted oil of A.
Malaccensis. It was found that at constant temperature of 40ºC, increasing the pressure from 20.7 to 34.5
MPa, has increased the cytotoxic properties of the sample to more than 50%. It was found that the
extracted sample at pressure of 20.7 MPa inhibited the growth of colon cancer cell by 41% and was
increased to 84% using the extracted sample at pressure of 34.5 MPa at constant temperature of 40ºC. On
the contrary, it was found that the highest cytotoxic extract was obtained at the lowest pressure of 20.7
MPa and 50ºC with 99% of cell growth inhibition. Iincreasing the pressure resulted in decreasing the
 A. H. Ibrahim et al. / Procedia Food Science 1 (2011) 1953 – 1959 1957

cytotoxic property of the extracted sample. It was found that the inhibition of the cancer cell growth
reduced from 99% for the extracted sample at 20.7 MPa to 94% for the extracted sample at pressure of
27.6 and 34.5 MPa (Figure 3).

Fig. 3. Cell growth inhibition of HCT116 cancer cell at 25
μg/ml of A. Malaccensis oil extracted by supercritical
extraction, at operating temperature of 40, 50ºC, pressure
20.7, 27.6 and 34.5 MPa, CO2 flow rate of ” 1ml/min,
particle size ” 500 μm for 30 min
Fig. 4. Cell growth inhibition of HCT116 cancer cell at 25
μg/ml of A. Malaccensis oil fractions by supercritical
extraction, F1; extracted oil at the first 10 min, F2; extracted
oil at the second 10 min, F3; extracted oil at the third 10 min

The most cytotoxic sample was then fractioned into three fractions using supercritical extraction with
10 min extraction time. The three fractions were then tested by MTT assay to investigate the most
cytotoxic fraction. The result of this experiment indicates that fraction obtained from the first 10 min (F1)
of A. Malaccensis oil, (50ºC, 20.7 MPa, CO2 flow rate ” 1 ml/min, particle size ” 500), has the highest
cytotoxic activity against the colon cancer cell HCT 116. It was found that using the concentration of 25
μg/ml of A. Malaccensis oil, the growth of colon cancer cell was inhibited with 94%, 16% and 14% using
the fractions F1, F2 and F3 respectively (Figure 4). The IC50 for (F1) the most cytotoxic fraction was 3.5
μg/ml which represents the amount of drug that can kill 50% of the cells, as shown in Figure 5.

Fig. 5. Dose-dependency eơects of A. Malaccensis extracts on the HCT116 cell Growth. Percentage of cell growth inhibition treated
with 2.5–25 ȝg/mL
1958 A. H. Ibrahim et al. / Procedia Food Science 1 (2011) 1953 – 1959

4. Conclusion

The finding of this study reveals that the supercritical extraction yield of A. Malaccensis increases with
increasing pressure and temperature. Supercritical extracte of A. Malaccensis is an effective cytotoxic
agent. Its toxicity increased with temperature increment up to 50ºC and lowering the pressure to 20.7
MPa. The first 10 min fraction of supercritical extraction possesses potent toxicity towards HCT116
cancer cells with an IC 50 of 4 ug/ml. This result demonstrates that temperature is an effective factor for
the extraction yield and for the extracting of cytotoxic compound.

Acknowledgements

The author would like to acknowledge with thanks the support provided by Universiti Sains Malaysia,
Penang, Malaysia. This research work was supported partially by the research chair funded by King Saud
University on drug targeting and treatment of cancer using nanoparticles.

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Presented at ICEF11 (May 22-26, 2011- Athens, Greece) as paper NFP690.