TABLE OF CONTENTS

NO.

CONTENTS

PAGE

ABSTRACT

INTRODUCTION

3-11

MATERIAL & APPARATUS

12-16

METHODOLOGY

17-24

RESULT

25-29

DISCUSSION

30-35

10

CONCLUSION

36

12

REFERENCES

37

ABSTRACT

The extraction process of seed oil using supercritical CO 2 is studied based on journals.
We choose pomegranate seeds

as our materials. The extraction process of the

pomegranate seeds was done by using Supercritical

CO2

(SC-CO2) at different

conditions. The range pressure of the extraction process are 15MPa to 30MPa (Liu et
al., 2012). We discuss on the effects such as effects of extraction pressure of
pomegranate seed oil (PSO) on seed oil yield, effects of extraction temperature of
pomegranate seed oil (PSO) on seed oil yield, effects of extraction pressure and
temperature on fatty acid composition of pomegranate seed oil (PSO), effects of
extraction pressure, temperature and time on tocopherols content of PSO, scavenging
abilities on DPPH radicals and scavenging abilities on ABTS radicals. Besides that, we
also make a comparison with extraction using superheated hexane of pomegranate
seed oil (PSO). The major factor affecting the oil yield is the extraction pressure. The
yield of PSO increased with the increase of extraction pressure. The fatty acid that were
identified were palmitic, stearic, oleic, linoleic and punicic acids. The composition of fatty
acids in the PSO were significantly affected by the extraction parameters.

INTRODUCTION

Extraction is the process of withdrawing of an active agent or a waste substance

from a solid or liquid mixture by using a liquid solvent. The solvent may be partial or
immiscible towards the solid or liquid. Through rigorous contact, the active agent
transfers raffinate to extract. The mixture of solid and liquid are then separated by
gravity or centrifugal force. There are several types of extraction depending on the
phases in the systems such as solid-liquid extraction, liquid-liquid extraction and gasgas extraction (Gamse, n.d).

Since vast research was done on extraction, more and more methods are
discovered in order to find the most efficient extraction process. One of the most
popular methods of extraction used nowadays is the supercritical fluid extraction (SFE).
Supercritical fluid extraction is a fast, selective and the most convenient method for
preparation of sample prior to the analysis of compounds in natural product matrices.
According to Modey, supercritical fluid extraction is usually performed with pure or
modified carbon dioxide, which facilitates off-line collection of extracts and on-line
coupling with other analytical methods such as gas and supercritical fluid
chromatography. A number of factors which influence the extraction yields are the
solubility of the solute in the fluid, the diffusion through the matrix and adsorption
processes in the matrix. This process enables a wide range of applications for the
extraction of carotenoids, lipids, flavour and fragrance compounds, steroids and
triterpenes, alkaloids and mycotoxins (Modey, Mulholland, et al., 1996).

The process of extraction by using supercritical fluid is well recognized for its
conventional method of usage to extract varieties of compounds. The extraction process
focused more on the properties between liquid and gas to intensify their solvation power
as the result of their high density, high diffusivity and low viscosity causing a significant
power of penetration into the solute matrix (Santos, Corra, Carvalho Jr, Costa and
Lannes, 2013). Extraction by using supercritical fluid is said to have more advantages
over traditional extraction process due to its process flexibility which enable the
possibility of continuous modulation of the solvent power, it also enables the elimination
of polluting

organic solvents and the expensive post-processing of the extracts for

solvent elimination (Mariod, Matthaus and Ismail, 2010).

The solvent used in the supercritical fluid extraction is called supercritical

fluid which is any substance exist above its critical temperature and pressure, where
typical liquid and gas phases do not exist. The temperature and pressure in some
supercritical fluids are assumed to be relatively low regardless the thermal stability.
Above its critical temperature, it does not condense or evaporate to form liquid or gas
but is a fluid with constantly changing properties from gas-like to liquid-like as the
pressure

increase.

As

result,

it

can effuse through

solids

like

gas,

and dissolve materials like a liquid. In addition, due to the close in distance with the
critical point, small changes in pressure or temperature will result in large changes
in density where it allows many properties of a supercritical fluid to be easily adjusted.
These types of solvents are suitable as a substitute for organic solvents in industrial and
laboratory processes. Carbon dioxide and water are the most commonly used
supercritical fluids mostly for decaffeination and power generation (Clifford, 1998).

Table 1. Critical properties of various solvents (Reid et al., 1987)

Molecular

Critical

Critical

Critical

weight

temperature

pressure

density

g/mol

MPa (atm)

g/cm3

44.01

304.1

7.38 (72.8)

0.469

18.015

647.096

Methane (CH4)

16.04

190.4

4.60 (45.4)

0.162

Ethane (C2H6)

30.07

305.3

4.87 (48.1)

0.203

Propane (C3H8)

44.09

369.8

4.25 (41.9)

0.217

Ethylene (C2H4)

28.05

282.4

5.04 (49.7)

0.215

Propylene (C3H6)

42.08

364.9

4.60 (45.4)

0.232

Methanol (CH3OH)

32.04

512.6

8.09 (79.8)

0.272

Ethanol (C2H5OH)

46.07

513.9

6.14 (60.6)

0.276

Acetone (C3H6O)

58.08

508.1

4.70 (46.4)

0.278

Solvent

Carbon dioxide (CO2)

Water (H2O)
(acc. IAPWS)

22.064
(217.755)

0.322

The most common used supercritical fluid is carbon dioxide. Supercritical carbon
dioxide is carbon dioxide under a pressure of above 74 bars and a temperature above
5

31 C (Clifford, Basile and Al-Saidi, 1999). It is widely used because it has a lot of
advantages namely due to its non-corrosive nature, non- toxicity and non-flammability,
and not to mention that it is easy to be removed from the extracted product. Even so,
carbon dioxide too has its disadvantages which are high investment costs for equipment
acquisition and the high energy demand of the CO 2 extraction unit (Moslavac, et al.,
2014).

Another method of extraction is the superheated liquid extraction (SHLE)

extraction by using aqueous or organic solvents at a high pressure and temperature
without reaching the critical point. It can be implemented in three modes: static (with a
fixed volume of extractant), dynamic (where the extractant flows continuously through
the sample) and staticdynamic (a combination of the two modes). Superheated liquid
extraction has been proven to save solvents, reduce manipulation, improve selectivity
and increase automatability; characteristics which have allowed superheated liquid
extraction to apply to remove polycyclic aromatic hydrocarbons (PAHs), polychlorinated
biphenyls (PCBs), herbicide, metals from soils and sediments, minor organic pollutants
(As, Se and Hg) from coal, metals from used industrial oils, essential oils from laurel
and fatty acids from grape seed, among others.

Seed
According to the OXFORD Dictionary, seed is a reproduction part of a flowering plant
which capable of developing into another such plant. It contain embryo that enclosed in
a protective outer covering called the seed coat, usually with some stored food. It is a
6

characteristic of spermatophytes (gymnosperm and angiosperm plants) and the product

of the ripened ovule which occurs after fertilization and some growth within the mother
plant. The formation of the seed completes the process of reproduction in seed plants
(started with the development of flowers and pollination), with the embryo developed
from the zygote and the seed coat from the integuments of the ovule.

Seeds have been an important development in the reproduction and spread of

gymnosperm and angiosperm plants, relative to more primitive plants such as ferns,
mosses and liverworts, which do not have seeds and use other means to propagate
themselves. This can be seen by the success of seed plants (both gymnosperms and
angiosperms) in dominating biological niches on land, from forests to grasslands both in
hot and cold climates.

Many structures commonly referred to as "seeds" are actually dry fruits. Plants
producing berries are called baccate. Sunflower seeds are sometimes sold
commercially while still enclosed within the hard wall of the fruit, which must be split
open to reach the seed. Different groups of plants have other modifications, the socalled stone fruits (such as the peach) have a hardened fruit layer (the endocarp) fused
to and surrounding the actual seed. Nuts are the one-seeded, hard-shelled fruit of some
plants with an indehiscent seed, such as an acorn or hazelnut.

Type of seed

Seed have been considered to occur in twelve separate types (Martin 1946). These are
based on a number of criteria, of which the dominant one is the "embryo to seed" size
ratios. This reflects the degree to which the developing cotyledons absorb the nutrients
of the endosperm, and this obliterate it. (The Seed Biology Place)

Six types occur amongst the monocotyledons, ten in the dicotyledons, and two in the
gymnosperms (Linear and spatulate). This classification is based on three
characteristics: embryo morphology, amount of endosperm and the position of the
embryo relative to the endosperm. These types are:

Broad (Bean)
Capitate
Lateral
Peripheral
Rudimentary
Dwarf
Micro
Linear
Spatulate
Investing
Bent
Folded

A seed oil is a vegetable oil that is obtained from the seed (endosperm) of some plant,
rather than the fruit (pericarp).

Most vegetable oils are seed oils. Some common examples are sunflower oil, canola oil,
and sesame oil.

Some important vegetable oils are not seed oils. Some examples are olive oil and
peanut oil.

plant
Almond
Argan
Borage
Canola
Castor
Coconut
Corn
Cotton
Flax
Grape
Hemp
Jojoba
Macadamia
Mustard
Neem
Oil palm
Pomagranate
Rapeseed
Safflower
Sesame
Shea
Sunflower
Tonka bean
Tung

oil
almond oil
argan oil
borage oil
canola oil
Castor oil
coconut oil
corn oil
cottonseed oil
linseed oil
grape seed oil
hemp oil
jojoba oil
macadamia oil
mustard oil
neem oil
palm kernel oil
Pomegranate oil
rapeseed oil
safflower oil
sesame oil
shea butter
sunflower oil
tonka bean oil
tung oil

Pomegranate

The pomegranate (Punica granatum), is a fruit-bearing deciduous shrub or small tree

growing between 5 and 8 m (1626 ft) tall. The fruit is typically in season from
September to February in the Northern Hemisphere, and in the Southern Hemisphere
from March to May. As intact arils or juice, pomegranates are used in cooking, baking,
meal garnishes, juice blends, smoothies, and alcoholic beverages, such as cocktails
and wine.

The pomegranate is said to be originated in the region between the himalayas and
Egypt, and has been cultivated since ancient times in India, Persia Mesopotamia,
Turkey and the Arabian Peninsula. In Babylonian texts and the Book of Exodus is the
ancient text that mentioned about pomegranate. It was introduced into Latin America
and California by Spanish settlers in 1769.

Today, it is widely cultivated throughout the Mediterranean region of southern Europe,

the Middle East and Caucasus region, northern Africa and tropical Africa, the Indian
subcontinent, Central Asia, and the drier parts of southeast Asia. It is also cultivated in
parts of California and Arizona.In recent years, it has become more common in the
commercial markets of Europe and the Western Hemisphere.

The pomegranate seeds show average contents of about 37143 g/kg of fruit. The
seeds are lipids rich seed, which vary between 140 and 270 g/kg dry matter (Melgarejo
et al., 1995; Al-Maiman and Ahmad, 2002). Pomegranate seed is a residue obtained
from juice and it contains vitamin E, sterols and punicic acid. Pomegranate seed oil is
has many health and beauty benefits such as prevention of heart attacks, high blood
pressure, atherosclerosis and other cardiovascular diseases, prevent cancer, help in
weight loss, protection against muscle ache and inflammation, maintain healthy and
flawless skin and treat other skin diseases.

10

MATERIALS AND METHOD

A) Materials and reagents
Pomegranate seeds were obtained from Huiyuan Juice Company. Firstly, the seeds
were ground with a high speed mill Model HY-200. The size of particle distribution (%,
w/w) of the ground seeds after the grinding process was varies as follows:
<0.3 mm (17.6%);
0.30.6 mm (42.6%);
0.60.9 mm (28.6%) and;
>0.9 mm (11.7%).
The samples of 2 g in mass, were dried to determine the moisture content within the
seeds at 108 0.5 C until constant weight and the final moisture content of the ground
pomegranate seeds was left 4.4%.
Reagents that were used are fatty acid methyl ester mixture standards, tocopherols
standards (-, - and -tocopherol), 6-hydroxy- 2, 5, 7, 8-tetramethylchroman-2carboxylic acid (Trolox), 2, 2 -azinobis-(3-ethylbenz-thiazoline-6-sulfonic) diammonium
salt (ABTS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH). They were acquired from
chemical distributor company, SigmaAldrich which were operated in Shanghai, China.
On the other hand, HPLC grade solvents were purchased from Merck and the other
chemicals (analytical grade) from Beijing Chemical Co. (Beijing, China). Last but not the
least was Carbon dioxide (99.5%), which was acquired from Pute Gas Co. from Beijing,
China.
11

12

Figure 1 Pomegranate seeds

Figure 2 Dry pomegranate seeds

Figure 3 Agilent 6890N GCFID (Agilent Technologies, DE, USA)

13

Figure 4 High speed mill (Model HY-200, Beijing Huanya Scientific Ltd., China)

Figure 5 HP-Innowax capillary column

Figure 6 Huaan supercritical fluid apparatus (Model HA220-50-06)

14

Figure 7 Shimadzu UV-1800 spectrophotometer (Jiangsu, China)

15

a) Methods
1) Supercritical

CO2

extraction of pomegranate seed oil

The first step was the extraction of pomegranate seeds. The extraction
process of pomegranate seeds was done by using Supercritical CO2 (SCCO2) at different conditions. Huaan supercritical fluid apparatus (Model
HA220-50-06) were used to implement all the SC-SO2 extraction trials.

A sample of 250.0 g of pomegranate seeds was prepared. The sample was

weighed accurately and placed into a 1 L extraction vessel.

Then, the extraction vessel was sealed completely. After the extraction vessel
was tightly sealed, carbon dioxide was pressurized.

After that, the pressurized carbon dioxide was pumped into a coil shaped
evaporator which was heated to the predefined temperature.

Carbon dioxide was pumped through the extraction vessel from bottom to the
top.

Next, carbon dioxide was pumped to the separators at given temperature and
pressure

Each extraction run was set to 2 hours due to the fact that longer extraction
times did not significantly increase the yield of oil.

16

There are 2 separator in the process. The first one was to collect the extracted
oil and the second one was to recover water at certain time intervals. On the
other hand, CO2 gas was recycled to the system.

Pomegranate seeds oil (PSO) was then weighed to obtain the yield. The PSO
yield was expressed as the ratio of the weight (g) of PSO to that of seed (g,
dry basis) placed into the extraction vessel.

Finally, the oil samples were stored at 20 C until further analysis.

17

2) Analysis of fatty acid methyl esters

The preparation of the fatty acid methyl esters (FAMEs) was done by
undergoing the extracted PSO through transesterification process. This
method was introduced by Xu et al. (2008)

Agilent 6890N GCFID was used to analyse FAMEs of the PSO. The
apparatus was equipped with a HP-Innowax capillary column (30 m 0.25
mm i.d., 0.32 m film thickness)

The column temperature was initially at 170 C. The temperature were

maintained for 14 min and then increased to 250 C at 10 C/min. The
temperature was held for 8 min.

The injector and detector temperature were 280 C and 300 C respectively.

18

The carrier gas that was used was Nitrogen (at a flow rate of 1.0 ml/min and
the split ratio was 100:1). The FAME peaks were identified using FAME
standards.

Each sample was analysed three times to acquire more accurate result.

The composition of the fatty acids was calculated from their peak areas.

19

3) Analysis of Tocopherols

The tocopherols content of PSO obtained by SC-CO2 extraction can be

determined according to the procedure reported in the literature (Xu et al.,
2008).

By using Agilent 1100 HPLC, the tocopherols content of PSO was analysed.
Agilent 1100 HPLC was equipped with a diode array detector.

The individual tocopherols were separated on an Agilent

NH 2

column (5.0

m, 250
4.6 mm i.d., Agilent Technologies, DE, USA) protected by a 10 mm guard
column.

At a flow rate of 0.9 ml/min, the mobile phase of n-hexane/isopropanol (95:5,

v/v) was used, and the peaks were detected at 292 nm.

At 30 C, the column temperature was maintained.

20

For the setup of calibration curves, tocopherol standards were used.

The analysis was carried out three times to obtain more accurate result.

21

4) Determination of DPPH radical scavenging activity (RSA)

With some modifications, method by von-Gadow et al. (1997) on scavenging

activity of PSO towards DPPH radical was determined.

2 mL of ethanolic DPPH (104 M) solution was added to 2 mL of ethanolic

solution of PSO. 2 mL of ethanol was used as the blank.

The mixture of the two solution was shaken vigorously. Then, the mixture was
immediately placed in a Shimadzu UV-1800 spectrophotometer to monitor the
decrease in absorbance at 517 nm.

The mixture was monitored continuously for 60 min until the reaction reached
a plateau.

Trolox and -tocopherol (SigmaAldrich) were used as a synthetic reference.

22

The RSA of the oil sample was expressed as percentage inhibition of DPPH,
% Inhibition = [1 (AiAj)] / Ac 100
where Ac is the absorbance of the blank; Ai is the absorbance of the sample
and Aj is the background absorbance of the sample.

The concentration of PSO that was able to constrain 50% of the initial DPPH
radicals (the IC 50 value), was expressed as mg/mL and calculated through
the interpolation of linear regression analysis.

23

RESULTS

Supercritical CO2 Extraction

Figure 1 - The effect of pressure on the extraction yield of PSO at 50 C.

Figure 2 The effect of temperature on the extraction yield of PSO at 30 MPa.

24

Figure 3 IC50 values of PSO through DPPH scavenging activity.

Figure 4 Antioxidant activity of PSO through ABTS scavenging.

25

Table 1 Fatty Acid composition (%, w/w) of PSOs extracted by SC-CO 2 for 2h.

Table 2 Fatty Acid composition (%, w/w) of PSOs extracted by SC-CO 2 at 30MPa and
50oC.

Table 3 Tocopherols content (mg/100g oil) of PSOs extracted by SC-CO 2 for 2h.

26

Table 4 Tocopherols content (mg/100g oil) of PSOs extracted by SC-CO 2 at 30MPa

and
50oC.

Superheated Hexane

Figure 5 Effect of temperature on SHHE (g/g) from 3.0g of pomegranate seeds.

Operating conditions: flow rate = 1.0mL/min, particle size = 0.50mm, pressure = 20bar,
and extraction time = 120min.

27

Figure 6 - Effect of particle size on SHHE (g/g) from 3.0g of pomegranate seeds.
Operating conditions: temperature = 80oC, flow rate = 1.0mL/min, and pressure = 20bar.

Figure 7 - Effect of flow rate on SHHE (g/g) from 3.0g of pomegranate seeds. Operating
conditions: temperature = 80oC, particle size = 0.25mm, and pressure = 20bar.

Table 5 The percentage compositions of pomegranate seed oil fatty acids (%)
extracted by SHHE, Soxhlet extraction and cold pressing.

28

DISCUSSION
Effects of Extraction Pressure of Pomegranate Seed Oil (PSO) on Seed Oil Yield

The yield of pomegranate seed oil increased at a certain time at a different elevation of
pressure (Liu, Xu, He, & Gao, 2012). The experiment on extraction of pomegranate
seed oil (PSO) has been conducted at a range of pressure of 15MPa to 30MPa ( Liu et
al., 2012). Based on the result obtained on Figure 1, within the 120 minutes of time and
at constant temperature of 50 degree Celsius, the extraction yield increased remarkably
with increasing value of pressure. When the extraction was conducted at 15 MPa, only
maximum yield of 20 percent at 45 MPa was achieved after 120 min of extraction. A
plateau in the extraction rate was achieved at pressure of 30 MPa. Further increasing
the pressure to 45 MPa led to a little positive effect on the extraction efficiency. It can be
shown that as time goes by, the yield percent increased slowly and reached its
maximum yield of 20 percent. This can be concluded that, for the experiment, the effect
29

of pressure had given a positive result throughout the whole process where it is most
likely due to the improvement of oil solubility resulted from the increased solvent
density.

Apart from that, high pressure is not really necessary being recommended due to
increased repulsing solute-solvent interactions resulting from highly compressed carbon
dioxide at high-pressure levels, which potentially induce complex extraction and difficult
analysis. Generally, with the increase of pressure, the density of supercritical fluid of
carbon dioxide (SF- CO2 ) increases, and thus the solubility of solute increases. The
extraction yield enhances significantly with the increase of pressure, due to the increase
of the solubility of the oil components. This is attributed to the increase of the carbon
dioxide density, which results in the increase of its dissolving ability.

At low pressure levels, the oil yield decreases with the rise of temperature, most likely
due to the reduced density of carbon dioxide at higher temperatures. At higher
pressures, however, the oil yield increases with the rise of temperature. The crossover
pressure, beyond which the effect of temperature on the oil yield begins to reverse, is
about 30 MPa. The extraction pressure is the main parameter that influenced the
extraction efficiency. It could be observed that the yield of oil significantly increases with
the increase of pressure at a given temperature, especially at low pressure and
temperature. If the given temperature is higher than a certain value (about 45 degree
Celsius), while pressure is rising, the oil yield increases at low-pressure levels. Once the
pressure reaches high levels, the oil yield slightly decreases.

Effects of Extraction Temperature of Pomegranate Seed Oil (PSO) on Seed Oil

Yield

30

According to the results illustrated in Figure 2, the extraction rate and the oil yield were
not significantly affected by the temperature at 30MPa. When the extraction
temperature elevated from 35 to 50 degree Celsius, the yield increased by 8.2 percent.
Based on the results experiment, the further elevation in temperature resulted in a
slower increased of the yield (Liu et al., 2012). Even though at different range of
temperature which is at 35, 50 and 65 degree Celsius, within 120 minutes of times the
increased of the yield occur constantly but slowly. The influence of temperature on
extraction is more difficult to predict than that of pressure, because of its two counter
effects on the yield of oil. First, the temperature elevation decreases the density of
carbon dioxide, leading to a reduction in the solvent power to dissolve the solute.
Second, the temperature rise increases the vapor pressure of the solutes, bringing
about the elevation in the solubility of oils in SF- CO2 . Consequently, the solubility of
the solute is likely to decrease, keep constant, or increase with rising temperatures at
constant pressure, which depends on whether the solvent density or the solute vapor
pressure is the predominant one.
The change of oil yield with the temperature is due to two kinds of effects. On one hand,
the increasing of temperature results in the decrease of solvent density thus decreases
the solubility of seed oil in supercritical fluid (SCF). On the other hand, the saturation
pressure of solute in SCF increases with the increase of temperature, which improves
the solubility.
The Effects of Extraction Pressure and Temperature on Fatty Acid Composition of
Pomegranate Seed Oil (PSO)
Based on Table 1 and 2, Punicic acid, 9c, 11t, 13c-octadecatrienoic acid, is a
conjugated
linolenic acid and has three conjugated double bonds. It was accounted for the highest
percentage which is around 60 percent of the fatty acid profile. There are different types
of fatty acid present in PSO which are oleic acid, linoleic acid, palmitic acid and also

31

stearic acid. Besides that there are also arachidic acid and gadoleic acid present in the
extracted oil. These two types of acids have higher carbon contents of fatty acids.
Based on the result obtained according to Table 1, in the extraction by supercritical
carbon dioxide within two hours, there was slightly increase in the content of punicic,
arachidic and gadoleic acid under higher extraction of pressure and temperature. Apart
from that, the fatty acid composition of seed oil extraction at different interval under
condition of 30 MPa and 50degree Celsius has also been observed during the
experiment and being demonstrated in Table 2. The amount of punicic acid showed that
the greatest individual variation increasing from 59.15 percent to 65.74 percent ( Liu et
al., 2012). However there are seems to be a decrease content of palmitic acid,
palmitoleic acid, stearic acid, oleic acid and linoleic acid when the extraction time have
been lengthen. In conclusion, it can be said that triglycerides with shorter chain length
have much higher solubility. Therefore, it can be extracted by using the super critical
carbon dioxide extraction.
The Effects of Extraction Pressure, Temperature and Time on Tocopherols
Content of PSO
The concentrations of tocopherols in the extracted oil at various extraction conditions
are being illustrated in Table 3. At a different range of pressure which are 15, 30 and 45
MPa and at constant temperature of 50 degree Celsius, the influence of the pressure on
the supercritical of carbon dioxide extraction of tocopherols from pomegranate seed
was estimated. At pressure of 15 MPa, it is observed the content of total tocopherols
was the highest. As the pressure increased throughout the experiment, the content of
total tocopherols began to decrease. Therefore, it is shown that, parameter temperature
affects

the

extraction

behaviors

between

tocopherols and

triglycerides. The

concentration of tocopherols in PSO at 65 degree Celsius was significantly higher than

at 35 degree Celsius and 50 degree Celsius where (P<0.05). The results are being
shown in Table 3. Based on Table 4, at different intervals of oil, the reduction in the
concentration of tocopherols is being observed during the process of extraction.

32

Scavenging Abilities on DPPH Radicals

According to Figure 3 the scavenging abilities of PSO is obtained under different
extraction conditions on DPPH radicals. In the first 10 minutes, PSO that is extracted at
30MPa and 50 degree Celsius had the highest scavenging capacity against DPPH and
the lowest IC50 of DPPH at 0.8 mg/ml. The scavenging ability of PSO dropped when
the IC50 went up to 5.2 mg/ml with the extension of extraction time. Meanwhile, PSO
with low extraction pressure (15 MPa) showed higher antioxidant ability against DPPH
radical than that with higher pressures. The changes of scavenging abilities on DPPH
radicals were the same with those of tocopherols levels in the extracted oil. These
changes can be shown in Figure 3, Table 3 and Table 4.
Scavenging Abilities on ABTS Radicals
Based on the figure shown in Figure 4, there is evident showing that PSO had the
scavenging abilities on ABTS radicals. The radicals scavenging abilities of each oil
samples were expresses in term of micro-gram of trolox equivalent (TE) per gram PSO.
PSO at low extraction pressure which is of 15 MPa show a higher scavenging activities
than that at higher pressure based on the results obtained in the DPPH test. During the
first 10 minutes, the oil sample extracted indicated the highest scavenging abilities with
respect to scavenging abilities of oils from different intervals.

The Comparison of Using Supercritical Carbon Dioxide Extraction and Extraction

Using Superheated Hexane of Pomegranate Seed Oil (PSO)
The superheated solvent extraction is a process that combines temperature and
pressure with liquid solvents to achieve rapid and efficient extraction of analytes from
several matrices. Supercritical fluid extraction using hexane extraction solvents are said
to be highly potential extraction techniques that can be applied, whereby high yield of oil
extraction could be achieved. This is because at supercritical state, the solvent exhibit
33

high extraction capabilities and more solute-solvent interactions can occur, thus
resulting a better solvent capacity Moreover, the oil extracted via supercritical fluid
extraction method will be relatively low in impurities.
Excessive heating gives rise to undesirable formation of free fatty acids from
triacylglycerols during extraction. The main factors affecting superheated hexane
(SHHE) were selected to be extraction temperature, mean particle size and solvent flow
rate each of them in three levels ranging from 80 to 120 degree Celsius, 0.25 to 1.00
mm, and 0.5 to 2.0 mL/min, respectively. Pressure is of minor importance. Pressure was
kept constant and equal to 20 bar to guarantee n-hexane being in liquid state at the
extraction temperatures.
Based on the experiment conducted, the effect of temperature has been the parameter
chosen to be observed. Usually positive effect of temperature on diffusion of solutes in
extraction processes. Thus, it is preferable to carry out superheated solvent extraction
process at the highest temperature. According to Figure 5, the extraction efficiency
increased generally with increasing temperature (Eikani et al., 2012). The resulted
cumulative values were 18.71, 23.84 and 31.27 wt% at 80, 100 and 120 degree Celsius,
respectively. Bright yellow pomegranate seed oil is obtained at 80 and 100 degree
Celsius and dark brown color was achieved at 120 degree Celsius. Such results may
due to degradation of constituents at the higher temperatures.
The mean ground seed particles were selected as 0.25, 0.5, and 1.0 mm. The effect of
mean particle size on the extraction of pomegranate seed oil as cumulative efficiency at
80 degree Celsius temperature, 1 mL/min flow rate, 20 bar pressure, and 120 min
extraction time has been shown in Figure 6. The extraction efficiencies obtained were
22.18, 18.71 and 11.26 wt% for 0.25, 0.50, and 1.00 mm, respectively. The extraction
efficiencies for 0.25 and 0.50 mm size particles were relatively close to each other. It is
a slightly different for larger 1.00 mm particles whereby the obtained efficiency was
much lower. It is proved that the SHHE process may be controlled by mass transfer of

34

oil for larger particle sizes. For further experiments, the optimum value for the mean
particle size was selected as 0.25 mm in order to achieve maximum efficiencies.
The effect of n-hexane flow rate within 0.52.0 mL/min on extraction efficiency of
pomegranate seed oil at 80 degree Celsius temperature, 0.25 mm particle size, 20 bar
pressure and 120 min extraction time has been shown in Figure 7. The values obtained
were 8.24, 22.18 and 29.37 wt% at 0.5, 1.0 and 2.0 mL/min, respectively. To prevent
slower extraction rate and longer extraction times and large amount of final extracts, 1.0
mL/min flow rate was selected to be the optimum value.
It can be concluded that from the result obtained, as the temperature increases, the
time of extraction also increases. It also indicated that the temperature is the main factor
affecting superheated hexane (SHHE). It is different compared to the supercritical
carbon dioxide extraction as in this type of extraction, pressure is the main factor
affecting the extraction yield but still both type of extraction gives a positive effect on the
process of extraction.

CONCLUSION
There are several benefits of using supercritical

CO2 , some of them are;

1. In order to achieve highly concentrated products, single step extraction is

tailored.
2. After using supercritical

CO2 , the extracts and used up biomass are free of

solvent residues.
35

3. Mild and average operating temperature ensures product stability and quality.
4. Supercritical

CO2

is commonly recognised as a biodegradable solvent and are

harmless to the environment.

5. Product recovery can be accomplished through a simple pressure reduction.
6. There are no hazardous solvent wastes as they are eliminated during the
extraction process.
7. By using the pressure dependent dissolving power of supercritical fluids,
compounds in a complex mixture can be selectively filtered.
8. Due to the fact that supercritical fluids have gas-like diffusion properties and the
absence of surface tension limitations, the extraction of supercritical fluids from
microporous substrates can be done.
9. Many extraction process is done by using supercritical

CO2

due to the lower

operating costs. Furthermore, compression energy is more efficient than

distillation energy.
10. Supercritical

CO2

processing can compete economically with traditional

extraction and separation processes because large scale supercritical fluid

extraction are low in price.

In order to extract pomegranate seed oil from pomegranate seed, supercritical

CO2 was used. The major factor affecting the oil yield is the extraction pressure.
The yield of PSO increased with the increase of extraction pressure. The fatty acid
that were identified were palmitic, stearic, oleic, linoleic and punicic acids. The
composition of fatty acids in the PSO were significantly affected by the extraction
parameters. We also found that superheated fluid such hexane can be used in
extraction to replace supercritical

CO2 .

REFERENCES

36

Eikani, H, M., Golmohammad, Fereshteh, Homami, & Saied, S. (2012). Extraction of

pomegranate (Punica granatum L.) seed oil using superheated hexane.
Liu, G., Xu, X., He, Y. G. L., & Gao, Y. (2012). Effects of supercritical CO2 extraction
parameters on chemical composition and free radical-scavenging activity of
pomegranate (Punica granatum L.) seed oil.

M.M.R. de Melo, A.J.D. Silvestre, C.M. Silva (2014). Supercritical fluid extraction of
vegetable matrices: Applications, trends and future perspectives of a convincing green
technology

Abdalbasit, mariod, ismail, et. Al, (2010) comparison of supercritical fluid and hexane
extraction methods in extracting kenaf seed oil.

Japon-lujan and castro, (2006) superheated liquid extraction of oleropein and related
biophenols from olive leaves.

37