Trends in Food Science & Technology 119 (2022) 201–214

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Trends in Food Science & Technology

journal homepage: www.elsevier.com/locate/tifs

Valorisation of plant seed as natural bioactive compounds by various

extraction methods: A review
Nurizzati Mohd Daud a, Nicky Rahmana Putra b, d, Roslina Jamaludin b, d,
Nur Salsabila Md Norodin b, d, Nurul Syaza Sarkawi b, Muhammad Hamiz Syukri Hamzah b,
Hasmida Mohd Nasir b, Dayang Norulfairuz Abang Zaidel b, c, Mohd Azizi Che Yunus b, d,
Liza Md Salleh b, d, *
a
School of Biomedical Engineering and Health Sciences, Faculty of Engineering, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia
b
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia
c
Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia
d
Centre of Lipids Engineering & Applied Research (CLEAR), Ibnu Sina Institute for Scientific and Industrial Research, Universiti Teknologi Malaysia, 81310, Johor
Bahru, Johor, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Background: The extraction of essential oil from plant seeds has a great economic potential for applications in the
Essential oil pharmaceutical, cosmeceutical and food industries. Oilseeds contain various phytochemical compounds such as
Plant seeds polyphenols, fatty acids and flavonoids, which are essential for the health maintenance and prevention of dis­
Soxhlet
ease. Seeds are generally regarded as waste in the agricultural industry; hence the maximum usage of seeds will
Supercritical carbon dioxide
Oil yield
greatly reduce the problem of final disposal.
Scope and approach: This review describes conventional; Soxhlet, Hydrodistillation, Microwave-Assisted and
modern; Supercritical Carbon Dioxide, Subcritical water, High Hydrostatic Pressure, Ultrasound-assisted
extraction methods with a focus on parameters that affect each technique. Comparisons of its oil yield and
the recovery of seeds from different plants are considered to assess the efficiency of the method of selection.
Parameters such as time, temperature, pressure and particle size which influence the production of oil yield and
its quality are also highlighted.
Key findings and conclusions: Conventional extraction methods are usually use polar or non-polar solvents, with
lower temperature and requires longer time to produce high yield. The application of high microwave power can
also obtain high yields in short period of time. While, modern method uses pressure or elevated temperature with
increasing flow rate can increase the oil yield in shorter time. Therefore, the selection of the most effective
methods of extracting oil from plant seeds is important to preserve the quality and achieving maximum effect.

1. Introduction various benefits such as lowering blood pressure, preventing cardio­

Medicinal plants have been used traditionally in folk medicine as
natural healing remedies. About 80% of the world population has been
estimated to relies primarily on herbal products as the main remedies
(Wangchuk, 2018). Approximately, 50 000 plant species are used
worldwide in traditional medicines with the majority of the medicine is
from Asia (Wangchuk, 2018). Plants are a rich source of potential
medicines, and in recent years, there has been a rising understanding of
the value of medicinal plants. Many bioactive compounds contribute to
vascular disease, reducing cancer risk, or reducing blood sugar. These
bioactive compounds can be found in different parts of medicinal plants
such as seed (grape, kiwi, Swietenia macrophylla), leaves (Moringa olei­
fera, Psidium guajava), roots (Eurycoma longifolia) and rhizomes (Cur­
cuma longa). Medicinal plants are also valuable as additives for the food
and cosmetics industry, due to their preservative effects caused by the
presence of antioxidants and antimicrobial activity.
Plant seeds have been used as sources of oil for dietary, medicinal
and industrial purposes. It can be derived directly from industrial fruit

* Corresponding author. Department of Bioprocess & Polymer Engineering, School of Chemical and Energy Engineering, Faculty of Engineering, UTM, 81310,
Johor Bahru, Johor, Malaysia.
E-mail addresses: r-liza@utm.my, i.liza@cheme.utm.my (L. Md Salleh).

https://doi.org/10.1016/j.tifs.2021.12.010
Received 2 March 2021; Received in revised form 17 November 2021; Accepted 5 December 2021
Available online 8 December 2021
0924-2244/© 2021 Published by Elsevier Ltd.
N.M. Daud et al. Trends in Food Science & Technology 119 (2022) 201–214

processing as a by-product. The procedures of the pre-extraction and
extraction process are essential for the recovery of bioactive components
from plant material. The seed is recovered by separating and sifting
technologies as it is typically discarded along with fruit vascular tissues
and skin, or is also known as pomace (Gómez-Mejía et al., 2020). Seeds
are one of the components with the highest concentration of bioactive
molecules in fruit and are generally considered to be waste in the
agro-industry, thus the utilisation of seeds will substantially reduce
problems with final disposal (Gómez-Mejía et al., 2020). The initial
crude extracts obtained by extraction procedures contain a complex
combination of numerous plant metabolites, including alkaloids, phe­
nolics, fatty acids and flavonoids, which vary depending on the kind of
seed. Traditionally, extraction such as hydrodistillation, Soxhlet and
microwave-assisted extraction is used in small settings research. This
extraction capacity relies entirely on the extracting strength of the sol­
vents, either organic or inorganic solvents, depending on the polarity
element for the target compounds (Lee et al., 2014; Pereira et al., 2019).
Contrarily, there are several extraction methods such as supercritical
fluid extraction (SFE) and subcritical water extraction (SWE) are per­
formed to meet the purpose of sustainable environment and
cost-effective technologies.
Despite the presence of bioactive compounds in other parts of plants,
this paper will focus on the seed extract, as there are not many studies on
this subject (Fig. 1a). As seen in the last five years, the publication on the
extract of seed continues to expand. Fig. 1 shows the trend for medicinal
seed, leaves and root extract and its chemical constituents, as mentioned
in the publication. The keywords search was done in a single survey, for
example, medicinal seed extracts and their chemical constituents, which

Fig. 1. (a) Publication on the medicinal seed, leaves and roots extract from 2015 to 2019 and (b) The chemical constituents mention in the publication based on the
medicinal seed, leaves and roots extract from 2015 to 2019 (Web of Science, database).

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was determined using Clarivate Analytics Web of Science database
search from 2015 to 2019. Flavonoids have the highest concentration in
the extract of the seed followed by fatty acids (Fig. 1b). These com­
pounds play an important medicinal role in human health. For example,
a raspberry seed has a high content of polyunsaturated fatty acids such
as Omega 3 and has been shown to have beneficial effects that can be
used in various food, cosmetic, and nutraceutical products (Marić et al.,
2020). Significance advances have been made in modern extraction to
increase yield with the green technology approach. For such a variety of
techniques available, a thorough assessment of the extraction method
selection is needed. Therefore, this paper discussed the various extrac­
tion method including conventional way; hydrodistillation, Soxhlet
extraction and microwave-assisted extraction and modern way; super­
critical fluid extraction (SFE), subcritical water extraction (SWE) and
high hydrostatic pressure extraction (HHPE) method for plant seed as
well as its effect on the parameter of each technique.
2. Plant seed – health benefits and bioactive components
Prior to the extraction process, seeds must undergo a pre-treatment
process to obtain oil including cleaning, seed coat removal, grinding
and preheating. The selected plant seed must be washed and dried to
remove arils, an outer coat of seeds (Regis et al., 2015). The removal of
arils is necessary because it agglomerates with seeds which can promote
fermentation and absorb moisture (Regis et al., 2015). Grinding seed is
important to ensure that oil-bearing embedding in fibrous structures is
broken to release the oil. The surface area will be increased thus, more
solvents can penetrate to bring out the oil efficiently (Efthymiopoulos
et al., 2018). Pre-treatment of seeds by microwave radiation can in­
crease the efficiency of oil extraction and increase the amount of to­
copherols and phytosterols (Fathi-Achachlouei et al., 2019). However,
pre-treatment of seeds by microwave irradiation can decrease the
polyunsaturated fatty acid (PUFA) content due to the degradation pro­
cess (Fathi-Achachlouei et al., 2019).
Plants oils are abundant in essential fatty acids and fat-soluble vi­
tamins, while also containing other plant components such as volatile
organic compounds (VOCs). The extraction of seeds can be further
analysed to identify the characteristics that are related to antioxidant,
antimicrobial, anti-inflammatory, antiviral, anti-diabetic and anti­
cancer. Table 1 shows the benefits of plant seed extracts exhibiting in
medicinal research. The seed of avocado and grape has a high antioxi­
dant and anti-inflammatory activity which are important to be used as
agro-industrial by-products for food and pharmaceutical purposes
(Ghouila et al., 2017; Tremocoldi et al., 2018). Anti-diabetic activity of
seed extracts can aid to regulate blood glucose levels by inhibiting the
carbohydrate digestion enzyme. Digestive enzymes in humans such as
α-amylase and α-glucosidase are responsible for increasing postprandial
Table 1
The benefits of plant seeds extract in medicinal research.
Seed Antioxidant Antimicrobial Anti- Antiviral
inflammatory
Avocado ✓ ✓ ✓ ✓
Grape ✓ ✓ ✓ ✓
Mahogany ✓ ✓ ✓ ✓
Mango ✓ ✓
Roselle ✓ ✓
Pomegranate ✓ ✓
Passionfruit ✓ ✓
Apricot ✓ ✓ ✓ (Kamel et al., 2018; Lee et al., 2014)
Papaya ✓ ✓
Anise ✓ ✓ ✓ (Kozłowska et al., 2016; Lee et al., 2011)
Trends in Food Science & Technology 119 (2022) 201–214
glucose levels. Large molecules like starch cannot pass the blood-brain
barrier, thus alpha-amylase catalyse the hydrolysis of starch which
converting starch into low-molecular-weight products such as glucose
units in order to cross it (Agarwal & Gupta, 2016). Excessive conversion
of glucose to sugars will increase the sugar level in the blood. Insulin will
then initiate the cells to metabolise the excess sugar moieties and store
them as energy sources such as glycogen (Agarwal & Gupta, 2016).
Swietenia macrophylla seeds extract has the potential to be used as
anti-diabetic treatment due to its strong inhibition of α-glucosidase
enzyme activity which is 98.4% but gives moderate inhibition of
α-amylase enzyme activity which is 34.9% (Md Norodin et al., 2018).
The ability of plant herbs in employing antiviral activity plays an
important role in the treatment of viral infections. Many pharmaceutical
companies are prone to develop drugs derived from herbal plants as an
alternative for antiviral development. Limonoids, a phytochemical of
the triterpenoid class contained in the seeds of Swietenia macrophylla
exhibited significant inhibitory activity against Dengue Virus (DENV-2),
the most highly transmitted dengue virus in Asia (Yuan-bin et al., 2014).
In recent findings, grape seed extract which is rich in proanthocyanins
shows the ability to fight Hepatitis A Virus (HAV) and Tulane virus in
combination with heat treatment (Patwardhan et al., 2020).
Plant seeds also demonstrate a vital source of bioactive compounds
that have high potential in showing cytotoxicity against cancer cells.
Most of the polyphenols that are highly accumulated in grape seeds,
ranging from 60% to 70% of the total extractable compounds can exert a
cytotoxic effect (Di Meo et al., 2019). Extracts from grape peel did not
inhibit the growth of malignant mesothelioma cells even in high con­
centrations, while extracts from grape seeds determined cell growth
inhibition in a concentration- and time-dependent manner (Di Meo
et al., 2019). Avocado seeds can induce apoptosis in Jurkat cells used as
a model of human T cell ALL (Acute Lymphocytic Leukemia) (Bonilla-­
Porras et al., 2014). Apoptosis or programmed cell death involves the
impairment of mitochondrial potential and phosphatidylserine exposure
indicative of cytoplasm membrane damage (Bonilla-Porras et al., 2014).
Based on Table 1, seeds from grape, avocado and Swietenia macrophylla
have shown potential therapeutic agents to be used in the pharmaceu­
tical, nutraceutical and food industries. Seeds considered as waste, needs
to undergo series of pre-treatment and the selection of suitable extrac­
tion methods are important. The quality of the seeds can be improved by
extraction into oil which is easy to consume.
3. Conventional extraction method
The yield, taste, flavour and chemical composition (amount and ratio
of components) of essential oil depends on several parameters, such as
plant variety, season, soil, environmental conditions, drying procedure,
storage conditions, distillation and extraction methods of active
Anti- Anti- Anticancer Reference
diabetic hypertensive
(Bonilla-Porras et al., 2014; Tremocoldi et al.,
2018)
✓ ✓ (Di Meo et al., 2019; Ghouila et al., 2017;
Patwardhan et al., 2020)
✓ (Md Norodin et al., 2018; Mohd-nasir et al.,
2018; Yuan-bin et al., 2014)
Nguyen et al. (2019)
Ali et al. (2014)
✓ (Asgary et al., 2014; Natolino & Da Porto,
2019)
✓ ✓ (Pereira et al., 2019; Prasertsri et al., 2019)
✓ (Gonçalves Rodrigues et al., 2019; Pathak et al.,
2014)
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N.M. Daud et al. Trends in Food Science & Technology 119 (2022) 201–214

compounds. In this section, extraction methods which are Soxhlet,
hydrodistillation and microwave-assisted, will be discussed with
focusing on their principle and effect of extraction parameter towards
the plant seed.
3.1. Soxhlet extraction – principles and application
Soxhlet extraction is the most common extraction process to assess
the efficiency of solid-liquid extraction methods and it is easy to
perform. The process takes place by putting a sample in a thimble. The
thimble is then placed inside the main chamber of the Soxhlet extractor.
The extraction process begins when the solvent is heated in the boiling
flask and causing vaporization of the solvent when it reaches its boiling
point. The vaporized solvent will enter the extraction chamber which
leads to condensation of solvent into the sample inside the thimble.
Condensation occurred when the vaporized hot solvent comes into
contact with the surface of the condenser and falling into the Soxhlet
extractor. When the solvent filled the Soxhlet extractor, the solvent will
aspirate back to the boiling flask. This is a time-consuming process
where the process runs repeatedly until completed. When bioactive
compounds are left for long hours at high temperatures, it will cause the
degradation of thermolabile compounds hence reduce their quality.
Solvent such as n-hexane (boiling point: 68.7 ◦ C) is preferable to extract
fats and oil from plant seed as a low boiling point requires less energy to
eliminate the solvent after the extraction process (Kumar et al., 2017).
The efficiency of Soxhlet extraction can be influenced by several factors
such as the average particle size, extraction time and the use of polar and
non-polar solvents.
Plant seed oils have been used in cosmetics, traditional medicine and
food products due to their rich source of bioactive compounds such as
sterol, fatty acids, phenolic and squalene. The pomegranate, cherry and
pumpkin seed was extracted by using diethyl ether as solvents for 6–14 h
to obtain a high percentage of oil yield, contains a complex mixture of
bioactive components and antioxidant compounds such as polyphenols,
carotenoids, sterols and unsaturated fatty acids, considered as nutra­
ceutical substance (Siano et al., 2016). Large quantities of seed
by-products from grapes in the winemaking sector have been studied as
a possible source of polyunsaturated acids such as linoleic and linolenic
acids which are essential for cardiovascular health by down-regulating
low-density lipoprotein cholesterol (Lachman et al., 2015).
3.1.1. Effect of solvents
Factors affecting the performance of solid-liquid extraction include
particle size, physical and chemical properties of the solvent, solubility
dependence on temperature, and solvent agitation (Eikani et al., 2012).
Physicochemical properties of a solvent such as boiling point, dielectric
constant and polarity are important as different types of solvent gives
significant impact on oil yield and bioactive compounds level. The se­
lection of polar or non-polar solvents is based on the evaluation of oil
extraction efficiency while the range in the boiling point of solvents
resulting in understanding the effect of temperature towards oil yield.
The utilisation of high boiling point solvent such as toluene can result in

Table 2
Different extraction condition for plant seeds in conventional method.
Plant Seeds Extraction Extraction Condition Outcomes References
method
Solvent Temperature Time
(◦ C) (Hours)

Passion fruit (Passiflora Soxhlet n-hexane 50 4 n-hexane Ethanol Pereira et al. (2019)
edulis Var. Flavicarpa) Ethanol Oil yield (%) 26.12 ± 20.46 ±
0.74 1.36
Fatty acid composition 84.07 86.63
(linoleic, oleic and α-linolenic
acid) (%)
Total tocopherol content (α-, 8.22 ± 2.98 ±
γ-, δ-) (mg/100 g) 0.01 0.03
Antioxidant activities (%) 53.57 ± 82.81 ±
0.30 0.49
Grapevine Soxhlet hexane 70 – - Linoleic acid = 68.10–78.18 g/100 g oil. - Oleic acid Lachman et al.
(MUFA) = 8.82 g/100 g–16.92 g/100 g (2015)
Almond Hydrodistillation – N/S 2.5 - Oil yield = 0.7% - Benzaldehyde (62.52%), Benzoic acid Geng et al. (2016)
(14.80%), Hexadecane (3.97%)
Apricot Hydrodistillation – N/S 3 Benzaldehyde = 90.6%, Mandelonitrile = 5.2%, Benzoic Lee et al. (2014)
acid = 4.1%
Anise (Pimpinella Soxhlet n-hexane 70 8 Oil yield = 7.40 ± 3.03% Kozłowska et al.
anisum) β-Sitosterol = 4034.53 ± 3.21% (2016)
Total phenolics = 0.42 ± 0.04 mg GA/g oil
DPPH = 3.44 ± 0.29 μmol TEAC/g
Hydrodistillation – N/S 1, 2, and Pimpinella in essential oils = A1: 94.4%, A2: 93.7% and A3: Anastasopoulou
3 97.3% et al. (2020)
Estragole = A1: 2.00%, A2: 3.39%, A3: 5.51%
trans-Anethole = A1: 88.3%, A2: 87.0%, A3: 89.9%)
γ-himachalene = A1: 2.53%, A2: 2.90%, A3: 1.31%
Coriander Soxhlet n-hexane 70 8 Oil yield = 20.00 ± 3.25% Kozłowska et al.
β-Sitosterol = 997.97 ± 8.07% (2016)
Total phenolics = 0.17 ± 0.15 mg GA/g oil
DPPH = 2.24 ± 0.21 μmol TEAC/g
Hydrodistillation – N/S 4 Oil yield at 160 min = 0.358% Zheljazkov et al.
Linalool at 240 min = 68% (2014)
Antioxidative capacity at 240 min = 35.9 μmol TEAC/g
Nutmeg (Myristica Soxhlet n-hexane 70 8 Oil yield = 7.43 ± 2.25% Kozłowska et al.
fragrans) β-Sitosterol = 1203.19 ± 1.67% (2016)
Total phenolics = 1.19 ± 0.12 mg GA/g oil
DPPH = 16.46 ± 0.77 μmol TEAC/g
Hydrodistillation – – 4 Oil yield = 5.811% Ibrahim et al. (2020)
Sabinene = 25.6%, Limonene = 5.84%, Myristicin =
13.16%

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N.M. Daud et al.
a high percentage of oil yield because of the long contact time between
the seed and the solvent, which increases the major amount of mass
transfer of oil from the seeds to solvent. Thus, more heat is needed for
toluene to form vapor in any distillation recovery process, compared to
other solvents which give issue on time-efficiency.
Solvents with a dielectric constant of less than 15 are considered to
be non-polar (Efthymiopoulos et al., 2018). Hexane is a non-polar sol­
vent with high volatility, a low boiling point, and is relatively easy to
remove from the solids and oil at low energy consumption (Krzycz­
kowska & Kozłowska, 2017). The percentage of oil yield and tocopherol
contents of passion fruit are higher when extracted using hexane
(Table 2). This is due to the non-polar solvent of hexane which has a
higher affinity to attract tocopherol, a natural non-polar lipophilic
antioxidant mainly found in vegetable oils (Pereira et al., 2019). Hexane
is preferred for extraction because of the low cost and the high solubility
of oils in hexane. However, the downside of using organic solvents is the
persistence of toxic solvent residues in the oil. Residues may be released
to the environment through improper disposal by users and have
harmful effects on mammals.
The combination of chloroform and methanol as a solvent to extract
seeds from coriander and nutmeg contributes to higher oil yield and
increased antioxidant activity (Kozłowska et al., 2016). Methanol which
is a polar solvent may extract polar materials such as phospholipids and
phenolic compounds. The presence of phenolics and polar lipids in oils
as the bioactive components and their possible interaction with other
constituents of oils may contribute to their stronger activity in scav­
enging free radicals (Kozłowska et al., 2016). However, the use of large
amounts of unsafe organic solvents such as chloroform arises the safety
issue. Prolonged exposure during inhalation with regular use will cause
toxicity to the human body especially the liver and kidney.
Other polar solvents such as ethanol are often used in Soxhlet
extraction because it is non-toxic, safe to use, renewable and it has a
similar capacity to n-hexane for oil extraction. Ethanol and water
contain hydroxyl groups that can form hydrogen bonding with the so­
lute. The extraction of passion fruit was performed to evaluate the in­
fluence of two different solvents; ethanol and n-hexane on antioxidant
activity and fatty acid components of the extracts (Table 2). Ethanolic
extracts give the highest antioxidant activity, phenolic compounds and
unsaturated fatty acids with a clear predominance of linoleic compo­
nents in the seed’s extracts (Pereira et al., 2019). The selection of
polarity-dependent solvents can increase the antioxidant activity and
phenolic compounds due to the high affinity of antioxidant compounds
in seeds towards more polar solvents as compared to nonpolar ones.
3.1.2. Effect of temperature and particle size
The optimum temperature is important in achieving optimum yield
during the extraction process. Even though higher temperatures may
increase the yield production, but the stability of antioxidant compo­
nents will degrade and the denaturation of membranes can happen at
temperatures >40 ◦ C (Sarkar & Ghosh, 2016). Table 2 shows that the
high temperature to extract anise, coriander and nutmeg seed caused
lower antioxidant activity. Raising the temperature contributes to the
increased efficiency of the process due to the decreased strength of
solvent intermolecular forces and allows the better penetration of sol­
vent into the particles. This will decrease the surface tension, thus fa­
voring extraction (Efthymiopoulos et al., 2018). High linoleic acid can
be extracted at 70 ◦ C from grape seed oils without allowing degradation
to occur (Lachman et al., 2015). Excessive heating may lead to the
increasing percentage of oil yield as well due to the undesirable for­
mation of free fatty acids from triacylglycerols during extraction. This
condition can be detected through the physical characteristics of seed oil
specifically by colour change (Eikani et al., 2012).
The mass transfer during the extraction of oil from the plant seeds
depending on intra-particle diffusion (transport of the oil component),
external diffusion (diffusion through the stagnant liquid environment
surrounding the solid seed) and removal of the oil to the adjacent solvent
205
Trends in Food Science & Technology 119 (2022) 201–214
(Kadurumba et al., 2018). The particle size of plant seeds is one of the
importance parameters in determining the efficiency of oil extraction.
The extraction of pomegranate seed shows that the smallest size (0.25
mm) obtain a high percentage of oil yield which is 22.18 wt% while the
largest size (1.0 mm) gives a low percentage of oil yield which is 11.26
wt% (Eikani et al., 2012). The reduction in particle size leads to an
increasing in oil yield due to the increase in surface area which allows
the mass transfer of oil from the solid phase (seed) to the liquid phase
(solvent). Intraparticle diffusion resistance becomes smaller for small
particle sizes due to reduced diffusion path (Eikani et al., 2012). The
transport of the oil component through the matrix pores is more pro­
nounced for larger particles which caused the decrease in oil extraction.
3.2. Hydrodistillation – principles and application
Hydrodistillation (HD) is the most common distillation system for
the extraction of essential oils from plants, equipped with the Clevenger
type apparatus for laboratory-scale studies. In HD process, the selected
plant seeds were placed in a still compartment. An appropriate amount
of water is added and boiled to remove bioactive compounds from the
plant cell. Then, a vapor mixture of oil and water is condensed to
separate essential oils from the water. This technique is not suitable for
heat-labile material as the compounds may be lost or degraded at a high
temperature. Various seeds from fruit, herbs and spices such as apricot,
almond, and black pepper utilised this technique for extraction of
different classes of volatile compounds in plants rich in essential oils.
The complex composition of the essential oil/extract and the variety of
chemical structures of their constituents may be responsible for a wide
range of antifungal, antimalarial, antioxidant and antibacterial
properties.
Ohmic heating-assisted hydrodistillation (OAHD) is the improved
technique of hydrodistillation, a conventional extraction method. OAHD
produces heat in the form of internal energy transformation within the
material and does not rely on conduction and convection modes of heat
transfer (Gavahian & Farahnaky, 2018). The ohmic heater flask can be
equipped with a Clevenger apparatus for essential oil distillation or a
convenient condenser for the distillation of water-soluble materials such
as ethanol distillation (Gavahian et al., 2016; Hashemi et al., 2017). The
electrical conductivity of the material can be adjusted to optimize the
OAHD technique. Shorter extraction time associated with OAHD tech­
nique could be correlated to a quick starting of the extraction process
(due to electroporation) and a higher temperature to reach the boiling
point (Hashemi et al., 2017). The elapsed time to obtain 1.1 mL of
essential oil using conventional HD was 95 min, whereas only 26 min
was enough to obtain the same amount using OAHD (200 V) (Hashemi
et al., 2017). This improved method has been used to enhance the mass
transfer effect and increasing diffusion due to the electroporation effect
(Karunanithi et al., 2019). Application of OAHD improves the oil yield
from seed regaining from sesame and tomato. To ensure the output of
high-quality distillate, it is important to determine the effect of electrical
conductivity and ion concentration of the feed mixture on the final
product requirements.
3.2.1. Effect of extraction time
The selection of optimum time is an important aspect of hydro­
distillation as it will give effects on the quality of oil yield, chemical
content and bioactivity capacity. The duration of time was found to
affect the essential oil yield of anise, dill, coriander, and caraway seed
and others (Anastasopoulou et al., 2020; Shiwakoti et al., 2016; Sintim
et al., 2015; Zheljazkov et al., 2014). Caraway seed oil with a higher
concentration of limonene can be obtained by sampling oil fractions in 2
min of HD process but the level was decreasing as the time increasing
until 100 min of extraction (Shiwakoti et al., 2016). The higher the ratio
of two major components of caraway seed; limonene and carvone, the
better oil yield quality. Therefore, the extraction time between 45 and
75 min will produce good quality oil with a high ratio of limonene and
N.M. Daud et al.
carvone of crushed caraway seed (Shiwakoti et al., 2016). Most of the oil
was eluted at the beginning of the hydrodistillation process; in the 0–2
min. This is due to the crushed seed prior to the HD process to extract
most of the essential oil. With the progression of time, less and less oil
was eluted at each subsequent sampling point.
Carvone and limonene are the major constituents of dill seed oils;
produced through cyclization of geranyl diphosphate to form D-limo­
nene which is then stored in the seed essential oil ducts (Sintim et al.,
2015). Higher antioxidant and antileishmanial activity can be obtained
may be attributed to D-limonene concentrations in dill seed essential oil
at 2 min HD process (Sintim et al., 2015). Anise seed showed the highest
oil yield and 28 identified chemical composition but lower estragole
content, after 1 h of extraction (Table 2). As the extraction time
increased to 3 h, the percentage of oil yield was also increased. However,
the estragole content at this time was also the highest and considered not
safe to use due to the report by Scientific Committee on Food concluded
that estragole is both genotoxic and carcinogenic at a certain level
(Anastasopoulou et al., 2020). Therefore, the determination of hydro­
distillation time is important in order to avoid any harmful chemical
constituents that could also be extracted at a higher level.
3.3. Microwave Assisted Extraction – principles and application
Microwave Assisted Extraction (MAE) is an emerging method of
extraction of diversified bioactive compounds from different plants in
recent years. MAE is a promising method as a green and safe technology
due to the low expenditure of energy and time, high efficiency of
extraction output as well as reduce the necessity of organic solvent used
(Barba et al., 2016). The high efficiency of this process has been
attributed to its use at an industrial scale for example in the extraction of
bioactive compounds mainly antioxidants, oils rich in carotenoids, or
polyphenol derived from agro-industrial residues of fruits or vegetables
(Araújo et al., 2020; Flórez et al., 2015). The principle of the extraction
using MAE is based on the electromagnetic spectrum of radiation, which
is microwave, ranging from 300 MHz (radio radiation) to 300 GHz
(infrared radiation). Microwave can penetrate certain materials and
interacts with the polar components to generate heat. The heating pro­
cess involved the microwave energy on dipole polarization and ion
conduction molecules (Fig. 2) (Flórez et al., 2015).
Dipole polarization leads to the disruption of weak hydrogen bonds
causing subsequent heating (Rodrigues et al., 2020). Microwave energy
can generate thermal effects by changing its frequencies hence easier
penetration by disrupting the cell walls of plant material leads to the
Fig. 2. Schematic setup of microwave-assisted extraction (MAE) (Castro-López et al., 2016).
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Trends in Food Science & Technology 119 (2022) 201–214
dissolution of solute and solvent (Chan et al., 2016; Flórez et al., 2015).
As compared to other conventional methods such as Soxhlet, MAE offers
high amounts of oil yield with significantly lower extraction time. Lower
extraction time results in higher oxidative stability which contributes to
efficient extraction of bioactive compounds such as polyunsaturated
fatty acids, phenols and carotene (Rezvankhah et al., 2019).
Meanwhile, the response of a material to microwaves can be
described by its dielectric properties. Mathematically, the complex
relative permittivity of the material, ℇr (Metaxas & Meredith, 1983):
ℇr=ℇ’ - jℇ’’ (1)
Where ℇ as a dielectric constant while ℇ’’ as a dielectric loss factor.
′
When solvent and biomass are heated volumetrically, the heat lost from
the process can only happen through the solvent. At a steady state, the
temperature difference between the biomass and solvent can only be
none or positive. Directly shown the selectivity of the microwave heat­
ing mechanism where the heating rate of the solvent and biomass is
affected by its respectively dielectric losses which do not account for the
heat loss to the surroundings. Hence, different microwave heating pro­
cesses will support the different electric field intensities. However, the
general principle is that the process variables that affect the heating rate
will show a similar threshold no matter what microwave heating process
is used (Mao et al., 2021).
3.3.1. Effect of microwave power
The use of microwave energy for the extraction of bioactive com­
pounds from plant seeds results in more effective and faster heating. The
waves are absorbed selectively by media possessing a high dielectric
constant resulting in more effective heating. The microwaves’ energy is
then converted into kinetic energy, thus enabling the selective heating of
the microwave-absorbent parts of the plant material. The volume
increased makes cells explode, releasing their content into the liquid
phase. When the liquid phase absorbs the microwaves, the kinetic en­
ergy of its molecules increases, and consequently, the diffusion rate in­
creases too, resulting in faster mass transfer (Gallo et al., 2010). As the
microwave power is higher, higher yields will be obtained in a relatively
short time. The extraction yield of carvone from caraway seeds was
increased as power increased from 50 to 150 W until reached a
maximum yield after 10 min (Chemat et al., 2005). The optimum mi­
crowave power is 287 W for 16 min were sufficient to extract high oil
yield from wet pomace of olive (Yanık, 2017).
N.M. Daud et al.
3.3.2. Effect of solvent-to-sample ratio
In MAE, both the solvent and the sample transform by electromag­
netic energy of high value based on ionic conduction and dipole rotation
(either permanent or induced by the electric field). Ionic conduction,
which is due to the electrophoretic migration of dissolved ions, increases
solvent penetration into the matrix and facilitates the solvation of tar­
geted compounds (Zin et al., 2020). The influence of solvent type on
microwave extraction of specific targeted compounds such as phenolic
has been investigated with several solvents such as pure water, pure and
aqueous forms of acetone, ethanol and methanol. The presence of a
suitable amount of water in the solvent somehow can improve the
swelling of the plant matrix, increasing the contact surface area of the
matrix with the solvent (Hayat et al., 2009).
Higher solvent-to-sample ratios led to higher oil yields. A significant
increase in oil yield between 6:1 and 8:1 was observed in the extraction
of Moringa oleifera seeds (Zhong et al., 2018). This is due to the higher
concentration gradient between the solvent and the material which
causes the solvent to rapidly diffuse into the cell to dissolve the oil, thus
increasing the oil yield (Zhong et al., 2018). The solvent-to-sample ratio
is also related to particle size and affects oil yield significantly. As re­
ported by Cavdar et al. (2017), the oil extraction yield of fine particles
(range 0.125–0.450 mm) of pomegranate seed was increased from 29 to
36% as the solvent-to-sample ratio increased from 2:1 to 10:1. While the
oil extraction yield of medium particle size (range 0.450–0.530 mm) was
increased from 20 to 26% as the solvent-to-sample ratio increased from
2:1 to 10:1 in 12.5 min of extraction time. However, when the coarse
particles (range 0.530–0.800 mm) of pomegranate seeds were extracted
under the same condition of solvent-to-sample ratio (2:1 to 10:1), the
extraction rate increased from 8% to 12%. This is due to the limited
penetration of microwaves into the interior of the coarse seeds. The oil
could not be exerted out from the seeds, thus, there was no extra oil to be
dissolved in the medium even though the amount of solvent increased
(Çavdar et al., 2017).
4. Modern extraction method
Modern extraction methods are important for the advanced devel­
opment of herbal remedies. It is suitable for thermolabile compounds,
has a high degree of automation, free of solvents and has several pa­
rameters that can be controlled at a time. The extraction of compounds
by using the modern method can obtain quality and higher yield with
less extraction time needed. The modern extraction methods are
including supercritical carbon dioxide (SC–CO2), subcritical water
extraction (SWE), high hydrostatic pressure extraction (HHPE) and ul­
trasound assisted extraction (UAE). Other extraction methods such as
pulsed electric field (PEF) technology serve as a pre-treatment process
and are followed by solvent extraction techniques for grape pomace to
obtain phenol compounds (Brianceau et al., 2015). PEF increases the
permeabilization of cell membranes due to the electroporation phe­
nomenon. The damaged of cell tissue caused by high electric field
strengths results in a release of ionic intracellular components to the
extracellular media.
4.1. Supercritical carbon dioxide (SC–CO2) extraction
Supercritical carbon dioxide (SC–CO2) extraction is one of the
available green technology due to the high purity of solute content from
an organic solvent and increases the speed of extraction. The SC-CO2
offers high amounts of bioactive compounds and high solubility of in­
terest compounds (fatty acids, tocopherol and antioxidant compounds)
compared to the conventional method (Putra et al., 2019). This is due to
its low critical temperature (31.1 ◦ C), thus the bioactive compounds are
not easily degraded during the extraction process (Putra et al., 2019). A
homogenous phase is achieved after the evaporation of the vapor phase
and the separation of liquid is beyond the critical point (Baskar et al.,
2019). Solvent removal following extraction is much easier as CO2 can
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Trends in Food Science & Technology 119 (2022) 201–214
easily evaporate, facilitating separation of the analyte from the solvent,
and allows for the CO2 to be recycled using a condenser (Mor­
adi-kheibari et al., 2019). Nevertheless, this method is only applicable to
extract non-polar compounds. The addition of ethanol/methanol as a
modifier into SC-CO2 is one of the development methods to extract the
polar compounds (Radfar & Ghoreishi, 2018). Table 3 shows the
different extraction conditions of various plant seeds and their
outcomes.
There are three mechanisms in the SC-CO2 namely constant extrac­
tion rate (CER), falling extraction rate (FER), and diffusion extraction
rate where the details of the mechanism of the SC-CO2 extraction (Putra
et al., 2019). The first stage or CER region describes the extraction of the
solute from the external or outer surface of particles at a constant rate.
The amount of the solute depends on the equilibrium solubility of fluid.
The convective mass transfer between the solute and solvent phase
occurred and was determined by the external mass transfer mechanism.
The external mass transfer known as the solubility region controls the
extraction of the solute. On the Y curve, this physical process is repre­
sented by a straight-line curve (a linear portion of the curve). At this
region, the physical characteristics are high solubility, rapid extraction
rate, and a minimal amount of dead volume in the associated and
extraction vessel.
The second stage is the FER region where the solute is depleted from
the external surface of the particles and the ‘tied solute’ is extracted at a
slower rate than during the first stage (Mohd-Nasir et al., 2021; Putra
et al., 2019). In this region, the extraction rates or kinetics of mass
transfer decreased substantially due to the depletion of the continuous
layer of the raw material on the particle surface. As an intermediate
region, the controlling step of the extraction process depends on the
increase in temperature. Furthermore, a transition to a
diffusion-controller also occurs in this region.
The third stage is the diffusion extraction rate which is also known as
the internal mass transfer coefficient. The diffusion region represents the
portion of extraction where the process is diffusion-limited (Aris et al.,
2018). The diffusion phenomenon is a condition where there is limited
mobility of the extract within the matrix sample and a limited access of
supercritical fluid to the targeted extract or the inner part of the matrix.
The third part of the yield curve shows that the curve approaches an
asymptotic yield, which represents the total amount of the solute that
has been extracted from the matrix of the material. In this region, in­
ternal mass transfers control the extraction process. The physical char­
acteristics in this region are the lower extraction rate, prolonged
extraction time to reach asymptotic yield and the diffusion-controlled
extraction process.
4.1.1. Effects of pre-treatment and particle size
The importance of pre-treatments such as drying and grinding to the
raw plant material can improve the extraction yield of lipophilic mole­
cules by SC-CO2. The high water content of raw material hinders the
fluid flow of CO2 through the seeds, thus reducing the surface contact
between solutes and solvent (Bimakr et al., 2016). Hot air, oven, freeze,
convective, microwave, and osmotic drying methods will affect the
drying kinetics and its chemical properties such as moisture, lipids, and
isoprenoids. Oven-drying is the most common dehydration process due
to its affordability but often leads to the degradation of thermolabile and
oxidizable substrates such as carotenoids and lipids (Inada et al., 2020).
The effect of different drying methods (by oven-dried or freeze-dried) of
pumpkin by using SC-CO2 and Soxhlet was reported by Durante et al.
(2014). Oven-drying, which is carried out under vacuum, at moderate
temperature (40–60 ◦ C) increased the extraction yields of oil, total
vitamin E and carotenoids (Durante et al., 2014). This method is much
cheaper than freeze-drying, serves a better quality that makes it suitable
for grinding and milling dehydrated materials into a fine powder
(Durante et al., 2014).
The recovery of interest compounds is also affected by the particle
size of the dried sample. Smaller particle sizes will increase the amount
N.M. Daud et al. Trends in Food Science & Technology 119 (2022) 201–214

Table 3
Different extraction condition for plant seeds in Supercritical Carbon Dioxide (SC–CO2).
Plant Seeds Extraction Condition Outcomes References
o
Pressure (MPa) Temperature ( C) Entrainer

Camelina sativa 30–45 40–60 10%–35% Ethanol Astaxanthin = 437 μg/g Xie et al. (2019)
Oil recovery = 421 ± 14 μg/g
Roselle 20–30 40–80 – γ-tocopherol = 1.6–5.6 mg/100 g Peng et al. (2020)
Oil recovery = 4.7%
Echium vulgare 20–40 30–70 – Oleic acid = 72.3% Bilgiç-Keleş et al. (2019)
Oil recovery = 16.2%
Pomegranate 24–32 40–60 Palmitic Acid = 3.3 ± 0.4 mg/mLoil Natolino and Da Porto (2019)
Stearic Acid = 1.49 ± 0.03 mg/mLoil
Oleic Acid = 3.9 ± 0.2 mg/mLoil
Linoleic Acid = 5.9 ± 0.2 mg/mLoil
Punicic Acid = 85.4 ± 0.4 mg/mLoil
Oil recovery = 0.18
Swietenia Macrophylla 20–30 40–80 – Solubility of oil = 1.93–4.77 mg/gCO2 Hilmi et al. (2020)
Grape 20–50 40–70 – γ -Tocopherols = 196 mg/kg Duba and Fiori (2016)
α-tocotrienols = 97 mg/kg.
Melon 25–70 40–70 – Linoleic acid = 67.06–68.22% Bouazzaoui et al. (2018)
Oleic acid = 21.63–22.45%
Palmitic acid = 5.57–6.23%
Stearic acid = 2.98–3.67%
Quinoa 25 40 20% (1:1 Ethanol-Water) Total Fatty Acids = 85% Wejnerowska and Ciaciuch (2018)
Linoleic acid = 50%
Oleic acid = 29%
Palmitic acid = 11%
Rosa canina 30 50 – Linolenic = 18.41% Jahongir et al. (2019)
Oleic acids = 18.42%

of extracted compounds due to the higher surface-to-volume ratio of
smaller particles and the reduction of the path length to reach the bulk
phase (Ekinci & Gürü, 2014). Jahongir et al. (2019) found that the
maximum oil of Rosa canina seed oil (0.097 g/g) was obtained at the
smallest particle sizes (0.27 mm) (Jahongir et al., 2019). The small
sample size might shorten the diffusion path into the seeds. The decre­
ment of extracted recovery may reduce the particle size because of the
reabsorption of the extract on the matrix surfaces, thus decreasing the
solute transportation (Dibagar et al., 2020). The smaller particle sizes or
powdered particles can excessively compact the extraction bed, thus the
channeling effect may occur (Bilgiç-Keleş et al., 2019).
4.1.2. Effects of pressure and temperature
Solubility and selectivity of supercritical fluid extraction are greatly
relied on the equilibrium between density and vapor pressure of solute,
whereas this condition is affected by temperature and pressure. The
higher temperature will decrease the density of SC-CO2 thus alleviating
its solvating power in constant pressure, but it enhances the vapor
pressure of compounds (Xie et al., 2019). Furthermore, the fluid density
increases with increasing pressure at a constant temperature, thus
enhance the solubility of the interest compounds (Natolino & Da Porto,
2019). Natolino and Da Porto (2019) found that the extraction yield of
pomegranate seed oil decreases by SC-CO2 from 0.16 to 0.12 g/g solid
with a temperature of 40–60 ◦ C and the constant pressure of 24 MPa.
This is due to higher temperature condition results in a decrease in CO2
density, which decrease its solvating power, thus reduce the extract
recovery. The highest roselle seed oil recovery by SC-CO2 was 16.17% at
modifier to the carbon dioxide can enhance the solvent power and
selectivity, depending on the molecular polarity of the modifier, hence
increase the solute solubility in supercritical fluid (Martins et al., 2016).
The swelling effect of the matrix caused by the modifier will increase the
contact surface area between the matrix and the supercritical fluid
(Machmudah et al., 2006). Ethanol is a preferable modifier due to its
polarity as shown in Table 4. This is due to the characteristic of ethanol
as a polar solvent, where ethanol can extract the polar compounds.
Furthermore, ethanol is available in the food grade, thus it is safe for the
human body.
Although the water has higher polarity index (9.0) and is safe, it has a
higher melting point (0 ◦ C) as compared to ethanol (− 114.1 ◦ C). The
water can easily transform into a solid-phase and clog the SC-CO2
apparatus. Xie et al. (2019) found that the highest Camila Sativa seed oil
recovery of 25.6% was obtained at 15% ethanol concentration, whereas
oil yield was only 18.1% with pure SC-CO2 (Xie et al., 2019). The total
yield of seed oil increased with increasing ethanol content thus
increased the rate of the extraction. The astaxanthin recovery of the oil
with pure SC-CO2 was 190 μg/g oil, while, the oil yield was 304 μg/g
when extracted with 15% ethanol. Wejnerowska and Ciaciuch (2018)
also found that the addition of ethanol/co-solvent to the quinoa
extraction by SC-CO2 gives a significant impact on the extracted oil. The
Table 4
Spectral polarity index of solvent (Sadek, 2002).
No. Solvent Polarity index

a temperature of 40 C and pressure of 30 MPa (Peng et al., 2020). This is

◦
1 Hexane 0.0
due to the increased density of the solvent, thus increased the solubility 2 Pentane 0.0
3 Toluene 2.4
of lipid. The vapor pressure was also elevated by higher pressure, thus
4 Xylene 2.5
enhance the probability of extract onto the surface particle. 5 Isopropyl alcohol 3.9
6 1-Butanol 4.0
4.1.3. Effects of Entrainer/modifier and Co-Matrices 7 Chloroform 4.1
The SC-CO2 extraction process has a limitation in the interest of 8 Methanol 5.1
9 Acetone 5.1
compounds. It is suitable for extracting triglycerides, fatty acids, ter­
10 Ethanol 5.2
penes and other non-polar compounds. However, it is quite challenging 11 Acetonitrile 5.8
to carry out flavanol, flavonoid and tannins which are polar compounds 12 Dimethyl formamide 6.4
from the seeds. Therefore, the modification of SC-CO2 extraction is 13 Dimethylsulfoxide 7.2
14 Water 9.0
needed to break this limitation. The addition of polar co-solvent or

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increase in the amount of co-solvent (1:1, Wethanol/Wmethanol) from 20%
to 27% contributes to the increased yield by 20% at a temperature of
80 ◦ C.
4.2. Subcritical water extraction (SWE) – principle and application
Subcritical water extraction is regarded as an eco-friendly extraction
method that uses only water as its main solvent, in isolating bioactive
compounds from plants and food materials. In recent years, SWE has
gained wide attention due to its technique which is cleaner, faster and
cheaper than the conventional methods (Asl & Khajenoori, 2013, pp.
459–487). There has been increased interest in valorisation of plant seed
by using SWE as compares to other conventional methods due to its
shorter extraction time, lower costs and environmental compatibility
which lead to a higher quality of the extract. Table 5 shows the different
extraction conditions of plant seeds by using SWE. Subcritical water
occurs when water maintains its liquid state at the temperature above its
boiling point (100 ◦ C) and below its critical point, Tc (374◦ ) under suf­
ficient pressure. Water has unique properties as it can behave differently
at various conditions which garnered strong interest especially using
subcritical water as solvent and reaction medium. It has high dielectric
constant, high polarity and has a disproportionately high boiling point
for its mass (Asl & Khajenoori, 2013, pp. 459–487). Water at room
temperature has high polarity and dielectric constant close to 80,
however, it can be decreased to 56 and 27 which is similar to organic
solvents, by increasing the pressure to 5 MPa and temperature to 100 ◦ C
and 250 ◦ C, respectively (Aliakbarian et al., 2012). Due to their polarity,
the most common compounds extracted are antioxidants, phenolic
compounds, and carbohydrates.
Subcritical water extraction operates by altering the dielectric con­
stant (polarity) of water as its main solvent with increasing the operating
temperature. The mechanism of the SWE process involves a transfer of
solutes from the various active sites in the sample matrix to the
extraction medium through diffusion, partitioning equilibrium, and
convection (Essien et al., 2020). The elution of the solutes from the
matrix (that is, external mass transfer through the liquid film) was said
to be the predominant mass transfer step whereas the solute partitioning
equilibrium between the solvent and the solid matrix was reported as the
rate-limiting mechanism for SWE (Asl & Khajenoori, 2013, pp.
459–487).
4.2.1. Effect of temperature
. There are few important extraction parameters in SWE that can
enhance its efficiency or hydrolysis of plant metabolite and have been
compared and optimized in several studies. Temperature is one of the
most important parameters affecting SWE efficiency. Increased tem­
perature can decrease permittivity, increase the diffusion rate and
decrease the viscosity and surface tension. Superior mass transfer
properties of subcritical water lead to high diffusivity and hence higher
extraction efficiency (Aliakbarian et al., 2012). Subcritical water
extraction process was more efficient than using organic solvent and
aqueous-based system at atmospheric pressure for recovery of antioxi­
dants. By increasing the temperature from 70 ◦ C to 150 ◦ C, the total
phenolic content (TPC) of papaya seeds enhanced 2.6 times from 34.7 ±
0.4 to 91.6 ± 3.0 mg GAE g− 1 (Gonçalves Rodrigues et al., 2019). The
optimum extraction conditions were at 140 ◦ C and 11.6 MPa to extract a
high amount of total polyphenols and flavonoids from grape pomace
(Aliakbarian et al., 2012).
The TPC of pomegranate seed increased with the extraction tem­
perature from 80 ◦ C to 220 ◦ C, gives the maximum TPC was 4854.7 (mg/
100 g DW) which is more than 10-folds than that obtained at 80 ◦ C (He
et al., 2012). When the temperature is increased from 80 to 220 ◦ C, the
decrement of the dielectric constant of subcritical water from 61 to 31
will occur, which is nearly closed to the dielectric constant of organic
solvents (He et al., 2012). Thus, phenolic compounds could dissolve in
subcritical water as much as they dissolve in the organic solvents at high
temperatures. However, TPC will decrease as the temperatures reach
above 260 ◦ C due to degradation of phenolic compounds and the
decreasing of dielectric constant provide less effectiveness to TPC (He
et al., 2012). The discovery of SWE can overcome the constraint in
conventional extraction methods such as time-consuming and costly.
Efficiency to extract nonpolar hydrophobic compounds than polar hy­
drophilic compounds can be achieved when thermal agitation increase
by SWE at higher temperature parameter which decrease the strength of
hydrogen bond (Ko et al., 2014).
Water as the main solvent in SWE has unique features which can
carry out various catalytic and hydrolytic reactions due to its high
reactivity where it becomes a medium rich in H+ and OH− ion (due to
water ionisation) and can act as acid or base catalyst when hot com­
pressed with constant heating (Švarc-Gajić et al., 2020). SWE can
hydrolyse plant matrices such as cellulose, hemicellulose and decom­
position of organic compounds including polymeric material. In the
study of durian seed using SWE hydrolysis as shown in Table 5, it was
found that temperature is the main factor in the hydrolysis of carbo­
hydrates into glucose (32.4% reducing sugar) at the optimum temper­
ature of 140 ◦ C for 3.58 h (Purnomo et al., 2016). Increasing extraction
time was found to show a significant hydrolytic effect when plant matrix
exposed to the ionic product of water (H3O+ and OH− ) and more

Table 5
Different extraction condition for plant seeds in subcritical water extraction (SWE).
Plant seeds Extraction Condition Outcomes Reference

Temperature Time Flow rate Pressure

(◦ C) (min) (mL/min) (MPa)

Pimpinella anisum (Anise: 100–175, 40–60 – 12.5 Trans-anethole: 7.56 mg/g Khajenoori et al.
seeds) (2015)
Durian 120, 140, 160 60–300 – 2–4 Reducing sugar: 32.37% Purnomo et al.
(2016)
Carica papaya L. (Papaya 70–150 5–40 4 10 Ferulic acid: 47.2%; 0.0227 mg/g, Mandelic acid:29.4%; Gonçalves
seeds) 0.1227 mg/g, Vanillic acid: 25.8%; 0.1080 mg/g Rodrigues et al.
(2019)
Grape pomace (skin & 100–140 30 1–2 8–15 Total polyphenols: 31.69 mgGAE/gDP Total flavonoids: 15.28 Aliakbarian et al.
seeds) mgCE/gDP (2012)
Defatted grape seed 80–120 20–120 2–5 10 Total polyphenols: 124 mgGAE/g Duba et al. (2015)
Punica granatum L. 80–280 15–120 – 6 Total phenolic content: 4854.7 mg/100 g DW, Total free amino He et al. (2012)
(Defatted pomegranate acid: 468.69 mg/g, Total reducing sugar: 123.94 mg/g DW, 5-
seed) HMF: 110 μg/g DW
Coriander seed 100–175 45 2 2 Linalool: 83% Eikani et al. (2007)
Aesculus hippocastanum 150–250 5–30 – – Extraction yield: 83%, Total phenols: 26.01 mg/g, Saponins: Gagić et al. (2021)
33.11 mg/g
Sunflower seed 60–130 5–120 3 – Oil yield: 44.3%, Linoleic acid: 25.82 mg/g, Oleic acid: 24.26 Ravber et al. (2015)
mg/g, Chlorogenic acid: 82.07 mg/g, 5-HMF: 8.42 mg/g

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N.M. Daud et al.
carbohydrate molecules can be hydrolysed until depleted, or degrada­
tion can occur when temperature raised above 140 ◦ C. In another study,
it was found that SWE was able to recover the phenolic compounds from
pomegranate residue at 160 ◦ C for 30 min which are 2164.3 mg/100 g
DW (He et al., 2012). This can be explained when lignin compounds in
pomegranate seeds could be hydrolysed with SWE at high temperature
thus breaking the ester and ether bonds and release phenolic compounds
from the seed matrix.
4.2.2. Effect of pressure and flow rate
Pressure has a negligible effect on extraction by using SWE. For each
level of temperature investigated in the present study, a distinct trend
was not observed between pressure changes and yield (Miller et al.,
1998). Hence, in optimizing the SWE of pectin, the extraction pressure
should be kept low provided that the liquid state of the solvent is
maintained (Liew et al., 2018). Although pressure has no significant
effect on extraction efficiency, it can aid the extraction process by
forcing water through sample pores that are unreachable at ambient
temperature (Essien et al., 2020). Pressure may not contribute a lot to
the parameter of SWE compared to temperature because water below
300 ◦ C is incompressible and has no significant effect on the physical
state of water (Rodrigues et al., 2020).
Flow rate is one of the significant factors in the extraction of oil from
plants seed. According to Eikani et al. (2007), the rate of linalool ex­
tractions from the essential oil of coriander seeds was very faster at the
higher flow rates which are 2 ml/min (Eikani et al., 2007). The
increasing flow rate can increase the superficial velocity and thus may
result in quicker mass transfer (Eikani et al., 2007). Although higher
flow rates give higher concentration gradients between the sample and
the solvent, the factor of time also had a major effect on the mass transfer
mechanisms (Duba et al., 2015). The total phenolic yield of defatted
grape seeds was decreased when the flow rate increase from 2 ml/min to
5 ml/min (Duba et al., 2015). In other work stated that, when the flow
rate was decreased from 3 to 2 mL/min, the polyphenol yield increased
from 17.0 to 38.1 mgGAE/g, indicating a higher quantity of phenols
passing from grape pomace to solvent (Pinelo et al., 2006). Thus, it can
be concluded that increasing the solvent flow rate will benefit only in the
initial extraction phase and will decrease substantially. The selection of
the optimum temperature, flow rate and pressure are important to
extract higher yields from a plant seed. Different plant seeds require
different optimum conditions.
4.3. High hydrostatic pressure extraction (HHPE) – principle and
application
High Hydrostatic Pressure Extraction (HHPE) uses super-high hy­
draulic pressure ranging from 100 to 800 MPa and can be operated at
room temperature without any heating treatment. HHPE has also been
used to extract bioactive compounds from peel, leaves and pomace from
grapes, berry and green tea. Recently, it has been used to extract com­
pounds such as phenolic acids, flavonoids and polysaccharides from
plant seeds of papaya, alfalfa and tomato (Briones-Labarca et al., 2015;
Neetoo & Chen, 2010; Strati et al., 2015). HHPE is differs from SWE
because it uses high pressure as the main mechanism in disruption of
plant matrices while the variety of solvent can be used at its reaction
medium. High-pressure treatment of 500 MPa for 2 min at 45 ◦ C applied
to the alfalfa seed during the extraction process can eliminate bacterial
contamination without decreasing the extraction quality of the seed
(Neetoo & Chen, 2010). This extraction technique will minimise the
processing time and can provide higher extraction yields (Xi & Luo,
2016). It has no adverse side effects on the activity or structure of
bioactive components which contribute to great potentials in the phar­
maceutical, metallurgical and food industries. High hydrostatic pressure
mainly affects non-covalent bonds (hydrogen, electrostatic, van der
Waals) which means that low molecular weight compounds, such as
vitamins, pigments, and flavonoids, are less affected (Huang et al.,
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Trends in Food Science & Technology 119 (2022) 201–214
2013). The HHPE method is isostatic, i.e., the pressure is transmitted
uniformly and instantly, and adiabatic, which means that no matter
what the biomaterial shape or size is, there is little variation in tem­
perature with increasing pressure (the temperature increases approxi­
mately 3 ◦ C per 100 MPa, depending on the composition of the
biomaterial) (Wilson et al., 2008).
4.3.1. Effect of pressure and time
During high-pressure treatment, the solubility of the soluble con­
stituents will also increase, thus enhanced the equilibrium concentration
in the extract. The deprotonation of charge groups and the disruption of
salt bridges and hydrophobic bonds will occur (Briones-Labarca et al.,
2015). This will result in conformational changes and protein denatur­
ation, which may lead to the high permeability of the cellular mem­
branes. As the hydrostatic pressure is higher, more solvents will enter
the cell. Consequently, more compounds will permeate the cell mem­
brane, which can result in a higher extraction yield (Briones-Labarca
et al., 2015). Different solvents were used depending on the types of
extracted compounds. Methanol was used as solvents to extract phenolic
and flavonoid compounds while n-hexane was used to extract oil and
fatty acids from the papaya seed (Briones-Labarca et al., 2015).
As the time increased from 5 to 15 min, the extraction yield was also
increased at constant pressure (500 Mpa). This is due to the high
diffusion speed of the solvent or the equilibrium of pressure between the
inside and outside of the cells is rapid (Shouqin et al., 2005). The higher
total phenolic, flavonoid, sulforaphane content and the stronger anti­
oxidant activity from papaya seed extracted within 15 min by HHPE
were also obtained when compared to conventional extraction which
requires more time (Briones-Labarca et al., 2015). Strati et al., 2015
demonstrated that HHPE can be performed at 700 MPa using lower
solvent volume at reduced processing times (10 min) without affecting
the extraction yields of tomato waste. High pressure will induce the
extracting solvent into cells, increased cell permeability and thus the
leaching-out rates of bioactive components are improved (Strati et al.,
2015). The denaturation of the carotenoid-binding protein induced by
pressure will damage the cell wall and facilitate the extraction of ca­
rotenoids from tomatoes (Strati et al., 2015).
4.4. Ultrasound-assisted extraction (UAE) – principle and application
Ultrasound-assisted extraction (UAE) is one of the green extraction
technologies that can reduce the use of solvent, energy, wastes and
environmental pollution while obtaining high yields of product at the
same time (Wen et al., 2018). UAE applied the principle of acoustic
cavitation which may cause cell walls disruption in the plant matrix and
thus promoting the discharge of bioactive compounds (Tiwari, 2015).
Cavitation is the development, expansion, and breakdown of gas–vapor
filled bubbles in a liquid, which can damage cell walls, reduce particle
size, and improve mass transfer across cell membranes. The cracks are
formed in the cell wall due to cavitation which enhances the plant tis­
sue’s permeability, allowing the solvent to enter the inner part of the
material and the extracts to be washed out more easily. The improve­
ment of permeability of the plant tissues enhances the quantum of sol­
vents that penetrate inside of the tissues (Shirsath et al., 2012). There are
different optimal values of operating parameters such as ultrasound
power, frequency, time and temperature on the recovery of maximum
oil yield from seed.
4.4.1. Effect of extraction time, temperature and power
The extraction time has a correlation with the extraction efficiency
due to the interaction between the cells of sample and solvent in UAE
process. Samaram et al. (2015) stated that higher oil recovery of papaya
seed can be obtained by prolonging the extraction time, elevating tem­
perature and ultrasound power, simultaneously. This is due to an in­
crease of contact area between the solvent and the sample leads to the
rupture of the cell wall during the sonication period, making the mass
N.M. Daud et al.
transfer rate increase. The mechanical vibration of ultrasound probe
related to ultrasound power resulted in wider contact surface area be­
tween solid and liquid phases. Moreover, the application of higher ul­
trasound power results in more extensive cavitations, where the bubbles
can explosively collapse and generate the localised pressure. The
enhanced pressure will disrupt the plant tissue and consequently
improve the release rate of intracellular substances from the sample
matrix into the solvent (Samaram et al., 2015). A greater amount of
phytosterols and tocopherols are also been extracted, thus greater
oxidative stability can be obtained.
As the temperature increases, it may cause a reduction in the vis­
cosity and surface tension, as well as a rise in vapor pressure. More
solvent vapours penetrate the bubble cavity and cavitation bubbles as
vapor pressure rises, resulting in a less dramatic collapse and reduced
sonication effects. The extraction rate constant will also be reduced
when applying high temperature. It was caused by the decrease in
cavitation intensity as a result of lower surface tension and greater
cavitation bubble vapor pressure (Chemat et al., 2017). Consequently,
the oil stability will be affected through chemical decomposition or
thermal degradation.
4.4.2. Effect of frequency
Ultrasound-assisted extraction (UAE) utilizes sound waves of 20 kHz
to 100 MHz to accelerate the heat and mass transfer of the extraction
process which leads to the damaged cell wall of the solute and releases a
higher amount of bioactive compounds (Roselló-Soto et al., 2015).
Chukwumah et al. (2009) found that different frequency can extract a
different kind of bioactive compounds and high frequency is not
favouring the extraction. It has been reported that at a lower frequency
of 25 kHz, higher extraction of daidzein and genistein were obtained and
at a higher frequency of 80 kHz, biochanin A and trans-resveratrol were
obtained (Chukwumah et al., 2009). However, a higher frequency (80
kHz) requires a longer extraction time. This is because of acoustic
cavitation will be more challenging to produce at higher frequencies
because cavitation bubbles take time to form during the rarefaction
cycle, and compression-rarefaction cycles are not enough to allow for
greater cavitation bubble growth. The length of cavitation bubbles to
grow is longer when the ultrasonic frequency is higher. Hence, it re­
quires larger intensities and amplitudes to produce cavitation at high
frequencies (Chemat et al., 2017).
5. Conclusion
This review emphasises the extraction parameters, such as temper­
ature and pressure, type of solvent and extraction time for complete
recovery of bioactive compounds from plant seeds using conventional
and modern extraction methods. The selection of the best extraction
method is critical to preserve its quality and increase the yield. Soxhlet
method is the most widely used extraction technique to obtain oil from
plant seeds. While Soxhlet presents high oil yields, the extraction time is
longer, and the usage of its solvents is not eco-friendly. The aspect of
time in the hydrodistillation method is important as the first few minutes
of extraction will obtain a high yield of essential oil from seeds and the
quality of yield will be decreased. The effect of particle size of seeds is
important in microwave-assisted extraction (MAE) to ensure micro­
waves can penetrate more into the interior of the seeds. Therefore, the
fine particle size of the seed is preferable to ensure a high extraction
yield can be obtained. Adaptation of green technologies, such as su­
percritical carbon dioxide (SC–CO2) and subcritical water becoming a
promising approach because of environmental, health and energy issues.
The usage of green solvents is effective to reduce downstream processing
steps without causing any effect on the desired yield. The extraction by
high hydrostatic pressure extraction (HHPE) can be explored more
especially for plant seeds as this technique is not only effective in
extracting compound but it can also eliminate bacterial contamination
due to its high-pressure treatment. Bioactive compounds in plant seed
211
Trends in Food Science & Technology 119 (2022) 201–214
such as fatty acids, flavonoids and phenols, have many benefits to be
applied in the pharmaceutical, cosmetic, or food sectors. Future research
findings should explore more through in vivo models to assess the
effectiveness of the extracts regarding their antilipidemic, anticancer,
antiviral and anti-hypertensive.
CRediT authorship contribution statement
Nurizzati Mohd Daud: Investigation, Methodology, Writing - Orig­
inal Draft, Writing - Review & Editing, Validation, Visualization. Nicky
Rahmana Putra: Investigation, Methodology, Writing - Original Draft,
Writing - Review & Editing. Roslina Jamaludin: Investigation, Method­
ology, Writing - Original Draft, Writing - Review & Editing. Nur Salsa­
bila Md Norodin: Investigation, Methodology, Writing - Original Draft,
Writing - Review & Editing. Nurul Syaza Sarkawi: Investigation, Meth­
odology, Writing - Original Draft, Writing - Review & Editing.
Muhammad Hamiz Syukri Hamzah: Investigation, Methodology,
Writing - Original Draft. Dayang Norulfairuz Abang Zaidel: Investiga­
tion, Writing - Original Draft. Hasmida Mohd Nasir: Writing - Review &
Editing, Mohd Azizi Che Yunus: Writing - Review & Editing. Liza Md
Salleh: Conceptualization, Project administration.
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgements
This research was supported by the Ministry of Higher Education
Malaysia, Fundamental Research Grant Scheme (Q.
J130000.2506.20H87) and Professional Development Research Uni­
versity (Q.J130000.21A2.05E03).
References
Agarwal, P., & Gupta, R. (2016). Alpha-amylase inhibition can treat diabetes mellitus.
Res. Rev. J. Med. Health Sci, 5(4), 1–8.
Aliakbarian, B., Fathi, A., Perego, P., & Dehghani, F. (2012). Extraction of antioxidants
from winery wastes using subcritical water. The Journal of Supercritical Fluids, 65,
18–24. https://doi.org/10.1016/j.supflu.2012.02.022
Ali, S. A. E., Mohamed, A. H., & Mohammed, G. E. E. (2014). Fatty acid composition,
anti-inflammatory and analgesic activities of Hibiscus sabdariffa Linn. seeds. Journal
of Advanced Veterinary and Animal Research, 1, 50–57. https://doi.org/10.5455/
javar.2014.a13
Anastasopoulou, E., Graikou, K., Ganos, C., Calapai, G., & Chinou, I. (2020). Pimpinella
anisum seeds essential oil from Lesvos island: Effect of hydrodistillation time,
comparison of its aromatic profile with other samples of the Greek market. Safe use.
Food and Chemical Toxicology, 135, 110875.
Araújo, R. G., Rodriguez-Jasso, R. M., Ruiz, H. A., Govea-Salas, M., Pintado, M. E., &
Aguilar, C. N. (2020). Process optimization of microwave-assisted extraction of
bioactive molecules from avocado seeds. Industrial Crops and Products. https://doi.
org/10.1016/j.indcrop.2020.112623
Aris, N. A., Zaini, A. S., Nasir, H. M., Idham, Z., Vellasamy, Y., & Yunus, M. A. C. (2018).
Effect of particle size and co-extractant in Momordica charantia extract yield and
diffusion coefficient using supercritical CO2. Malaysian Journal of Fundamental and
Applied Sciences, 14(3), 368–373.
Asgary, S., Sahebkar, A., Afshani, M. R., Keshvari, M., Haghjooyjavanmard, S., &
Rafieian-Kopaei, M. (2014). Clinical evaluation of blood pressure lowering,
endothelial function improving, hypolipidemic and anti-inflammatory effects of
pomegranate juice in hypertensive subjects. Phytotherapy Research, 28(2), 193–199.
Asl, A. H., & Khajenoori, M. (2013). Subcritical water extraction. Mass Transfer-
Advances in sustainable energy and environment oriented numerical modeling, -.
Barba, F. J., Zhu, Z., Koubaa, M., Sant’Ana, A. S., & Orlien, V. (2016). Green alternative
methods for the extraction of antioxidant bioactive compounds from winery wastes
and by-products: A review. Trends in Food Science & Technology. https://doi.org/
10.1016/j.tifs.2016.01.006
Baskar, G., Kalavathy, G., Aiswarya, R., & Abarnaebenezer Selvakumari, I. (2019). 7 -
advances in bio-oil extraction from nonedible oil seeds and algal biomass. In K. Azad
(Ed.), Advances in eco-fuels for a sustainable environment (pp. 187–210). Woodhead
Publishing. https://doi.org/10.1016/B978-0-08-102728-8.00007-3.
Bilgiç-Keleş, S., Şahin-Yeşilçubuk, N., Barla-Demirkoz, A., & Karakaş, M. (2019).
Response surface optimization and modelling for supercritical carbon dioxide
extraction of Echium vulgare seed oil [Article]. The Journal of Supercritical Fluids,
143, 365–369. https://doi.org/10.1016/j.supflu.2018.09.008
N.M. Daud et al.
Bimakr, M., Rahman, R. A., Ganjloo, A., Taip, F. S., Adzahan, N. M., & Sarker, M. Z. I.
(2016). Characterization of valuable compounds from winter melon (Benincasa
hispida (Thunb.) Cogn.) seeds using supercritical carbon dioxide extraction
combined with pressure swing technique [journal article]. Food and Bioprocess
Technology, 9(3), 396–406. https://doi.org/10.1007/s11947-015-1636-3
Bonilla-Porras, A. R., Salazar-Ospina, A., Jimenez-Del-Rio, M., Pereañez-Jimenez, A., &
Velez-Pardo, C. (2014). Pro-apoptotic effect of Persea americana var. Hass (avocado)
on Jurkat lymphoblastic leukemia cells. Pharmaceutical Biology, 52, 458–465.
https://doi.org/10.3109/13880209.2013.842599
Bouazzaoui, N., Bouajila, J., Camy, S., Mulengi, J. K., & Condoret, J.-S. (2018). Fatty acid
composition, cytotoxicity and anti-inflammatory evaluation of melon (Cucumis melo
L. Inodorus) seed oil extracted by supercritical carbon dioxide. Separation Science and
Technology, 53(16), 2622–2627.
Brianceau, S., Turk, M., Vitrac, X., & Vorobiev, E. (2015). Combined densification and
pulsed electric field treatment for selective polyphenols recovery from fermented
grape pomace. Innovative Food Science & Emerging Technologies, 29, 2–8. https://doi.
org/10.1016/j.ifset.2014.07.010
Briones-Labarca, V., Plaza-Morales, M., Giovagnoli-Vicuna, C., & Jamett, F. (2015). High
hydrostatic pressure and ultrasound extractions of antioxidant compounds,
sulforaphane and fatty acids from Chilean papaya (Vasconcellea pubescens) seeds:
Effects of extraction conditions and methods. LWT-Food Science and Technology, 60
(1), 525–534.
Castro-López, C., Rojas, R., Sánchez-Alejo, E. J., Niño-Medina, G., & Martínez-Ávila, G. C.
(2016). Phenolic compounds recovery from grape fruit and by-products: An
overview of extraction methods. Grape and wine biotechnology, 103–123.
Çavdar, H. K., Yanık, D. K., Gök, U., & Göğüş, F. (2017). Optimisation of microwave-
assisted extraction of pomegranate (Punica granatum L.) seed oil and evaluation of
its physicochemical and bioactive properties. Food Technology and Biotechnology, 55
(1), 86–94.
Chan, C. H., Yeoh, H. K., Yusoff, R., & Ngoh, G. C. (2016). A first-principles model for
plant cell rupture in microwave-assisted extraction of bioactive compounds. Journal
of Food Engineering. https://doi.org/10.1016/j.jfoodeng.2016.05.017
Chemat, S., Aït-Amar, H., Lagha, A., & Esveld, D. (2005). Microwave-assisted extraction
kinetics of terpenes from caraway seeds. Chemical Engineering and Processing: Process
Intensification, 44(12), 1320–1326.
Chemat, F., Rombaut, N., Sicaire, A.-G., Meullemiestre, A., Fabiano-Tixier, A.-S., & Abert-
Vian, M. (2017). Ultrasound assisted extraction of food and natural products.
Mechanisms, techniques, combinations, protocols and applications. A review.
Ultrasonics Sonochemistry, 34, 540–560.
Chukwumah, Y. C., Walker, L. T., Verghese, M., & Ogutu, S. (2009). Effect of frequency
and duration of ultrasonication on the extraction efficiency of selected isoflavones
and trans-resveratrol from peanuts (Arachis hypogaea). Ultrasonics Sonochemistry, 16
(2), 293–299.
Di Meo, F., Aversano, R., Diretto, G., Demurtas, O. C., Villano, C., Cozzolino, S., Filosa, S.,
Carputo, D., & Crispi, S. (2019). Anti-cancer activity of grape seed semi-polar
extracts in human mesothelioma cell lines. Journal of Functional Foods, 61, 103515.
https://doi.org/10.1016/j.jff.2019.103515
Dibagar, N., Kowalski, S. J., Chayjan, R. A., & Figiel, A. (2020). Accelerated convective
drying of sunflower seeds by high-power ultrasound: Experimental assessment and
optimization approach. Food and Bioproducts Processing.
Duba, K. S., Casazza, A. A., Mohamed, H. B., Perego, P., & Fiori, L. (2015). Extraction of
polyphenols from grape skins and defatted grape seeds using subcritical water:
Experiments and modeling. Food and Bioproducts Processing, 94, 29–38. https://doi.
org/10.1016/j.fbp.2015.01.001
Duba, K. S., & Fiori, L. (2016). Solubility of grape seed oil in supercritical CO2:
Experiments and modeling. The Journal of Chemical Thermodynamics, 100, 44–52.
https://doi.org/10.1016/j.jct.2016.04.010
Durante, M., Lenucci, M. S., D’Amico, L., Piro, G., & Mita, G. (2014). Effect of drying and
co-matrix addition on the yield and quality of supercritical CO2 extracted pumpkin
(Cucurbita moschata Duch.) oil. Food Chemistry, 148, 314–320. https://doi.org/
10.1016/j.foodchem.2013.10.051
Efthymiopoulos, I., Hellier, P., Ladommatos, N., Russo-Profili, A., Eveleigh, A., Aliev, A.,
Kay, A., & Mills-Lamptey, B. (2018). Influence of solvent selection and extraction
temperature on yield and composition of lipids extracted from spent coffee grounds.
Industrial Crops and Products, 119, 49–56.
Eikani, M. H., Golmohammad, F., & Homami, S. S. (2012). Extraction of pomegranate
(Punica granatum L.) seed oil using superheated hexane. Food and Bioproducts
Processing, 90(1), 32–36. https://doi.org/10.1016/j.fbp.2011.01.002
Eikani, M. H., Golmohammad, F., & Rowshanzamir, S. (2007). Subcritical water
extraction of essential oils from coriander seeds (Coriandrum sativum L.). Journal of
Food Engineering, 80(2), 735–740. https://doi.org/10.1016/j.jfoodeng.2006.05.015
Ekinci, M. S., & Gürü, M. (2014). Extraction of oil and β-sitosterol from peach (Prunus
persica) seeds using supercritical carbon dioxide. The Journal of Supercritical Fluids,
92, 319–323. https://doi.org/10.1016/j.supflu.2014.06.004
Essien, S. O., Young, B., & Baroutian, S. (2020). Recent advances in subcritical water and
supercritical carbon dioxide extraction of bioactive compounds from plant materials.
Trends in Food Science & Technology, 97, 156–169. https://doi.org/10.1016/j.
tifs.2020.01.014
Fathi-Achachlouei, B., Azadmard-Damirchi, S., Zahedi, Y., & Shaddel, R. (2019).
Microwave pretreatment as a promising strategy for increment of nutraceutical
content and extraction yield of oil from milk thistle seed. Industrial Crops and
Products, 128, 527–533. https://doi.org/10.1016/j.indcrop.2018.11.034
Flórez, N., Conde, E., & Domínguez, H. (2015). Microwave assisted water extraction of
plant compounds. Journal of Chemical Technology and Biotechnology. https://doi.org/
10.1002/jctb.4519
212
Trends in Food Science & Technology 119 (2022) 201–214
Gagić, T., Knez, Ž., & Škerget, M. (2021). Subcritical water extraction of horse chestnut
(Aesculus hippocastanum) tree parts. Journal of the Serbian Chemical Society, 86(6),
603–613.
Gallo, M., Ferracane, R., Graziani, G., Ritieni, A., & Fogliano, V. (2010). Microwave
assisted extraction of phenolic compounds from four different spices. Molecules, 15
(9), 6365–6374.
Gavahian, M., & Farahnaky, A. (2018). Ohmic-assisted hydrodistillation technology: A
review. Trends in Food Science & Technology, 72, 153–161.
Gavahian, M., Farahnaky, A., Shavezipur, M., & Sastry, S. (2016). Ethanol concentration
of fermented broth by ohmic-assisted hydrodistillation. Innovative Food Science &
Emerging Technologies, 35, 45–51.
Geng, H., Yu, X., Lu, A., Cao, H., Zhou, B., Zhou, L., & Zhao, Z. (2016). Extraction,
chemical composition, and antifungal activity of essential oil of bitter almond.
International Journal of Molecular Sciences, 17(9), 1421.
Ghouila, Z., Laurent, S., Boutry, S., Vander Elst, L., Nateche, F., Muller, R., &
Baaliouamer, A. (2017). Antioxidant, antibacterial and cell toxicity effects of
polyphenols Fromahmeur bouamer grape seed extracts. Journal of Fundamental and
Applied Sciences, 9(1), 392–420.
Gómez-Mejía, E., Roriz, C. L., Heleno, S. A., Calhelha, R., Dias, M. I., Pinela, J., Rosales-
Conrado, N., León-González, M. E., Ferreira, I. C., & Barros, L. (2020). Valorisation of
black mulberry and grape seeds: Chemical characterization and bioactive potential.
Food Chemistry, 337, 127998.
Gonçalves Rodrigues, L. G., Mazzutti, S., Vitali, L., Micke, G. A., & Ferreira, S. R. S.
(2019). Recovery of bioactive phenolic compounds from papaya seeds agroindustrial
residue using subcritical water extraction. Biocatalysis and Agricultural Biotechnology,
22, 101367. https://doi.org/10.1016/j.bcab.2019.101367
Hashemi, S. M. B., Nikmaram, N., Esteghlal, S., Khaneghah, A. M., Niakousari, M.,
Barba, F. J., Roohinejad, S., & Koubaa, M. (2017). Efficiency of Ohmic assisted
hydrodistillation for the extraction of essential oil from oregano (Origanum vulgare
subsp. viride) spices. Innovative Food Science & Emerging Technologies, 41, 172–178.
Hayat, K., Hussain, S., Abbas, S., Farooq, U., Ding, B., Xia, S., Jia, C., Zhang, X., & Xia, W.
(2009). Optimized microwave-assisted extraction of phenolic acids from citrus
Mandarin peels and evaluation of antioxidant activity in vitro. Separation and
Purification Technology, 70(1), 63–70.
He, L., Zhang, X., Xu, H., Xu, C., Yuan, F., Knez, Ž., Novak, Z., & Gao, Y. (2012).
Subcritical water extraction of phenolic compounds from pomegranate (Punica
granatum L.) seed residues and investigation into their antioxidant activities with
HPLC–ABTS+ assay. Food and Bioproducts Processing, 90(2), 215–223.
Hilmi, M. L. B., Salleh, L. M., Jamaludin, R., Norodin, N. S. M., & Zaidel, D. N. A. (2020).
Solubility of swietenia macrophylla seeds in supercritical carbon dioxide extraction.
IOP Conference Series: Materials Science and Engineering.
Huang, H.-W., Hsu, C.-P., Yang, B. B., & Wang, C.-Y. (2013). Advances in the extraction
of natural ingredients by high pressure extraction technology. Trends in Food Science
& Technology, 33(1), 54–62.
Ibrahim, M. A., Cantrell, C. L., Jeliazkova, E. A., Astatkie, T., & Zheljazkov, V. D. (2020).
Utilization of nutmeg (Myristica fragrans Houtt.) seed hydrodistillation time to
produce essential oil fractions with varied compositions and pharmacological effects.
Molecules, 25(3), 565.
Inada, K. O. P., Nunes, S., Martínez-Blázquez, J. A., Tomás-Barberán, F. A., Perrone, D., &
Monteiro, M. (2020). Effect of high hydrostatic pressure and drying methods on
phenolic compounds profile of jabuticaba (Myrciaria jaboticaba) peel and seed. Food
Chemistry, 309, 125794.
Jahongir, H., Miansong, Z., Amankeldi, I., Yu, Z., & Changheng, L. (2019). The influence
of particle size on supercritical extraction of dog rose (Rosa canina) seed oil. Journal
of King Saud University - Engineering Sciences, 31(2), 140–143. https://doi.org/
10.1016/j.jksues.2018.04.004
Kadurumba, C. H., Orakwue, C. C., & Agu, C. M. (2018). Kinetics, thermodynamics and
process parameter impact on solvent extraction of oil from Colocynthis vulgaris
Shrad (melon) seeds. Journal of the Chinese Advanced Materials Society, 6(2),
186–206.
Kamel, G., Awad, N. E., & Shokry, A. A. (2018). Phytochemical screening, acute toxicity,
analgesic and anti-inflammatory effects of apricot seeds ethanolic extracts. Journal of
Applied Veterinary Sciences, 3(1), 26–33.
Karunanithi, S., Pare, A., Sunil, C., & Loganathan, M. (2019). Optimization of process
parameters of ohmic heating for improving yield and quality of tomato seed oil. Int.
J. Pure App. Biosci, 7(3), 104–114.
Khajenoori, M., Asl, A. H., & Eikani, M. H. (2015). Optimization of subcritical water
extraction of pimpinella anisum seeds. Journal of Essential Oil Bearing Plants, 18(6),
1310–1320. https://doi.org/10.1080/0972060X.2013.831564
Ko, M.-J., Cheigh, C.-I., & Chung, M.-S. (2014). Relationship analysis between flavonoids
structure and subcritical water extraction (SWE). Food Chemistry, 143, 147–155.
Kozłowska, M., Gruczyńska, E., Ścibisz, I., & Rudzińska, M. (2016). Fatty acids and
sterols composition, and antioxidant activity of oils extracted from plant seeds. Food
Chemistry, 213, 450–456.
Krzyczkowska, J., & Kozłowska, M. (2017). Effect of oils extracted from plant seeds on
the growth and lipolytic activity of Yarrowia lipolytica yeast. Journal of the American
Oil Chemists’ Society, 94(5), 661–671.
Kumar, S. J., Prasad, S. R., Banerjee, R., Agarwal, D. K., Kulkarni, K. S., & Ramesh, K.
(2017). Green solvents and technologies for oil extraction from oilseeds. Chemistry
Central Journal, 11(1), 1–7.
Lachman, J., Hejtmánková, A., Táborský, J., Kotíková, Z., Pivec, V., Střalková, R.,
Vollmannová, A., Bojňanská, T., & Dědina, M. (2015). Evaluation of oil content and
fatty acid composition in the seed of grapevine varieties. LWT-Food Science and
Technology, 63(1), 620–625.
N.M. Daud et al.
Lee, H.h., Ahn, J. H., Kwon, A. R., Lee, E. S., Kwak, J. H., & Min, Y. H. (2014). Chemical
composition and antimicrobial activity of the essential oil of apricot seed.
Phytotherapy Research, 28(12), 1867–1872.
Lee, J.-B., Yamagishi, C., Hayashi, K., & Hayashi, T. (2011). Antiviral and
immunostimulating effects of lignin-carbohydrate-protein complexes from
Pimpinella anisum. Bioscience Biotechnology and Biochemistry, 1101242356-
1101242356.
Liew, S. Q., Teoh, W. H., Tan, C. K., Yusoff, R., & Ngoh, G. C. (2018). Subcritical water
extraction of low methoxyl pectin from pomelo (Citrus grandis (L.) Osbeck) peels.
International Journal of Biological Macromolecules, 116, 128–135.
Machmudah, S., Shotipruk, A., Goto, M., Sasaki, M., & Hirose, T. (2006). Extraction of
astaxanthin from Haematococcus p luvialis using supercritical CO2 and ethanol as
entrainer. Industrial & Engineering Chemistry Research, 45(10), 3652–3657.
Mao, Y., Robinson, J., & Binner, E. (2021). Understanding heat and mass transfer
processes during microwave-assisted and conventional solvent extraction. Chemical
Engineering Science, 233, 116418.
Marić, B., Abramović, B., Ilić, N., Krulj, J., Kojić, J., Perović, J., Bodroža-Solarov, M., &
Teslić, N. (2020). Valorization of red raspberry (Rubus idaeus L.) seeds as a source of
health beneficial compounds: Extraction by different methods. Journal of Food
Processing and Preservation, 1–8. https://doi.org/10.1111/jfpp.14744
Martins, M. A., Domańska, U., Schröder, B., Coutinho, J. A., & Pinho, S.o. P. (2016).
Selection of ionic liquids to be used as separation agents for terpenes and terpenoids.
ACS Sustainable Chemistry & Engineering, 4(2), 548–556.
Md Norodin, N. S., Md Salleh, L., Yusof, N., Mustapha, N. M., Kamarulzaman, F.,
Zahari, M. A. M., & Bakeri, N. A. (2018). Inhibitory effects of swietenia mahagoni
seeds extract on А-glucosidase and А-amylase. International Journal of Engineering,
Transactions B: Applications, 31, 1308–1317. https://doi.org/10.5829/
ije.2018.31.08b.20
Metaxas, A.a., & Meredith, R. J. (1983). Industrial microwave heating. IET.
Miller, D. J., Hawthorne, S. B., Gizir, A. M., & Clifford, A. A. (1998). Solubility of
polycyclic aromatic hydrocarbons in subcritical water from 298 K to 498 K. Journal
of Chemical & Engineering Data, 43(6), 1043–1047.
Evaluation of Swietenia mahagoni Jacq seed extracts in Mohd-nasir, Hasmida, Liza
Salleh, Irma Suryani, and Azila Abd. 2018 Mohd-nasir, H., Salleh, L., Suryani, I., &
Abd, A. (2018), 4. Evaluation of Swietenia mahagoni jacq seed extracts in promoting
wound healing properties (Vol. 14, pp. 432–436). promoting wound healing 14, 432-
436.
Mohd-Nasir, H., Putra, N. R., Chuo, S. C., Daud, N. M., Hartati, H., Bakeri, N.,
Ruslan, M. S., Mohd-Setapar, S. H., Ahmad, A., & Md Salleh, L. (2021). Optimization
of the supercritical carbon dioxide extraction of Quercus infectoria galls extracts and
its bioactivities. Journal of Food Processing and Preservation, 45(2), Article e15156.
Moradi-kheibari, N., Ahmadzadeh, H., Talebi, A. F., Hosseini, M., & Murry, M. A. (2019).
Chapter 10 - recent advances in lipid extraction for Biodiesel production. In
M. Hosseini (Ed.), Advances in feedstock conversion technologies for alternative fuels and
bioproducts (pp. 179–198). Woodhead Publishing. https://doi.org/10.1016/B978-0-
12-817937-6.00010-2.
Natolino, A., & Da Porto, C. (2019). Supercritical carbon dioxide extraction of
pomegranate (Punica granatum L.) seed oil: Kinetic modelling and solubility
evaluation. The Journal of Supercritical Fluids, 151, 30–39.
Neetoo, H., & Chen, H. (2010). Inactivation of Salmonella and Escherichia coli O157:H7
on artificially contaminated alfalfa seeds using high hydrostatic pressure. Food
Microbiology, 27(3), 332–338. https://doi.org/10.1016/j.fm.2009.11.003
Nguyen, N. M. P., Le, T. T., Vissenaekens, H., Gonzales, G. B., Van Camp, J., Smagghe, G.,
& Raes, K. (2019). In vitro antioxidant activity and phenolic profiles of tropical fruit
by-products. International Journal of Food Science and Technology, 54, 1169–1178.
https://doi.org/10.1111/ijfs.14093
Pathak, N., Khan, S., Bhargava, A., Raghuram, G. V., Jain, D., Panwar, H.,
Samarth, R. M., Jain, S. K., Maudar, K. K., Mishra, D. K., & Mishra, P. K. (2014).
Cancer Chemopreventive effects of the flavonoid-rich fraction isolated from papaya
seeds. Nutrition and Cancer, 66(5), 857–871. https://doi.org/10.1080/
01635581.2014.904912
Patwardhan, M., Morgan, M. T., Dia, V., & D’Souza, D. H. (2020). Heat sensitization of
hepatitis A virus and Tulane virus using grape seed extract, gingerol and curcumin.
Food Microbiology, 90, 103461.
Peng, W. L., Mohd-Nasir, H., Setapar, S. H. M., Ahmad, A., & Lokhat, D. (2020).
Optimization of process variables using response surface methodology for tocopherol
extraction from Roselle seed oil by supercritical carbon dioxide. Industrial Crops and
Products, 143, 111886.
Pereira, M. G., Maciel, G. M., Haminiuk, C. W. I., Bach, F., Hamerski, F., de Paula
Scheer, A., & Corazza, M. L. (2019). Effect of extraction process on composition,
antioxidant and antibacterial activity of oil from yellow passion fruit (Passiflora
edulis Var. Flavicarpa) seeds. Waste and Biomass Valorization, 10(9), 2611–2625.
Pinelo, M., Sineiro, J., & Núñez, M.a. J. (2006). Mass transfer during continuous
solid–liquid extraction of antioxidants from grape byproducts. Journal of Food
Engineering, 77(1), 57–63. https://doi.org/10.1016/j.jfoodeng.2005.06.021
Prasertsri, P., Booranasuksakul, U., Naravoratham, K., & Trongtosak, P. (2019). Acute
effects of passion fruit juice supplementation on cardiac autonomic function and
blood glucose in healthy subjects. Preventive nutrition and food science, 24(3), 245.
Purnomo, A., Yudiantoro, Y. A. W., Putro, J. N., Nugraha, A. T., Irawaty, W., & Ismadji, S.
(2016). Subcritical water hydrolysis of durian seeds waste for bioethanol production.
International Journal of Integrated Care, 7(1), 29–37.
Putra, N. R., Che Yunus, M. A., & Machmudah, S. (2019). Solubility model of arachis
hypogea skin oil by modified supercritical carbon dioxide. Separation Science and
Technology, 54(5), 731–740.
Radfar, S., & Ghoreishi, S. M. (2018). Experimental extraction of L-Carnitine from oyster
mushroom with supercritical carbon dioxide and methanol as co-solvent: Modeling
213
Trends in Food Science & Technology 119 (2022) 201–214
and optimization. The Journal of Supercritical Fluids, 140, 207–217. https://doi.org/
10.1016/j.supflu.2018.06.014
Ravber, M., Knez, Ž., & Škerget, M. (2015). Simultaneous extraction of oil- and water-
soluble phase from sunflower seeds with subcritical water. Food Chemistry, 166,
316–323. https://doi.org/10.1016/j.foodchem.2014.06.025
Regis, S. A., Resende, E. D.d., & Antoniassi, R. (2015). Oil quality of passion fruit seeds
subjected to a pulp-waste purification process. Ciência Rural, 45(6), 977–984.
Rezvankhah, A., Emam-Djomeh, Z., Safari, M., Askari, G., & Salami, M. (2019).
Microwave-assisted extraction of hempseed oil: Studying and comparing of fatty acid
composition, antioxidant activity, physiochemical and thermal properties with
Soxhlet extraction. Journal of Food Science and Technology, 56(9), 4198–4210.
Rodrigues, L. G. G., Mazzutti, S., Siddique, I., da Silva, M., Vitali, L., & Ferreira, S. R. S.
(2020). Subcritical water extraction and microwave-assisted extraction applied for
the recovery of bioactive components from Chaya (Cnidoscolus aconitifolius Mill.).
The Journal of Supercritical Fluids, 165, 104976.
Roselló-Soto, E., Koubaa, M., Moubarik, A., Lopes, R. P., Saraiva, J. A., Boussetta, N.,
Grimi, N., & Barba, F. J. (2015). Emerging opportunities for the effective valorization
of wastes and by-products generated during olive oil production process: Non-
conventional methods for the recovery of high-added value compounds. Trends in
Food Science & Technology, 45(2), 296–310.
Sadek, P. C. (2002). HPLC solvent guide. Wiley-Interscience.
Samaram, S., Mirhosseini, H., Tan, C. P., Ghazali, H. M., Bordbar, S., & Serjouie, A.
(2015). Optimisation of ultrasound-assisted extraction of oil from papaya seed by
response surface methodology: Oil recovery, radical scavenging antioxidant activity,
and oxidation stability. Food Chemistry, 172, 7–17. https://doi.org/10.1016/j.
foodchem.2014.08.068
Sarkar, A., & Ghosh, U. (2016). Classical single factor optimisation of parameters for
phenolic antioxidant extraction from tamarind seed (Tamarindus indica). Plant
Science Today, 3(3), 258–266.
Shirsath, S., Sonawane, S., & Gogate, P. (2012). Intensification of extraction of natural
products using ultrasonic irradiations—a review of current status. Chemical
Engineering and Processing: Process Intensification, 53, 10–23.
Shiwakoti, S., Poudyal, S., Saleh, O., Astatkie, T., & Zheljazkov, V. D. (2016). Method for
attaining caraway seed oil fractions with different composition. Chemistry and
Biodiversity, 13(6), 695–699.
Shouqin, Z., Jun, X., & Changzheng, W. (2005). High hydrostatic pressure extraction of
flavonoids from propolis. Journal of Chemical Technology and Biotechnology:
International Research in Process, Environmental & Clean Technology, 80(1), 50–54.
Siano, F., Straccia, M. C., Paolucci, M., Fasulo, G., Boscaino, F., & Volpe, M. G. (2016).
Physico-chemical properties and fatty acid composition of pomegranate, cherry and
pumpkin seed oils. Journal of the Science of Food and Agriculture, 96(5), 1730–1735.
Sintim, H. Y., Burkhardt, A., Gawde, A., Cantrell, C. L., Astatkie, T., Obour, A. E.,
Zheljazkov, V. D., & Schlegel, V. (2015). Hydrodistillation time affects dill seed
essential oil yield, composition, and bioactivity. Industrial Crops and Products, 63,
190–196.
Strati, I. F., Gogou, E., & Oreopoulou, V. (2015). Enzyme and high pressure assisted
extraction of carotenoids from tomato waste. Food and Bioproducts Processing, 94,
668–674.
Švarc-Gajić, J., Morais, S., Delerue-Matos, C., Vieira, E. F., & Spigno, G. (2020).
Valorization potential of oilseed Cakes by subcritical water extraction. Applied
Sciences, 10(24), 8815.
Tiwari, B. K. (2015). Ultrasound: A clean, green extraction technology. TRAC Trends in
Analytical Chemistry, 71, 100–109.
Tremocoldi, M. A., Rosalen, P. L., Franchin, M., Massarioli, A. P., Denny, C., Daiuto, É. R.,
Paschoal, J. A. R., Melo, P. S., & De Alencar, S. M. (2018). Exploration of avocado by-
products as natural sources of bioactive compounds. PLoS One, 13, 1–12. https://doi.
org/10.1371/journal.pone.0192577
Wangchuk, P. (2018). Therapeutic applications of natural products in herbal medicines,
biodiscovery programs, and biomedicine. Journal of Biologically Active Products from
Nature, 8(1), 1–20.
Wejnerowska, G., & Ciaciuch, A. (2018). Optimisation of oil extraction from quinoa seeds
with supercritical carbon dioxide with co-solvents. Czech Journal of Food Sciences, 36
(1), 81–87.
Wen, C., Zhang, J., Zhang, H., Dzah, C. S., Zandile, M., Duan, Y., Ma, H., & Luo, X.
(2018). Advances in ultrasound assisted extraction of bioactive compounds from
cash crops–A review. Ultrasonics Sonochemistry, 48, 538–549.
Wilson, D. R., Dabrowski, L., Stringer, S., Moezelaar, R., & Brocklehurst, T. F. (2008).
High pressure in combination with elevated temperature as a method for the
sterilisation of food. Trends in Food Science & Technology, 19(6), 289–299.
Xie, L., Cahoon, E., Zhang, Y., & Ciftci, O. N. (2019). Extraction of astaxanthin from
engineered Camelina sativa seed using ethanol-modified supercritical carbon
dioxide. The Journal of Supercritical Fluids, 143, 171–178. https://doi.org/10.1016/j.
supflu.2018.08.013
Xi, J., & Luo, S. (2016). The mechanism for enhancing extraction of ferulic acid from
Radix Angelica sinensis by high hydrostatic pressure. Separation and Purification
Technology, 165, 208–213. https://doi.org/10.1016/j.seppur.2016.04.011
Yanık, D. K. (2017). Alternative to traditional olive pomace oil extraction systems:
Microwave-assisted solvent extraction of oil from wet olive pomace. LWT, 77, 45–51.
https://doi.org/10.1016/j.lwt.2016.11.020
Yuan-bin, C., Yi-ting, C., Jin-ching, L., Chin-kai, T., Hui-chun, W., I-wen, L., Yi-hung, W.,
Sheng-yang, W., Yang-chang, W., & Fang-rong, C. (2014). Limonoids from the seeds
of swietenia macrophylla with inhibitory activity against dengue virus 2. Journal of
Natural Products, 20, 18551–18564.
N.M. Daud et al.
Zheljazkov, V. D., Astatkie, T., & Schlegel, V. (2014). Hydrodistillation extraction time
effect on essential oil yield, composition, and bioactivity of coriander oil. Journal of
Oleo Science, Article ess14014.
Zhong, J., Wang, Y., Yang, R., Liu, X., Yang, Q., & Qin, X. (2018). The application of
ultrasound and microwave to increase oil extraction from Moringa oleifera seeds.
214
Trends in Food Science & Technology 119 (2022) 201–214
Industrial Crops and Products, 120, 1–10. https://doi.org/10.1016/j.
indcrop.2018.04.028
Zin, M. M., Anucha, C. B., & Bánvölgyi, S. (2020). Recovery of phytochemicals via
electromagnetic irradiation (microwave-assisted-extraction): Betalain and phenolic
compounds in perspective. Foods, 9(7), 918.
 The Colloquium 11 (2017) pp.7-10

In vitro toxicology test on Swietenia Mahagoni essential oil extracted by using

supercritical fluid extraction

Noor Fadzilah Abu Bakar 1,2*, Nor Asyiqin Bakeri 1,2, Liza Md Salleh 1,2, Dayang Norulfairuz Abang Zaidel 2
1
Centre of Lipids Engineering & Applied Research (CLEAR), Ibnu Sina Institute for Scientific & Industrial
Research, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia.
2
Bioprocess and Polymer Engineering Department,Faculty of Chemical and Energy Engineering, Universiti
Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia

KEYWORDS ABSTRACT
Swietenia Mahagoni In this paper, the investigation of extraction method selection is crucial
Supercritical Fluid Extraction to ensure the extract of high purity, rich with components and non-toxic.
(SFE) Swietenia mahagoni seed has potentials as antimicrobial activity,
Toxicology test antioxidant activity, cytotoxic activity, antiulcer activity, antifungal
Cell viability activity, anti-HIV activity, anti-inflammatory, antipyretic activity and
hypoglycemic activity. The toxicity content of the extracted Swietenia
mahagoni essential oil by using supercritical fluid extraction (SFE)
should be investigated. In vitro toxicology test on human skin fibroblast
1184 (HSF 1184) was done to examine the toxicity of Swietenia
mahagoni seed oil. With the cell viability above 80 % of different
extract concentrations (10, 1, 0.1, 0.01, 0.001 and 0.0001 mg/ml),
Swietenia mahagoni essential oil extracted using supercritical carbon
dioxide extraction was proved to be non-toxic by using MTT assay.

1. Introduction extracted Swietenia mahagoni essential oil by

Swietenia mahagoni comes from Meliaceae
family is a large, deciduous, and economically
important timber tree native to the West Indies
which is commonly known as mahogani.
Swietenia mahagoni is a medium-sized semi-
evergreen tree growing to 30–35 meters tall.
The leaves are pinnate, 12 to 25 centimeters
long, with four to eight leaflets. The flowers
are small, produced in panicles. The fruit is a
woody capsule 5 to 10 centimeters long and 3
to 6 centimeters broad, containing numerous
winged seeds [1]. Swietenia mahagoni has
been used traditionally as treatments such as
diabetes, asthma, eczema, premenstrual
syndrome and migraine. Especially seed, it has
potential of antioxidant activity, antimicrobial
activity, antifungal activity and hyperglycemic
activity [2]. Supercritical Fluid Extraction
(SFE) has many beneficial as it has low
toxicity, inexpensive and does not harm the
nature [3]. This is because, the physical
properties of carbon dioxide at supercritical
state include low critical temperature and
critical pressure made supercritical carbon
dioxide environmentally friendly and green
solvent. The selection of extraction method is
crucial to ensure the extract in high purity, rich
with components and non-toxic. Thus, the
objective to investigate the toxicity content of
using supercritical fluid extraction (SFE)
should be accomplished for further application.
2. Experimental procedure
2.1 In Vitro Toxicology Test on Human Skin
Fibroblast 1184 (HSF 1184)
The study was prior to indicate the
toxicology of Swietenia mahagoni seed extract
on human skin fibroblast 1184 (HSF 1184).
This part covered on cell recovery, cell
subculture, cell cryopreservation, cell viability
and toxicology test using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay.
2.1.1 Cell Recovery
Cryogenic vial containing human skin
fibroblast 1184 (HSF 1184) cells were
removed from the liquid nitrogen storage. The
cells and the media which was modified eagle
medium (MEM) was thawed bath (Edelstahl)
at 37 oC for 1 to 5 min or until its completely
thawed. After that, the thawed cells were
culture in MEM using t-flask. Lastly, the cells
are maintained at 37 oC in a humidifier
atmosphere of 5 % CO2 (Branstead, USA)
until its achieved 90 to 95 % confluent
monolayer.

* Corresponding author:
noorfadzilahabubakar@gmail.com
 The Colloquium 11 (2017) pp.7-10

2.1.2 Cell Subculture Where;

The subculture procedures were done
immediately after the cells achieved 90 to 95% C = Concentration of cells (cells/ml)
confluent monolayer. First, all the media were NA = Average number of cells counted
aspirated and the cells in the t-flask were D = Dilution factor
washed with 5 ml phosphate buffer saline While, the percentage of cell viability was
(PBS) to remove any cellular debris or serum calculated by using the equation 2.2:
which could retarded the action of trypsin.
Then, the PBS was removed and 3 ml of Percent of viability (%) ;
trypsin-EDTA was added. After that, the flask
incubated for 5 min at 37 oC. Then, when cells %=
"#$%&' () *&++, #-,./0-&1
x 100 (2.2)
begun to round and detach under the .(./+ -#$%&' () *&++,

microscope, equal volume of MEM was added

2.1.5 MTT Assay
to neutralize the trypsin. Next, all the cell
The effect of Swietenia mahagoni seed
mixture was placed into the centrifuge tube
extracts on cell viability was assessed by using
and then centrifuged (Hettich, rotina 420R) for
the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-
10 min at 3300 rpm. After that, the supernatant
tetra-zolium bromide (MTT) assay according
were discarded and the pellets were diluted
to [5]. A monolayer of fibroblast was
with MEM to the volume required. Lastly, the
trypsinized and neutralized. After that, the cell
cells were incubated at 37 oC in 5 % CO2 air
suspension was centrifuged for 10 min at 3300
atmosphere [4].
rpm. The supernatant of the cells was
discarded and the pellet was collected and
2.1.3 Cell Cryopreservation
resuspended with MEM and the cells were
Cells at log phase were used for freezing.
counted. After that, the cells were seed at a
Cells were trysinized and centrifuged. The
density of 2 x 105 cells/well in 96-well plate,
freezing medium containing 90 % fetal bovine
excluded the first row, and incubated in
serum (FBS) and 10 % DMSO were slowly
humidified atmosphere for 24 hours prior to
added to the cells suspension. After that, cells
treatment. The test samples were prepared by
were aliquoates into 1.2 ml prelabel cryogenic
dissolving the extracts in MEM to yield the
vials and the vials were placed into the
final concentrations of the crude extracts at 10,
NalgeNunc freezing container (for adaptation
1, 0.1, 0.01, 0.001, 0.0001 mg/ml respectively.
process) with temperature of -70 oC for 24
The medium was replaced after 24 hours
hours. Lastly, the vials were transferred to
with 200µL of MEM contain of 10 % FBS and
liquid nitrogen storage [4].
1% antibiotic mixture and serial dilution of
plant extracts. Meanwhile, the last well was
2.1.4 Cell Viability
loaded with MEM containing 10 % FBS 1 %
To determine the number of viable cells
antibiotic mixture without the addition of plant
present in cell suspension, the cells need were
extracts as a negative control. The cells with
counted using dye exclusion test. The principle
and without serial dilution of extracts were
of this test is the live cells own intact cell
incubated again in humidify atmosphere for 24
membrane which will exclude certain dyes
hours. After incubation, the cells were washed
while dead cells do not. To implement cells
using PBS. Then, PBS was removed and 20 µL
counting Trypan blue exclusion test and
of freshly prepare MTT solution (5 mg/ml
Neubauer improved bright line
dissolved in PBS) was added into each well
haemocytometer (Fortuna®, Germany) were
and the cells were incubated again at 37 oC for
used. The dead cells accumulated trypan blue
5 hours. The MTT solution was removed and
whereas live cells excluded it. Firstly, a clean
replaced with 200 µL DMSO for 15 minutes to
coverslip was placed on the top of
allow dissolution of the purple MTT formation
haemocytometer slide. Next, 50 µL of cell
crystal. The absorbance was measured at 540
suspension was mix with 50 µL of trypan blue.
nm and reference wavelength at 630 nm using
Then, the mixture was carefully transferred
ELISA plate reader (BIOTEK, ELx-808). The
into the edge of the coverslip. The number of
percentage of cell viability was calculated by
unstained (live cells) and stained (dead cells)
using the following equation 2.3:
were counted under a light microscope (Zeiss,
axiovert 40CFL, Germany). The concentration
Cell viability (%);
of viable cells was calculated using the
following equation 2.1: 2&/- /%,('%/-*& () &34&'0$&-./+ *&++,
%= x 100
2&/- /%,('%/-*& () *(-.'(+ *&++,
4
C = NA x D x 10 (2.1)
(2.3)

8
 The Colloquium 11 (2017) pp.7-10
3. Results and discussion
3.1 In Vitro Toxicity Analysis of Swietenia
mahagoni seed oil
The study is prior to indicate the toxicology
level of Swietenia mahagoni seed oil on
human skin fibroblast (HSF 1184). The study
covered the cell viability and toxicology test
using 3-(4,5-dimethylthiazol-2-yl)2,5diphenyl-
tetrazolium bromide (MTT) assay. The MTT
assay consists in the fact that only viable cells
are able to reduce and convert the relatively
colorless MTT compound into formazan,
which is a dark purple (Angius and Floris,
2015). The toxicology test result is presented
on Figure 1. Different concentrations of
Swietenia mahagoni seed oil are achieved by
dissolving the extract in MEM till achieve
concentration of 10, 1, 0.1, 0.01, 0.001 and
0.0001 mg/ml respectively. The cell viability
of different concentrations Swietenia
mahagoni seed oil is compared to negative
control without extract. The absorbance
readings are recorded as measured at 540 nm
and reference wavelength at 630 nm using
ELISA plate reader (BIOTEK, ELx-808). The
percentage of cell viability is calculated to
discover the toxicology of Swietenia mahagoni
seed oil.
Figure 1: Cell viability on different
concentrations of Swietenia mahagoni seed
extract.
The extract is not toxic as the cell viability
reaches above control. This is because, human
skin cell manage to keep growth even exposed
to extract. If the calculation on cell viability
reaches below control, it indicates that there
are dead cell as it exposed to extract.
Therefore, from Figure 3.1 it shows that
extract is non-toxic at concentration level of 1,
0.1 and 0.01 mg/ml. At concentration 0.001
mg/ml, the cell viability slightly below control
meanwhile, at concentration of 0.0001 mg/ml
the cell viability reaches above control. Which
mean, at concentration of 0.0001 mg/ml it is
non-toxic. Furthermore, Swietenia mahagoni
seed oil has enhanced cell growth as the cell
viability increase compared to control.
According to [6], it stated that ISO 10993-5
that the cell viability above 80 % is non-toxic
but below 40 % cell viability it is strongly
toxic. This result can be a preliminary study
for studying the cell growth and wound
healing potential of Swietenia mahagoni seed.
4. Conclusion
The extract from Swietenia Mahagoni
proved to be non-toxic by analysis on cell
viability toxicity MTT assay. With the cell
viability above 80 % of extract concentrations
(10, 1, 0.1, 0.01, 0.001 and 0.0001 mg/ml), it
shows that Swietenia mahagoni seed extract
using supercritical carbon dioxide extraction is
non-toxic. Further study on potential of
Swietenia mahagoni extracted using
supercritical carbon dioxide extraction can be
proceed on wound healing and antidiabetic
potentials. Since, MTT assay is the starting
procedure for further in vivo toxicity study on
acute and sub acute toxicity at high dosage up
to 5000 mg/ml extract.
Acknowledgement
The authors would like to acknowledge the
Ministry of Higher Education Malaysia and
research grant Q.J130000.2546.16H78 and
Q.J130000.2546.14H06.
Reference
[1] Khare Divya, Pradeep H.R., Kumar K.
K., Hari Venkatesh K.R, J. T. (2012)
‘Herbal Drug Swietenia Mahagoni Jacq.
- A Review’, 1(10), Pp. 557–567.
[2] Bhurat, M. R., Bavaskar, S. R., Agrawal,
A. D. And Bagad, Y. M. (2011)
‘Swietenia Mahagoni Linn. – A
Phytopharmacological Review’, Asian
Journal Of Pharmacuetical Research,
1(1), Pp. 1–4.
[3] Khaw, K.-Y., Parat, M.-O., Shaw, P. N.
and Falconer, J. R. (2017) ‘Solvent
Supercritical Fluid Technologies to
Extract Bioactive Compounds from
Natural Sources: A Review’, Molecules,
22(7), p. 1186. doi:
10.3390/molecules22071186.
[4] Freshney, R. I. (2005) ‘Culture of
Specific Cell Types’, Culture of Animal
Cells, pp. 375–420. doi:
10.1002/0471747599.cac023.
[5] Bakorova, M., Backor, M., Mikes, J.,
Jendzelovsky, R. and Fedorocko, P.
(2011) ‘Variable responses of different
human cancer cells to the lichen
compounds parietin, atranorin, usnic acid
and gyrophoric acid’, Toxicology in
Vitro, 25(1), pp. 37–44. doi:
10.1016/j.tiv.2010.09.004.
9
 The Colloquium 11 (2017) pp.7-10

[6] López-García, J., Lehocký, M.,

Humpolíček, P. and Sáha, P. (2014)
‘HaCaT Keratinocytes Response on
Antimicrobial Atelocollagen Substrates:
Extent of Cytotoxicity, Cell Viability and
Proliferation’, Journal of Functional
Biomaterials, 5(2), pp. 43–57. doi:
10.3390/jfb5020043.

10
IOP Conference Series: Materials Science and Engineering

PAPER • OPEN ACCESS

Experimental Measurements and Solubility Correlation of Swietenia

macrophylla Seeds Oil in Supercritical CO2
To cite this article: Nur Salsabila Md Norodin et al 2020 IOP Conf. Ser.: Mater. Sci. Eng. 932 012029

View the article online for updates and enhancements.

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1st International Conference on Science, Engineering and Technology (ICSET) 2020 IOP Publishing
IOP Conf. Series: Materials Science and Engineering 932 (2020) 012029 doi:10.1088/1757-899X/932/1/012029

Experimental Measurements and Solubility Correlation of

Swietenia macrophylla Seeds Oil in Supercritical CO2

Nur Salsabila Md Norodin1,2, Ahmad Ramdan Ismail1,2, Ahmad Syahmi Zaini1,2,

Nor Faadila Idrus1,2, Hartati3, Mohammad Lokman Hilmi1,2, Liza Md Salleh1,2*
1
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti
Teknologi, Malaysia.
2
Centre of Lipid Engineering and Applied Research (CLEAR), Ibnu Sina Institute for
Scientific and Industrial Research (ISI-SIR), Universiti Teknologi Malaysia.
3
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas
Negeri Makassar, Indonesia.

Email: r-liza@utm.my

Abstract. The aim of this study was to determine the oil recovery from Swietenia macrophylla
seeds and its solubility in Supercritical CO2 extraction process. Experimentally, the oil
recovery from the seeds were conducted at 15-35 MPa and 40-60°C by Supercritical CO2
extraction process. Density-based models such as Chrastil and del Valle and Aguilera models
were used to correlate the solubility data of Swietenia macrophylla seeds oil. The high oil
recovery obtained at 35 MPa and 60°C was 6.609 mg oil/g CO2 while the lowest oil recovery
was at 15 MPa and 40°C (0.810 mg oil/g CO2). Chrastil model provide the best correlation
between experimental and calculated solubility data for oil recovery from Swietenia
macrophylla seeds with lowest value of average absolute percent deviation (AAPD) which is
0.33% compared to del Valle and Aguilera model (0.39%).

1. Introduction
Swietenia macrophylla, also known as ‘tunjuk langit’ in Malaysia is used traditionally to treat various
diseases such as diabetes and high blood pressure [1]. Swietenia macrophylla seeds have been reported
to have various biological activities such as anti-inflammatory, anticancer and antitumor [2], wound
healing properties [3,4] as well as antidiabetic activities [5]. The main component in Swietenia
macrophylla seeds oil is fatty acid which cannot be synthesized by the human body, but it is essential
for normal metabolism. The example of these fatty acids is linoleic acid. Linoleic acid extracted from
Swietenia macrophylla seeds was 37.58% [6], contributed to antidiabetic [5] and wound healing
properties [3,4].
The definition of extraction is a mass transfer process between two components from one phase to
another where the extracting solvent can be in the form of liquid, solid or supercritical fluid [7].
Supercritical fluids (SF) describe as a single substance at above its critical point (pressure and
temperature) with physicochemical properties indistinguishable between gases and liquid [8].

___________________________
1
To whom any correspondence should be addressed.

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The advantage of SF, supercritical carbon dioxide (CO2) is the transport phenomena that can be
altered accordance to its supercritical region. The disadvantage of SF accounted to its high operating
cost, handling with high pressure and temperature in its processing. Adaption of SF in various
technologies such as extraction is a greener and effective approach on both industrial and analytical
scale.
The utilization of supercritical fluid extraction process in the industry for separation process
included the needs of correlation or prediction of the solubility solutes in supercritical solvents.
Solubility refer to the solvating capability of supercritical solvent where the maximum amount of
solute f7that can be extracted at saturation equilibrium [9]. Density-based approach able to correlate
the pressure, temperature and density of solvent in order to predict the solute solubility in supercritical
fluid. [10]. To the best of our knowledge, there is no information in the available literature on
evaluation of Swietenia macrophylla seeds oil solubility in supercritical CO2. In the present study,
supercritical CO2 extraction process was used for the recovery of oil from Swietenia macrophylla
seeds. The aim was to correlate the solubility and its influence toward recovery of oil.

2. Experimental Details
2.1. Chemicals
Commercial grade liquid carbon dioxide (purity 99.99%) used in supercritical carbon dioxide
extraction was purchased from Kras, Instrument and Services, Johor, Malaysia.

2.2. Plant Material

The Authentication of the Swietenia macrophylla seeds was performed by Forest Research Institute
Malaysia (FRIM) (Sample No.: PID 270818-25). The seeds were oven dried dried for a whole day
using an oven (Memmert, Germany). Then, the seeds were grinded into 500 µm mean particle size
using commercial blender (Waring, US) and sieved using Endecotts Octagon 200 Digital Sieve
Shaker. Lastly, the samples were kept in a tight sealed storage plastic stored in freezer at -20 °C for
further analysis.

2.3. Extraction Procedure’

Supercritical fluid extraction (SFE) unit in Center of Lipids Engineering and Applied Research
(CLEAR), Universiti Teknologi Malaysia consisted of CO2 gas cylinder, CO2 controller pump (Lab
Alliance), co-solvent pump (Lab Alliance), oven (Memmert, Germany), 10 ml stainless steel
extraction vessel, pressure gauge (Swagelockk, Germany), automatic back pressure regulator (Jasco
BP 2080- Plus) and restrictor valve. A schematic diagram of SFE apparatus is illustrated in Figure 1.

Figure 1. Schematic Diagram of SFE

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2.4. For each experiment, 3 grams of sample matrix was placed in 10 ml stainless steel extraction
vessel and sealed tightly in the oven. The flow rate of supercritical CO2 was maintained at 1.82 g/min.
The extraction process was conducted at 15-35 MPa and 40-60°C. The oil yields were collected after
120 minutes in a vial and refrigerated at -4°C until further analysis. The expression of the extracted oil
yield of Swietenia macrophylla can be expressed as:

Where Woil is the mass of the extracted oil yield (mg), Wi is the mass of the sample after the extraction
(mg) and Wf is the mass of the sample before the extraction (mg).

2.5. Solubility Modelling

The experimental solubility (Ys) data were calculated from the initial slope of the first period of
extraction curve at 15-35 MPa and 40-60°C. Chrastil and del Valle and Aguilera models were
modelled based on the equations.

Chrastil equation was formed for the solubility in dense gas considering the interaction of solvato
complex [11] is given as follows:

Where Ys is the solubility of solute in dense gas (g/L), k is the association, ρCO2 is the CO2 density
(g/L), a and b are constants.

del Valle and Aguilera equation is a modification of Chrastil equation to compensate the variations of
the solute’s heat of vaporization with temperature [12] is given as follows:

2.6. Statistical analysis

Analysis of average absolute percent deviation (AAPD) was used to determine the accuracy of these
models, as shown in equation below:

( ) ∑| |

Where where n is is the total experimental data and Yexp and Ycalc are the data obtained from
experiment and model equations, respectively, at i condition. Aside to AAPD, R2, analysis of
determination coefficient value was also used in evaluating the consistency of the solubility curves.

3. Results and Discussion

The density of CO2 and the solubility of Swietenia macrophylla seed oil in Supercritical CO2 at
various conditions are presented in Table 1. The correlation of solubility data was further studied
based on density-based models which are Chrastil and del Valle and Aguilera models.

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Table 1. Solubility of Swietenia macrophylla seed oil in Supercritical CO2

Pressure Temperature Density of CO2 Solubility
(MPa) (°C) (g/mL) (mg oil/g CO2) (g oil/L CO2)
40 0.790 0.810 0.640
15 50 0.708 1.032 0.731
60 0.606 1.800 1.091
40 0.848 1.905 1.615
20 50 0.791 2.703 2.138
60 0.730 3.077 2.246
40 0.886 3.148 2.789
25 50 0.841 3.333 2.803
60 0.792 3.529 2.795
40 0.919 3.778 3.472
30 50 0.880 3.968 3.492
60 0.837 4.800 4.018
40 0.942 5.185 4.884
35 50 0.906 5.376 4.871
60 0.869 6.609 5.743

Figure 2 represents the solubility of Swietenia macrophylla seed oil in Supercritical CO2 as a
function of pressure at each constant temperature of 40°C, 50°C and 60°C. The pattern from the graph
shows that the solubility of oil is increased proportionally as the pressure is increased at constant
temperature. The highest solubility of oil is observed at highest temperature of 60 °C and pressure of
35 MPa, which is 6.609 mg oil per g CO 2. Meanwhile at lowest temperature of 40 °C and pressure of
15 MPa, the lowest solubility of 0.81 mg oil per g CO2 oil is obtained.

8
Ys (mg oil/g CO₂)

6
40 °C
4
50 °C
60 °C
2

0
10 15 20 25 30 35 40
Pressure (MPa)

Figure 2. Solubility of Swietenia macrophylla seed oil as a function of pressure

at different temperature

The solubility of Pithecellobium jiringan (Jack) prain seeds was evaluated at 40-70°C and
27.58-44.81 MPa [13]. It showed that the solubility of oil yield is slightly increased with the increases
of CO2 density when the pressure is increased. Figure 3 shows the effect of temperature on solubility
of Swietenia macrophylla oil yield in supercritical CO2 extraction at constant pressure. It is observed
that the solubility of Swietenia macrophylla oil is increased with the increases of temperature, even
though the solvent density was decreased. The solubility of palm oil at 40-60°C and 4-10 MPa shows

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the increment of the extraction yield with the temperature [14]. It is also stated that increasing of
solubility as a function of temperature was much faster than the increment of solubility as a function
of pressure.

7
6
YS (mg oil/g CO₂)

15 MPa
5
20 MPa
4
25 MPa
3
30 MPa
2 35 MPa
1
0
30 40 50 60 70
Temperature (°C)

Figure 3. Solubility of Swietenia macrophylla seed oil as a function of

temperature at different pressure

The solubility parameters for both Chrastil and del Valle and Aguilera equations are presented
in Table 2. The parameters of k, a and b in Chrastil equation represents average number of molecules
that form the solvate-complex, the heat of salvation and vaporization of the solute, and the molecular
weight and melting point of solute respectively. Meanwhile the parameters of k, a, b and c in del Valle
and Aguilera equation represents the same function as in Chrastil equation with additional of constant
c in the equation. The correlation of solubility data of Pithecellobium Jiringan (Jack) prain seeds at
40-70°C and 27.58-44.82 MPa shows that Chrastil model as the best model that fit the experimental
data due to its lower AAPD (0.206%) value compared to del Valle and Aguilera model (0.582%) [13].
But since the R2 values for the both models were similar. Hence, the modification in de Valle and
Aguilera model has no significant changes in the experimental data compared to Chrastil model [16].

Table 2. Coefficient parameters for Chrastil and del Valle and Aguilera equations
Equations Constants AAPD (%) R2
k a b c
Chrastil 4.70401 -6368.44 -10.9353 0.33 0.9764
Del Valle and 5.66111 -9811.87 -35.8699 -6.73102 0.39 0.9764
Aguilera

Figure 4 shows the correlation of Swietenia macrophylla oil solubility experimental data with
Chrastil and del Valle and Aguilera model. From the graph, both models are closed to the
experimental data, but it was observed that the correlation using Chrastil model is more precise and
fitted with the experimental data compared to del Valle and Aguilera model but the comparison
between both models itself does not shows any significant difference in statistical evaluation.

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2.5
2
Solubility, ln Y* (g/L)

1.5
1
0.5
0
6.3 6.4 6.5 6.6 6.7 6.8 6.9
-0.5
-1
ln ρCO₂ (g/L)
313.15 K 323.15 K
333.15 K Chrastil model
del Valle and Aguilera model
Figure 4. Correlation of Swietenia macrophylla oil solubility in Supercritical CO2 between
experimental data with Chrastil and del Valle and Aguilera models.

4. Conclusion
The highest solubility of Swietenia macrophylla oil was obtained at 35 MPa and 60°C which is 6.609
mg oil/g CO2 while the lowest solubility of Swietenia macrophylla oil was obtained at 15 MPa and
40°C which is 0.810 mg oil/g CO2. The solubility of Swietenia macrophylla oil is increased with
increasing pressure and CO2 density at constant temperature. On the other hand, the solubility of
Swietenia macrophylla oil is increased with increasing temperature at constant pressure, even though
the CO2 density is decreased. The correlation of solubility experimental data with density-based model
was conducted using Chrastil and del Valle and Aguilera models. From the result, the best model that
fitted to the experimental data was Chrastil model due to the lowest value of average absolute percent
deviation (AAPD) which is 0.33% compared to del Valle and Aguilera model (0.39%) but the R 2 for
both models were similar (0.9764).

References
[1] Moghadamtousi S Z, Goh B H, Chan C K, Shabab T and Kadir H A 2013 Molecules 18(9)
10465-83.
[2] Sayyad M, Tiang N, Kumari Y, Goh B H 2017 Saudi Pharm. J..25(2) 196-205.
[3] Hartati H, Mohd-Nasir H, Md Salleh L, Idris I S and Abd Aziz A 2018 Mal. J. Fund. Appl. Sci.
14(4) 432-436
[4] Hartati, Liza M S, Irma S I and Azis A A 2019 J. Teknol. 81 119–123.
[5] Md Norodin N S, Md Salleh L, Yusof N, Mustapha N M 2018 Int. J. Eng. Trans. B Appl.
31(8) 1308-1317.

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1st International Conference on Science, Engineering and Technology (ICSET) 2020 IOP Publishing
IOP Conf. Series: Materials Science and Engineering 932 (2020) 012029 doi:10.1088/1757-899X/932/1/012029

[6] Hartati, Salleh L M, Pagarra H and Rachmawaty 2018 IOP J. of Physics: Conf. Series 1028
012011 doi :10.1088/1742-6596/1028/1/012011
[7] Hartati, Salleh L M , Idris I S, and Azis A A 2019 J. Teknol. 81 119–123.
[8] Reverchon E, and De Marco I 2006 J. Supercrit. Fluids 38(2) 146-166.
[9] da Silva R P F F, Rocha-Santos T A P, and Duarte A C 2016 TrAC - Trends Anal. Chem.76
40-51.
[10] Soetaredjo F E, Ismadji S, Yuliana Liauw M, Angkawijaya A E and Ju Y H 2013 Fluid Phase
Equilib. 358 220-225.
[11] Bruno T J and Ely J F 2017 Supercritical fluid technology: Reviews in modem theory and
applications.Boca Raton Publisher CRC Press.
[12] Chrastil J 1982. J. Phys. Chem. 3016–3021.
[13] del Valle J M, and Aguilera J M 1988 Ind. Eng. Chem. Res.1551–1553.
[14] Yunus M A C, Arsad N H, Zhari S and Idham Z 2013 J. Teknol. (Sciences Eng). 45–50.
[15] Md Norodin N S, Md Salleh L, Machmudah S and Mustafa N M 2018 Malay. J. Fundam.
Appl. Sci. 14 411–417.
[16] Kostrzewa D, Dobrzyńska-Inger A, and Turczyn A 2019 Molecules 24 1–13.
[17] Mustapa A N, Manan Z A, Mohd Azizi C Y, Nik Norulaini N A and Omar A K M 2009 J.
Food Eng. 606–616.

Acknowledgments
The authors gratefully acknowledge the financial support by the Zamalah, Universiti Teknologi
Malaysia (UTM). Acknowledgement also extended to UTM Research University Grants 16H97,
04G03, 01M65 and 4C242 for funding this study

7
Journal of Physics: Conference Series

PAPER • OPEN ACCESS

The effect of the swietenia mahagoni seed extracts on the production of

collagen in human fibroblast cell (HSF1184)
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ICOMSET2018 IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1317 (2019) 012071 doi:10.1088/1742-6596/1317/1/012071

The effect of the swietenia mahagoni seed extracts on the

production of collagen in human fibroblast cell (HSF1184)

Hartati1*, Liza Md Salleh2, Azizi Che Yunus3, Azila Abd Azis2, Halifah Pagarra1
and Rachmawaty1
1
Biology Department, FMIPA, Universitas Negeri Makassar, South Sulawesi,
Indonesia.
2
Bioprocess Engineering Department, Faculty of Chemical Engineering, Universiti
Teknologi Malaysia,Johor Bahru, Malaysia
3
Centre of Lipid Engineering Applied Research (CLEAR), Universiti Teknologi
Malaysia, Johor Bahru, Malaysia

*hartati@unm.ac.id

Abstract. We have used the in vitro sircol collagen test to investigate the effects of Swietenia
mahagoni seed extract on collagen production in human fibroblast cells (HSF1184). The results
showed that Swietenia mahagoni seed extract with concentrations of 0.001, 0.01 and 0.1 mg /
mL caused stimulation in the synthesis of type I collagen in HSF1184 cells. Although, three
concentration of SC-CO2 and soxhlet that showed no statistically significant of collagen on the
HSF1184 when compared with the negative control, while LA (fibroblast cells with pure
Linoleic acid as supplement in culture medium) gave significant effect on the collagen
production.

1. Introduction
Swietenia mahagoni is commonly used as traditional medicine in several countries including,
Indonesia, Malaysia, India and China. The S.mahagoni seeds studied have several uses as herbal
medicines and are proven to have antifungal and antibacterial properties [1] and can inhibit platelet
aggregation induced by platelet activating factor [2] and has anti-human immunodeficiency virus
activity [3]. Ethanol extract from S.mahagoni seeds has antioxidant activity in vitro [4]. Linoleic acid
as an important fatty acid that must be consumed for good health care. Linoleic acid has become
popular in the beauty products industry in skin care. Research shows that linoleic acid is anti-
inflammatory, reductive and can moisturize when applied topically to the skin [5]. Swietenia
mahagoni has higher linoleic acid content of 30.1% [6].
Fibroblasts are found in inflammatory lesions due to tissue injury by various different agents. They
influence the repair of damaged tissue by synthesizing and placing extracellular components that form
scars. The mechanism by which special effector cells are attracted to the site of tissue injury and which
is inflamed. Collagen is an important component in the wound healing stage. Collagen is produced
from fibroblast cells to give strength to all tissues and play an important role for tissue growth and
recovery. Fibroblast are responsible for the synthesis of collagen and other protein regenerated during
the repair process [7].

Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
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2. Method

2.1. Fibroblast Cell Culture

Human Skin Fibroblast 1184 (HSF 1184) was used in this study. HSF 118 was cultured by seeding the
cells using 3 × 105 cells/cm2 cells density with MEM medium. Firstly, the MEM media was mixed
with 1% penicillin streptomycin and 10% Fetal Bovine Serum (FBS) and than cells cultured in a 75
cm2 T-flask and maintained at 37°C, 5% humidified CO2 incubator until cells were 90-95% confluent.
The medium in the flask was changed every two or three days until cells become 90-95% confluent.
The subculture procedure was conducting immediately when cells achieved 90-95% confluent
monolayer. Firstly, the medium in 75 cm2 T-flask was aspirated and rinsed three times with 5 ml
phosphate buffer saline (PBS) to get rid of cellular debris or serum which could interfered the action
of trypsin. Then, remove PBS and add 3 ml of trypsin-EDTA. The flask was incubated at temperature
37° C for three minute. Then, a flask was observed under microscope to ensure the cells detach from
the flask’s surface. 3 ml of MEM was added to neutralize the trypsin. Next, the cell mixture in the
flask was pour centrifuged tube and was centrifuged at 3300 rpm for 10 minutes. Then, the supernatant
is removed and the pellet is diluted MEM medium. Lastly, Fibroblasts cells (HSF 1184) were
incubated in 5% CO2 air atmosphere at 37°C.

2.2. Cell Cryopreservation

Cells in the log phase are stored to be frozen. Cells are mixed and centrifuged. Clotting media
containing 90% fetal bovine serum and 10% Dimethyl Sulfoxide slowly added to cell suspension.
After that, the cells were aliquoted into 1.2 ml of the cryogenic vial that had been labeled before and
the vials was putted in to the Nalge Nunc freezing container (for adaptation process) with temperature
of -70°C for 24 hour. Then, a vial containing cells is stored in liquid nitrogen storage [8].

2.3. Cell Viability

Method of determining the number of living cells, these cells need to be calculated using the dye
exclusion test. The principle of this test is that living cells have intact cell membranes that will exclude
certain dyes while dead cells do not. Trypan blue and Neubauer exclusion tests increase bright line
hemocytometers (Fortuna®, Germany) used to apply cell counts. The dead cells will accumulate
trypan blue whereas live cells will exclude it. Firstly, a clean coverslip was placed on the top of
haemocytometer slide. Then, 50μL suspension of fibroblast cells mixed with dye 50μL trypan blue.
After that, the mixture is transferred to the edge of the coverslip. Then, the amount that is not stained
(living cell) and stained (dead cell) were counted under a light microscope (Zeiss, axiovert 40CFL,
Germany). The concentration of viable cells was calculated using the following equation:
C = NA × D × 104 (1)
C = Concentration of cells (Cells/mL)
NA = The average number of living cells
D = Dilution factor
Whereas, percentage of cell viability was calculated using the following formula:
Percent of viability (%) = (Number of cells unstained)/(Total number of cells) x 100 (2)

2.4. Growth Profile

A growth profile or growth curve can be useful in assessing the growth characteristic of fibroblast.
From the growth curve, information of lag phase, exponential phase, saturation density and stationary
phase can be easily determined.
Ten 25cm2 t-flasks were prepared and labeled for the cell culture. Each of the flasks was seeded
with similar concentration of fibroblast which is 5 x 105 cells/ml. Other than that, this procedure also
involved basic handling of cells such as washing, trypsinization and centrifugation so that the pellet
can be obtained for cell counting. After 24 hours, one of the flasks were taken out and the number of
fibroblast cells were counted using by method described. All steps were repeated until day 10.

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IOP Conf. Series: Journal of Physics: Conf. Series 1317 (2019) 012071 doi:10.1088/1742-6596/1317/1/012071

2.5. Sircol Collagen Assay

Sircol Collagen Assay that can be used to evaluate and monitor the amount of collagen produced in
cell culture in vitro Cultured human skin fibroblast cell (HSF1184), standard reagent blank (100 ml)
mixed with Sircol Dye reagent (1 ml) into a microcentrifuge tube and left for 30 minutes. The
collagen-dye complex forms precipitation from the solution. The complex was packaged firmly to the
bottom of the tube by centrifugation at 12,000 rpm for 10 minutes. Unbound dyes are removed by
turning and drying the tube. Washing cold acid salts (750 μl) was added gently to remove unbound
dyes from the surface of the pellets and microcentrifuge tubes. The tube was centrifuged at 12,000 rpm
for 10 minutes and the washing was drained. Bound dye is released and dissolved through the addition
of alkaline reagents (250 μl or 1000 μl depending on the size of the pellet) and mixing the vortex for
10 minutes. The dyes released are measured spectrophotometry at 555 nm or colorimetry with blue-
green filters using multi-wall plate readers [9].

3. Result and Discussion

3.1 The Growth Curve of Human Skin Fibroblast (HSF1184) Cells Line
Fibroblasts are a type of primary cell located within the dermis of the skin. They present an important
role in wound healing through collagen synthesis and extracellular matrix (ECM) in connective tissue.
Furthermore, they also capable to produce growth factor such as PDGF, TGF-B and KGF-1 which
plays a role in proliferation, migration, and differentiation of other cells that help the wound healing
process [10].
Thus, the growth cycle of fibroblast was investigated as its provide the necessary information of
cell that is handle such as seeding concentrations, the duration of growth before subculture and the
duration of the experiments [11]. The growth curve of fibroblast was implemented using ten 25cm2 T-
flask without medium replenishment. The density used was 5 x 104 Cells/mL and the number of
fibroblasts cells was calculated daily by haemocytometer. Figure 1 presented the growth curve of
fibroblasts cell line.

80
Cells concentration (cells/ml) x 10⁴

70
60
50
40
30
20
10
0
0 2 4 6 8 10 12
Day

Figure 1. The normal curve growth of human skin fibroblast cells (HSF 1184)

Normally, the growth cycle of fibroblast consist three phases which are lag phase, log phase and
stationary phase [11]. The figure 1 show that the lag phase occurs for 2 days. The lag phase from
fibroblasts were determined because of extrapolated the intercept between the straight line from the
log phase and cell density at day 0 (5 x 104 cells/ml). During this phase, the cells tried to adapt with
the new growth environment such as Fetal bovine serum which needed for the cells growth.
Furthermore, at day 1 and 2, the cells were unable to duplicate themselves and the number of cell

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ICOMSET2018 IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1317 (2019) 012071 doi:10.1088/1742-6596/1317/1/012071

constant due to incapable to survive with the new environment. However, the remaining cells that
survived started to duplicate themselves and increased the cell number.
The cells started to enter log phase after 2 days and continued increasing exponentially until day 6.
At this phase, the growth of the cells is high (usually 90-100%) and cells were in the most
reproducible form. Thus, time optimum for sampling is at this phase due to it showed the most
consistent and high viability of the cells. At the end of this phase, the cells become confluent and all
the available surface of the growth was occupied.
After that, at day 6 until day 10 the cells started to decrease and get into the stationary stage. The
saturation density of the cells was estimated to be 62.25 x 104 cells/mL. This saturation density
showed the maximal population growth achieved using particular growth condition. At this phase the
cells proliferation was decelerated due to lack of medium for essential nutrient and also the division of
the cells was balanced with the death of the cells [12]. However, regular media changes will make the
cell continue to proliferate (although at reduce rate) well beyond confluence which resulted in
multilayer of cells [11].

3.2. The Effect of Swietenia mahagoni seed on collagen production in human fibroblast cells
(HSF1184)

Collagen is an important component in all phases of wound healing. Collagen is synthesized by

fibroblast cells, they provide integrity and strength for all tissues and play an important role in
increasing the proliferation and remodeling phase. Collagen is the basis for intracellular matrix
formation in wounds.
In this study, the SircolTM Collagen Assay was used to evaluate the collagen synthesis in the human
fibroblast cell (HSF 1184).

0.1 mg/ml
8
* 0.01 mg/ml
7
6 0.001 mg/ml
Collagen (µg)

5
4
3
2
1
0
SC-CO₂ Soxhlet LA PDGF-BB Control
Swietenia mahagoni seed extracts

Figure 2. The effect of Swietenia mahagoni seed extracts on the collagen synthesis in human skin
fibroblast cell (HSF 1184). LA (linoleic acid) and PDGF-BB (positive control) and MEM ( negative
control).

Increasing fibroblast cells are accompanied by new capillary growth in the wound to provide for
metabolic processes. The strength and vascular integrity of the new capillary layer are caused by
collagen production and cross-linking. Cross-linking of one of the collagen fibers has been assessed
for postoperative bleeding in patients with normal coagulation parameters. In the initial phase of
proliferation, fibroblast cell activity occurs in cellular replication and migration. On the third day after
the wound, the mass of growing fibroblast cells begins to synthesize and release collagen production.
Collagen levels rise continuously for about three weeks. The amount of collagen released during this

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ICOMSET2018 IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1317 (2019) 012071 doi:10.1088/1742-6596/1317/1/012071

period determines the tensile strength of the wound. Increased collagen production is an important
factor for wound healing. Type-I collagen is the main collagen of bones, tendons, skin, and wounds
that have just healed [13].
Figure 2 shows the effect of extract from S. mahagoni seeds on collagen synthesis from fibroblast
cells (HSF1184). In this study only three concentrations were chosen by reason of these three
concentrations proved to be consistent in stimulating the growth of fibroblast cells (HSF1184) based
on the results of previous studies. The graph in Figure 2 the three concentrations cause stimulation in
the synthesis of type I collagen in HSF1184 cells. Although, three concentration of SC-CO2 and
soxhlet that showed no statistically significant of collagen on the HSF1184 when compared with the
negative control, while LA (fibroblast cells with pure Linoleic acid as supplement in culture medium)
gave significant effect on the collagen production (p < 0.05).

4. Conclusion
Swietenia mahagoni seed extract with concentrations of 0.001, 0.01 and 0.1 mg / mL caused
stimulation in the synthesis of type I collagen in HSF1184 cells. Although, three concentrations of SC-
CO2 and soxhlet are showed no statistically significant of collagen on the HSF1184 when compared
with the negative control, while linoleic acid (fibroblast cells with pure Linoleic acid as supplement in
culture medium) gave significant effect on the collagen production.

Acknowledgment
The authors thank the financial support of the Indonesian Ministry of Research and Higher Education,
and also to Universiti Teknologi Malaysia (UTM) and Universitas Negeri Makassar (UNM) for the
use of laboratory instruments.

References
[1] Goun E, Cunningham G, Chu D, Nguyen C, Miles D.(2003). Antibacterial and antifungal
activity of Indonesian ethnomedical plants. Fitoterapia, 74: 592-596.
[2] Ekimoto H, Irie Y, Araki Y, Han GQ, Kadota S., Kikuchi T. (1991). Platelet Aggregation
inhibitors from the seeds of Swietenia mahagoni, Inhibition of in vitro and in vivo platelet-
activating factor induced effects of tetranortriterpenoids related to Swietenine and
switenolide. Planta Med., 57: 56-58.
[3] Rahman, A.K.M.S. and Chowdhury, A.K.A. (2009). Antibacterial Activity Of Two Limnoids
From Swietenia Mahagoni Against Multiple-Drug-Resistant (MDR) Bacterial Strains. J.Nat.
Med., 63: 41-45
[4] Hajra S, Mehta A, Pandey P, Vyas SP. (2011). Antioxidant and antidiabetic potential of
ethanolic extract of Swietenia mahagoni Linn Seeds. Int. J. Pharmaceuti.Res. Develop. 3(3):
180-186.
[5] Letawa, C., Boone, M., Pierard, GE. (1998). Digital image Analysis of The Effect of Topically
Applied Linoleic Acid on Acne Microcomedones. Clinical & Experimental Dermatology,
23(2): 56-58.
[6] Ali, M.A., Sayeed, M.A., Islam, M.S., Yeasmin, M.S., Khan, G.R.M.A.M. and Ida, I.M. (2011).
Physicochemical and Antimicrobial Properties of Trichosanthes anguina and Swietenia
mahagoni seeds. Bull.Chem. Soc. Ethiop., 25(3): 427-436.
[7] Ignotz RA, Massaugue J., (1986). Transforming growth caftor beta stimulates the expression of
fibronectin and into the extracellular matrix, J. Biol Chem, 261: 4337-4345.
[8] Freshney. R., I. (2005). Freshney’s Culture of Animal Cells. A Manual of Basic Technique. (5th
ed). Willey Publication.
[9] Choi, H., Kim, D., Kim, J.W., Ngadiran, S., Sarmidi, M.R., Park C. S. (2010). Labisia pumila
extract protects skin cells from photoaging caused by UVB irradiation. Journal of Bioscience
and Bioengineering, 109(3), 291-296.
[10] Houghton, P.J., Hylands, P.J., Mensah, A.Y., Hensel, A., Deters, A.M. (2005). In vitro Tests

5
ICOMSET2018 IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1317 (2019) 012071 doi:10.1088/1742-6596/1317/1/012071

and Ethnopharmacological Investigations; Wound Healing as an Example. Journal of

Ethnopharmacology, 100: 100-107.
[11] Freshney. R., I. (2000). Freshney’s Culture of Animal Cells. A Manual of Basic Technique.
(2nd ed). Willey Publication.
[12] Davis., J. (2011) Animal cell culture Essential method (1st edition). Wiley publication.
[13] Aramwit, P., Kanokpanont, S., De-Eknamul, W., Kamei, K. and Srichana, T. (2009). The Effect
of Sericin With Variable Amino-Acid Content From Different Silk Strains on The
Production of Collagen and Nitric Oxide. J. Biomater. Sci. Polym. Ed., 20:1295-1306

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 Hartati et al. / Malaysian Journal of Fundamental and Applied Sciences Vol. 14, No. 4 (2018) 432-436

RESEARCH ARTICLE

Evaluation of Swietenia mahagoni Jacq seed extracts in promoting

wound healing properties
Hartati a, b, *, Hasmida Mohd-Nasir b, Liza Md Salleh b, c, Irma Suryani Idris a, Azila Abd Aziz c
a
Biology Department, Universitas Negeri Makassar, South Sulawesi, Indonesia
b
Centre of Lipids Engineering & Applied Research (CLEAR), Ibnu Sina Institute for Scientific & Industrial Research, Universiti Teknologi Malaysia,
81310 UTM Johor Bahru, Johor, Malaysia
c
Bioprocess and Polymer Engineering Department, Faculty of Chemical & Energy Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor
Bahru, Johor, Malaysia

* Corresponding author: hartati@unm.ac.id

Article history Abstract

Received 28 March 2018
Revised 1 April 2018 Swietenia mahagoni or known as tunjuk langit is a widely known plant to possess good properties
Accepted 20 May 2018 in treating diseases as well as a wound treatment. The purpose of this work was to examine the
Published Online 3 December 2018 wound healing ability of the seed extracts in term of its ability to promote cell proliferation and
migration. The extracts from two extraction methods, i.e. supercritical carbon dioxide and Soxhlet,
were evaluate using cytotoxicity and scratch assays on human skin fibroblast cells. The findings
showed that the extraction yield using supercritical fluid extraction was lower than Soxhlet method
with 48.9% yield recovery. In addition, the seed extracts were able to stimulate cell growth and
migratory effect. This information can be used as a basis to performed subsequent study to report
wound healing activity of this plant material.

Keywords: Swietenia mahagoni, cell proliferation, cell migration, extraction methods, wound healing

© 2018 Penerbit UTM Press. All rights reserved

INTRODUCTION
Wound healing is a process involving inflammation as well as the
formation and remodeling of new tissue (Guartner et al., 2008; Fronza
et al., 2009). The first phase is re-epithelization, where the processes
such as migration of keratinocytes of the injured epidermis and hair
follicles followed by proliferation of these cells at the wound edge, are
observed at the beginning of new tissue formation. Keratinocytes
redifferentiation is then occurred to restore the barrier function.
Additionally, fibroblast is important in the repair of the injured dermis.
These cells proliferation are expanding and migrate into the wound
area, synthesize new extracellular matrix (ECM), as well as express
thick actin bundles as myofibroblasts (Schafer and Werner, 2007;
Guartner et al., 2008; Fronza et al., 2009).
The use of plants and preparations thereof to accelerate the wound
healing process was reported years ago (Reuters et al., 2009; Schmidt
et al., 2009). Their use is often based on traditional, without any
scientific evidence of efficacy and little knowledge about putative
active compounds or their mode of actions. As wound healing is a
complex biological process, several in vitro and in vivo assays are
presented. Among these, Liang et al. (2007) stated that the scratch assay
has been proven as a valuable and inexpensive tool to obtain first
insights into how plant preparations or their isolated compounds can
positively influence the formation of new tissue.
When wounded or scratched, cell monolayers respond to the
disruption of cell-cell contacts by increasing the concentration of
growth factors and cytokines at the wound edge, thus initiating
proliferation and migration of the cell. When performing a scratch
assay, an artificial gap, a so-called ”scratch” is created in a cell
monolayer with a sharp object such as a syringe needle or pipette tip.
The assay is performed on individual coverslips or in a multi-well plate.
The monolayers recovery and wound healing occur in a process that
can be monitored over time. The wound heals in a patterned fashion-
cells polarize toward the wound, initiate protrusion, migrate, and close
the wound. The progress of these events can be observed by manually
imaging samples fixed at different time points, or by time-lapse
microscopy (Liang et al., 2007; Fronza et al., 2009).
Swietenia mahagoni (Linn.) Jacq. is a plant mainly grows in
tropical areas of Asia, for example, Malaysia, India, Indonesia and
southern mainland China. The seeds have been used as folk medicine
for hypertension, diabetes, and malaria treatments, while the decoction
of its bark has been applied as a febrifuge (Chen et al., 2007). The
therapeutic effects associated with the seeds are mainly caused by the
biologically active ingredients, fatty acids and etranortriterpenoids
(Bacsal et al., 1997). There are reports of S. mahagoni seeds having
anti-inflammatory, antimutagenicity, and antitumour activities
(Guevara et al., 1996). The plant extracts have been accounted to
possess antibacterial and antifungal activities. Limonoid obtained from
S. mahagoni has antifungal activity and diabetes therapy (Ardahe et al.,
2010). The seeds of S. mahagoni are good agricultural product and have
been found potentially rich in fat (64.9%) (Ali et al., 2011).
The extraction methods are also playing an important role in
evaluating the effectiveness of the plant extract. The traditional
procedures for plant extractions include hydrodistillation and organic
solvent extraction (percolation, maceration or Soxhlet methods).
However, there are drawbacks with these methods such as time and
labour consuming operation, and involves large volumes of hazardous
solvents. Nevertheless, increasing solvent acquisition and disposal
costs; and regulatory restrictions have triggered the interest in
alternative extraction methods with less organic solvent consumption
(Yamini et al., 2008). Therefore, it is highly desired to develop
alternative extraction procedures with better efficiency and selectivity.

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Consequently, supercritical fluid extraction (SFE) is extensively
studied for separation of active compounds from herbs and other plants
as it is environmentally responsible and efficient extraction technique
for solid materials. Lang and Wai (2001) have reviewed several
advantages of SFE which are: (1) Shorter extraction time when using
supercritical fluids, since they have relatively higher diffusivity and
lower viscosity, (2) Continuous fluid flowing though samples could
provide quantitative or complete extraction, (3) SFE is usually
performed at low temperature, (4) The solvent power can be adjusted
by varying the pressure and temperature, (5) Separation of solutes
dissolved in supercritical fluid can be easily done by depressurization,
(6) Only a little quantity of sample is needed, (7) Zero or little amount
of organic solvent is needed, which is environmentally responsible. In
addition, SFE provides more quality extract compare to conventional
extraction methods as discussed by Hasmida et al. (2015).
The objective of the present study was to evaluate the ability of
Swietenia mahagoni seed extracts to promote cells proliferation into
and migration to the wounded monolayer which provide a basis on
wound healing properties of the plant material. Human skin fibroblasts
(HSF1184) were used, and platelet derived growth factor (PDGF)
served as positive control. Two extraction methods were applied in
order to compare the effectiveness of wound closure rate of the extracts
which were supercritical carbon dioxide (SC-CO2) and Soxhlet
extraction with hexane as solvent.
EXPERIMENTAL
Plant material preparation
S. mahagoni seeds were collected from Indonesia. The seeds were
rinsed with tap water to remove any foreign particles and dirt prior to
drying. Then, the cleaned seeds were cut into small pieces and dried in
an oven at temperature of 50°C for one week to remove its moisture.
The seeds were ground by a blender (Panasonic) with average particle
sizes varied from 0.25 to 0.75 mm.
Chemicals
Supercritical carbon dioxide (SC-CO2) extraction
The SC-CO2 extraction equipment used in this study was similar to
those of Mohd Nasir et al. (2017). The ground sample of 5 g was placed
into an extractor vessel. The extracts were collected into a glass vial
placed in the separator at ambient temperature and pressure. A flow rate
of CO2 was 2 mL/min. The investigated values of pressure,
temperature, and particle size were varied from 20 to 30 MPa, 40 to
60°C, and 0.25 to 0.75 mm, respectively. After each extraction, the
obtained extract was placed into glass vials, sealed and stored at 4°C to
prevent any possible degradation.
Soxhlet extraction
The extraction of Swietenia mahagoni seed was carried out as
described by Markom et al. (2007) with slight modifications. The
extraction of Swietenia mahagoni was implemented by using Soxhlet
extraction technique. To prepare the extract, 5 g of powdered S.
mahagoni seed were weight and placed in a Whatmann 25 mm x 100
mm cellulose thimble while 150 mL of hexane (100%) were placed at
the bottom of the apparatus. The extraction process was done for 6
hours at temperature 65°C. Then, the extraction yield was put in the
rotary evaporator (BUCHI rotavapor, R-114) at 40°C for 2 hours to
remove the solvent. The extracts were then placed in room temperature
condition before weighing gravimetrically to calculate the yields. The
samples were stored at 4°C in refrigerator for further analysis.
Expression of yield
The extraction yield was calculated using the following equation
where mo and m1 were mass of sample and extract in grams (g),
respectively.
𝑚
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑦𝑖𝑒𝑙𝑑 (%) = 𝑚1 × 100 (1)
0 compared to Soxhlet method.
Cell proliferation assay
The method described by Ranzato et al. (2011) was used. The
proliferation activity of human skin fibroblast (HSF1184) cells after
treated with crude extracts was determined by methylthiazol
tetrazolium (MTT) colorimetric assay. All cells were cultured in
minimum essential medium (MEM) + 10% fetal bovine serum (FBS) +
1% pen Strype (PS) under 5% CO2 humidified incubator. The cells
were seeded at a density of 2 x 105 cells/well in 96-well plate and
incubated for 24 hr prior to treatment. The test samples were prepared
by dissolving S. mahagoni extract in MEM to yield the final
concentration of 10, 1, 0.1, 0.01, 0.001 and 0.0001 mg/mL. The
medium was replaced after 24 hours with 200 µL of MEM containing
10% FBS and 1% PS; and serial dilution of plant extracts. After
incubation, the cells were washed with phosphate buffered saline
(PBS), 20 µL freshly prepared MTT solution (5 mg/mL) was added into
each well and cells were incubated at 37°C for 5 hours. The MTT
solution was then removed and replaced with 200 µL DMSO to allow
dissolution of the purple MTT formazan crystal. The absorbance was
measured at 540 nm using ELISA plate reader.
Cell migration assay
The wound closure of the HSF1184 was evaluated using scratch
wound assay which measures the expansion of a cell population on
surfaces modified from Fronza et al. (2009). The cells were cultured
into 12-well plate at a concentration of 3 x 105 cells until confluent.
Then, a linear wound was made on the monolayer with a sterile 200 µL
pipette tip. Any cellular debris was removed by washing the cells with
PBS. MEM (control), platelet derived growth factor (positive control),
the crude extracts (0.01 mg/mL) was added to the cells and incubated
for 24 hours at 37°C. Two representatives images from each well of the
scratched areas under each condition were photographed at 0, 12 and
24 hours to measure the wound closure. The data were analysed with
NIH ImageJ software (Ranzato et al., 2011). Wound closure was
determined by the difference in wound width between 0 hour and 24
hours.
Statistical analysis
Data were expressed as the mean ± S.E.M. Results were submitted
to the one-way analysis of variance ANOVA, followed by the post hoc
Tukey’s test, using the SPSS 16.0 software. Values of р < 0.05 were
considered statistically significant.
RESULTS AND DISCUSSION
Extracted oil yield of S. mahagoni seeds
The extraction of S. mahagoni seeds was done using two different
extraction methods which were supercritical carbon dioxide (SC-CO2)
and Soxhlet extraction. The comparison of both extraction methods was
shown in Fig. 1. Generally, the figure shows that Soxhlet extraction
gave the highest percentage oil yield (41.08% ± 0.98) compared to SC-
CO2 extraction which was 20.07% ± 0.48. However, taking into
account the extraction time, SC-CO2 offer better extraction process
since Soxhlet extraction required 6 hours of extraction time while SC-
CO2 extraction process only takes 3 hours to obtain the highest
extracted oil yield.
The higher oil yield obtained using Soxhlet extraction compared to
the SC-CO2 extraction method is due to the high temperature and long
extraction time applied during the extraction procedure. High
temperatures can increase the volatility of the solute hence enhancing
the solute solubility in hexane. Besides, longer extraction time increase
the contact time between the solute and solvent molecules resulting in
high extracted oil yield. The drawback of this properties is that the
method will extract all of the solutes, including impurities within the
particle. On the other hand, SC-CO2 extraction technique offers better
selectivity ability and it was proven by only 48.9% yield recovery
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 Hartati et al. / Malaysian Journal of Fundamental and Applied Sciences Vol. 14, No. 4 (2018) 432-436
50
40
Oi Yield (%)
30
20
10
0 reduction of the MTT can only be implemented by the metabolically
SC-CO₂
Extraction Methods
Fig. 1 Percentage of S. mahagoni seed oil extract using SC-CO2 and
Soxhlet extraction
The comparison between these two extraction methods had been
done previously as the researchers proved the effectiveness of SC-CO2
extraction to obtain more pure substances. For example, Zhao and
Zhang (2014) stated that the Soxhlet had the highest yield, followed by
SFE and hydrosilation. They explained that the nature of Soxhlet in
which it extracts high molecular weight molecule is one of the reasons
for the obtained result. A similar result also published by Liza et al.
(2012) where the flavonoid content was detected in significant quantity
when extracted using SC-CO2 method.
Cell proliferation analysis of S. mahagoni seed extracts
Cytotoxicity test using (3-(4,5-dimethyl-tiazol-2y) 2,5-diphenyl-
tetrazolium bromide (MTT) assay was done to obtain the suitable
extracts concentration which gives non-toxic effect towards cell
200
Relative Cell VIability (%)
* *
150
100
50
0
0.0001
Figure 2 Effect of S. mahagoni extracts using SC-CO₂ and Soxhlet extraction on human skin fibroblast (HSF1184) cells evaluated for 24 hours.
Referring to Ranzato et al. (2009) and Wang et al. (2011), relative
cell viability higher than control is considered to have a growth
promoting activity, while lower values than control show a toxic effect.
Based on the data obtained from this analysis, it can be concluded that
all extract’s concentrations ranging from 0.1 to 0.001 mg/mL did not
show any toxicity effects on HSF1184, even promoting fibroblast
proliferation as the cell's survival rate was higher than the control. The
concentrations dose higher than that is not suitable to be applied might
be due to its high antioxidant properties which will provide harmful
effect towards the fibroblast cells (Mbata, 2014). Previously, Ghosh et
al. (2009) found that two doses of methanolic extract of S. mahagoni
seeds (50 and 100 mg/kg) did not show any significant toxic effect up
to 1.2 g/kg of rat weight within 24 hours. According to other studies
conducted using brine shrimp lethality assay, LD50 oral acute toxicity
for S. mahagoni methanolic extracts exceed 2,500 mg/kg suggests that
the extract of this plant was relatively nontoxic. The results of the study
coincide with the fact that this plant can be used as traditional medicine
(Sahgal et al., 2010).
Although in vitro cytotoxicity studies conducted on monolayer
culture (fibroblasts) cannot be directly linked to in vivo assays, this
finding provides an important basis for subsequent studies on specific
bioactive compounds and mechanisms that act as proliferation agent.
Besides, the data of this study can be used as a quick assessment of the
cytotoxic effects of S. mahagoni seed extract on fibroblast cells.
434
proliferation. MTT assay is useful in vitro model for testing
cytotoxicity because it can show the ability of the compound to
stimulate the proliferation of the fibroblast. The toxic compounds will
affect the basic functions of the cells and the toxicity can be determined
by assessing cellular damage. MTT will enter the cell and passes the
mitochondria and the ring of MTT will be reduced to a dark purple
formazan product which is insoluble in the aqueous solution. The
Soxhlet active cells, and therefore this reduction can directly measure the
viability of the cell. In other words, this screening can assist for the
identification of the suitable amount of extracts that can be utilized for
next cell culture study.
Fig. 2 shows the relative viability of fibroblast cells after being
treated with S. mahagoni extracts for 24 hours at different
concentrations ranging from 0.0001 to 10 mg/mL for both extraction
methods. Generally, all extracts showed the same pattern of relative
viability at 0.1, 0.01 and 0.001 mg/mL extracts’ concentrations. The
highest relative cell viability was indicated by linoleic acid (LA), which
was the standard chemical marker in this study, at 0.1, 0.01 and 0.001
mg/mL with 145% ± 0.015, 190% ± 0.037 and 165% ± 0.037,
respectively, provide an idea that the high quality compound without
impurities shows a favourable response to cell proliferation rate.
Meanwhile, the SC-CO₂ and Soxhlet extracts showed only a slight
difference in cell proliferation between each other at concentrations of
0.1, 0.01 and 0.001 mg/mL. However, the extracts obtained from
Soxhlet extraction demonstrates relatively low cell viability compared
to others suggesting that the SC-CO₂ extraction from S. mahagoni seeds
results in a better effect on the rate of cell proliferation.
*
* *
* *
*
SC-CO2
*
*
soxhlet
LA
0.001 0.01 0.1 1 10 Positive
Control
Concentrations of S. mahagoni seed extracts (mg/mL)
Therefore, based on these result (Figure 2), the concentration of 0.1,
0.01, and 0.001 mg/mL were selected as the appropriate concentrations
to be applied in the next assay which was scratch assay to evaluate the
ability of the extracts in promoting cell migration.
Cell migration analysis of S. mahagoni seed extracts
Scratch assay have been previously used to study the characteristics
of proliferation and migration of various cell types after being treated
with extracts from different species such as Astragalus, Calendula,
Hemigraphic, Matricaria and Simmondsia (Fronza et al., 2009; Edwin
and Nair, 2011 ; Ranzato et al., 2011; Sevimli-Gur et al., 2011). The
reported optimum incubation period after the sctraching and cell
treatment for both fibroblasts and keratinocytes with plant extracts is
12-72 hours, depending on the type of cells, extracts and scratches. The
application of plant extracts with an optimum concentration of specific
compounds can reduce or decrease the rates of cell proliferation and
migration. High extract concentrations of legume genus Astragalus (10
mg/mL) and jojoba (Simmondsia chinensis) seed (wax, 5% v/v) are less
favorable for proliferation and migration of cells than less concentrated
extracts (1 mg/mL; 1%, v/v) (Ranzato et al., 2011; Sevimli-Gur et al.,
2011). To confirm the healing properties of S. Mahagoni seed extracts
discussed in the previous section, the ability of this extract to encourage
fibroblast migration has been studied.
 Hartati et al. / Malaysian Journal of Fundamental and Applied Sciences Vol. 14, No. 4 (2018) 432-436

In this study, migration of fibroblast cells was studied after 24 hours
of wounded cells and treated with two different S. mahagoni extracts.
Fig. 3 shows the effects of different S. mahagoni samples based on the
methods and concentration of extracts towards fibroblast cells. The
chart illustrates that SC-CO2 extract at concentration of 0.001 mg/mL
and Soxhlet extract at 0.1 and 0.01 mg/mL showed significant effect
on cell migration compared to negative control (MEM + 10% FBS +
1% antibiotic mixture) (p < 0.05), with the values of 85.306% ± 0.966,
93.496% ± 3.314, and 83.904% ± 2.901, respectively, and not
statistically different compared to LA and PDGF-BB (positive control).
However, there is no significant difference noticed on the trend of
wound closure percentage when treated with S. mahagoni extracts from
both extraction methods, applying that the extracts of both methods can
promote cell migration.

120
Wound Closure (%)

100 * *
* * *
80
60 0.1 mg/mL

40 0.01 mg/mL
0.001 mg/mL
20

0
SC-CO₂ Soxhlet LA PDGF-BB Kawalan
Control

S. mahagoni seed extracts

Fig. 3 Cell migration after wounded and treatment using different S. mahagoni seed extracts at concentration 0.1, 0.01 and 0.001 mg/mL, standard
marker, positive and negative controls for 24 hours.

The extracts from S. mahagoni seed, LA and PDGF repaired the The extract at a concentration of 0.1 mg/mL showed a higher
cells to the > 90% confluent level within 24 hours, in contrast with percentage of wound closure than positive control (2 mg/mL of PDGF).
negative control (Fig. 4). This finding explains that S. mahagoni seed extracts can promote
similar wound healing activity as PDGF at selected concentrations. The
0 hour 24 hour ability of the extracts in accelerating wound closure process might be
due to the linoleic acid content which acts as an essential tissue remedy
(a) as it promotes chemotactic and angiogenesis by maintaining moisture
in the environment and speeding up the tissue granulation process
(Ferreira et al., 2012). Linoleic acid also plays an important role of
chemotaxis for macrophages, and it is very crucial in the expression of
components for the fibrinolysis system (controlling the production of
collagenase). Ferreira et al. (2012) reported high concentrations of
linoleic acid (65%) was one of the factors that contribute to positive
(b) results in wound healing.

CONCLUSION

The ability of Swietenia mahagoni seed extracts in possessing

wound healing properties was accessed in term of cell proliferation and
(c) migration using cytotoxicity and scratch assays, respectively. The
findings suggest that all extracts can stimulate fibroblast growth in
which could play a vital role in its effect on tissue repair. These
assessments can be used as a basis to report wound healing activity of
plant material and subsequent study should be made especially in vivo
assay to determine the suitable dosage for human consumption.

(d)
ACKNOWLEDGEMENT

The authors gratefully acknowledge the financial support by the

Ministry of Research and Higher Education Indonesia, and also to
Universitas Negeri Makassar (UNM) and Universiti Teknologi
Malaysia (UTM) for the use of laboratory instruments.
Acknowledgement also extended to Research University Grants 12H93
(e) and 06H30 for funding this study.

REFERENCES

Adetutu, A., Morgan, W.A., Corcoran, O. 2011. Ethnopharmacological survey

and in vitro evaluation of wound-healing plants used in South-Western
Nigeria. J. Ethnopharmacol. 137, 50-56.
Ali, M.A., Sayeed, M.A., Islam, M.S., Yeasmin, M.S., Khan, G.R.M.A.M., Ida,
Fig. 4 Cell migration of (a) negative control, (b) positive control, (c) I.M. 2011. Physicochemical and antimicrobial properties of Trichosanthes
Linoleic acid (LA), (d) SC-CO2 extract of S. mahagoni, (e) Soxhlet extract anguina and Swietenia mahagoni seeds. Bull. Chem. Soc. Ethiop. 25(3),
of S. mahagoni at 0 and 24 hours. 427-436.

435
 Hartati et al. / Malaysian Journal of Fundamental and Applied Sciences Vol. 14, No. 4 (2018) 432-436
Alrdahe, S.S., Abdulla, M.A., Razak, S.A., Kadir, F.A., Hassandarvish, P. 2010.
Gastroprotective activity of Swietenia mahagoni seed extract on ethanol-
induced gastric mucosal injury in rats. World Acad. Sci. Eng. Technol. 67,
883-887.
Balekar, N., Gangadhar, K.N., Nakpheng, T., Jehtae, K., Srichana, T. 2012.
Evaluation of the wound healing potential of Wedelia trilobata (L) leaves.
J. Ethnopharmacol. 141, 817-824.
Barile, F.A., Dierick, P.J, Kristen, U. 1994. In vitro cytotoxicity testing for
prediction of acute human toxicity. J. Cell Biol. Toxicol. 10, 155-162.
Bascal, K., Chavez, L., Diaz, I., Espina, S., Javillo, J., Manzanilla, H., Motalban,
J., Panganiban, C., Rodriguez, A., Sumpaico, C., Talip, B., Yap, S. 1997.
The effect of Swietenia mahagoni (mahogany) seed extract on
indomethacin-induced gastric ulcers in female Sprague-Dawley rats. Acta
Med. Philipp. 3, 127-139.
Casas, I., Mantell, C., Rodroguez, M., Torres, A., Macias, F.A., De la Ossa,
E.J.M. 2008. Supercritical fluid extraction of bioactive compounds from
sunflower leaves with carbon dioxide and water on a pilot plant scale. J.
Supercrit. Fluids. 45, 37-42.
Chen, Y.Y., Wang, X.N., Fan, C.Q., Yin, S., Yue, J.M. 2007. Swiemahogins A
and B, two novel limnoids from Swietenia mahogany. Tetrahedron Lett.
48, 7480-7484.
Ekpo, M., Mbagwu, H., Jackson, C., Eno, M. 2011. Antimicrobial and wound
healing activities of Centrosema pubescens (Leguminosae). J. Phys.
Chem. Solids. 1,1-6.
Fronza, M., Heinzmann, B., Hamburger, M., Laufer, S., Merfort, I. 2009.
Determination of the wound healing effect of Calendula extract using the
scratch assay with 3T3 fibroblasts. J. Ethnopharmacol. 126, 463-467.
Guevara, A.P., Apilado, A., Sakurai, H., Kozuka, M., Tokuda, H. 1996. Anti-
inflammatory, antimutagenic and antitumor promoting activities of
mahogany seeds, Swietenia macrophylla (Meliaceae). Philipp. J. Sci. 125,
271-278.
Hasmida, M.N., Md. Salleh, L., Nur Syukriah, A.R., Harisun, Y., Che Yunus,
M.A., Abdul Majid, F.A. 2015. Total phenolic content and antioxidant
activity of Quercus infectoria galls using supercritical CO2 extraction
436
technique and its comparison with Soxhlet extraction. Pertanika J. Sci.
Technol. 23 (2), 287-295.
Joseph, B., Justin, R.S. 2011. A comparative study on various properties of five
medicinally important plants. Int. J. Pharm. 7(2), 206-211.
Lang, Q.Y., Wai, C.M. 2001. Supercritical fluid extraction in herbal and natural
product studies – a practical review. Talanta. 53, 771-782.
Liu, J., Han, B., Liu, G.Li., He, .J., Yang, G. 2001. Solubility of the non-ionic
surfactant tetramethylene glycol n-laurel ether in supercritical CO2 with n-
pentanol. Fluid Phase Equilib. 15, 247-254.
Liza, M.S., Abdul Rahman, R., Mandana, B., Jinap, S., Rahmat, A., Zaidul,
I.S.M., Hamid, A. 2012. Supercritical fluid extraction of bioactive
flavonoid from Strobilanthes crispus (pecah kaca) and its comparison with
solvent extraction. Int. Food Res.J. 19(2), 503-508.
Mbata, T. 2014. Antioxidant nutrients: beneficial or harmful. Internet Journal
of Food Safety. V, 29-33.
Mohd Nasir, H., Md Salleh, L., Ismail, A.R., Machmudah, S. 2017. Solubility
correlation of gall (Quercus infectoria) extract in supercritical CO2 using
semi-empirical equations. Asia-Pac. J. Chem. Eng. 12(5), 790-797.
Nagalakshmi, M.A.H., Thangadurai, D., Rao, D.M., Pullailah, T. 2001.
Phytochemical and antimicrobial study of Churasia tabularis leaves.
Fitoterapia. 72, 62-64.
Ranzato, E., Martinotti, S., Burlando, B. 2011. Wound healing properties of
jojoba liquid wax: an in vitro study. J. Ethnopharmacol. 134, 443-449.
Sahgal, G., Ramanathan, S., Sasidharan, S., Mordi, M.N., Ismail, S. Mansor,
S.M. 2009. Phytochemical and antimicrobial activity of Swietenia
mahagoni crude methanolic seed extract. Trop. Biomed. 26 (3), 274-279.
Wang, Z., Pan, Z., Ma, H., Atungulu, G. G. 2011. Extract of phenolics from
pomegranate peels. The Open Food Science Journal. 5, 17-25.
Yamini, Y., Khajeh, M., Ghasemi, E., Mirza, M., Javidnia, K. 2008. Comparison
of essential oil compositions of Salvia mirzayanii obtained by supercritical
carbon dioxide extraction and hydrodistillation methods. Food Chem. 108,
341-346.
Zhao, S., Zhang, D. 2014. Supercritical CO2 extraction of Eucalyptus leaves oil
and comparison with Soxhlet extraction and hydro-distillation methods.
Sep. Purif. Technol. 133, 443-451.
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Supercritical Fluid Extraction of Swietenia mahagoni Seed: Antioxidant and

Antimicrobial Activities

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DOI: 10.11113/jt.v67.2799

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Teknologi

Supercritical Fluid Extraction of Swietenia mahagoni Seed: Antioxidant

and Antimicrobial Activities
Hartatia,c*, Liza Md Salleha,b, Ahmad Ramdan Ismaila, Mohd. Azizi Che Yunusb, Azila Abd. Aziza
aDepartment of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia
bCentre of Lipid Engineering and Applied Research (CLEAR), Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru,
Johor, Malaysia
cDepartment of Biology, Universitas Negeri Makassar, South Sulawesi, Indonesia

*Corresponding author: tati_biounm@yahoo.co.id

Article history Abstract

Received :14 October 2013
Received in revised form :
21 January 2014
Accepted :24 February 2014
Graphical abstract
Swietenia mahagoni seeds have been used as folk medicine for a treatment of hypertension, malaria and
diabetes. This research was conducted to obtain extracts from the Swietenia mahagoni using supercritical
fluid extraction (SFE) with pure CO2 solvent, in order to evaluate the high- pressure method in terms of
process yield and biological activity. The various conditions namely flow rate of CO2 were set up at 2, 3,
and 4 ml/min; at constant pressures (P) and temperature (T). The sample extracts obtained by SFE with
CO2 flow rate of 4 ml/min showed the highest percentage yield (19.67%) compared to the others. The
antioxidant potential of the extracts was evaluated by the DPPH method and Folin- Ciocalteu method.
The solvent flow rate of 2 ml/min gave the lowest percentage of yield, but good results in antioxidant
activity and total phenolic content. The antimicrobial activity of the extracts against Gram-positive
(Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli) was evaluated based on
the inhibition zone using disc diffusion assay. These results ensured that Swietenia mahagoni seed extract
had inhibitory effects on the growth of Bacillus subtilis, Staphylococcus aureus and Escherichia coli at
concentration of 100 mg/ml.

Keywords: Supercritical fluid extraction (SFE); Swietenia mahagoni; antioxidant; antimicrobial

© 2014 Penerbit UTM Press. All rights reserved.

1.0 INTRODUCTION expeditiousness, automation and environmental safety as the

Swietenia mahagoni seeds have been applied as folk medicine for
treatment of hypertension, malaria, and diabetes [1]. The
therapeutic effects associated with the seeds are mainly caused by
the biologically active ingredients, fatty acids and
tetranortriterpenoids [2]. There are reports of S. mahagoni seeds
having anti-inflammatory, antimutagenecity, and antitumor
activities [3]. The plant extracts have been accounted to possess
antibacterial and antifungal activities. Therefore, in the last
several years, studies regarding extraction of natural compounds,
capable to avoid microorganism’s development, present special
interest, especially related to microorganisms responsible for the
deterioration of foods and ones responsible for a great number of
diseases [4].
Supercritical fluid extraction (SFE) is based on the use of
solvents in conditions above the critical point, resulting in a liquid
like density, gas like viscosity and the diffusivity values within
two orders of magnitude higher than that for typical liquids. This
technology presents several advantages over traditional liquid-
solvent-based methods including improved selectivity,
solvent can easily be removed from the solutes by expansion to
ambient pressure [5]. Carbon dioxide (CO2) is commonly used in
SFE because, in contrast with organic solvents, it is non-toxic,
inexpensive and volatile, with moderate critical conditions, thus
no thermal or chemical degradations of bioactive substance are
expected [6]. The quality and the composition of the extracts are
strongly dependant on the extraction technique, the solvent used,
the origin of the raw material, the part of the plant used (skin,
seeds, leaves, etc), its storage condition and the pre-treatment
applied [7,8]. The quality of the extract can be represented by its
properties, such as its biological activities. In this study, the
extract yield, antioxidant and antimicrobial activities of the
extracts from S. mahagoni seed obtained by SFE with pure CO2 of
different flow rate.

67:4 (2014) 59–62 | www.jurnalteknologi.utm.my | eISSN 2180–3722 |

60 Hartati et al. / Jurnal Teknologi (Sciences & Engineering) 67:4 (2014), 59–62
2.0 MATERIAL AND METHODS
2.1 Raw Material and Sample Preparation
The S. mahagoni seeds were collected from Indonesia. Then, the
seeds were rinsed with tap water to remove any foreign particles
and dirt prior to drying. Then, the cleaned seeds were cut into
small pieces and dried by using oven at temperature of 50 oC for
one week to remove moisture. The seeds were powdered by using
a blender (Merck Panasonic).
2.2 Supercritical Fluid Extraction (SFE)
Extraction was conducted under pressures of 30 MPa, temperature
of 40oC and CO2 flow rate of 2, 3, 4 ml/min. 5 g of powder
sample of S. mahagoni seed was kept in the extraction vessel. The
cotton is placed at the end to avoid any possible residue of solid
material. The vessel is placed in an oven to maintain operating
temperature. The extraction yield is collected in vial and placed in
an oven to allow for evaporation solvent. And then, the extract is
weighted and calculation of concentration yield is done based on
cumulative mass of extract and stored at -20oC before further
analysis for the extract yield and bioactive components.
2.3 Antioxidant Potential
2.3.1 Free Radical Scavenging Activity (DPPH)
The free radical scavenging of the S. mahagoni extracts was
evaluated using the 1.1-diphenyl-2-picrylhydrazil (DPPH)
method, as described [9] with a slight modification. Extract
solution were prepared by dissolving 0.025 g of dry extract in 10
ml of methanol to give final concentration at 2.5 mg/ml. Then, 77
μL of the extract solution were mixed with 3 ml of 6 x 10 -5 M
methanolic solution of DPPH. After 30 min at room temperature,
the absorbance values were measured at 517 nm in
spectrophotometer. The DPPH radical concentration was
calculated by using the following equation,
A Control−A Sample
DPPH radical concentration (%) = x 100
A Control
where A Control is the absorbance value of the control reaction and
A Sample is the absorbance value with the presence of the tested
extracts in the sample.
2.3.2 Total Phenolic Content (TPC)
The TPC was determined according to the Folin-Ciocalteau
method [10] with slight modification. Briefly, the reaction
mixture was composed by 1 mL of the extract (concentration of
0.01 g/mL), 5 mL of Folin-Ciocalteu reagent and 4 mL of sodium
carbonate (75 g/L) and was allowed for 1 hour in the dark at room
temperature. The absorbance was measured at 765 nm against a
reagent blank (containing all test reagents except for sample). The
TPC was calculated according to a standard curve. The
concentration of total phenolic compounds in the extract was
expressed as milligrams of Gallic acid equivalent per gram of dry
weight (mg GAE/g) of extract. The content of phenolic
compounds in the plant extracts were calculated using the
following formula: C = A / B, Where C is expressed as mgGAE/g
dry weight of the extract, A is the equivalent concentration of
Gallic acid established from calibration curve (mg), and B is the
dry weight of the extract (g) [10].
2.4 Antimicrobial Activity
The agar diffusion method Murray et al. [11] were used to
evaluate the antimicrobial activity of the subjected extracts.
Inoculum of 100 μL suspension containing 10 8 CFU/mL of
bacteria was spread on Mueller Hinton Agar. The discs (9 mm in
diameter) impregnated with 20 μL of 100 mg/mL extracts were
placed on seeded agar medium. Streptomycin (10μg/disc) was
used as positive control for bacteria. After that, the experiments
were conducted in triplicate and the test plates were incubated 24
hours at 37º C for bacteria. Then, the diameters of zone of
inhibition measured in mm [12].
2.5 Statistical Analysis
The data presented were analyzed by using SPSS 16.00 for
Windows (SPSS Inc. Chicago, IL). Values were as mean ±
standard deviation with three independent experiments. One-way
analysis of variance (ANOVA) using Tukey’s test at 95%
confidence level were used to determine the significance
difference between the samples.
3.0 RESULTS AND DISCUSSION
3.1 Percentage of Extraction Yield by SC-CO2
The yield of S. mahagoni seed extract is given in Figure 1. The
extractive value indicates CO2 flow rate 4 mL/min gives the
maximum yield (19.67 %) amongst the other extracts. On the
other hand, 2 mL/min flow rate CO2 resulted in lowest extraction
yield.
Figure 1 Percentage of extraction yield on different CO2 flow rate
The extracted S.mahagoni oil yield increased from 9.58% to
19.67% with an increase in flow rate from 2 to 4 mL/min at
pressure 30 MPa and temperature 40oC. The effect of flow rate
was also studied for the extraction of hazelnut oil [13]. At a low
pressure (15 MPa), an increase in flow rate from 0.5 to 2 ml/min
(measured at extractor pressure and 10oC) did not cause a
significant difference in the extraction yield of hazelnut oil.
However, at high pressure (30 MPa ), the oil yield increased more
than three-fold [13].
3.2 Free Radical Scavenging Activity (DPPH)
The presented Table 1 shows the DPPH free radical scavenging
assay result of three different carbon dioxide flow rate. The CO2
61 Hartati et al. / Jurnal Teknologi (Sciences & Engineering) 67:4 (2014), 59–62
flow rate 2 ml/min (94.40±5.38) gives highest DPPH radical
scavenging activity compared to 3 mL/min (91.13± 4.84) and 4
mL/min (90.38± 1.53). This indicates the antioxidant ability of
CO2 flow rate 2 ml/min oils is stronger than the other two oils.
The radical scavenging activity of the extracts could be related to
the nature of phenolics, thus contributing to their electron
transfer/hydrogen donating ability. Methanolic extract of the seed
of S. mahagoni contain phenol and flavonoid which give
antioxidant activity in vitro. The extract has potent antioxidant
activity against free radical scavenging activity (DPPH) assays
[14]. To date, the antioxidant activity of S.mahagoni seed extracts
using types of CO2 flow rate has not been well documented but
from the results obtained, types of CO2 flow rate does not give
significant differences towards the scavenging of free radicals
(p>0.05).
Table 1 DPPH free radical scavenging and TPC of S. mahagoni seed
extract of different CO2 flow rate
CO2 Flow rate DPPH TPC
(mL/min)*
2 94.40±5.38 72.75± 8.83
3 91.13± 4.84 67.98± 7.84
4 90.38± 1.53 62.20± 1.65
*
Temperature 40oC; Pressure 30 MPa, value are mean ± standard deviation (n=3)
3.3 Total Phenolic Content (TPC)
Total phenolic content, as determined by the Folin-Ciocalteu
method, is reported as gallic acid equivalents by reference to
standard curve (y = 0.00064x – 0.03186) and r2 = 0.986). The
total phenolic content extract is shown in Table 1. As can be seen
from Table 1, CO2 flow rate 2 mL/min have higher phenolic
content (72.75±8.83 mg GAE/g sample) compared to other CO2
flow rate 3 mL/min (67.98±7.84 mg GAE/g sample) and 4
mL/min (62.20±1.65 mg GAE/g sample). That may be
responsible for the antioxidative activities of this extract. Phenols
and polyphenolic compounds, such as flavonoid, are widely found
in food products derived from plant sources, and they have been
shown to possess significant antioxidant activities [10]. Different
types of phenolic compounds have different antioxidant activity,
which mainly depends on their structure as extract contains
different types of phenolic compounds which have different
antioxidant capacities. The statistical analysis indicated that types
of CO2 flow rate does not give significant differences towards the
total phenolic content (p>0.05). In this study, obtained the
positive linier correlation between antioxidant activity and total
phenolic content.
3.4 Antimicrobial Activity
The antibacterial activity of S.mahagoni seed extracts using
different CO2 flow rate was studied by using disc diffusion
method and the result are summarized in Table 2. This method
involved the measurement of the inhibition zone by selected
microorganism after 24 hours incubation.
Table 2 Antimicrobial activity for S. mahagoni seed extract of different
CO2 flow rate
CO2 Flow rate E.coli B.Subtilis S.aureus
(mL/min)*
2 9.5±0.71a 8.5±2.12a 9.5±0.71a
3 13.5±2.12a 12.0±1.41a 11.5±0.71a
4 12.5±2.12a 9.0±2.83a 12.5±2.12a
Streptomycin 15.5±0.71a 15.0±0.00a 13.0±1.41a
*
Temperature 40oC; Pressure 30 MPa, value are mean ± standard deviation (n=3)
As can be seen from Table 2, all extracts showed inhibitory
effects which were not significantly different (p>0.05) against
each bacterial species tested. In other words, the size inhibitory
zones showed by all CO2 flow rate did not differ significantly
against all tested bacteria. After 24 hours, that CO2 flow rate 3
mL/min extract was found to be active against the bacteria like
Escherchia coli (13.5±2.12) and Bacillus subtilis (12.0±1.41). The
results of disc diffusion assay of the crude extract were compared
with that standart antibiotic Streptomycin (10µg/disc). Results
also proved that CO2 flow rate 3 mL/min extract has more
effectiveness than that of CO2 flow rate 2 mL/min and CO2 flow
rate 4 mL/min extract against subjected bacteria strains.
4.0 CONCLUSION
Supercritical CO2 extraction of S. mahagoni seeds was carried out
and effects of CO2 flow rate on the yields were studied. The yield
of S. mahagoni seeds extract contained total phenolic compounds
and were capable of inhibiting, quenching free radicals to
terminate the free radical chain reaction, and acting as reducing
agents. In the present study, a linear relationship was found
between the antioxidant activity and phenolic content, indicating
that phenolic compound could be major contributors to
antioxidant activity. So it can be concluded that the seeds of S.
mahagoni possess significant antimicrobial activity in terms of
antibacterial properties.
Acknowledgement
The authors are highly grateful to the financial supports from
Minister of Higher education (MOHE) and acknowledgement is
also extended to Universiti Teknologi Malaysia for the use of
Laboratory instrument/equipments and research grant GUP
(Q.J130000.7125.02H01) during this study
References
[1] Chen, Y. Y., X.N. Wang, C. Q. Fan, S. Yin and J. M. Yue. 2007.
Swiemahogins A and B, Two Novel Limnoids from Swietenia
mahogany. Tetrahedron Letters. 48: 7480–7484.
[2] Bacsal, K., L. Chavez, I. Diaz, S. Espina, J. Javillo, H. Manzanilla, J.
Motalban, C. Panganiban, A. Rodriguez, C. Sumpaico, B. Talip and S.
Yap. 1997. The Effect os Swietenia mahagoni (Mahogany) Seed Extract
on Indomethacin-Induced Gastric Ulcers in Female Sprague-Dawley
Rats. Acta Med. Philipp. 3: 127–139.
[3] Guevara, A. P., A. Apilado, H. Sakurai, M. Kozuka and H. Tokuda.
1996. Anti-Inflammatory, Antimutagenic and Antitumor Promoting
Activities of Mahogany Seeds, Swietenia macrophylla (Meliaceae).
Philippine Journal of Science. 125: 271–278.
[4] Michielin, E., A. Salvador, C. Riehl, E. Smania and S. Ferreira. 2009.
Chemical Composition and Antibacterial Activity of Cordia Verbenaceae
 62 Hartati et al. / Jurnal Teknologi (Sciences & Engineering) 67:4 (2014), 59–62
Extracts Obtained by Different Methods. Bioresource Technology. 100:
6615–6623.
[5] Brunner, G. 2005. Suprcritical Fluids: Technology and Application to
Food Processing. Journal of Food Engineering. 67: 21–33.
[6] Pinelo, M., A. Ruiz-Rodrigues, J. Sineiro, F.J. Senorans, G. Reglero and
M.J. Nunez. 2007. Supercritical Fluid and Solid-Liquid Extraction of
Phenolic Antioxidants from Grape Pomace: A Comparative Study. Eur.
Food Res. Technol. 226: 199–205.
[7] Moure A., J. M. Cruz, D. Franco, J. M. Dominguez, J. Sineiro, H.
Dominguezet, MJ. Nunez and J. Carlos Paraju. 2001. Natural
Antioxidants from Residual Sources. Food Chem. 72: 145–171.
[8] Louli, V., N. Ragoussis and K. Magoulas. 2004. Recovery of Phenolic
Antioxidants From Wine Industry By-Products. Bioresource Technology.
92: 201–208.
[9] Miliauskas, G., P.R. Venskutonis and T.A. Van Beek. 2004. Screening of
Radical Scavenging Activity of some Medicinal and Aromatic Plant
Extracts. Food Chemistry. 85: 231–237.
View publication stats
[10] Sahgal, G., S. Ramanathan, S. Sasidharan, M. N. Mordi, S. Ismail and
S.M. Mansor. 2009. In Vitro Antioxidant and Xanthine Oxidase
Inhibitory Activities of Methanolic Swietenia mahagoni Seed Extracts.
Molecules. 14: 4476–4485.
[11] Murray, P. R., E. J. Baron, M. A. Pfallar, FC. Tenover and R. H. Yolke.
1995. Manual of Clinical Microbiology. Washington DC. 214–215.
[12] Mandal, S. C., A. Nandy, M. P. Pal and B. P. Saha. 2000. Evaluation of
Antimicrobial Activity of Asperagus recemosus Willd Root. Phytother.
Res. 14: 118–119.
[13] Ozkal, S. G., U. Salgin and M. E. Yener. 2005. Supercritical Carbon
Dioxide Extraction of Hazelnut oil. J. Food Eng. 69: 217–223.
[14] Bhurat, M. R., R. B. Sunil, A. D. Agrawal and M. B. Yogesh. 2011.
Swietenia mahagoni Linn.- A Phytopharmacological Review. Asian J.
Pharm. Res. 1: 1–4.
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Optimization of Supercritical Co2 Extraction of Swietenia Mahagoni Seed by

Response Surface Methodology

Article in Jurnal Teknologi · March 2014

DOI: 10.11113/jt.v67.2166

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 Jurnal Full paper
Teknologi

Optimization of Supercritical Co2 Extraction of Swietenia Mahagoni Seed

by Response Surface Methodology
Hartatia,c, Liza Md Salleha,b*, Mohd. Azizi Che Yunusb, Azila Abd. Aziza
aDepartment of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia
bCentre of Lipid Engineering and Applied Research (CLEAR), c/o Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru,
Johor, Malaysia
cDepartment of Biology, University State of Makassar, South Sulawesi, Indonesia

*Corresponding author: i.liza@cheme.utm.my

Article history Abstract

Received :28 October 2013
Received in revised form :
6 December 2014
Accepted :15 January 2014
Graphical abstract
Three operating parameters were pressure, temperature and particle size of supercritical carbon dioxide
extraction of oil from Swietenia mahagoni have been optimized by response surface methodology to obtain
high yield of oil. Results showed that data were adequately fitted into the second-order polynomial model.
The linear and quadratic terms of independent variables of temperature, pressure and particle size have
significant effects on the oil yield. Optimum conditions for oil yield within the experimental range of the
studied variables were 29.99 MPa, 55.29oC and 0.75 mm, and the oil yield was predicted to be 20.76%.

Keywords: Swietenia mahagoni seed; supercritical CO2 extraction; response surface methodology

Abstrak

Minyak Swietenia mahagoni telah diekstrak menggunakan kaedah superkritikal karbon dioksida di mana
tiga parameter iaitu tekanan, suhu dan sais zarah telah dioptimumkan menggunakan teknik gerak balas
permukaan untuk mendapat hasil minyak yang tinggi. Keputusan telah diperolehi dengan memasukkan data
kedalam model polinomial tertib kedua. Terma linear dan kuadratik menunjukkan bahawa pemboleh ubah
tekanan, suhu dan saiz zarah mempunyai peranan yang penting pada penghasilan minyak. Keadaan
optimum dalam julat eksperimen ialah 29.99 MPa, 55.29℃, 0.75 mm dan hasil minyak yang diramal ialah
20.76%.

Kata kunci: Benih Swietenia mahagoni; pengeluaran CO2 superkritikal; kaedah permukaan response

© 2014 Penerbit UTM Press. All rights reserved.

1.0 INTRODUCTION solvents. Nevertheless, there is increasing interest in alternative

Swietenia mahagoni (Linn.) Jacq. (Meliaceae) grows mainly in
tropical areas of Asia, such as India, Malaysia, Indonesia and
southern mainland China. The seeds have been applied as
traditional medicine for treatment of hypertension, diabetes, and
malaria, while the decoction of its bark has been used as a
febrifuge [1]. The therapeutic effects associated with the seeds
are mainly caused by the biologically active ingredients, fatty
acids and tetranortriterpenoids [2]. There are reports of S.
mahagoni seeds having anti-inflammatory, antimutagenicity,
and antitumour activities [3]. The plant extracts have been
accounted to possess antibacterial and antifungal activities.
Limnoid obtained from S.mahagoni has antifungal activity and
diabetes therapy [4]. The seeds of S. mahagoni are good
agricultural products and have been found to be potentially rich
in fat (64.9%) [5].
Conventional procedures for the extraction of plant
materials include hydrodistillation and organic solvent extraction
using percolation, maceration or Soxhlet techniques. However,
there are drawbacks with these methods such as time and labour
consuming operation, and involves large volumes of hazardous
extraction methods that consume smaller quantities of organic
solvent due to the rising solvent acquisition and disposal costs,as
well as regulatory restrictions [6]. Therefore, it is highly desired
to develop alternative extraction techniques with better
selectivity and efficiency. Consequently, supercritical fluid
extraction (SFE) as an environmentally responsible and efficient
extraction technique for solid materials was introduced and
extensively studied for separation of active compounds from
herbs and other plants [7].
Carbon dioxide (CO2) is the most common choice of
supercritical fluid due to its advantages of being non-toxic, non-
flammable, cost effective, and can be easily removed from the
extract following decompression [8, 9]. Currently, SFE has
become an acceptable extraction technique and being used in
many different areas. SFE of active natural products from herbs
or more generally, from plant materials has become one of the
most important areas of application. Supercritical carbon dioxide
(SC-CO2) was successfully used in the extraction of edible oils
from a wide range of seeds, including amaranth [10], hiprose
[11], cuphea [12], flax [13], sunflower and rape [14].

67:1 (2014) 15–20 | www.jurnalteknologi.utm.my | eISSN 2180–3722 |

16 Liza Md Salleh et al. / Jurnal Teknologi (Sciences & Engineering) 67:1 (2014), 15–20
Response surface methodology (RSM), originally described by
Box and Wilson [15], is a collection of mathematical and
statistical techniques useful for modeling and analysis of
problems in which a response of interest is influenced by several
variables and the objective is to optimize this response [16].
Recently, RSM has been successfully applied to optimize SC-
CO2extraction of oils from Salvia mirzayanii[17], silkworm
pupae [18], Passiflora seed [19], wheat germ [20], cottonseed
[21], Curcuma longa [22], rosehip seed [23], Cyperus
rotundus[24] and amaranth seed [25].
In the present study, SC-CO2 was used to extract the oil
from S.mahagoni seed. The aim was to optimize the processing
conditions, including pressure, temperature and particle size by
applying response surface methodology. The response variables
were examined with respect to the yield of oil under different
operating conditions.
Figure 1 A schematic design of the supercritical fluid extraction (SFE) unit
2.3 Experimental Design
Box-Behnken design (BBD) was applied to determine the
optimum extraction pressure, temperature and particle size for
supercritical CO2 extraction of Swietenia mahagoni seed. The
pressure (A), temperature (B) and particle size (C) were
independent variables studied to optimize the oil yield (Y) from
S.mahagoni seed. The CO2 flow rate was constant.
Box-Behnken design requires an experiment number (N)
according to the following equation:
N = 2k(k-1)+cp (1)
2.0 MATERIALS AND METHODS
2.1. Plant Material Preparation
S.mahagoni seeds were collected from Indonesia. The seeds
were rinsed with tap water to remove any foreign particles and
dirt prior to drying. Then, the cleaned seeds were cut into small
pieces and dried in an oven at the temperature of 50oC for one
week to remove the moisture. The seeds were then ground by a
blender MX-337 (Panasonic Malaysia Sdn Bhd).
2.2 Supercritical CO2 Extraction
A schematic diagram of SFE apparatus for extraction of
S.mahagoni seed is illustrated in Figure 1. The ground sample of
5 g was placed in an extractor vessel. The extracts were collected
in a glass vial placed in the separator at ambient temperature and
pressure. A flow rate of CO2 was 2 mL/min. The investigated
values of pressure, temperature, and particle size were varied
from 20 to 30 MPa, 40 to 60oC, and 0.25 to 0.75 mm,
respectively. After each extraction, the obtained extract was
placed into glass vials, sealed and stored at 4oC to prevent any
possible degradation.
Where k is the factor number and cp is the replicate number of
the central point. There are three levels of design (-1, 0, +1)
with equally spaced intervals between these levels. The
investigated factors and tested levels are reported in Table 1.
17 Liza Md Salleh et al. / Jurnal Teknologi (Sciences & Engineering) 67:1 (2014), 15–20

Table 1 The coded and uncoded levels of independent variables used 3.0 RESULTS AND DISCUSSION
in RSM design
3.1 Response surface analysis
Independent Symbol Level
variable Low (-1) Middle High (+1)
(0)
Since various parameters potentially affect the extraction
Pressure (MPa) A 20 25 30 process, the optimization of the experimental conditions
Temperature B 40 50 60 represents a critical step in the application of SFE method. The
(oC) experimental design was adopted on the basis of coded level
Particle size C 0.25 0.50 0.75 from three variables (Table 1), resulting in seventeen
(mm) simplified experimental sets (Table 2) with five replicates for
the central point. The selected factors were extraction
The experimental data were fitted with the second order temperature (in oC), pressure (in MPa) and particle size (in
response surface model of the following form: mm) with consideration that these factors are important in the
extraction process.
The effect of linear, quadratic or interaction coefficients
on the response was tested for significance by analysis of
variance (ANOVA). The degree of significance of each factor
are represented in Table 3 by its probability (Prob) > F. When
(2) the values of “Prob>F” was less than 0.05, the factor has a
significant influence on the process (for a confidence level of
Where y is the response (extraction yield in %); β0, βj, βjj, 95%) [23].
βij are constant coefficients of intercept, linear, quadratic, and Table 3 shows that the linear term of pressure and particle
interaction terms, respectively;and Xi and Xj are coded size had significant effect on the extraction yield, followed by
independent variables (pressure, temperature or particle size). the quadratic term of temperature and interaction of pressure
Analysis was performed using commercial software Design- and temperature. Meanwhile, the interaction between pressure
Expert® v.6.0.4 and particle size, and temperature and particle size on the yield
The analysis of variance (ANOVA) was also used to of S. mahagoni seed extraction were not statistically significant
evaluate the quality of the fitted model. The test of statistical (“Prob>F” more than 0.05).
difference was based on the total error criteria with a
confidence level of 95%.

Table 2 Experimental matrix and values of the observed response

Run Pressure Temperature Particle Coded Coded Coded Observed

(MPa) (oC) size (mm) pressure temperature particle extraction
variable variable size yield (%)
1 20 40 0.50 -1 -1 0 15.52
2 30 40 0.50 +1 -1 0 15.47
3 20 60 0.50 -1 +1 0 7.76
4 30 60 0.50 +1 +1 0 20.68
5 20 50 0.25 -1 0 -1 6.19
6 30 50 0.25 +1 0 -1 11.64
7 20 50 0.75 -1 0 +1 10.97
8 30 50 0.75 +1 0 +1 18.26
9 25 40 0.25 0 -1 -1 5.07
10 25 60 0.25 0 +1 -1 13.42
11 25 40 0.75 0 -1 +1 16.35
12 25 60 0.75 0 +1 +1 17.74
13 25 50 0.50 0 0 0 11.12
14 25 50 0.50 0 0 0 10.91
15 25 50 0.50 0 0 0 10.52
16 25 50 0.50 0 0 0 11.04
17 25 50 0.50 0 0 0 10.82

The second order polynomial model used to express the Table 3 Response surface of yield obtained by SC-CO2
total extraction yield as a function of independent variables (in
terms of coded values) is shown below: Variable Coefficients Standard F- Prob>F
error value
Intercept 10.88 0.78
Y=149.623-5.381A-3.861B+45.736C+0.051A2+0.026B2-
A 3.20 0.62 26.94 0.0013
6.636C2+0.064AB+0.368AC-0.696BC (3) B 0.90 0.62 2.12 0.1884
C 3.38 0.62 29.94 0.0009
A2 1.30 0.85 2.33 0.1707
B2 2.68 0.85 9.92 0.0162
C2 -0.41 0.85 0.24 0.6406
AB 3.24 0.87 13.82 0.0075
AC 0.46 0.87 0.28 0.6142
BC -1.74 0.87 3.98 0.0863
18 Liza Md Salleh et al. / Jurnal Teknologi (Sciences & Engineering) 67:1 (2014), 15–20

Table 4 Analysis of variance (ANOVA) for the response surface effect on the oil yield. As the pressure was raised, the density
quadratic model for the yield of S.mahagony seed obtained by SC-CO2 of SC-CO2 would increase, resulting in enhanced solubility of
extraction SC-CO2. However, there was a negative quadratic effect at
high pressure (Table 3). A possible reason was that highly
Source Sum of Degree Mean F- Prob>F
squares of square value
compressed CO2 facilitates solute-solvent repulsion [18]. Thus,
freedom high pressure is not always recommended, as it can potentially
Model 273.67 9 30.41 9.99 0.0031 induce complex extraction and complicate the analysis [27].
Residual 21.30 7 3.04 The effect of particle size was not important on the total extract
Lack of 21.09 3 7.03 129.40 0.0002 yield for particle size of 0.75 mm; however the oil yield
fit 0.22 4 0.054 increased by increasing particle size (Figure 3). Particle size
Pure 294.98 16 needs to be evaluated case by case based on the type of material
error to be processed. In the case of spice and seeds processing,
Total
particle size is generally between 30 and 60 Mesh [28].
According to previous investigations [29, 30], we came to a
conclusion that particle size has no influence on the extraction
Analysis of variance (ANOVA) results of the model are rate in the two outermost cases: fine milled material and
shown in Table 4. The regression model for the oil yield was coarsely ground plant material. Effects of temperature and
significant with “prob>F” of less than 0.05 and satisfactory particle size on the oil yield at a fixed pressure of 25 MPa are
coefficient of determination (R2) of 0.9278. Three dimensional shown in Figure 4. Particle size had positive linear effects on
(3D) response surface plots as a function of two factors while the yield, while the interaction between particle size and
keeping other factors at fixed levels are helpful in temperature was negatively correlated with yield.
understanding the effect on the response and the interaction It was more difficult to predict the influence of
effects of these two factors [27]. Thus, in order to gain a better temperature on SC-CO2 extraction than for pressure because
understanding of the influence of independent variables and temperature has two opposing effects on the yield of oil. As the
their interactions on the dependent variable, 3D response temperature increases, the density of CO2 decreases, which
surface plots for the measured responses were produced based leads to a reduced solvent power. Conversely, higher
on the model equation (3) in this study. Figure 2-4 show the 3D temperature increases the solute vapour pressure, resulting in
response surface plots as the functions of two variables with enhanced SC-CO2 solubility. Hence, the solubility of SC-CO2
another variable is kept constant at the center level. is inclined to increase, remain constant, or decrease with
Figure 2 illustrates the response surface and contour plot increasing temperature at constant pressure, depending
for the influence of pressure and temperature on the yield of oil whether solute vapour pressure or solvent density dominates
for a fixed 0.50 mm particle size. The yield increased at lower [17, 31].
pressure and temperature. Lower pressure had a positive linear

Figure 2 Surface plot of oil yield from S.mahagoni as a function of pressure and temperature at constant particle size of 0.50 mm

Figure 3 Surface plot of oil yield from S.mahagoni as a function of pressure and particle size at constant temperature of 50 oC
19 Liza Md Salleh et al. / Jurnal Teknologi (Sciences & Engineering) 67:1 (2014), 15–20
Figure 4 Surface plot of oil yield from S.mahagoni as a function of temperature and particle size at constant pressure of 25 MPa
3.2 Optimization of Extraction Parameters
In general, reducing the independent variable will optimize the
ratio of oil yield to production cost. The independent variable
was further minimized for optimum oil yield using the
numerical optimization function of the Design Expert
programme. The numerical range specified was set to minimize
temperature between 40 and 60oC, pressure between 20 and 30
MPa, and particle size between 0.25 and 0.75 mm, while
maximizing oil yield to 5.07-20.76%. Design Expert
programme indicated that for an oil yield of 20.76%, the
optimum values for each independent variable were
temperature of 55.29oC, pressure of 29.99 MPa, and particle
size of 0.75 mm. Under these optimum conditions, the
experimental value was 20.07%, which is in agreement with
those predicted by Design Expert programme.
4.0 CONCLUSION
Current results showed that second-order polynomial model
was sufficient to describe and predict the response variable of
the Swietenia mahagoni seed yield obtained by SC-CO2
extraction within the experimental ranges. The linear and
quadratic terms of pressure, temperature and particle size
significantly affected the yield. Based on the proposed model,
the optimum conditions for Swietenia mahagoni seed yield
within the experimental range were found to be 29.99 MPa,
55.29 oC and 0.75 mm, and the predicted yield was found to be
20.76 %. Under these optimum conditions, the experimental
values were in agreement with the predicted values.
Acknowledgment
The authors are highly grateful to the financial support from
Universiti Teknologi Malaysia for the research grant GUP
(Q.J130000.7125.02H01).
References
[1] Chen, Y.Y., X.N. Wang, C.Q. Fan, S. Yin, and J. M. Yue. 2007.
Swiemahogins A and B, Two Novel Limnoids from Swietenia
mahogany. Tetrahedron Letters. 48: 7480–7484.
[2] Bacsal, K., L. Chavez, I. Diaz, S. Espina, J. Javillo, H. Manzanilla, J.
Motalban, C. Panganiban, A. Rodriguez, C. Sumpaico, B. Talip, and
S. Yap. 1997. The Effect of Swietenia mahagoni (Mahogany) Seed
Extract on Indomethacin-induced Gastric Ulcers in Female Sprague-
dawley Rats. ActaMed. Philipp. 3:127–139.
[3] Guevara, A.P., A. Apilado, H. Sakurai, M. Kozuka, and H. Tokuda.
1996. Anti-Inflammatory, Antimutagenic and Antitumor Promoting
Activities of Mahogany Seeds, Swietenia macrophylla (Meliaceae).
Philippine Journal of Science. 125: 271–278.
[4] Ardahe, S. S., M. A. Abdulla, S. A. Razak, F. A. Kadir, and P.
Hassandarvish. 2010. Gastroprotective Activity of Swietenia
Mahagoni Seed Extract on Ethanol-Induced Gastric Mucosal Injury
in Rats. World Academyca of Science, Engineering and Technology.
67: 883–887.
[5] Ali, M. A., M. A. Sayeed, M. S. Islam, M. S. Yeasmin, G.R.M.A.M.
Khan, and I. M. Ida, 2011. Physicochemical and Antimicrobial
Properties of Trichosanthes anguina and Swietenia mahagoni seeds.
Bull.Chem. Soc. Ethiop. 25: 427–436.
[6] Khajeh, M. 2011. Optimization of Process Variable for Essential Oil
Components from Satureja hortensis by Supercritical Fluid
Extraction using Box-Behnken Experimental Design. The Journal of
Supercritical Fluids. 55: 944–948.
[7] Lang, Q., and C. M. Wai. 2001. Supercritical Fluid Extraction in
Herbal and Natural Product Studies- a Practical Review. Talanta. 53:
771–782.
[8] Casas, I., C.Mantell, M. Rodroguez, A. Torres, F.A. Macias, and E.
J. M. De la Ossa. 2008. Supercritical Fluid Extraction of Bioactive
Compounds from Sunflower Leaves with Carbon dioxide and Water
on a Pilot Plant Scale. J. Supercritical Fluids. 45: 37–42.
[9] Liu, J., B. Han, G. Li. Liu, J. He, and G. Yang. 2001. Solubility of the
Non-Ionic Surfactant Tetraethylene Glycol N-Laurel Ether in
Supercritical CO2 With N-Pentanol. Fluid Phase Equilibrium. 15:
247–254.
[10] Westerman, D., R. C. D. Santos, J. A. Bosley, J. S. Roger, and B. Al-
Duri. 2006. Extraction of Amaranth Seed Oil by Supercritical Carbon
Dioxide. The Journal of Supercritical Fluids. 37: 38–52.
[11] Reverchon, E., A. Kaziunas, and C. Marrone. 2000. Supercritical CO2
Extraction of Hiprose Seed Oil: Experiments and Mathematical
Modeling. Chemical Engineering Science. 55: 2195–2201.
[12] Eller, F. J., S. C. Cermak, and S. I. Taylor. 2011. Supercritical Carbon
Dioxide Extraction of Cuphea Seed Oil. Industrial Crops and
Products. 33: 554–557.
[13] Ozkal, S. G. 2009. Response Surface Analysis and Modeling of Flax
Seed Oil Yield in Supercritical Carbon Dioxide. Journal of the
American Oil Chemists Society. 86: 1129–1135.
[14] Boutin, O., and E. Badens. 2009. Extraction from Oleaginous Seeds
using Supercritical CO2: Experimental Design and Products Quality.
Journal of Food Engineering. 92: 396–402.
[15] Box, G. E. P., and K. B. Wilson. 1951. On the Experimental
Attainment of Optimum Conditions. J. Roy. Stat. Soc. 13: 1–45.
[16] Montgomery, D. C. 1991. Design and Analysis of Experiments. John
Willey & Sons, Inc., New York.
[17] Wei, Z. J., A. M. Liao, H. X. Zhang, J. Liu, and S. T. Jiang. 2009.
Optimization of Supercritical Carbon Dioxide Extraction Silkworm
Pupal Oil Applying the Response Surface Methodology. Bioresource
Technology. 100: 4214–4219.
[18] Liu, S., F. Yang, C. Zhang, H. Ji, P. Hong, and C. Deng. 2009.
Optimization of Process Parameters for Supercritical Carbon Dioxide
Extraction of Passiflora Seed Oil by Response Surface Methodology.
The Journal of Supercritical Fluids. 48: 9–14.
[19] Ku, C. S., and S. P. Mun. 2008. Optimization of the Extraction of
Anthocyanin from Bokbunja (Rubusco reanus miq) Marc Produced
During Traditional Wine Processing and Characterization of the
Extracts. Bioresource Technology. 99: 8325–8330.
[20] Shao, P., P.Sun, and Y. Ying. 2008. Response Surface Optimization
of Wheat Germ Oil Yield by Supercritical Carbon Dioxide
Extraction. Food and Bioproducts Processing. 86: 227–231.
 20 Liza Md Salleh et al. / Jurnal Teknologi (Sciences & Engineering) 67:1 (2014), 15–20
[21] Bhattacharjee, P., R.S. Singhal, and S.R. Tiwari. 2007. Supercritical
Carbon Dioxide Extraction of Cotton Seed Oil. Journal of Food
Engineering. 79: 892–898.
[22] Chang, L., T. Jong, H. Huang, Y. Nien, and C.J. Chang. 2006.
Supercritical Carbon Dioxide Extraction of Turmeric oil from
Curcuma Longa Linn. and Purification of Turmerones. Separation
and Purification Technolog. 47: 119–125.
[23] Machmudah, S., Y. Kawahito, M. Sasaki, and M. Goto. 2007.
Supercritical CO2 Extraction of Rosehip Seed Oil: Fatty Acids
Composition and Process Optimization. J. Supercrit. Fluid. 41: 421–
428.
[24] Wang, H., Y. Liu, S. Wei, and Z. Yan. 2012. Application Of Response
Surface Methodology to Optimize Supercritical Carbon Dioxide
Extraction of Essential Oil from Cyperus rotundus Linn. Food
Chemistry. 132: 582–587.
[25] Kraujalis, P., and P.R. Venskutonis. 2013. Optimisation of
Supercritical Carbon Dioxide Extraction of Amaranth Seeds by
Response Surface Methodology and Characterization of Extracts
Isolated from Different Plant Cultivars. The Journal of Supercritical
Fluids. 73: 80–86.
[26] Yetilmezsoy, K., S. Demirel, and R. J. Vanderbei. 2009. Response
Surface Modeling of Pb(II) Removal from Aqueous Solution by
View publication stats
Pistaciavera L: Box-Behnken Experimental Design. J. Hazardous
Materials. 171: 551–562.
[27] Yamini, Y., M. Khajeh, E. Ghasemi, M. Mirza and K. Javidnia. 2008.
Comparison of Essential Oil Compositions of Salvia Mirzayanii
Obtained by Supercritical Carbon Dioxide Extraction and
Hydrodistillation Methods. Food Chemistry. 108: 341–346.
[28] Martinez, J. L. 2008. Supercritical Fluid Extraction of Nutraceuticals
and Bioactive Compounds. Taylor & Francis Group, CRC Press is an
imprint of Taylor & Francis Group, an informa business. New York.
[29] Glisic, S., D. Misic, M. Stamenic, I Zizovic, R. Asanin, and D. Skala.
2007. Supercritical Carbon Dioxide Extraction of Carrot Fruit
Essential Oil: Chemical Composition and Antimicrobial Activity.
Food. Chem. 105: 346–352.
[30] Zizovic, I., M. Stamenic, and A. Orlovic. 2007. Supercritical Carbon
Dioxide Extraction of Essential Oils from Plants with Secretory
Ducts: Mathematical Modeling on the Micro-Scale. J. Supercrit.
Fluid. 37: 338–346.
[31] Thana, P., S. Machmudah, M. Goto, M. Sasaki, P. Pavasant and
A.Shotipruk. 2008. Response Surface Methodology to Supercritical
Carbon Dioxide Extraction of Astaxanthin from Haematococcus
pluvialis. Bioresources Technology. 99: 3110–3115.
 IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317

International Journal of Engineering

Journal Homepage: www.ije.ir

Inhibitory Effects of Swietenia Mahagoni Seeds Extract on Α-Glucosidase and Α-

Amylase
N. S. Md Norodina,b, L. Md Salleh*a,b, N. Yusofc, N. M. Mustaphad, F. Kamarulzamand, M. A. Mohamed Zaharia,b, N. A.
Bakeria,b
a Center of Lipids Engineering and Applied Research (CLEAR), Ibnu Sina Institute Scientific & Industrial Research (Ibnu Sina ISIR), Universiti
Teknologi Malaysia, Johor, Malaysia
b Department of Bioprocess and Polymer Engineering, School of Chemical and Energy Engineering, University Teknologi Malaysia, Johor,

Malaysia
c Advanced Membrane Technology Research Centre (AMTEC), Universiti Teknologi Malaysia, Skudai, Johor, Malaysia
d Natural Product Division, Forest Research Institute Malaysia (FRIM), Selangor Darul Ehsan, Malaysia

PAPER INFO A B S T R A C T

Paper history:
Received 16 December 2017
Received in revised form 25 January 2018
Accepted 08 February 2018
Keywords:
Swietenia Mahagoni Seed,
Α-Glucosidase Enzyme,
Α-Amylase Enzyme,
Supercritical Carbon Dioxide (SC-CO2)
Extraction
This study analyzed the inhibition activity of Swietenia mahagoni seeds extract on α-glucosidase and
α-amylase enzymes inhibition assays. Swietenia mahagoni seeds were extracted by using supercritical
carbon dioxide (SC-CO2) extraction at pressures of 20- 30 MPa and temperatures of 40- 60°C. The oil
yields obtained were analyzed with α- glucosidase and α- amylase enzymes inhibition assays. All data
obtained were expressed as mean ± standard deviation for triplicate experiments. One way analysis
was used for statistical significance by using statistica software version 7.0 (StartSoft, EUA) and IC 50
(extract concentration causing 50% enzyme inhibitory) was determined by using GraphPad Prism 6.0
software. Swietenia mahagoni seeds extract have a strong inhibition of α-glucosidase enzyme activity
(98.4% ± 0.2) but a moderate inhibition of α- amylase enzyme activity (34.9% ± 1.2). These findings
implied that Swietenia mahagoni seeds extract could be an effective natural antidiabetic agent.
doi: 10.5829/ije.2018.31.08b.20

1. INTRODUCTION1 delay the release of d-glucose from carbohydrate which

Currently, 117 million diabetes cases were reported and
expected to rise to 336 million by the end of 2030 [1].
Diabetes is a prevalent disease depict by distorted
glucose level in blood also known as hyperglycemia [2].
Hyperglycemia can be depict by the disorder of β-cells,
insulin inadequacy and distorted glucose level in blood
[3]. In managing diabetes mellitus, one of efficacious
way is to delay the glucose level and to achieve this is
by the inhibition of carbohydrate-digesting enzymes
such as α-glucosidase and α-amylase [2]. In the final
digestive process, α-glucosidase act as vital enzyme in
catalyzing the disaccharides and oligosaccharides into
glucose [4]. Meanwhile, α-amylase imply in catalyzing
starch to disaccharides and oligosaccharides. Thus, α-
glucosidase and α-amylase inhibitors can be used to
*Corresponding Author Email: i.liza@cheme.utm.my (L. Md Salleh)
also delaying the absorption of glucose in the small
intestine [5, 6]. Hence, reducing the glucose level in
blood and repression of postpandrial hyperglycemia
(PPHG). By retaining the reduction of hyperglycemia,
risk of developing microvascular and macrovascular
complications can be reduce [7].
According to World Health Organization (WHO),
approximately 80% of world population prefer
traditional medicine rather than modern approach.
Relatable, synthetic oral antidiabetic agents such as
acarbose was reported with side effect and failure to
reduce diabetes complications [8]. Therefore, interest
for natural oral antidiabetic agent from medicinal plants
is in demand. Swietenia mahagoni also known locally
as ‘tunjuk langit’ in Malaysia is used traditionally to
treat various diseases such as diabetes and high blood
pressure [9]. S. mahagoni seeds also reported to have
various biological activities such as anti-inflammatory

Please cite this article as: N. S. Md Norodin, L. Md Salleh, N. Yusof, N. M. Mustapha, F. Kamarulzaman, M. A. Mohamed Zahari, N. A. Bakeri,
Inhibitory Effects of Swietenia Mahagoni Seeds Extract on Α-Glucosidase and Α-Amylase, International Journal of Engineering (IJE), IJE
TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317
1309 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317
activity, anticancer and antitumor activity [10] and also
antidiabetic activity [11]. In Malaysia, the raw seeds
have been used for hypertension and diabetes [12].
Previous research on S. mahagoni seeds had proven the
antidiabetic activity as shown in Table 1.
As summarized in Table 1, conventional methods
of extraction were used for the antidiabetic study in vivo
and in vitro diabetic model. Limited study on
antidiabetic study using advance extraction method such
as supercritical carbon dioxide (SC-CO2) extraction for
S. mahagoni was studied. SC-CO2 extraction is a
separation process of matters by using supercritical
carbon dioxide as solvent. Carbon dioxide is the most
frequent solvent used that is an environmentally friendly
(fairly non-toxic), low cost and can easily been removed
from the extract [13]. Moreover, the extraction rate of
SC-CO2 can be easily manipulated or controlled by
pressure and temperature which also influence the
solvency power [14]. Also, by using SC-CO2 extraction,
degradation of thermolabile compounds are prevented
due to the reduced operating temperature in the
extraction [15].
Moreover, quantification of β-sitosterol was
determined and the correlation of β-sitosterol and the
inhibitory activities of α-glucosidase and α-amylase
were evaluated. β-sitosterol (Figure 1) is one of the
diversified group of compounds in phytosterols.
Phytosterols are well known as plant sterols, one of the
vital components of plant membranes [16]. The most
ample compound in natural sterols is β-sitosterol [17]
and it can be found in seeds, nuts, vegetables and fruits.
Furthermore, β-sitosterol have been reported to have
various pharmacological activities such as anti-
inflammatory activity [18], chemopreventive effects
[19], hypocholesterolemic activity [20], antioxidant
effects [21] and also antidiabetic effects [22]. Previous
study had shown that β-sitosterol may responsible to the
antidiabetic activity shown in Table 2.
In this study, the aim is to evaluate the antidiabetic
activity of Swientenia mahagoni seed extract from SC-
CO2 extraction. The antidiabetic activity of S. mahagoni
seeds extract was analyzed by in vitro diabetic model
(inhibition of carbohydrate-digesting enzymes).
Moreover, the correlation of β-sitosterol and the
inhibitory activities of α-glucosidase and α-amylase
were also evaluated.
2. EXPERIMENTAL
2. 1. Materials Commercial grade liquid carbon
dioxide (purity 99.99%) used in supercritical carbon
dioxide extraction was purchased from Kras, Instrument
and Services, Johor, Malaysia. Acarbose, 1-
deoxynojirimycin, p-nitrophenyl-α-D-glucopyranoside
(pNPG), sodium dihydrogen phosphate, sodium
phosphate tartrate, disodium hydrogen phosphate,
sodium hydroxide, dimethyl sulfoxide (DMSO),
potassium chloride, 3,5-dinitrosalicylic acid (DNS),
starch (soluble), α-amylase from porcine pancreas and
α-glucosidase from Saccharomyces cerevisiae supplied
by Sigma-Aldrich (St. Louis MO, USA). Sodium
chloride (Bendosen Laboratory Chemicals, Bendosen,
Norway. Potassium phosphate monobasic supplied by
Merck, Millipore, Billerica, (Massachusetss, USA).
2. 2. Sample Preparation of Swietenia Mahagoni
Seeds Swietenia mahagoni seeds were bought in the
local market. The seeds were rinsed with tap water to
remove any foreign particles and dirt prior to drying.
Then, the cleaned seeds were cut into small pieces and
dried by using oven at temperature of 50°C for a week
to remove moistures. The seeds were ground by using a
blender (Merck, Panasonic) and sieved to approximate
0.50 mm of particle size.
2. 3. Supercritical Carbon dioxide (SC-CO2)
extraction Clear supercritical fluid extraction (SFE)
machine in Center of Lipids Engineering and Applied
Research (CLEAR), Universiti Teknologi Malaysia
consisted of CO2 gas cylinder, CO2 controller pump
(Lab Alliance), co-solvent pump (Lab Alliance), oven
(Memmert, Germany), 10 ml stainless steel extraction
vessel, pressure gauge (Swagelockk, Germany),
automatic back pressure regulator (Jasco BP 2080- Plus)
and restrictor valve. A schematic diagram of CLEAR
SFE apparatus is illustrated in Figure 2.
The parameters and constant parameters used in
extraction process are presented in Table 3. Five gram
of sample was placed in 10 ml stainless steel extraction
vessel and sealed tightly in the oven. Set all the
parameters (temperature, pressure and flowrate of CO2),
the extraction process started after all the parameters
were attained. Lastly, depressurized the system and the
oil yields were collected after 120 minute extraction
time.
Figure 1. Structure of β-sitosterol [17]
 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317
Experimental Model
(In vitro)
In vitro studies
In vitro bioassay
(glucose utilization
assay)
In vitro glucose
absorption by isolated Petroleum ether
everted intestine
In vitro α-amylase
Petroleum ether
inhibitory activity
In vitro (non-cell based
enzymatic assay)
NA = Not available
Experimental Model
In vivo studies
Streptozotocin-induced
diabetic rats
Alloxan-induced diabetic
mice
TABLE 3. The process parameters for SC-CO2 extraction
Parameter
Temperature (°C)
Pressure (MPa)
Flowrate of CO2 (ml/min)
Particle size (mm)
Mass of sample (g)
Extraction time (min)
Figure 2. Schematic diagram of CLEAR supercritical fluid
extraction (SFE) machine
1310
TABLE 1. Summary of studies on antidiabetic effects of Swietenia Mahagoni
Extraction Suggested mechanism(s)
Results Reference
method of action
Exhibited a significant improvement (p<0.005) of
Chloroform Insulin mimetic [23]
peripheral glucose utilization
Did not significantly inhibit glucose absorption
NA* [24]
through everted intestinal
Showed the highest inhibitory activity, measured 86.81 Inhibition of digestive
[25]
percent inhibition enzyme activities
Showed the highest inhibitory activity, measured Inhibition of digestive
Ethanolic [6]
18.647 percent inhibition enzyme activities
TABLE 2. Summary of studies on antidiabetic effects of β-sitosterol
Suggested mechanism(s) of
Results Reference
action
Decreases in glycated hemoglobin, serum glucose,
Insulin mimetic and β-cell
and nitric oxide, with concomitant increases in serum [22]
regeneration
insulin levels
Decrease glucose
Lowered the serum glucose level in diabetic mice [26]
production at liver
Collected oil yield was calculated as percentage of oil
Range/ value yield by using equation below:
𝑀
40-60 𝑂𝑖𝑙𝑌𝑖𝑒𝑙𝑑(%) = ( 1) × 100 (1)
𝑀2
20-30 Where, M1 is the mass of oil extract in gram and M2 is
the mass of the sample in gram.
2.00
0.50 2. 4. High Performance Liquid Chromatography
5.00 (HPLC) Analysis β-sitosterol was analyzed [27]
with a slight modification by using a Waters HPLC
120
system (Milford, MA, USA) consisting of a pump and
system controller (Model Waters e2695) and photo-
diode array detector (Model 2998). The compound
separation was carried out by a C18 reserved phase
Kinetex Biphenyl column (5 µm, 4.6 × 150 mm) with a
flow rate of 1.0 ml/min. The mobile phase was
consisted of Methanol (60%)/ Acetonitrile (40%), in an
isocratic program. The injection volume is 20 µL. All
samples were filtered with 0.45 µm nylon filters prior to
injection. The detection was monitored at 210 nm and
data were integrated by Empower 3 software (Waters)
(Milford, MA, USA). Figure 3 shows the calibration
curve for β-sitosterol and the equation obtained was
used to calculate the β-sitosterol content in each
extracts.
1311 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317

absorbance at 540 nm. Final reaction mixture contain

100 µg/mL sample, 1.0 U/mL enzyme and 0.25% w/v
starch. Inhibition of α-amylase (%) was calculated by
using equation below:
𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴 𝑆𝑎𝑚𝑝𝑙𝑒
𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛(%) = ( ) × 100 (3)
𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙

where A is the absorbance of test mixture of wavelength

of 540 nm.

2. 7. Statistical Analysis Data obtained were

Figure 3. Calibration curve of β-sitosterol
2. 5. α-glucosidase Inhibition Assay α-
glucosidase inhibition activity was determined
following previous methods [28-30] with modifications.
10 μL of sample with various concentrations (0- 100
μg/ml), acarbose (positive control) and DMSO (negative
control) were transferred to 96-well plate (Greiner Bio-
one) using micropipettes (Eppendorf, ResearchPlus).
After that, 20 μL of α-glucosidase, 40 μLof phosphate
buffer saline (PBS) pH 6.5 and 20 μL of distilled water
were pipetted into each wells and incubated at 37°C for
10 minutes (pre-incubation) by using thermo-shaker
incubator (Allsheng, MSC-100). Next, 10 μL of pNPG
was added into all wells in dark and immediately the
first absorbance was read at 405 nm (A 0 min) using
spectrophotometer (BMG, FLUOstar Omega). Then, the
plate was incubated at 37°C for 30 minutes and the
absorbance was read again at 405 nm (A30 min). Final
reaction mixture contain 0.1 U/mL enzyme and 1.25
mM pNPG substrate. Inhibition of α-glucosidase (%)
was calculated using equation below:
𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴 𝑆𝑎𝑚𝑝𝑙𝑒
𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛(%) = ( ) × 100
𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙
where A is the absorbance of test mixture of wavelength
of 405 nm.
expressed as mean ± SEM from triplicate experiments.
One-way analysis of variance was used to test for
statistical significance between means using Statistica
software version 7.0 (StatSoft, EUA) and IC50 (extract
concentration causing 50% enzyme inhibitory) was
determined using GraphPad Prism 6.0 software. p<
0.01 and p< 0.05 were considered as significant. The
Pearson correlation test was performed to obtain
correlation value (r) between β-sitosterol concentration
and α-glucosidase and α-amylase inhibition.
3. RESULTS AND DISCUSSION
3. 1. Supercritical Carbon Dioxide (SC-CO2)
Extraction The extraction of the essential oil of
Swietiena mahagoni seedsby using SC-CO2 extraction
showed that the highest yield (14.45 %) obtained was at
the maximum level (30MPa, 60°C) meanwhile lowest
(1.49%) at (20MPa, 60°C) as shown in Table 4. At high
temperature, the decomposition of cell walls occurred
thus extracted oil produced is also high [33]. Operating
at higher pressure will influence the solvent density,
thus enhanced the solvency power (the interaction of
(2) inter-molecules and solutes increase) [34]. Similar result
was reported in the extraction of the essential oil of
Swietiena mahagoni seeds using SC-CO2 extraction
[35].

2. 6. α-amylase Inhibition Assay α-amylase

inhibition activity was determined following previous TABLE 4. Extraction of the essential oil of Swietiena
methods [28, 31, 32] with modifications. 10 μL of mahagoni seedsat different conditions (pressure, P and
sample, acarbose (positive control) and 5% DMSO temperature, T)
(negative control) were transfer to test and blank well in Condition (s) Oil yield (%)
96-well plate (Greiner Bio-one) using micropipettes P= 20MPa, T= 40°C 6.56
(Eppendorf, ResearchPlus). After that, 50 μL of α-
P= 20MPa, T= 50°C 3.68
amylase solution (4.0 U/mL) and 40 μL of distilled
water were added into each wells (add 90 μL of distilled P= 20MPa, T= 60°C 1.49
water at blank wells) and the plate was incubated at P= 25MPa, T= 40°C 6.64
25°C for 5 minutes (pre-incubation) by using thermo-
P= 25MPa, T= 50°C 4.95
shaker incubator (Allsheng, MSC-100). Then, 100 μL of
starch (0.5 % w/v) solution was added into every wells P= 25MPa, T= 60°C 4.56
and incubated at 25°C for 7 minutes. Next, 100 μL of P= 30MPa, T= 40°C 7.02
DNS color solution was added into all wells in dark and
P= 30MPa, T= 50°C 8.61
incubate at 85°C for 30 minutes. The plate was allowed
to cool to room temperature before measuring the P= 30MPa, T= 60°C 14.45
 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317
3. 2. Quantification of β-sitosterol Content β-
sitosterol content of S. mahagoni seeds extract at
different conditions in SC-CO2 extraction were
identified and quantified. The highest β-sitosterol
obtained was 9.20 mg/g at 30 MPa and 40°C meanwhile
the lowest (3.12 mg/g) was obtained at 20 MPa and
50°C as summarized in Table 5.
β-sitosterol is a non-polar compound [36] and
supercritical carbon dioxide (SC-CO2) extraction is
more appropriate to extract non-polar nature compounds
[37] than solvent extraction. From Table 5, the highest
yield of β-sitosterol was obtained at the lowest
temperature studied. This shows that the domination of
solute vapor pressure at low temperature [38].
Meanwhile at high pressure, the recovery of β-sitosterol
was higher. This result also accordance with previous
research in the extraction of β-sitosterol by using SC-
CO2 extraction [39-42]. It shows that the increase of
SC-CO2 density cause the increase in the solubility of β-
sitosterol in SC-CO2. Figures 4 and 5 show the HPLC
chromatograms of the standard (β-sitosterol) at
concentration of 80 ppm and β-sitosterol compound
detected in S. mahagoni oil extract accordingly.
TABLE 4. Extraction of the essential oil of Swietiena
mahagoni seedsat different conditions (pressure, P and
temperature, T)
Condition (s) Β-sitosterol content (mg/g)
P= 20MPa, T= 40°C 3.52
P= 20MPa, T= 50°C 3.12
P= 20MPa, T= 60°C 5.91
P= 25MPa, T= 40°C 7.03
P= 25MPa, T= 50°C 6.23
P= 25MPa, T= 60°C 8.72
P= 30MPa, T= 40°C 9.20
P= 30MPa, T= 50°C 3.67
P= 30MPa, T= 60°C 6.70
Figure 4. HPLC chromatography of the standard ((β-
sitosterol) at concentration of 80 ppm.
1312
Figure 5. HPLC chromatogram of β-sitosterol compound
detected in S. mahagoni oil extract at 30 MPa and 40°C.
3. 3. α-glucosidase activity Inhibitory activity of
α-glucosidase from S. mahagoni seeds extract at
different condition is shown in Table 6. All conditions
achieved IC50 value (extract concentration causing 50%
enzyme inhibitory). The stronger the α-glucosidase
inhibitory activity, the lowest the IC50. In this study, the
lowest IC50 value achieved was 11.92 ± 0.23 µg/mL (p<
0.01) at 20 MPa and 60°C and the highest IC50 achieved
was 39.62 ± 2.95 µg/mL (p< 0.05) at 25 MPa and 50°C.
Currently, no studies were found for different conditions
of SC-CO2 extraction on inhibitory activity of
carbohydrate-digesting enzymes.
Previous research on α-glucosidase inhibitory
activity of S. mahagoni is shown in Table 7 with
comparison with the result in this study. It shows that
by using SC-CO2 extraction, the percent inhibition is
much higher than using conventional methods. This
may due to the reduced operating temperature in the
SC-CO2 extraction that prevent the degradation of
thermolabile [15]. Despite the fact that conventional
extraction methods with solvents could achieve the
target, but during elimination of solvent, oxidative
deteriorations may occur [43]. This may resulting in the
low percent inhibition of α-glucosidase activity using
the conventional methods mention in Table 7.
Moreover,effect of pressure and temperature of SC-
CO2 also affect on α-glucosidase inhibitory activity.
Figure 6 shows the effect of pressure and temperature
on IC50 of α-glucosidase inhibitory activity. The
evaluation for the effect of temperature toward α-
glucosidase inhibitory activity showed that at constant
pressure of 20, 25 and 30 MPa, the IC50 value increases
as temperature increase from 40 to 50°C and decrease as
its reached 60°C. This is because as temperature further
increase, the recovery of compound responsible to the
inhibitory activity of α-glucosidase may decrease due to
vaporization or decomposition of the volatile compound
during the extraction.
1313 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317

TABLE 6. IC50 data of α-glucosidase inhibitory activity of S. as pressure increase from 20 to 25 MPa and decrease as
mahagoni seeds extract at different conditions (pressure, P and its reached 30 MPa. Lastly, at constant temperature of
temperature, T). The values presented are expressed as mean 60°C, as pressure increases, the IC50 value increases. In
± standard error of the mean of triplicate experiments this study, β-sitosterol in the extract was quantified by
Condition (s) IC50(µg/mL) high performance liquid chromatography (HPLC). The
Positive control 62.45 ± 1.79 correlation of β-sitosterol content and α-glucosidase
inhibitory activity was studied and shown in Figure 7.
P= 20MPa, T= 40°C 31.11 ± 2.25**
From Figure 7, the β-sitosterol concentration in
P= 20MPa, T= 50°C 34.55 ± 2.61** extracts showed a negative correlation toward the
P= 20MPa, T= 60°C 11.92 ± 0.23* activity of α-glucosidase at constant pressure of 20, 25
P= 25MPa, T= 40°C 27.53 ± 2.07*
and 30 MPa. Meanwhile at constant temperature of
60°C shows no correlation at all but at constant
P= 25MPa, T= 50°C 39.62 ± 2.95** temperature of 40°C and 50°C shows negative and
P= 25MPa, T= 60°C 13.9 ± 0.33* positive correlation respectively. This result shows
P= 30MPa, T= 40°C 17.56 ± 0.19*
inconsistency of β-sitosterol concentration in extracts
correlate with the activity of α-glucosidase. Thus,
P= 30MPa, T= 50°C 34.70 ± 1.15** synergy of all the compounds in the extract may
P= 30MPa, T= 60°C 19.28 ± 1.13* responsible to the activity instead of single compound.
*p< 0.01 as compared with positive control
**p< 0.05 as compared with positive control 3. 4. α-amylase activity Inhibitory activity of α-
amylase from S. mahagoni seeds extract at different
condition shown in Table 8. In this study, the lowest
TABLE 7. Comparison of extraction methods on % inhibitory percent inhibition was 2.12 ± 1.24 (p< 0.01) at 25 MPa
activity of α-glucosidase inhibitory from S. mahagoni seeds and 50°C and the highest was 34.89 ± 1.23 (p< 0.05) at
extract at concentration of 100 μg/ml. 30 MPa and 40°C. At high pressure and low
Swietenia mahagoni temperature, the density of solvent increase [13, 34].
seeds extraction % Inhibition Reference
method
This will also enhanced the solvency power and
increase the solute solubility [34] resulting in higher
Aquaeos maceration 4.376 ± 0.192 recovery of compound responsible to the activity of α-
Ethanol maceration 18.647 ± 3.86 [6] amylase.
Previous research on α-amylase inhibitory activity
Aquaeos reflux 5.309 ± 0.514
of S. mahagoni is shown in Table 9 with comparison
Ethanol reflux 14.313 ± 3.522 with the result in this study. It shows that moderate
SC-CO2 93.773 ± 1.306 This study percent inhibition was obtained compared with
Subhadip et al. [25]. However, moderate α–amylase
inhibition with strong α-glucosidase inhibition may
offer better therapeutic strategy that could slow the
availability of dietary carbohydrate substrate for glucose
production in the gut [44]. Furthermore,the effect of
pressure and temperature of SC-CO2 also affect the α-
amylase inhibitory activity.

Figure 6. The effect of pressure and temperature on IC50 of α-

glucosidase inhibitory activity

Next, at constant temperature of 40°C shows

decreasing IC50 of α-glucosidase inhibitory activity. As
mentioned, the stronger the α-glucosidase inhibitory
activity, the lowest IC50. At low temperature, the
degradation of compounds can be avoided. Meanwhile Figure 7. Correlation of IC50 of α-glucosidase (μg/ml) and β-
at constant temperature of 50°C, the IC50 value increases sitosterol concentration (% w/w)
 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317 1314

Figure 8 shows the effect of pressure and TABLE 8. α-Amylase inhibitory activity of S. mahagoni
temperature on percent inhibition of α-amylase seeds extract at different conditions (pressure, P and
inhibitory activity. At constant pressure of 20 MPa, the temperature, T). The values presented are expressed as mean
temperature increases as the α-amylase inhibitory ± standard error of the mean of triplicate experiments.
activity increases meanwhile at 25 and 30 MPa, the α- Condition (s) Inhibition (%)
amylase inhibitory activity increases as temperature Positive control 94.34 ± 0.62
increase from 40 to 50°C and decrease as its reached
P= 20MPa, T= 40°C 6.86 ± 1.30*
60°C. This is because as temperature further increase,
the recovery of compound responsible to the inhibitory P= 20MPa, T= 50°C 22.44 ± 2.82*
activity of α-amylase may decrease due to vaporization P= 20MPa, T= 60°C 34.21 ± 0.48*
or decomposition of the volatile compound during the P= 25MPa, T= 40°C 18.50 ± 0.67*
extraction.
Next, at constant temperature of 40°C shows the P= 25MPa, T= 50°C 2.12 ± 1.24*
increase of the inhibition activity as pressure increase P= 25MPa, T= 60°C 16.82 ± 1.23*
from 20 to 30 MPa. Meanwhile, at constant temperature P= 30MPa, T= 40°C 34.89 ± 1.23*
of 50 and 60°C, the α-amylase inhibitory activity
decrease as pressure decrease from 20 to 25 MPa but P= 30MPa, T= 50°C 5.69 ± 3.88*
increase slight at pressure 30 MPa. The correlation of P= 30MPa, T= 60°C 25.58 ± 3.11*
β-sitosterol content and α-amylase inhibitory activity
was studied and shown in Figure 9.
The β-sitosterol concentration in extracts showed a
TABLE 9. Comparison of extraction method on % inhibitory
positive correlation toward the activity of α-amylase at
activity of α-amylase inhibitory from S. mahagoni seeds
constant pressure of 20, 25 and 30 MPa. Meanwhile at extract at concentration of 100 μg/ml.
constant temperature of 40°C shows positive correlation
but at constant temperature of 50°C and 60°C shows Swietenia mahagoni seeds
% Inhibition Reference
extraction method
negative correlation. This result shows inconsistency of
β-sitosterol concentration in extracts correlate with the Petroleum ether maceration 64.84 ± 0.52 [25]
activity of α-amylase. Previous research showed that β- SC-CO2 34.89 ± 1.23 This study
sitosterol may not affect α-amylase inhibitory activity
[45].

4. CONCLUSION

Swietenia mahagoni seeds extract exhibit a strong α-

glucosidase activity with mild α-amylase activity. In
addition, the relationship of β-sitosterol content in
extract and the inhibitory activities of α-glucosidase and
α-amylase shows that β-sitosterol do not contribute to
the activities but may be the synergize of all compounds
in the extract.
Figure 8. The effect of pressure and temperature on α-amylase
inhibitory activity
5. ACKNOWLEDGEMENT

The authors would like to acknowledge the financial

support from the Ministry of Higher Education
Malaysia and Universiti Teknologi Malaysia under
Fundamental Research Grant Scheme
(R.J130000.7846.4F929), GUP grant
(Q.J130000.2546.16H29), and Higher Institution Centre
of Excellence (HiCOE) grant (R.J090301.7846.4J180).
The authors would also like to acknowledge the
technical and management support from Research
Figure 9. The effect of pressure and temperature on α-amylase Management Centre (RMC), Universiti Teknologi
inhibitory activity Malaysia.
1315 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317
6. REFERENCE
1. Adefegha, S.A., Oboh, G., Adefegha, O.M., Boligon, A.A. and
Athayde, M.L., "Antihyperglycemic, hypolipidemic,
hepatoprotective and antioxidative effects of dietary clove
(szyzgium aromaticum) bud powder in a high‐fat
diet/streptozotocin‐induced diabetes rat model", Journal of the
Science of Food and Agriculture, Vol. 94, No. 13, (2014),
2726-2737.
2. Kumar, S., Narwal, S., Kumar, V. and Prakash, O., "Α-
glucosidase inhibitors from plants: A natural approach to treat
diabetes", Pharmacognosy Reviews, Vol. 5, No. 9, (2011), 19.
3. Association, A.D., "Diagnosis and classification of diabetes
mellitus", Diabetes Care, Vol. 37, No. Supplement 1, (2014),
S81-S90.
4. Cheng, A.Y. and Fantus, I.G., "Oral antihyperglycemic therapy
for type 2 diabetes mellitus", Canadian Medical Association
Journal, Vol. 172, No. 2, (2005), 213-226.
5. Casirola, D.M. and Ferraris, R.P., "Α-glucosidase inhibitors
prevent diet-induced increases in intestinal sugar transport in
diabetic mice", Metabolism, Vol. 55, No. 6, (2006), 832-841.
6. WRESDIYATI, T., SA'DIAH, S., Winarto, A. and Febriyani,
V., "Alpha-glucosidase inhibition and hypoglycemic activities
of sweitenia mahagoni seed extract", HAYATI Journal of
Biosciences, Vol. 22, No. 2, (2015), 73-78.
7. Tas, S., Sarandol, E., Ziyanok, S., Aslan, K. and Dirican, M.,
"Effects of green tea on serum paraoxonase/arylesterase
activities in streptozotocin-induced diabetic rats", Nutrition
research, Vol. 25, No. 12, (2005), 1061-1074.
8. Triggle, C.R. and Ding, H., "Cardiovascular impact of drugs
used in the treatment of diabetes", Therapeutic advances in
chronic disease, Vol. 5, No. 6, (2014), 245-268.
9. Goh, B.H., Abdul Kadir, H., Abdul Malek, S. and Ng, S.W.,
"Swietenolide diacetate from the seeds of swietenia
macrophylla", Acta Crystallographica Section E: Structure
Reports Online, Vol. 66, No. 6, (2010), o1396-o1396.
10. Goh, B.H. and Kadir, H.A., "In vitro cytotoxic potential of
swietenia macrophylla king seeds against human carcinoma cell
lines", Journal of Medicinal Plants Research, Vol. 5, No. 8,
(2011), 1395-1404.
11. Maiti, A., Dewanjee, S., Kundu, M. and Mandal, S.C.,
"Evaluation of antidiabetic activity of the seeds of swietenia
macrophylla in diabetic rats", Pharmaceutical Biology, Vol.
47, No. 2, (2009), 132-136.
12. Balijepalli, M.K., Suppaiah, V., Chin, A.-m., Buru, A.S.,
Sagineedu, S.R. and Pichika, M.R., "Acute oral toxicity studies
of swietenia macrophylla seeds in sprague dawley rats",
Pharmacognosy Research, Vol. 7, No. 1, (2015), 38.
13. Liza, M., Rahman, R.A., Mandana, B., Jinap, S., Rahmat, A.,
Zaidul, I. and Hamid, A., "Supercritical carbon dioxide
extraction of bioactive flavonoid from strobilanthes crispus
(pecah kaca)", Food and Bioproducts Processing, Vol. 88, No.
2-3, (2010), 319-326.
14. Liza, M.S., Russly, A.R., Jinap, S., Azizah, H. and Md, Z.,
"Optimization of extraction condition for supercritical carbon
dioxide (scco 2) extraction of strobhilantes crispus (pecah kaca)
leaves by response surface methodology", Journal of Food
Processing and Technology, Vol. 4, No. 1, (2013).
15. Foster, N., Mammucari, R., Dehghani, F., Barrett, A.,
Bezanehtak, K., Coen, E., Combes, G., Meure, L., Ng, A. and
Regtop, H.L., "Processing pharmaceutical compounds using
dense gas technology", Industrial & Engineering Chemistry
Research, Vol. 42, No. 25, (2003), 6476-6493.
16. Schuler, I., Milon, A., Nakatani, Y., Ourisson, G., Albrecht, A.-
M., Benveniste, P. and Hartman, M.-A., "Differential effects of
plant sterols on water permeability and on acyl chain ordering of
soybean phosphatidylcholine bilayers", Proceedings of the
National Academy of Sciences, Vol. 88, No. 16, (1991), 6926-
6930.
17. Saeidnia, S., Manayi, A., Gohari, A.R. and Abdollahi, M., "The
story of beta-sitosterol-a review", European Journal of
Medicinal Plants, Vol. 4, No. 5, (2014), 590.
18. Loizou, S., Lekakis, I., Chrousos, G.P. and Moutsatsou, P.,
"Β‐sitosterol exhibits anti‐inflammatory activity in human aortic
endothelial cells", Molecular Nutrition & Food Research, Vol.
54, No. 4, (2010), 551-558.
19. Ju, Y.H., Clausen, L.M., Allred, K.F., Almada, A.L. and
Helferich, W.G., "Β-sitosterol, β-sitosterol glucoside, and a
mixture of β-sitosterol and β-sitosterol glucoside modulate the
growth of estrogen-responsive breast cancer cells in vitro and in
ovariectomized athymic mice", The Journal of Nutrition, Vol.
134, No. 5, (2004), 1145-1151.
20. Miettinen, T.A. and Gylling, H., "Ineffective decrease of serum
cholesterol by simvastatin in a subgroup of
hypercholesterolemic coronary patients", Atherosclerosis, Vol.
164, No. 1, (2002), 147-152.
21. Vivancos, M. and Moreno, J.J., "Β-sitosterol modulates
antioxidant enzyme response in raw 264.7 macrophages", Free
Radical Biology and Medicine, Vol. 39, No. 1, (2005), 91-97.
22. Gupta, R., Sharma, A.K., Dobhal, M., Sharma, M. and Gupta,
R., "Antidiabetic and antioxidant potential of β‐sitosterol in
streptozotocin‐induced experimental hyperglycemia", Journal
of Diabetes, Vol. 3, No. 1, (2011), 29-37.
23. Maiti, A., Dewanjee, S. and Sahu, R., "Isolation of
hypoglycemic phytoconstituent from swietenia macrophylla
seeds", Phytotherapy Research, Vol. 23, No. 12, (2009), 1731-
1733.
24. Hashim, M.A., Yam, M.F., Hor, S.Y., Lim, C.P.P., Asmawi,
M.Z. and Sadikun, A., "Anti-hyperglycaemic activity of
swietenia macrophylla king (meliaceae) seed extracts in
normoglycaemic rats undergoing glucose tolerance tests",
Chinese Medicine, Vol. 8, No. 1, (2013), 11.
25. Hajra, S., Mehta, A., Pandey, P., John, J., Mehta, P. and Vyas,
S., "Free radical scavenging and α-amylase inhibitory activity of
swietenia mahagoni seeds oil", International Journal of
Pharmacognosy and Phytochemical Research, Vol. 5, No. 1,
(2013), 51-56.
26. Karan, S.K., Mishra, S.K., Pal, D. and Mondal, A., "Isolation of-
sitosterol and evaluation of antidiabetic activity of aristolochia
indica in alloxan-induced diabetic mice with a reference to in-
vitro antioxidant activity", Journal of Medicinal Plants
Research, Vol. 6, No. 7, (2012), 1219-1223.
27. Sánchez‐Machado, D., López‐Hernández, J., Paseiro‐Losada, P.
and López‐Cervantes, J., "An hplc method for the quantification
of sterols in edible seaweeds", Biomedical Chromatography,
Vol. 18, No. 3, (2004), 183-190.
28. Jemain, M.M., Musa'adah, M.N., Rohaya, A., Rashid, L.A. and
Hadiani, I.N., "In vitro antihyperglycaemic effects of some
malaysian plants", Journal of Tropical Forest Science, Vol.
23, No. 4, (2011), 467-472.
29. Bothon, F.T., Debiton, E., Avlessi, F., Forestier, C., Teulade, J.-
C. and Sohounhloue, D.K., "In vitro biological effects of two
anti-diabetic medicinal plants used in benin as folk medicine",
BMC Complementary and Alternative Medicine, Vol. 13, No.
1, (2013), 51-58.
30. Rivera-Chávez, J., González-Andrade, M., Del Carmen
González, M., Glenn, A. E., and Mata, R., , "A-glucosidase
inhibitors from mexu 27095, an endophytic fungus from
hintonia latiflora", Phytochemistry, Vol. 94, (2013), 198-205.
 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317
31. Miller, G.L., "Use of dinitrosalicylic acid reagent for
determination of reducing sugar", Analytical Chemistry, Vol.
31, No. 3, (1959), 426-428.
32. Ali, H., Houghton, P. and Soumyanath, A., "Α-amylase
inhibitory activity of some malaysian plants used to treat
diabetes; with particular reference to phyllanthus amarus",
Journal of Ethnopharmacology, Vol. 107, No. 3, (2006), 449-
455.
33. Machmudah, S., Kawahito, Y., Sasaki, M. and Goto, M.,
"Supercritical co2 extraction of rosehip seed oil: Fatty acids
composition and process optimization", The Journal of
supercritical fluids, Vol. 41, No. 3, (2007), 421-428.
34. Pereira, C.G. and Meireles, M.A.A., "Supercritical fluid
extraction of bioactive compounds: Fundamentals, applications
and economic perspectives", Food and Bioprocess Technology,
Vol. 3, No. 3, (2010), 340-372.
35. Hartati Salleh, L., Yunus, A. and Aziz, A., "Optimization of
supercritical co2 extraction of swietenia mahagoni seed by
response surface methodology", Journal of Teknology and
Sciences Engineering, Vol. 67, No. 1, (2014), 15-20.
36. Sajfrtová, M., Ličková, I., Wimmerová, M., Sovová, H. and
Wimmer, Z., "Β-sitosterol: Supercritical carbon dioxide
extraction from sea buckthorn (hippophae rhamnoides l.) seeds",
International Journal of Molecular Sciences, Vol. 11, No. 4,
(2010), 1842-1850.
37. Vilegas, J.H., de Marchi, E. and Lancas, F.M., "Extraction of
low‐polarity compounds (with emphasis on coumarin and
kaurenoic acid) from mikania glomerata (‘guaco’) leaves",
Phytochemical Analysis: An International Journal of Plant
Chemical and Biochemical Techniques, Vol. 8, No. 5, (1997),
266-270.
38. Kawahito, Y., Kondo, M., Machmudah, S., Sibano, K., Sasaki,
M. and Goto, M., "Supercritical co2 extraction of biological
active compounds from loquat seed", Separation and
Purification Technology, Vol. 61, No. 2, (2008), 130-135.
1316
39. Catchpole, O., Perry, N., Da Silva, B., Grey, J. and Smallfield,
B., "Supercritical extraction of herbs i: Saw palmetto, st john's
wort, kava root, and echinacea", The Journal of Supercritical
Fluids, Vol. 22, No. 2, (2002), 129-138.
40. Simandi, B., Kristo, S.T., Kery, A., Selmeczi, L., Kmecz, I. and
Kemeny, S., "Supercritical fluid extraction of dandelion leaves",
The Journal of Supercritical Fluids, Vol. 23, No. 2, (2002),
135-142.
41. András, C.D., Simándi, B., Örsi, F., Lambrou, C.,
Missopolinou‐Tatala, D., Panayiotou, C., Domokos, J. and
Doleschall, F., "Supercritical carbon dioxide extraction of okra
(hibiscus esculentus l) seeds", Journal of the Science of Food
and Agriculture, Vol. 85, No. 8, (2005), 1415-1419.
42. Cossuta, D., Simándi, B., Vági, E., Hohmann, J., Prechl, A.,
Lemberkovics, É., Kéry, Á. and Keve, T., "Supercritical fluid
extraction of vitex agnus castus fruit", The Journal of
Supercritical Fluids, Vol. 47, No. 2, (2008), 188-194.
43. Díaz-Reinoso, B., Moure, A., Domínguez, H. and Parajó, J.C.,
"Supercritical co2 extraction and purification of compounds
with antioxidant activity", Journal of Agricultural and Food
Chemistry, Vol. 54, No. 7, (2006), 2441-2469.
44. Kajaria, D., Ranjana, J.T., Tripathi, Y.B. and Tiwari, S., "In-
vitro α amylase and glycosidase inhibitory effect of ethanolic
extract of antiasthmatic drug—shirishadi", Journal of Advanced
Pharmaceutical Technology & Research, Vol. 4, No. 4,
(2013), 206.
45. Safamansouri, H., Nikan, M., Amin, G., Sarkhail, P., Gohari,
A.R., Kurepaz-Mahmoodabadi, M. and Saeidnia, S., "Α-amylase
inhibitory activity of some traditionally used medicinal species
of labiatae", Journal of Diabetes & Metabolic Disorders, Vol.
13, No. 1, (2014), 114.
1317 N. S. Md Norodin et al. / IJE TRANSACTIONS B: Applications Vol. 31, No. 8, (August 2018) 1308-1317

Inhibitory Effects of Swietenia Mahagoni Seeds Extract on Α-Glucosidase and Α-

Amylase
N. S. Md Norodina,b, L. Md Salleha,b, N. Yusofc, N. M. Mustaphad, F. Kamarulzamand, M. A. Mohamed Zaharia,b, N. A.
Bakeria,b
a Center of Lipids Engineering and Applied Research (CLEAR), Ibnu Sina Institute Scientific & Industrial Research (Ibnu Sina ISIR), Universiti
Teknologi Malaysia, Johor, Malaysia
b Department of Bioprocess and Polymer Engineering, School of Chemical and Energy Engineering, UniversitiTeknologi Malaysia, Johor, Malaysia
c Advanced Membrane Technology Research Centre (AMTEC), Universiti Teknologi Malaysia, Skudai, Johor, Malaysia
d Natural Product Division, Forest Research Institute Malaysia (FRIM), Selangor Darul Ehsan, Malaysia

PAPER INFO ‫چكيده‬

Paper history:
Received 16 December 2017
Received in revised form 25 January 2018
Accepted 08 February 2018
Keywords:
Swietenia Mahagoni Seed,
Α-Glucosidase Enzyme,
Α-Amylase Enzyme,
Supercritical Carbon Dioxide (SC-CO2)
Extraction
‫مطالعه حاضر به بررسی فعالیت مهارکننده استخراج عصاره گیاه ماهون در آزمایش مهار آنزیم 𝜶 گلوکز و 𝜶 آمیالز‬
‫ درجه‬40‫ـ‬60 ‫ مگاپاسکال و دماهای‬20‫ـ‬30 ‫ عصاره گیاه ماهون با استفاده از دی اکسید کربن فوق بحرانی با فشار‬.‫میپردازد‬
‫ تمامی داده ها در‬.‫ بازدهی روغن حاصل از آنالیز با آنزیم 𝜶 گلوکز و 𝜶 آمیالز آنالیز می شود‬.‫سانتیگراد استخراج میشوند‬
‫ برای معنی‬7 ‫ نسخه‬Statistica ‫ از نرم افزار‬.‫ انحراف معیار برای آزمایشهای سه گانه تفسیر شده اند‬± ‫قالب میانگین‬
GraphPad ‫ درصد آنزیم مهارکننده با استفاده از نرم افزار‬50 ‫دارشدن آماری آنالیز یکطرفه استفاده شد و غظلت عصاره‬
‫) شدید و در‬98.4% ± 0.2( ‫ قدرت مهارکنندگی عصاره گیاه ماهون در فعالیت آنزیم 𝜶 گلوکز‬.‫ تعیین شد‬6.0 Prism
‫ این یافته ها نشان ميدهد که عصاره گياه ماهون ميتواند ماده‬.‫) است‬34.9% ± 1.2( ‫فعالیت آنزیم 𝜶 آمیالز متوسط‬
.‫ضد دیابت طبيعی موثر باشد‬
doi: 10.5829/ije.2018.31.08b.20
Journal of Physics: Conference Series

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This content was downloaded from IP address 161.139.126.248 on 29/12/2020 at 00:32

2nd International Conference on Statistics, Mathematics, Teaching, and Research IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1028 (2018)
1234567890 ‘’“” 012011 doi:10.1088/1742-6596/1028/1/012011

Response Surfaces of Linoleic Acid of Swietenia Mahagoni in

Supercritical Carbon Dioxide

Hartati1*, Liza Md Salleh2, Halifah Pagarra1, Rachmawaty1

1
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas
Negeri Makassar, Makassar 90222, Indonesia
2
Department of Bioprocess Engineering, Faculty of Chemical Engineering,
UniversitiTeknologi Malaysia, 81310 UTM Johor Bahru, Malaysia

*
hartati@unm.ac.id

Abstract. The process variables pressure, temperature and particle size were studied for
optimization of linoleic acid by response surface methodology following a Box-Behnken
design of experiments. The results indicated the effect of extraction condition on linoleic acid
of the extracts produced SC-CO2 gave the different effect. But, Analysis of the variance of the
data indicated that there was no statistically significant difference between the other sample.
Although there was greater variation within the sample, there was still no statistically
significant effect of temperature and pressure on the extraction. The optimum conditions for
linoleic acid yield from Swieteniamahagoni seed within the experimental range were found to
be pressure of 29.02 MPa, a temperature of 67.88ºC and particle size 0.75 mm, and the
predicted linoleic acid was found to be 34.91%.

1. Introduction
Swietenia mahagoni seeds have been applied as folk medicine for the treatment of hypertension,
malaria, and diabetes [1]. There have also been reports of S. mahagoni seeds having anti-
Inflammatory, antimutagenecity, antitumor [2], antoxidant and antimicrobial activities [3]. The
therapeutic effects associated with the seeds are mainly caused by the biologically active ingredients;
fatty acids and tetranortriterpenoids [4].
Supercritical carbon dioxide extraction (SC-CO2) is an alternative technique to conventional
extraction of lipids with organics solvents. Moreover, carbon dioxide as a solvent possesses many
advantages (nontoxic, nonflammable, inexpensive and yields high purity oil) which can be
successfully explored in food and pharmaceutical application [5,6,7]. SC-CO2 was successfully used in
the extraction of edible oils from a wide range of seeds, including hiprose [8], cuphea [9], flax [10],
amaranth [11], sunflower and rape [12], Swietenia mahagoni [13]. Previous studies on SC-CO2 of S.
mahagoni seeds were mainly focused on the determination of total oil contents in ground seeds [13].
Response surface methodology (RSM) is a statistical technique, which is used to evaluate the effect
of multiple factors and their interaction on one or more response variables. Recently, RSM has been
successfully applied to optimize SC-CO2 extraction of oils from Swietenia mahagoni seed [13], Salvia
mirzayanii [14], Passiflora seed [15], Silkworm pupae [16], wheat germ [17], cotton seed [18],
Curcuma longa [19], rosehip seed [20], Cyperus rotundus [21], and amaranth seed [22]. In the present

Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd 1
2nd International Conference on Statistics, Mathematics, Teaching, and Research IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1028 (2018)
1234567890 ‘’“” 012011 doi:10.1088/1742-6596/1028/1/012011

study, SC-CO2 was used to extract the oil from S. mahagoni seed. The aim was to investigated the
rbon dioxide parameter on the linoleic acid on S. mahagoni seeds extract.
influence of supercritical carbon

2. Experimental Details
A schematic flow diagram of the extraction apparatus is shown in Figure 1. S. mahagoni seed oil was
extracted with supercritical carbon dioxide. The ground samplee of 5 g was placed in an extractor
vessel. The extracts were collected in a glass vial placed in the separator at ambient temperature and
pressure. A flow rate of CO2 was 2 mL/min. The investigated values of pressure, temperature, and
particle size were varied from 20 to 30 MPa. 40 to 60ºC, and 0.25 to 0.75 mm, respectively. After each
extraction, the obtained extract was placed into glass vials, sealed and store at 4º
4ºC to prevent any
possible degradation.

Figure 1. A schematic design of the supercritical fluid extraction (SFE) unit

unit.

The experimental design chosen for this study was that of Box and Behnken (BBD). BBD was
applied to determine optimum extraction pressure,
pressure, temperature and particle size for supercritical CO2
extraction of S mahagoni seed. The pressure (A), temperature (B), and particle size (C) were
independent variables studied to optimize the linoleic acid (Y) from S. mahagoni seed. The CO2 flow
rate was constant.
Box-Behnken
Behnken design requires an experiment number (N) according to the following equation.
N=2k(k-1)+cp
Where k is the factor number and cp is the replicate number of the central point. Three levels (low,
medium, and high denoted as -1,1, 0, and +1,
+1, respectively of variables chosen for the experiments are
given in Table 1. Analysis was performed using commercial software Design-Expert®
Design Expert® v.6.0.4
v.6.0.4.
Table 1. Levels of variables chosen.
Variables Levels
High Middle Low (-)
(+) (0)
A; Pressure (MPa) 30 25 20
B; Temperature (ºC) 60 50 40
C; Particle size (mm) 0.75 0.50 0.25

The analysis of variance (ANOVA) was also used to evaluate the quality of the fitted model. The
test of statistical difference was based on the total error criteria with a confidence level 95%.
Determination of active constituents from extracted compounds were examined using Gas
Chromatography-Mass Mass Spectrometry (GC-MS)
(G as described by Kandhro, et al al. [23]. with slight
modification. In order to evaluate the quality of extracted compounds, all the sample were analyzed by
using gas chromatography-mass mass spectrometry. The GC-MS
GC MS analysis for fatty acid methyl ester
(FAMEs) was performed on Agilent 1909Is-433. 1909Is 433. A capillary column HP HP-5MS (5% phenyl

2
2nd International Conference on Statistics, Mathematics, Teaching, and Research IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1028 (2018)
1234567890 ‘’“” 012011 doi:10.1088/1742-6596/1028/1/012011

methylsiloxane) with dimension of 30 m x 0.25 mm i.d x 0.25 µm film thickness (Agilent

Technologies, Palo Alto, CA, USA) were used for separation of fatty acid methyl esters. The initial
temperature of 150ºC was maintained for 2 min raised to 230ºC at the rate of 4ºC/min, and kept at
230ºC for 5 min. The split ratio was 1:50, and helium were used carrier gas with the flow rate of 0.8
ml/min. The injector and detector temperature are 240 and 260ºC, respectively. The mass spectrometer
was operated in the electron impact mode at 70 eV in the scan range of 50-550 m/z.

3. Results and Discussion

Since various parameters potentially affect the extraction process, the optimization of the experimental
conditions represents a critical step in the application of the SFE method. The experimental design was
adopted on the basis of coded level from three variables (Table 1), resulting in seventeen simplified
experimental sets (Table 2) with five replicates for the central point. The selected factors were
extraction pressure (in MPa), temperature (in ºC) and particle size (in mm) with the consideration that
these factors are important in the extraction process.
Table 2. Experimental matrix and values of the observed response.
Run A B C Coded Coded Coded Observed Predicted
A B C Linoleic acid of linoleic
variable variable variable (peak area %) acid
1 20 40 0.50 -1 -1 0 25.61 27.15
2 30 40 0.50 +1 -1 0 19.92 21.40
3 20 60 0.50 -1 +1 0 23.74 22.26
4 30 60 0.50 +1 +1 0 33.58 32.04
5 20 50 0.25 -1 0 -1 27.30 26.00
6 30 50 0.25 +1 0 -1 31.53 30.29
7 20 50 0.75 -1 0 +1 31.10 32.35
8 30 50 0.75 +1 0 +1 30.78 32.09
9 25 40 0.25 0 -1 -1 26.71 26.47
10 25 60 0.25 0 +1 -1 24.03 26.82
11 25 40 0.75 0 -1 +1 30.81 28.02
12 25 60 0.75 0 +1 +1 33.18 33.42
13 25 50 0.50 0 0 0 30.80 33.58
14 25 50 0.50 0 0 0 26.88 33.58
15 25 50 0.50 0 0 0 37.58 33.58
16 25 50 0.50 0 0 0 37.15 33.58
17 25 50 0.50 0 0 0 35.47 33.58

The second order polynomial model used to express the total extraction linoleic acid (LA) of S.
mahagoni as a function of independent variables (in terms of coded values) is shown below:
LA=33.58+1.01*A+1.44*B+2.04*C-3.18*A2-4.68*B2-0.21*C2+3.88*A*B-
1.14*A*C+1.26*B*C
Assessment of extracts and linoleic acid from S. mahagoni seed at extreme carbon dioxide
extraction was carried out at pressures (20, 25 and 30 MPa), temperature (40, 50 and 60ºC) and
particle size (0.25, 0.50 and 0.75 mm).The study proved that the optimum yield of S. mahagoni seed
was linoleic acid content was 34.91% at 29.02 MPa pressure, temperature 67.88ºC and particle size
0.75 mm with the equivalent value of this condition was 0.92 (desirability). The accuracy of the
predicted value can be seen from the desirability value. Determination of desirability values serves to
state the degree of optimum result of precision, which is closer to value 1, the higher the optimization
value of precision [24]. The validation of the accuracy of this extraction condition was obtained from
the results of the experimental extract of 20.07% and linoleic acid yield of 34.26%, this result shows
agreement with the value predicted by Design Expert software

3
2nd International Conference on Statistics, Mathematics, Teaching, and Research IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1028 (2018)
1234567890 ‘’“” 012011 doi:10.1088/1742-6596/1028/1/012011

The effect of extraction pressure and temperature on linoleic acid yield at constant particle size, the
effect of extraction pressure and particle size on linoleic
linoleic acid yield at constant temperature and also the
effect of extraction temperature and particle size on linoleic acid yield at constant pressure were
illustrated in Figure 2, Figure 3 and Figure 4 respectively. As shown in Figure 2, linoleic acid
increasedd with the increased in pressure from 20 MPa to 25 MPa and temperature 40ºC to 50ºC at
constant particle size (0.5 mm). However, further increase in pressure from 25 MPa to 30 MPa and
temperature from 50ºC to 60ºC resulted in decreasing of linoleiclinol acid. Meanwhile,
eanwhile, in Figure 33, it
shows that at constant temperature, linoleic acid increased with icreased pressure from 20 MPa to 30
MPa and particle size 0.25 mm to 0.75 mm. Furthermore, in Figure 4 showed linoleic acid increased
with the increased in temperature re from 40ºC to 50ºC and started to decrease when further increased
from 50ºC to 60ºC at constant pressure (25 MPa). The study shows that the effect of SC SC-CO2
parameters on the linoleic acid did not have the same traits as the effect of the SC SC-CO2 parameter on
the extracted oil.

Figure 2. Surface plot of linoleic Figure 3. Surface plot of linoleic

acid from S mahagoni as a function acid from S mahagoni as a function
of pressure and temperature at of pressure and particle size at
constant particle size of 0.50 mm.
mm constant temperature of 50ºC

Figure 4. Surface plot of linoleic acid yield from S mahagoni as a function of temperature and particle
size at constant pressure of 25 MPa

Effect of extraction condition

tion on linoleic acid showed that linoleic acid of the extracts produced
SC-CO2 gave the different effect. But, Analysis of a variance of the data indicated that there was no
statistically significant (р>0.05) difference between the other sample. Although there was greater
variation within the sample, there was still no statistically significant effect of temperature and
pressure on the extraction. The reason for the variation within the extract produced by SC SC-CO2
extraction was due to the changes in the solubility
solubility of the linoleic acid with the changing extraction
conditions.

4
2nd International Conference on Statistics, Mathematics, Teaching, and Research IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1028 (2018)
1234567890 ‘’“” 012011 doi:10.1088/1742-6596/1028/1/012011

The solubility of the oil in SC-CO

SC 2 is mainly determined by the SC-CO2 density and the volatility
of the oil components. In general, SC-CO
SC 2 density increases with pressure at constant ttemperature and
decreases with temperature at constant pressure, where the density decrease becomes smaller at higher
pressures. On the other hand, the volatility of oil components increases with temperature. These two
opposing effects of temperature on density
den and volatility lead to the well-established
established crossover
behavior of solubility isotherms. A temperature increase may also cause breakdown of cell structure
and increase the diffusion rate of the oil in the particles, therefore accelerating the extractio
extraction process
[25].
The fatty acid of S. mahagoni seed extracted SC-CO2 were tested for GC-MS MS analysis (Table 2).
There is one significant peak in the GC spectrums of the samples (Figure 55). Fatty acids have
important roles in immune and inflammatory responses [26]. Monounsaturated and polyunsaturated
fatty acids are present in plasma membranes, which are capable of stimulating cellular proliferation
and angiogenesis, thus exert an important role in the process healing. The topical administration of
linolenic (n-3) and linoleic (n-6)
6) acids essential and oleic (n-9)
(n 9) acid nonessential fatty acids modulate
the closure of surgically induced skin wounds [27]. The use of n-66 fatty acid may increase pro pro-
inflammatory cytokines production in wound sites, stimulating the the cutaneous wound healing process
[28].

Figure 5. GC spectrums of fatty acid from S mahagoni seed oils

oils.

4. Conclusion
The extraction of linoleic acid in supercritical carbon dioxide was measured as a function of pressure,
temperature and particle size. Effect
Effect of extraction condition on linoleic acid of the extracts produced
SC-CO2 gave different effect. But, Analysis of variance of the data indicated that there was no
statistically significant difference between the other sample. Although there was greater variation
within the sample, there was still no statistically significant effect of temperature and pressure on the
extraction. The optimum conditions for linoleic acid yield from S mahagoni seed within the
experimental range were found to be pressure of 29.02 29.02 MPa, a temperature of 67.88ºC and particle
size 0.75 mm, and the predicted linoleic acid was found to be 34.91%. Under these optimum
conditions, the experimental values were in agreement with the predicted values.
values

Acknowledgments
We gratefully acknowledge
ledge the financial support by Ministry of Research and Higher Education
Indonesia, and acknowledgement is also extended to Universitas Negeri Makassar and Universiti
Teknologi Malaysia for the use of laboratory instruments.

5
2nd International Conference on Statistics, Mathematics, Teaching, and Research IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 1028 (2018)
1234567890 ‘’“” 012011 doi:10.1088/1742-6596/1028/1/012011

References
[1] Chen Y Y, Wang X N, Fan C Q, Yin S and Yue J M 2007 Tetrahedron Letters 48 7480-7484.
[2] Guevara A P, Apilado A, Sakurai H, Kozuka M and Tokuda H 1996 Philippine Journal of
Science 125 271-278
[3] Hartati, Liza M S, Ahmad R S, Azizi C Y and Azila A A 2014 Jurnal Teknologi Science and
Engineering 67:4 59-62
[4] Bascal K, Chavez L, Diaz I, Espira S, Javillo J, Manzarilla H, Motalban J, Panganiban C,.
Rodriquez A, Sumpaico C, Talip B and Yap S 2005 ActaMed. Philipp 3 127-1391997
[5] Brunner G 2005 Journal of food engineering 67 21-33
[6] Sahena F, Zaidul L M, Jinap S, Karim A A, Abbas K A, Norulaini N A N and Omar A K M
2009 Journal of Food Engineering 95 240-243
[7] Reverchon E and De Marco I 2006 The Journal of Supercritical Fluids 38 146-166
[8] Reverchon E, Kaziunas A and Marrone C 2000 Chemical Engineering Science 55 2195-2201
[9] Eller S C F J, Cermark and Taylor S I 2011 Industrial Crops and products 33 554-557.
[10] Ozkal S G 2009 Journal of the American Oil Chemists Society 86 1129-1135
[11] Westerman D, Santos R C D, Bosley J A, Roger J S and .Al-Duri B 2006 The Journal of
Supercritical Fluids 37 38-52
[12] Boutin O and Badens E 2009 Journal of Food Engineering 92 396-402
[13] Hartati, Liza M S, Yunus M A and Azila A A 2014 JurnalTeknologi (Science and Engineering
67 1. 15-20
[14] Yamini Y, Khajeh M, Ghasemi E, Mirza M and Javidnia K 2008 Food Chemistry 108 341-346
[15] Liu S,Yang F, Zhang C, Ji H, Hong P and Deng C 2009 The Journal of Supercritical Fluids 48
9-14
[16] Wei Z J, Liao A M, Zhang H X, Liu J and Jiang S T 2009 Bioresource Technology 100 4214-
4219
[17] Shao P, Sun P and Ying Y 2008 Food and Bioproducts Processing 86 227-231
[18] Bhattacharjee P, Singhal R S and Tiwari S.R 2007 Journal of Food Engineering. 79: 892-898
[19] Chang L, Jong T, Huang H, Nien Y, Chang C J 2006 Separation and Purification Technolog 47
119-125
[20] Machmudah S, Kawahito Y, Sasaki M and Goto M 2007 J. Supercrit. Fluid 41 421-428
[21] Wang H, Liu Y, Wei S and Yan Z 2012 Food Chemistry 132 582-587
[22] Kraujalis P and Venskutonis P R 2013 The Journal of Supercritical Fluids 73 80-86
[23] Kandhro A, Sherazi S T H, Mahesar S.A, Bhanger M I, Talpur M Y and Rauf A 2008 Food
Chemistry 109 207-211
[24] Montgomery D C 2005 Design and Analysis of Experiments, 6th ed., (John Wiley & Son, Inc ).
[25] Martinez J L 2008 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds.
(CRC Press Taylor & Francis Group LLC)
[26] McClusker M M, Jane M M D and Grat-Kels M D 2010 Clinics Dermatology 28 440-451.
[27] Cardoso C R, Souza M A, Ferro E A., Favoreto J R and Pera J D S 2004 Wound Repair Regen
12 235-243
[28] Pereira L M, Hatanaka E and Martins E F 2008 Cell BiochemFunct 26 197-204

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 Md Norodin et al. / Malaysian Journal of Fundamental and Applied Sciences Vol. 14, No. 3 (2018) 411-417

RESEARCH ARTICLE

Extraction of β-sitosterol from Swietenia mahagoni seeds by using

supercritical carbon dioxide (SC-CO2) extraction
Nur Salsabila Md Norodin a, b, Liza Md Salleh a, b, *, Siti Machmudah c, Nik Musaadah Mustafa d,
Hartati e, Ramdan Ismail a, b
a
Center of Lipids Engineering and Applied Research (CLEAR), Ibnu Sina Institute Scientific & Industrial Research (Ibnu Sina ISIR), Universiti
Teknologi Malaysia, 81300 Johor Bahru, Johor, Malaysia
b
Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, UniversitiTeknologi Malaysia, 81310 UTM
Johor Bahru, Johor, Malaysia
c
Chemical Engineering Department, Sepuluh Nopember Institute of Technology, 60111 Surabaya, Indonesia
d
Natural Product Division, Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor Darul Ehsan, Malaysia
e
Department of Biology, Universitas Negeri Makassar, South Sulawesi, Indonesia

* Corresponding author: i.liza@cheme.utm.my

Article history Abstract

Submitted 6 March 2018
Revised 19 March 2018
Accepted 1 April 2018
Published Online 3 September 2018
Graphical abstract
This work investigates the effect of supercritical carbon dioxide (SC-CO2) extraction conditions (pressure
and temperature) on the oil yield and β-sitosterol content extracted from Swietenia mahagoni seeds by
using response surface methodology (RSM). The experimental data obtained were fitted to a second-
order polynomial model and the obtained oil yields were 1.49-14.45%, while β-sitosterol content obtained
were 3.12-9.20 mg/g. The best conditions within the ranges studied were 30 MPa and 40°C to extract β-
sitosterol in the highest amount. The present findings show that S. mahagoni seeds extract has a high
concentration of β-sitosterol.

Keywords: Swietenia mahagoni seeds, β-sitosterol, supercritical CO2 extraction, response surface
methodology

© 2018 Penerbit UTM Press. All rights reserved

INTRODUCTION dioxide as a solvent. In this case, thermolabile and non-polar

Swietenia mahagoni is also known as ‘tunjuk langit’ in Malaysia
(Fig. 1) is used traditionally to treat various diseases such as diabetes
and high blood pressure (Goh et al., 2010). Swietenia mahagoni tree is
30 meters or taller (Eid et al., 2013) and the wood, usually being used
for making furniture (Falah et al., 2008). Meanwhile, the bark can be
used for natural colorant (Haque et al., 2013). The fruit of Swietenia
mahagoni is woody and consisting of capsules containing winged seeds
(Blundell et al., 2003). Whereas, the seed of Swietenia mahagoni can
be obtained by removing the wing. In Malaysia, the raw seeds were
used for treating hypertension and diabetes (Balijepalli et al., 2014). In
addition, Swietenia mahagoni seeds have been reported to have various
biological activities such as anti-inflammatory activity, anticancer and
antitumor activity (Goh et al., 2011) and also antidiabetic activity
(Maiti et al., 2009). Moreover, the seeds contain a number of bioactive
compounds as has been noted by Hashim et al., (2013) and presented
in Table 1.
To date, no study was found on the quantification of β-sitosterol
from Swietenia mahagoni seeds using high performance liquid
chromatography (HPLC). Recently, attention on the importance of
natural compounds from plants and herbs has been reassessing. As a
matter of fact, bioactive compounds from plant sources are chemically
sensitive and present in low concentration, hence supercritical carbon
dioxide (SC-CO2) extraction is an appropriate extraction method to use.
SC-CO2 is a separation process of matters by using supercritical carbon
compounds can be extracted by using SC-CO2 extraction due to the low
operating temperature of 30°C without any degradation. It cannot be
used to extract polar compounds since SC-CO2 extraction is more
appropriate to extract non-polar nature compounds (Vilegas et al.,
1997). Previously, β-sitosterol has been extracted from various plants
using SC-CO2 since β-sitosterol is a non-polar compound. Therefore,
no co-solvent is needed in the extraction of β-sitosterol by using SC-
CO2 extraction.
(a) (b) (c) (d)
Fig. 1 Swietenia mahagoni also known as ‘tunjuk langit’ in Malaysia (a)
tree, (b) fruit, (c) winged seeds and (d) seeds.
Moreover, carbon dioxide (CO2) is the most frequently solvent
used because it is environmental friendly (fairly non-toxic), low cost
and can be easily removed from the extract (Liza et al., 2010). The
elimination of CO2 is easily achieved since CO2 is in a gas state at room

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temperature. In addition, CO2 in the supercritical state is in a moderate

critical temperature (31.3°C) and pressure (7.38 MPa). Supercritical
state is when gas and liquid are indistinguishable where at this state it
is compressible but possessing a density of a liquid. In a word,
supercritical CO2 makes a good solvent because of the gas-like state
that attributed the low viscosity and high diffusion coefficient and the
liquid-like state that gave the solvating power (Aionicesei et al., 2008).

Table 1 Primary compounds found in S. mahagoni seeds determined by

gas chromatography-mass spectrometry (Hashim et al., (2013).

Molecular
Compounds
formula
Hexadecanoic acid, methyl ester C17H34O2
n-Hexadecanoic acid C16H32O2
9-Octadecenoic acid (Z)- methyl ester C19H36O2 Fig. 2 Schematic diagram of CLEAR supercritical fluid extraction (SFE)
9,12-Octadecadienoic acid (Z,Z)-Linoleic acid C18H32O2 machine
Gamma-tocopherol C28H48O2
Fucosterol C29H48O The parameters and constant parameters used in extraction process
β- sitosterol C29H50O are presented in Table 2. Five gram of sample was placed in 10 ml
stainless steel extraction vessel and sealed tightly in the oven. All the
Furthermore, the extraction of β-sitosterol from various plants parameters (temperature, pressure and flowrate of CO2) was fixed, and
using SC-CO2 extraction have been reported in the extraction of saw the extraction process was started after all the parameters were attained.
palmetto berries (Catchpole et al., 2002), Vitex agnus castus fruit The extract was collected by depressurizing the system. The oil yields
(Cossuta et al., 2008) and sea buckthorn seeds (Sajfrtová et al., 2010). were collected after 120 minute extraction time.
Sajfrtová et al., (2010) has reported that low temperature in the
extraction of β-sitosterol as low as 50°C didn’t cause the degradation Table 2 The process parameters for SC-CO2 extraction.
of β-sitosterol since the degradation was occurred at temperature Parameter Range/value
exceeding the temperature mentioned. Also, the yield of β-sitosterol
Temperature (°C) 40-60
increased slightly as pressure increased and the highest yield found Pressure (MPa) 20-30
from Vitex agnus castus fruit was 1.1 mg/g at a pressure of 45 MPa and Flowrate of CO2 (ml/min) 2.00
a temperature of 40°C (Cossuta et al., 2008). In this context, the Particle size (mm) 0.50
extraction of β-sitosterol can be manipulated or controlled by pressure Mass of sample (g) 5.00
and temperature. Pressure and temperature are the most relevant Extraction time (min) 120
parameters in supercritical carbon dioxide (SC-CO2) extraction. In
The oil yield was calculated as percentage of oil yield using Eq.
general, quantitative recovery of analytes influence by the increase in
(1) as follow:
pressure lead to the increase in solvent power. Solvent power is
described as the solvent density in any given conditions. Significantly, Oil Yield (%) = (M0/ M1) × 100 (1)
high pressure and moderate temperature favor the extraction of β-
sitosterol from plants using SC-CO2. where M0 is the mass of oil extract in gram and M1 is the mass of sample
Therefore, the aim of this work is to determine the effect of pressure in gram.
and temperature of supercritical carbon dioxide (SC-CO2) extraction on
the oil yield and β-sitosterol content from Swietenia mahagoni seeds by Design of experimental for response surface methodology
using response surface methodology (RSM). (RSM)
Response surface methodology (RSM) is a technique used to
EXPERIMENTAL describe the behavior of a set of data. The main purpose is to optimize
the variables so that the best system performance could be obtained.
Three-level factorial design was employed to optimize the oil yield and
Materials
β-sitosterol content from Swietenia mahagoni seed. The number of
Swietenia mahagoni seeds were bought in the local market of Johor,
experiments is calculated by expression of Eq. (2) (Bezerra et al., 2008)
Malaysia. Commercial grade liquid carbon dioxide (purity 99.99%)
below :
used in SC-CO2 extraction was purchased from Kras, Instrument and
Services, Johor, Malaysia. Methanol grade HPLC and β-sitosterol N = 3k (2)
standard were purchased from Sigma-Aldrich, Germany
where N is the number of experiment and k is the number of factor.
Sample preparation Three-level factorial is suitable for second-order polynomial model
The seeds were rinsed with tap water to remove any foreign of two factors. In supercritical fluid extraction, three level factorial
particles and dirt prior to drying. Then, the cleaned seeds were cut into usually been used to optimize the number of factors for obtaining the
small pieces and dried by using oven at temperature of 50°C for a highest yield of extract (Sharif et al., 2014). The coded and un-coded
week to remove moistures. The seeds were ground by using a blender values are shown in Table 3. Moreover, the analysis of variance
(Waring® Commercial blender) and sieved to approximately 0.50 mm (ANOVA) and the regression analysis were all obtained by using
of particle size. Statistica software version 7.0 (STatSoft, EUA). ANOVA analysis was
used to analyze the significance of the results at 95% of confidence
level.
Supercritical carbon dioxide (SC-CO2) extraction
Supercritical fluid extraction (SFE) machine in Center of Lipids Table 3 The extraction process variables in coded and un-coded levels.
Engineering and Applied Research (CLEAR), Universiti Teknologi
Malaysia is consisted of CO2 gas cylinder, CO2 controller pump (Lab Un-coded factors level
Coded
Alliance), co-solvent pump (Lab Alliance), oven (Memmert, factors level Pressure, X1 Temperature, X2
Germany), 10 ml stainless steel extraction vessel, pressure gauge (MPa) (°C)
(Swagelockk, Germany), automatic back pressure regulator (Jasco BP Low (-1) 20 40
2080- Plus) and restrictor valve. A schematic diagram of CLEAR SFE Middle (0) 25 50
apparatus is illustrated in Fig. 2. High (+1) 30 60

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High performance liquid chromatography (HPLC) analysis (β-sitosterol) at a concentration of 80 ppm and β-sitosterol compound
Identification of β-sitosterol was conducted by using a Waters detected in Swietenia mahagoni oil extract, respectively.
HPLC system (Milford, MA, USA) consisting of a pump and system
controller (Model Waters e2695) with photo-diode array detector
(Model 2998). The method of identification for β-sitosterol was
referred to the previous method (Sánchez-Machado et al., 2004) with a
slight modification. C18 reserved phase Kinetex Biphenyl column (5
μm, 4.6 × 150 mm) with a flow rate of 1.0 ml/min was used for
compound separation. The mobile phase was consisted of methanol
(60%)/ acetonitrile (40%), in an isocratic program. The injection
volume of sample was 20 μL and all samples were filtered with 0.45
μm nylon filters prior to injection. The detection was monitored at 210
nm and data were integrated by Empower 3 software (Waters) (Milford,
MA, USA).

Fig. 3 HPLC chromatogram of the standard (β-sitosterol) at

RESULTS AND DISCUSSION concentration of 80 ppm

β-sitosterol content
The β-sitosterol content of Swietenia mahagoni seeds extract with
different conditions in SC-CO2 extraction were identified and
quantified. The highest β-sitosterol content was 9.2 mg/g obtained at
30 MPa and 40°C, meanwhile the lowest one (3.12 mg/g) was obtained
at 20 MPa and 50°C. Previous researches on the β-sitosterol content of
other plants using SC-CO2 extraction were compared with the result in
this study as shown in Table 4. Notably, the temperature of 40°C shows
better extraction of β-sitosterol from plants since low temperature can
avoid the degradation of compound. The temperature in SC-CO2
extraction influenced the yield of β-sitosterol because of the solvent
density changed. The solvent density increases with decreasing
temperature, hence the solubility of β-sitosterol increases by increasing Fig. 4 HPLC chromatogram of β-sitosterol compound detected in S.
the solvating power. Moreover, high pressure also increased the solvent mahagoni oil extracted at 30 MPa and 40°C
density.
This finding is accordance with previous researches in the Optimization of supercritical carbon dioxide (SC-CO2)
extraction of β-sitosterol by using SC-CO2 extraction (Catchpole et al., extraction
2002, Simandi et al., 2002, Andras et al., 2005, Cossuta et al., 2008). Optimization in experimental design for supercritical fluid
Catchpole et al., (2002) reported the extraction of β-sitosterol from saw extraction referred to as a separation performance to achieve high
palmetto berries using SC-CO2 at pressures of 25 and 28 MPa and extraction efficiency by improving different operating conditions of
temperature of 40°C. The maximum β-sitosterol content was achieved various processes (Sharif et al., 2014). Experimental design for
at 28 MPa and 40°C. It can be stated that high pressure and low Swietenia mahagoni seed was based on three level factorial with 13 set
temperature favor to be applied in the extraction of β-sitosterol from of experiments with four repetition at middle point, as shown on Table
plants. Fig. 3 and 4 shows the HPLC chromatograms of the standard 5.

Table 4 Extraction of β-sitosterol by SC-CO2 extraction.

Extraction conditions
β-sitosterol content
Raw material Pressure Temperature Reference
(mg/g)
(MPa) (°C)
Saw palmetto berries 28 40 2.3 [13]
Vitex agnus castus fruit 45 40 1.1 [14]
Sea Buckthorn seeds 15 40 5.0 [15]
Swietenia mahagoni seeds 30 40 9.2 This study

Table 5 Experimental matrix and values of the observed responses

Pressure, Temperature, Extraction yield (%) β-sitosterol concentration (%)

Run Coded level
X1 (MPa) X2 (°C) Actual Predicted Actual Predicted
1 20 40 -1 -1 6.56 7.28 0.35 0.35
2 20 50 -1 0 3.68 3.31 0.31 0.27
3 20 60 -1 +1 1.49 1.13 0.59 0.64
4 25 40 0 -1 6.64 5.78 0.70 0.81
5 25 50 0 0 4.79 4.93 0.56 0.61
6 25 60 0 +1 4.56 5.87 0.87 0.86
7 30 40 +1 -1 7.02 7.16 0.92 0.82
8 30 50 +1 0 8.61 9.43 0.37 0.50
9 30 60 +1 +1 14.45 13.50 0.67 0.63
10 25 50 0 0 4.95 4.93 0.56 0.61
11 25 50 0 0 6.03 4.93 0.69 0.61
12 25 50 0 0 5.06 4.93 0.64 0.61
13 25 50 0 0 4.28 4.93 0.67 0.61

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Fitting the response surface model
The selection of a model for the experimental data was selected
based on correlation coefficient (R2) and Fisher F-test (Rastogi et al.,
1999). In addition, R2 can be expressed as a proportion of variance in a
set of data explained by a statistical model. When R2 value approaching
or approximately 1, the model can be said well fitted to the actual data
(Sin et al., 2006). Typically, R2 value more than 0.75 is considered
accurate in developing statistical model or equation (Henika., 1982).
Fig. 5 and 6 are illustration of the experimental data (observed) and
predicted values of oil yield and β-sitosterol content, respectively. The
R2 values for oil yield and β-sitosterol concentration at 95% confident
level were 0.94 and 0.85, respectively.
Based on the analysis of variance for both oil yield and β-sitosterol
content fitted in the second-order polynomial model, the calculated F-
values are 23.08 and 7.81, respectively. To determine the significant
of the calculated F-values obtained, the tabulated F-values from the
table of the critical value of F with 0.05 of significance level were
compared. Thereby, the calculated F-value obtained are greater than
tabulated F (5, 7, 0.05) obtained which is 3.97. It indicates the significance
between independent variables with the responses at 95% confidence
level. Hence, second-order polynomial model was chosen to depict the
relationship between the oil yield and β-sitosterol content with the
independent variables (temperature and pressure) The second-order
polynomial model equations for oil yield, Y1, and β-sitosterol content,
Y2 (dependent variables), with pressure, X 1, and temperature, X2,
(independent variables) are shown in Eq. (3) and (4), respectively:

Y1 = 125.7830 - 5.3882 X1 + 0.0575 X12 - 2.4506 X2 + 0.0089 X22 +

0.0625 X1X2 (3)

Y2 = - 3.0321 + 0.5881 X1 – 0.0088 X12 – 0.1628 X2 + 0.0023 X22 +

0.0025 X1X2 (4)

The multiple regression coefficients (individual linear, quadratic

Fig. 5 Experimental data (observed) versus predicted values for S.
mahagoni seeds oil yield
and interaction terms) of the oil yield and β-sitosterol content were
determined and summarized in Figure 7 and 8, respectively, together
with the Pareto charts. Regression coefficients indicate the ability of
any term(s) toward the response variable(s) (Mironeasa et al., 2016).
All the terms in the polynomial were analyzed by the degree of
significance (p-value) of each term. Thus, the term that is considered
significant (p <0.05) has an influenced on the process (Cvjetko., 2012).
Based on Fig. 7, the oil yield regression coefficients were
significant except for temperature in quadratic (X 22) and linear terms
(X2) with p >0.05. Therefore, the temperature has no influence on the
oil extraction. The pressure in a linear term (X 1) showed a negative
effect on the response (oil yield) with p <0.05. While the pressure in
the quadratic term (X12) and interaction of pressure and temperature
term (X1X2) gave a positive effect on oil yield with p >0.05 and p >0.01,
respectively. Hence, pressure is a dominant factor on the oil yield. The
solvent density increases with increasing pressure hence the interaction
of inter-molecules and solutes increase (Pereira and Meireles., 2009).

Fig. 6 Experimental data (observed) versus predicted values for β-

sitosterol

Furthermore, the F-calculated values from ANOVA for oil yield

and β-sitosterol content were also considered in selecting an adequate
model for the process. Table 6 and 7 show the analysis of variance for
oil yield and β-sitosterol, respectively, fitted in the second-order
polynomial model. The calculated F-value defined as the ratio of the
mean square of model or regression to the mean square of residual. The
larger the F-value, the greater significance of the model or equation in
the set of data (Vogel and Todaro., 1997).

Table 6 Analysis of variance (ANOVA) for the response surface second-

order polynomial model for the yield of S. mahagoni seed obtained by
SC-CO2 extraction Fig. 7 Multiple regression coefficients and Pareto chart of the oil yield

Source Sum of Degree of Mean Fcalculated Pareto chart in statistical analysis is used to demonstrate the effect
squares freedom square of the factor to the response (Nei et al., 2009). When the bars that
Due to 107.66 5 21.53 represent each independent variables exceeding the line at p =0.05
Regression 23.08 indicate that the independent variables are significant at 95%
Residual 6.53 7 0.93 confidence level (Rodriguez-Nogales et al., 2005). Based on the Pareto
Total 114.20 12 chart, the most influence independent variable is pressure in a linear
term (X1), meanwhile to least influence is pressure in a quadratic term
Table 7 Analysis of variance (ANOVA) for the response surface second- (X12). The temperature in a linear term (X 2) is not significance to the
order polynomial model for β-sitosterol obtained by SC-CO2 extraction response.
Subsequently, the regression coefficients for β-sitosterol content in
Source Sum of Degree of Mean Fcalculated
Fig. 8 also shows that all the terms were significant except for
squares freedom square
temperature in a linear term (X2) with p >0.05 and temperature in a
Due to 0.35 5 0.069 quadratic term (X22) with p >0.01 . Thus, the temperature in linear and
Regression 7.81 quadratic terms do not affect the β-sitosterol concentration. The
Residual 0.062 7 0.009 pressure in quadratic (X12) and interaction of pressure and temperature
Total 0.41 12 (X1X2) terms shows a positive effect on β-sitosterol concentration with

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p >0.01 and p >0.05, respectively. Inversely, the pressure in a linear
term (X1) shows the negative effect on β-sitosterol content with p
>0.05. Hence, pressure in the recovery of β-sitosterol is crucial.
Fig. 8 Multiple regression coefficients and Pareto chart of the β-sitosterol
content
Pressure is significant to the recovery of β-sitosterol. Theoretically,
by increasing the pressure, the density of solvent also increase (Pereira
and Meireles., 2009, Liza et al., 2010). This will also enhance the
solvating power and increase the solute solubility (Pereira and
Meireles., 2009) resulting in a higher recovery of β-sitosterol. Based
on the Pareto chart, the most influence independent variable is the
pressure in a quadratic term (X12). Whereas, the temperature in a linear
term (X2) and temperature in a quadratic term (X22) are not significance
to the response.
Analysis of response surface
Fig. 9 show the surface plot for the response of the oil yield. When
the temperature decreases from 60-40°C, the oil yield slightly
increases, while as pressure increases from 20-30 MPa, the oil yield
increases. It concluded that pressure is a dominant factor for the
extraction of oil yield from Swietenia mahagoni seeds, whereas
temperature has a minimal effect on the oil yield. According to Qiuhui
et al., (2007), the extraction of Chlorella pyrenoidosa resulted in the
increase of oil yield as pressure increase from 25 to 40 MPa due to the
change in solubility of oil in SC-CO2. The increase in solubility of oil
in the solvent will increase the extraction rate because of the solvating
power. De-Castro et al., (1994) stated solvating power is the interaction
of intermolecular solvent and solute.
Fig. 9 Surface plot of oil yield from S. mahagoni as a function of pressure
and temperature
In addition, Mustapa et al., (2009) reported that the increase in
intermolecular interactions of solvent and solute resulted in the increase
of solvent density, thereby the extraction rate increases. Similar finding
was reported by de Azevedo et al., (2008), the extraction of green
coffee oil ranging pressures from 15.2 to 35.2 MPa found that the
extraction rate correlates with the increase in solvent density. The
authors added that pressure also attributed to the increase in solvating
power and the intermolecular physical interactions between solvent and
solute.
Moreover, similar trends were reported in the extraction of Vitex
agnus castus (Cossuta et al., 2008) and virgin coconut oil (Nik
Norulaini et al., 2009) where the extraction rate increases as pressure
increases due to the solvent power. In the extraction of Vitex agnus
castus fruit at the pressure of 10 to 45 MPa increased the extraction
rate. The authors also related the solubility parameter in the study with
the solvent power of SC-CO2 that increased significantly as the pressure
increased from 10 to 27.5 MPa. Meanwhile, the effect of pressure in the
extraction of virgin coconut oil found that yield obtained also depended
on the pressure, where a 100% oil yield was obtained at the highest
pressure.
The study of the extraction of bottle gourd seed oil by Said et al.,
(2014) reported that the direct relationship of pressure and SC-CO2
gave the dominant effect of pressure toward the mass transfer rate as
well as the extraction rate. Viganó et al., (2016) stated that the recovery
of extraction yield is related to the solvent power where the increase in
pressure at constant temperature resulted in the increase of extraction
yield due to the increase of CO2 density as well as solvent power.
Subsequently, the minimal effect of temperature in the extraction
of Swietenia mahagoni seeds as the drop of temperature from 60-40°C,
increases the extraction yield. This phenomena can be related to the
study of Lee et al., (1991), where the solvent solubility increased at the
lower temperature due to the changes in density. Jerry et al., (2001) also
reported that the maximum oil yield was extracted at lower temperature
in the extraction of Vernonia galamensis seeds. This is due to the
increase in density of extraction fluid (SC-CO2) when the temperature
decreases from 100-40°C.
Azizi et al., (2007) reported the similar result in the extraction of
Parkia Speciosa seeds using SC-CO2. The oil yield decreased as the
temperature increased due to the retrograde vaporization behavior.
This behavior referred to the increase in the solvent solubility at lower
temperature up to cross over pressure zone as the density increases.
Meanwhile, in the extraction of Vitex agnus castus fruit by Cossuta et
al., (2008) found that as temperature increases, the solubility parameter
also decreases as well as the extraction yield. Solubility parameter in
the author’s study refer to the relative solvency behavior of SC-CO2.
This finding can be related to the study in the extraction of passion fruit
bagasse by Viganó et al., (2016), where the reduction in oil yield as
temperature increase because of the density of CO2 decrease.
Fig. 10 shows the response surface plot of β-sitosterol content as a
function of pressure and temperature. The effect of pressure on the
extraction of β-sitosterol shows a positive quadratic effect. As pressure
increases from 20 to 25 MPa, the β-sitosterol content in extract
increases as the solubility of β-sitosterol in the solvent but decreases as
it reaches 30 MPa, which shows the interaction of repulsive solute-
solvent increases (Liu et al., 2009). This may be due to the compressed
solvent at high pressure in the extractor.
Fig. 10 Surface plot of β-sitosterol content from S. mahagoni as a
function of pressure and temperature
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Similar finding reported by Hartati et al., (2014) in the extraction
of Swietenia mahagoni seeds. The negative quadratic effect at high
pressure is resulted from the highly compressed CO2 that facilitates
solute-solvent repulsion. The authors suggested that high pressure is
not always recommended because it can potentially induce the complex
extraction. Catchpole et al., (2002) performed the extraction of β-
sitosterol from saw palmetto berries at the pressure of 25 and 28 MPa
and at the temperature of 40°C. The highest concentration of β-
sitosterol found in the extract was at 28 MPa and 40°C.
According to Cossuta et al., (2008), the increase of the pressure
slightly increased the yield of the β-sitosterol. Pressure had the
strongest effect on the concentration of phytostreol in roselle seed
(Nyam et al., 2010). At high pressure, the CO2 density increases hence
the solvent power to dissolve the analyte also increases (Machmudah et
Fig. 11 The correlation of oil yield and β-sitosterol content in the extracts
Correlation of oil yield and β-sitosterol
Moreover, several studies had suggested the act of oil as co-solvent
in the extraction of compound (Vasapollo et al., 2004, Sun et al., 2006,
Krichnavaruk et al., 2008, Viganó et al., 2016). Thus, the correlation
of oil yield and β-sitosterol content of extracts was examined as shown
in Fig. 11. At the constant pressure of 25 and 30 MPa, it shows no
correlation between extraction of oil yield and β-sitosterol content since
r value is nearer to zero. On the contrary, at the constant pressure of 20
MPa it shows a negative correlation. A negative correlation means that
the oil yield decreases, while the β-sitosterol content increases. This
result is the opposite as the other studies mentioned. This may be due
to the low pressure of extraction led to the decrease in the density of the
fluid due to the distance between the molecules, that resulted in reduce
in the solubility of β -sitosterol content.
Meanwhile, at the constant temperature of 50°C and 60°C it shows
no correlation between extraction of the oil yield and the β-sitosterol
content, however at the constant temperature of 40°C, the r value is 0.88
that shows a positive correlation. A positive correlation means that the
oil yield increases, and the β-sitosterol content also increases. This
shows that the domination of solute vapor pressure at low temperature
(Kawahito et al., 2008). Hence, it is proven that the influence of
extracted oil as co-solvent in SC-CO2 was influenced by pressure and
temperature in the extraction of the compound.
CONCLUSION
Supercritical carbon dioxide (SC-CO2) extraction applied to extract
β-sitosterol from Swietenia mahagoni seeds shows that pressure and
temperature influenced the extraction of β-sitosterol. The maximum
yield of the extract was 14.45% obtained at 30 MPa and 60°C, and the
416
al., 2007). On the other hand, at higher temperature, the concentration
of β-sitosterol decrease in both studies. Similarly, the concentration of
β-sitosterol decreased with an increase in temperature of 40-80°C in
Kalahari melon seed oil (Nyam et al., 2010).
The evaluation of the effect of temperature toward the extraction of
β-sitosterol is much more complex due to the dual effects. In the Fig.
10, it shows the negative quadratic effect of temperature. Dual effects
of temperature are when the temperature at 40°C to 50°C, the decrease
in β-sitosterol content due to the reduce in the solubility of β-sitosterol
in the solvent but as temperature further increasing to 60°C, the β-
sitosterol content in the extract increases. This is because of the mass
transfer of β-sitosterol in the solvent as the solubility of mentioned
analyte increases.
maximum of β-sitosterol content was 0.9204% obtained at 30 MPa and
40°C. This work is the first to report the quantification of β-sitosterol
from Swietenia mahagoni seeds and had succedded in obtaining the
optimized parameters for obtaining highest valued of β-sitosterol from
Swietenia mahagoni seeds.
ACKNOWLEDGEMENT
The authors are highly grateful to the financial support from
Universiti Teknologi Malaysia for the Research University Grant
Scheme, GUP (Q.J130000.2546.12H93 and Q.J130000.2546.16H97).
The authors would also like to acknowledge the technical and
management support from Research Management Centre (RMC),
Universiti Teknologi Malaysia.
REFERENCES
Aionicesei, E., Škerget, M., and Knez, Ž. 2008. Measurement of CO2 solubility
and diffusivity in poly(1-lactide) and poly(d,1-lactide-co-glycolide) by
magnetic suspension balance. J. Supercri. Fluids. 47, 296-301.
Andras, C. D., Simandi, B., Orsi, F., Lambrou, C., Missopdinou-Tatala, D.,
Panayiotou, C., Dmokus, J., and Doleschall, F. 2005. Supercrtical carbon
dioxide extraction of okra (Hibiscus esculentus L.) seeds. J. Sci. Food. Agri.
85, 1415-1419.
Balijepalli. M. K., Suppaiah, V., Chin, A. M., Buru, A. S., Saqineedu, S. R., and
Pichika, M. R. 2014. Acute oral toxicity studies of Swietenia macrophylla
seeds in sprague dawley rats. Pharmacognosy Res. 7, 38-44.
Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., and Escaleira, L. A.
2008. Response surface methodology (RSM) as a tool for optimization in
analytical chemistry. Talanta. 76, 965-977.
 Md Norodin et al. / Malaysian Journal of Fundamental and Applied Sciences Vol. 14, No. 3 (2018) 411-417
Blundell, A. G., and Gullison, R. E. 2003. Poor regulatory capacity limits the
ability of science to influence the management of mahogany. For. Policy
Econ. 5, 395-405.
Catchpole, O. J., Perry, N. B., Da Silva, B. M. T., Grey, J. B., and Smallfield, B.
M. 2002. Supercritical extraction of herbs I: Saw Palmetto, St John’s Wort,
Kava root, and Echinacea. J. Supercrit. Fluids. 22, 129-138.
Cossuta, D., Simandi, B., Vagi, E., Hohmann, J., Prechl, A., Lemberkovics, E.,
Kery, A., and Keve, T. 2008. Supercritcal fluid extraction of Vitex agnus
castus fruit. J. Supercrit. Fluids. 47, 188-194.
Cvjetko, M. 2012. Optimization of the supercritical CO2 extraction of oil from
rapeseed using response surface methodology. Food Technology and
Biotechnology. 50, 2, 208-215.
De Azevedo, A. B. A., Mazzafera, P., Mohamed, R. S., Vieira De Melo, S. A.
B., Kieckbusch, T. G. 2008. Extraction of caffeine, cholorogenic acids and
lipids from green coffee beans using supercritical carbon dioxide and co-
solvents. Brazilian J. Chem. Eng. 25, 543-552.
Eid, A. M. M., Elmarzugi, N. A., El-Enshasy, H. A. 2013. A review on the
phytopharmacological effect of Swietenia macrophylla. Int. J. Pharma. Sci. 5,
47-53.
Falah, S., Suzuki, T., Katayama, T. 2008. Chemical constituents from Swietenia
macrophylla bark and their antioxidant activity. Pakistan J. Biol. Sci. 11,
2007-2012.
Goh, B. H., Abdul Kadir, H., Abdul Malek, S., Ng, S.W. 2010. Swietenolide
diacetate from the seeds of Swietenia macrophylla. Acta Crystallogr. Sect. E
Struct. Report Online. 66, 6, o1396.
Goh, B. H., and Kadir, H. A. 2011. In vitro cytotoxic potential of Swietenia
macrophylla King seeds against human carcinoma cell lines. J. Med. Plants
Res. 5, 1395-1404.
Hartati, Salleh, L. M., Mohd Yunus, A. C., and Aziz, A. A. 2014. Optimization
of supercritical CO2 extraction of Swietenia mahagoni seed by response
surface methodology. J. Teknol. 67, 15-20.
Hashim, M. A., Yam, M. F., Hor, S. Y., and Lim, C. P. 2013. Anti-
hyperglycemic activity of Swietenia macrophylla King (meliaceae) seed
extracts in normoglycaemic rats undergoing glucose tolerance test. Chin. Med.
8, 11.
Haque, M. A., Khan, G. M. A., Razzaque, S. M. A., Khatun, K., Chakraborty,
A. K., and Alam, M. S. 2013. Extraction of rubiadin dye from Swietenia
mahagoni and its dyeing characterictics onto silk fabric using metallic
mordants. Indian J. Fibre Text. Res. 38, 280-284.
Henika. R. G. 1982. Use of response-surface methodology in sensory evaluation.
Food Technology. 36, 96-100.
Hu, Q., Pan, B., Xu, J., Sheng, J., and Shi, Y. 2007. Effects of supercritical
carbon dioxide extraction conditions on yields and antioxidant activity of
Chlorella pyrenoidosa extracts. J. Food Eng. 80, 997-1001.
Kawahito, Y., Kondo, M., Machmudah, S., Sibano, K., Sasaki, M., and Goto,
M. 2008. Supercritical CO2 extraction of biological active compounds from
loquat seed. Sep. Purif. Technol. 61, 130–135.
King, J. W., Mohamed, A., Taylor, S. L., Mebrahtu, T., and Paul, C. 2001.
Supercritical fluid extraction of Vernonia galamensis seeds. Ind. Crops Prod.
14, 241-249.
Krichnavaruk, S., Shotipruk, A., Goto, M., and Pavasant, P. 2008. Supercritical
carbon dioxide extraction of astaxanthin from Haematococcus pluvialis with
vegetable oils as co-solvent. Bioresour. Technol. 99, 5556–5560.
Lee, B. C., Kim, J. D., Hwang, K. Y., and Lee, Y. Y. 1991. Extraction of oil
from evening primrose seed with supercritical carbon dioxide. Supercrit.
Fluid Process. Biomater. 168-180.
Liu, S., Yang, F., Zhang, C., Ji, H., Hong, P., and Deng, C. 2009. Optimization
of parameters for supercritical carbon dioxide extraction of Passiflora seeds
oil by response surface methodology. J. Supercrit. Fluids. 48, 9-14.
Liza, M. S., Abdul Rahman, R., Mandana, B., Jinap, S., Rahmat, A., Zaidul, I.
S. M. and Hamid, A. 2010. Supercritical carbon dioxide extraction of
bioactive flavonoid from Strobilanthes cripus (Pecah kaca). Food Bioprod.
Process. 88, 319-326.
Luque de Castro, M. D., Valcarcel, M., and Tena, M. T. 1994. Analytical
Supercritical Fluid Extraction, Germany: Springer-Verlag.
Machmudah, S., Kawahito, Y., Sasaki, M., and Goto, M. 2007. Supercritical
CO2 extraction of rosehip seed oil: Fatty acids composition and process
optimization. J. Supercrit. Fluids. 41, 421–428.
Maiti, A., Dewanjee, S., Kundu, M., and Mandal, S. C. 2009. Evaluation of
antidiabetic activity of the seeds of Swietenia macrophylla in diabetic rats.
Pharma. Biol. 47, 132-136.
Mironeasa, S., Codină, G. G., and Mironeasa, C. 2016. Optimization of wheat-
grape seed composite flour to improve alpha-amylase activity and dough
rheological behavior. Int. J. Food. Prop. 19, 859-872.
Mohd Azizi, C. Y., Salman, Z., Nik Norulaini, N. A., and Mohd Omar, A. K.
2007. Extraction and identification of compounds from Parkia speciose seeds
by supercritical carbon dioxide. J. Chem. Nat. Resour. Eng. Spec. Ed. 153-
163.
Mustpha, A. N., Manan, Z. A., Mohd Azizi, C. Y., Nik Norulaini, N. A., and
Omar, A. K. M. 2009. Effects of parameters on yield for sub-critical R134a
extraction of palm oil. J. Food Eng. 95, 606-616.
Nei, H. Z. N., Fatemi, S., Salimi, A. R., Vatanara, A., and Najafabadi, A. R.
2009. Enrichment of omega 3 fatty acids from Tyulka oil by supercritical CO2
extraction. J. Chem. Technol. Biotechnol. 84, 1854-1859.
Nik Norulaini, N. A., Setianto, W. B., Zaidul, I. S. M., and Nawi, A. H. 2009.
Effects of supercritical carbon dioxide extraction parameters on virgin
coconut oil yield and medium-chain triglyceride content. Food Chem. 116,
193-197.
Nyam, K. L., Tan, C. P., Karim, R., Lai, O. M., Long, K., and Che Man, Y. B.
2010. Extraction of tocopherol-enriched oils from Kalahari melon and roselle
seeds by supercritical fluid extraction (SFE-CO2). Food Chem. 119, 1278–
1283.
Nyam, K. L., Tan, C. P., Lai, O. M., Long, K., and Che Man, Y. B. 2010.
Optimization of supercritical fluid extraction of phytosterol from roselle seeds
with a central composite design model. Food Bioprod. Process. 88, 239–246.
Pereira, C. G., and Meireles, M. A. A. 2009. Supercritical fluid extraction of
bioactive compounds: Fundamentals, application and economic perspectives.
Food Bioprocess Technol. 3, 340-372.
Rastogi, N. K., and Rashmi, K. R. 1999. Optimization of enzymatic liquefaction
of mango pulp by response surface methodology. Eur. Food. Res. Technol.
209, 57-62.
Rodriguez-Nogales, J. M., Roura, E., and Contreras, E. 2005. Biosynthesis of
ethyl butyrate using immobilized lipase: A statistical approach. Process
Biochem. 40, 63-68.
Said, P. P., Sharma, N., Naik, B., and Pradhan, R. C. 2014. Effect of pressure,
temperature and flow rate on supercritical carbon dioxide extraction of bottle
gourd seed oil. Int. J. Food. Nutr. Sci. 3, 14-17.
Sánchez-Machado, D. I., López-Hernández, J., Paeiro-Losada, P., and López-
Cervantes, J. 2004. An HPLC method for the quantification of sterols in edible
seaweeds. Biomed. Chromatogr. 18, 183-190.
Sajfrtová, M., Licková, I., Wimmerová, M., Sovová, H., and Wimmer, Z. 2010.
β-Sitosterol supercritical carbon dioxide extraction from sea buckthorn
(Hippophae rhamnoides L.) seeds. Int. J. Mol. Sci. 11, 1842-1850.
Sharif, K. M., Rahman, M. M., Azmir, J., Mohamed, A., Jahuru, M. H. A.,
Sahena, F., Zaidul, I. S. M. 2014. Experimental design of supercritical fluid
extraction – A review. J. Food Eng. 124, 105-116.
Simandi, B., Kristo, S. T., Kery, A., Selmeczi, L. K., Kmecz, I., and Kemény,
S. 2002. Supercritical fluid extraction of dandelion leaves.
Sin, H. N., Yusof, S., Hamid, N. S. A., and Rahman, R. A. 2006. Optimization
of enzymatic clarification of sapodilla juice using response surface
methodology. J. Food. Eng. 73, 313-319.
Sun, M., and Temelli, F. 2006. Supercritical carbon dioxide extraction of
carotenoids from carrot using canola oil as a continuous co-solvent. J.
Supercrit. Fluids. 37, 397-408.
Vasapollo, G., Longo, L., Rescio, L., and Ciurlia, L. 2004. Innovative
supercritical CO2 extraction of lycopene from tomato in the presence of
vegetable oil as co-solvent. J. Supercrit. Fluids. 29, 87–96.
Viganó, J., Coutinho, J. P., Souza, D. S., Baroni, N. A. F., Helena, T. G., Juliana,
A., and Julian, M. 2016. Exploring the selectivity of supercritical CO2 to
obtain nonpolar fractions of passion fruit bagasse extracts. J. Supercrit. Fluids.
100, 1-10.
Vilegas, J. H. Y., de Marchi, E., and Lancas, F. M. 1997. Extraction of low
polarity compounds (with emphasis on coumarin and kaurenoic acid) from
Mikania glomerata (“guaco”) leaves. Phytochem. Anal. 8, 266-270.
Vogel, H. C., and Todaro, C. L. 1997. Fermentation and Biochemical
Engineering Handbook. (3rd ed.). Westwood, New Jersey, U.S.A. Elsevier Inc.
417
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 Second International Conference on Chemical Engineering (ICCE) UNPAR IOP Publishing
IOP Conf. Series: Materials Science and Engineering 162 (2017) 012030 doi:10.1088/1757-899X/162/1/012030

Supercritical carbon dioxide (SC-CO2) extraction of essential oil

from Swietenia mahagoni seeds

N S M Norodin1, a *, L M Salleh2, b , Hartati3,c and N M Mustafa4,d

1
Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy
Engineering, UniversitiTeknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia
2
Center of Lipids Engineering and Applied Research (CLEAR), Ibnu Sina Institute
Scientific & Industrial Research (Ibnu Sina ISIR), Universiti Teknologi Malaysia, 81300
Johor Bahru, Johor, Malaysia
3
Department of Biology, Universitas Negeri Makassar, South Sulawesi, Indonesia
4
Natural Product Division, Forest Research Institute Malaysia (FRIM), 52109 Kepong,
Selangor Darul Ehsan, Malaysia

E-mail : anursalsabilamdnorodin@gmail.com, bi.liza@cheme.utm.my,

c
tati_biounm@yahoo.co.id, dmusaadah@frim.gov.my

Abstract. Swietenia mahagoni (Mahogany) is a traditional plant that is rich with bioactive
compounds. In this study, process parameters such as particle size, extraction time, solvent
flowrate, temperature and pressure were studied on the extraction of essential oil from
Swietenia mahagoni seeds by using supercritical carbon dioxide (SC-CO2) extraction.
Swietenia mahagoni seeds was extracted at a pressure of 20-30 MPa and a temperature of
40-60°C. The effect of particle size on overall extraction of essential oil was done at 30
MPa and 50°C while the extraction time of essential oil at various temperatures and at a
constant pressure of 30 MPa was studied. Meanwhile, the effect of flowrate CO 2 was
determined at the flowrate of 2, 3 and 4 ml/min. From the experimental data, the extraction
time of 120 minutes, particle size of 0.5 mm, the flowrate of CO 2 of 4 ml/min, at a pressure
of 30 MPa and the temperature of 60°C were the best conditions to obtain the highest yield
of essential oil.

1. Introduction
Swietenia mahagoni is also known as ‘tunjuk langit’ in Malaysia is used traditionally to treat various
diseases such as diabetes and high blood pressure [1]. S. mahagoni tree is 30 meters or taller [2] and the
wood, usually being used for making furniture [3]. Meanwhile, the bark can be used for natural colorant [4].
The fruit of S. mahagoni is woody and consisting of capsules containing winged seeds [5]. Whereas, the
seed of S. mahagoni can be obtained by removing the wing. In Malaysia, the raw seeds were used for
treating hypertension and diabetes [6]. In addition, S. mahagoni seeds have been reported to have various
biological activities such as anti-inflammatory activity, anticancer and antitumor activity [7] and also
antidiabetic activity [8].
Also, carbon dioxide (CO2) is the most frequent solvent used that is environmental friendly (fairly non-
toxic), low cost and can be easily removed from the extract [9]. The elimination of CO2 is easily achieved
since CO2 is in a gas state at room temperature. In addition, CO 2 in the supercritical state is in a moderate
critical temperature (31.3°C) and pressure (7.38 MPa). Supercritical state is when gas and liquid are
indistinguishable where at this state it is compressible but possessing a density of a liquid. In a word,
supercritical CO2 makes a good solvent because of the gas-like state that attributed the low viscosity and
high diffusion coefficient and the liquid-like state that gave the solvating power [10].

2. Methodology
2.1. Materials and reagents
Swietenia mahagoni seeds were bought in the local market. The seeds were rinsed with tap water to remove
any foreign particles and dirt prior to drying. Then, the cleaned seeds were cut into small pieces and dried by
using oven at temperature of 50°C for a week to remove moistures. The seeds were ground by using a
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
blender (merckofPanasonic) and sieved to approximate 0.25, 0.50 and 0.75 mm of particle size. Commercial
this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd 1
 Second International Conference on Chemical Engineering (ICCE) UNPAR IOP Publishing
IOP Conf. Series: Materials Science and Engineering 162 (2017) 012030 doi:10.1088/1757-899X/162/1/012030

grade liquid carbon dioxide (purity 99.99%) used in supercritical carbon dioxide extraction was purchased
from Kras, Instrument and Services, Johor, Malaysia.

2.2. Supercritical Carbon dioxide (SC-CO2) extraction

CLEAR supercritical fluid extraction (SFE) machine in Center of Lipids Engineering and Applied Research
(CLEAR), Universiti Teknologi Malaysia consisted of CO2 gas cylinder, CO2 controller pump (Lab
Alliance), co-solvent pump (Lab Alliance), oven (Memmert, Germany), 10 ml stainless steel extraction
vessel, pressure gauge (Swagelockk, Germany), automatic back pressure regulator (Jasco BP 2080- Plus) and
restrictor valve. A schematic diagram of CLEAR SC-CO2 unit is illustrated in figure 1.

Figure 1. The schematic design of the SC-CO2 unit

Five gram of sample was placed in 10 ml stainless steel extraction vessel and sealed tightly in the oven.
Set all the parameters (temperature, pressure and flowrate of CO 2), the extraction process started after all the
parameters were attained. Lastly, depressurized the system and the oil yields were collected. The
parameters used in extraction process is presented in table 1.

Table 1. The process parameters for SC-CO2 extraction

Parameter Range/ value
Temperature (°C) 40-60
Pressure (MPa) 20-30
Flowrate of CO2 (ml/min) 2.00-4.00
Particle size (mm) 0.25-0.75
Mass of sample (g) 5.00
Extraction time (min) 0-180

Collected oil yield was calculated as percentage of oil yield by using equation below:

(Eq. 1)

3. Result and Discussion

3.1. Particle size
Particle size is one of the important factors to be considered in the extraction of S. mahagoni seeds. In a
word, as the surface area increases, the extraction rate also increases. Hence, smaller particle size will
increase the extraction yield because the mass transfer will increase with smaller particle size of the sample.
But the downside is as the sample is ground to small, the extraction rate may decrease due to channeling
2
 Second International Conference on Chemical Engineering (ICCE) UNPAR IOP Publishing
IOP Conf. Series: Materials Science and Engineering 162 (2017) 012030 doi:10.1088/1757-899X/162/1/012030

effect inside the extraction vessel [11]. Channeling effect in the extraction vessel is due to over fine particle
size of the sample that reduce the contact between the sample and solvent. Supported by Salleh (2010), fine
powder with smaller particle size can increase the extraction rate but it is also lead to the difficulty in
maintaining the flow rate to obtain a good optimization of SC-CO2 extraction [12].

Figure 2. Effect of particle size on extraction yield

Figure 2 shows that particle size of 0.5 mm gave the highest percentage of oil yield which is 20.68% ±
5.25 compared to 0.75 mm and 0.25 mm. From the figure, it shows that when the particle size is too small,
the extraction yield is decreased. This is because when the particle size is too fine, the porosity of the sample
is reduced and slower CO2 penetration into the sample, hence lower the extraction yield. In this study, S.
mahagoni seeds are naturally oily thus it may easily clumped together in the extraction vessel hence reduce
the surface area. Similarly with the study in the extraction of rubber seed, smaller particle size (≤0.355 mm)
can easily clump together, however larger particle size (0.50mm) do not clump together easily and provide
larger surface area [13]. It is resulted that the particle size of 0.5 mm is considered for this SC-CO2
extraction.

3.2. Extraction time

The determination of the extraction time for the extraction of S. mahagoni seeds by using SC-CO2 extraction
was performed. The conditions used were pressure of 20, 25 and 30 MPa, with the temperature of 50°C.
The flow rate used in this study was 2 ml/min. The yield of extract was collected every 20 minutes over 180
minutes of extraction time. The results of the best extraction time are presented in Figure 3.

3
 Second International Conference on Chemical Engineering (ICCE) UNPAR IOP Publishing
IOP Conf. Series: Materials Science and Engineering 162 (2017) 012030 doi:10.1088/1757-899X/162/1/012030

Figure 3. The effect of extraction time on extraction yield of SC-CO2 at 20, 25 and 30 MPa and the
temperature of 50°C

The figure displays the oil yield in percent versus the extraction time in minutes. At pressure of 20 MPa,
it took more than 120 minutes to achieve the asymptomatic value. The highest yield (0.4306%) was at the
highest pressure of 30 MPa within 120 minutes. Lina et al., (2010) also obtained similar result with this
study where the seed oil of Microula sikkimensis increased as extraction time increased [14]. The same trend
was found in the study of saffron extract by Lozano et al. (2000) [15]. In conclusion, the extraction time of
S. mahagoni seeds was performed at 120 min constantly for each sample throughout this study in order to
obtain asymptotic value to ensure maximum extraction yield was extracted.

3.3. Flowrate
Flow rate of solvent (example: CO2) is another factor affecting the extraction process to produce a high yield
of extract by using supercritical carbon dioxide (CO2) [16]. By increasing the flow rate of solvent, also
reduce the extraction time and mass transfer resistance.

Figure 4. Effect of flow rate on the overall oil yield against extraction time at the pressure of 30 MPa and
temperature of 50ºC

As shown in figure 4, the overall oil yield increase with the increasing of flow rate at constant pressure of
30 MPa and temperature of 50ºC. The extraction of borage oil by SC-CO2 extraction showed higher flow
rate gives higher yield of borage seed oil [17]. Similarly, in the extraction of flaxseed showed that at higher
4
 Second International Conference on Chemical Engineering (ICCE) UNPAR IOP Publishing
IOP Conf. Series: Materials Science and Engineering 162 (2017) 012030 doi:10.1088/1757-899X/162/1/012030

flow rate (3 L/min) recovered higher oil yield [18]. Furthermore, by increasing the SC-CO2 flowrate, the
intermolecular interaction between solvent and the solute increase due to the increase of CO2 molecules per
volume in the extraction vessel [19]. In this condition, the mass transfer increases as the flowrate increases.
Hence, 4 ml/min of CO2 was selected as the best flowrate for the extraction of S. mahagoni seeds.

3.4. Temperature and Pressure

The extraction of S. mahagoni seeds by using SC-CO2 extraction shows that the highest yield (14.45 %)
obtained was at the maximum level (30MPa, 60°C) meanwhile lowest (1.49%) at (20MPa, 60°C) as shown
in Table 2. At higher temperature, the decomposition of cell walls occurred thus extracted oil produced is
higher [19]. Operating at higher pressure will influence the solvent density, thus enhanced the solvating
power (the interaction of inter-molecules and solutes increase) [11]. Similar result was reported in the
extraction of S. mahagoni seeds by using SC-CO2 extraction [20].

Table 2. Extraction of S. mahagoni seeds at different conditions (pressure, P and temperature, T)

Condition (s) Oil yield (%)
P= 20MPa, T= 40°C 6.556
P= 20MPa, T= 50°C 3.676
P= 20MPa, T= 60°C 1.490
P= 25MPa, T= 40°C 6.640
P= 25MPa, T= 50°C 4.954
P= 25MPa, T= 60°C 4.558
P= 30MPa, T= 40°C 7.024
P= 30MPa, T= 50°C 8.612
P= 30MPa, T= 60°C 14.45

Figure 5 shows the effect of pressure and temperature on oil extraction. At constant temperature, oil yield
increases as pressure increases (20-30 MPa). This is due to the increase of density and thus increase the
solvating power of SC-CO2. Effect of pressure influenced both solvating power and intermolecular
interaction strength [21,22]. Similar result was reported in SC-CO2 extraction of green coffee oil [23],
wheat bran [24] and S. mahagoni seeds [20].

Figure 5. The effect of pressure and temperature on extraction yield

5
 Second International Conference on Chemical Engineering (ICCE) UNPAR IOP Publishing
IOP Conf. Series: Materials Science and Engineering 162 (2017) 012030 doi:10.1088/1757-899X/162/1/012030

Next, at constant pressure (20 and 25 MPa), oil yield decreases as temperature increases (40-60 ºC). But
at constant pressure (30 MPa), oil yield increases as temperature increases. Effect of temperature in
extraction process is much more difficult to evaluate unlike pressure. This is due to the dual effect of
temperature toward extraction process depending on the domination of solute vapour pressure or solvent
density [19,25,26]. Decreasing solvent density with the increase in temperature resulted in higher extracted
yield because of the increase in vapour pressure. Similar effect were reported on extraction green coffee oil
[23] and silkworm pupal oil [26].

4. Conclusion
Supercritical carbon dioxide (SC-CO2) extraction applied to extract Swietenia mahagoni. From the
experimental data, the extraction time of 120 minutes, particle size of 0.5 mm, the flowrate of CO 2 of 4
ml/min, at a pressure of 30 MPa and the temperature of 60°C were the best conditions to obtain the highest
yield of essential oil.

Acknowledgements
The authors gratefully acknowledge the Universiti Teknologi Malaysia for awarding with the Research
University Grant Scheme, GUP (Q.J130000.2546.10H50).

References
[1] Goh B H, Kadir H A, Malek S N A, Ng S W 2010 Swietenolide diacetate from the seeds of Swietenia
macrophylla Acta Crystallogr. Sect. E Struct. Reports Online 66
[2] Eid A M M, Elmarzugi N A, El-Enshasy H A 2013 A review on the phytopharmacological effect of
Swietenia macrophylla Int. J. Pharm. Pharm. Sci. 5 47–53
[3] Falah S, Suzuki T, Katayama T 2008 Chemical constituents from Swietenia macrophylla bark and
their antioxidant activity Pakistan J. Biol. Sci. 11 2007–2012
[4] Haque M A, Khan G M A, Razzaque S M A, Khatun K, et al. 2013 Extraction of rubiadin dye from
Swietenia mahagoni and its dyeing characteristics onto silk fabric using metallic mordants Indian J.
Fibre Text. Res. 38 280–284
[5] Blundell A G, Gullison R E 2003 For. Policy Econ. 5 395–405
[6] Balijepalli M K, Suppaiah V, Chin A-M, Buru A S, et al. 2014 Acute oral toxicity studies of swietenia
macrophylla seeds in sprague dawley rats Pharmacognosy Res. 7 38–44
[7] Goh B H, Kadir H A 2011 In vitro cytotoxic potential of Swietenia macrophylla King seeds against
human carcinoma cell lines J. Med. Plants Res. 5 1395–1404
[8] Maiti A, Dewanjee S, Kundu M, Mandal S C 2009 Evaluation of antidiabetic activity of the seeds of
Swietenia macrophylla in diabetic rats Pharm. Biol. 47 132–136
[9] Liza M S, Rahman, R A, Mandana B, Jinap S, et al. Supercritical carbon dioxide extraction of
bioactive flavonoid from Strobilanthes crispus (Pecah Kaca) Food Bioprod. Process 88 319–326
[10] Aionicesei E, Škerget M, Knez Ž 2008 Measurement of CO2 solubility and diffusivity in poly(l-
lactide) and poly(d,l-lactide-co-glycolide) by magnetic suspension balance J. Supercrit. Fluids 47
296–301
[11] Pereira C G, Meireles M A A 2009 Supercritical Fluid Extraction of Bioactive Compounds:
Fundamentals, Applications and Economic Perspectives Food Bioprocess Technol. 3 340–372
[12] Salleh L M 2010 Extraction of bioactive flavonoid compounds from Pecah Kaca (Strobilanthes
Crispus) using supercritical carbon dioxide Ph. D. Thesis Universiti Putra Malaysia
[13] Yian L N 2015 Supercritical carbon dioxide extraction of rubber seed oil rich in alpha-linolenic acid
Ph.D. Thesis Universiti Teknologi Malaysia
[14] Lina S, Fei R, Xudong Z, Yangong D, Fa H 2010 Supercritical carbon dioxide extraction of microula
sikkimensis seed oil. J. Am. Oil Chem. Soc 87 1221–1226
[15] Lozano P, Delgado D, Gómez D, Rubio M, Iborra J L 2000 A non-destructive method to determine
the safranal content of saffron (Crocus sativus L.) by supercritical carbon dioxide extraction combined
with high-performance liquid chromatography and gas chromatography J. Biochem. Biophys. Methods
43 367–378
[16] Leo L, Rescio L, Ciurlia L, Zacheo G 2005 Supercritical carbon dioxide extraction of oil and α-
tocopherol from almond seeds J. Sci. Food Agric. 85 2167–2174
[17] Gómez A M, de la Ossa E M 2002 Quality of borage seed oil extracted by liquid and supercritical
6
 Second International Conference on Chemical Engineering (ICCE) UNPAR IOP Publishing
IOP Conf. Series: Materials Science and Engineering 162 (2017) 012030 doi:10.1088/1757-899X/162/1/012030

carbon dioxide Chem. Eng. J. 88 103–109

[18] Bozan B, Temelli F 2002 Supercritical CO2 extraction of flaxseed J. Am. Oil Chem. Soc. 79 231–235
[19] Machmudah S, Kawahito Y, Sasaki M, Goto M 2007 Supercritical CO2 extraction of rosehip seed oil:
Fatty acids composition and process optimization J. Supercrit. Fluids 41 421–428
[20] Hartati, Salleh L M, Yunus, A C M, Aziz A A 2014 Optimization of supercritical CO2 extraction of
Swietenia mahagoni seed by response surface methodology J. Teknol. Sciences Eng. 67 15–20
[21] Bulgarevich D S, Sako T, Sugeta T, Otake K, et al. 2002 The role of general and hydrogen-bonding
interactions in the solvation processes of organic compounds by supercritical CO2 / n-Alcohol
Mixtures Ind. Eng. Chem. Res 41 2074–2081
[22] Morita A, Kajimoto O 1990 Solute-solvent interaction in nonpolar supercritical fluid: a clustering
model and size distribution J. Phys. Chem. 94 6420–6425
[23] de Azevedo A B A, Kieckbush T G, Tashima A K, Mohamed R S, et al. 2008 Extraction of green
coffee oil using supercritical carbon dioxide J. Supercrit. Fluids 44 186–192
[24] Kwon K, Uddin S, Jung G, Sim J, Chun B 2010 Supercritical carbon dioxide extraction of phenolics
and tocopherols enriched oil from wheat bran Int. J. Biol. Life Sci. 6 117–122
[25] Thana P, Machmudah S, Goto M, Sasaki M, et al. 2008 Response surface methodology to supercritical
carbon dioxide extraction of astaxanthin from Haematococcus pluvialis Bioresour. Technol. 99 3110–
3115
[26] Wei Z J, Liao A M, Zhang H X, Liu J, Jiang S T 2009 Optimization of supercritical carbon dioxide
extraction of silkworm pupal oil applying the response surface methodology Bioresour. Technol. 100
4214–4219

7
 Jurnal Full paper
Teknologi

Antioxidant Activity and Total Phenolic Contents in Methanol Extracts

from Swietenia Mahagoni and Andrographis Paniculata
Liza Md Salleha,b*, Hartatia,c, Roslina Jamaludina, Mohd. Azizi Che Yunusb, Harisun Yakubd, Azila Abd. Aziza,d
aBioprocess Engineering Department, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia
bCentre of Lipid Engineering Applied Research (CLEAR), Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru,
Johor, Malaysia
cBiology Department, University State of Makassar, South Sulawesi, Indonesia
dInstitute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia

*Corresponding author: i.liza@cheme.utm.my

Article history Abstract

Received :4 October 2013 Traditional medicinal plants such as Swietenia mahagoni and Andrographis paniculata are rich with
Received in revised form : bioactive compounds. The phenolic composition and antioxidant activity of methanol extract from
23 April 2014 Swietenia mahagoni and Andrographis paniculata were studied. They were extracted by Soxhlet
Accepted :8 May 2014 extraction of 70% methanol solvent. The results obtained from this study showed Andrographis
paniculata gave highest percentage yield by 19.94% compared to Swietenia mahagoni at 11.86%. In
Graphical abstract antioxidant activity, Andrographis paniculata and Swietenia mahagoni showed 89.93% and 60.77%
respectively. While for total phenolic content highest in extract of Swietenia mahagoni by 55.0 mg/g and
Andrographis paniculata only 7.7 mg/g.

Keywords: Antioxidant; total phenolic contents; methanol extracts; Swietenia mahagoni; Andrographis
paniculata

© 2014 Penerbit UTM Press. All rights reserved.

1.0 INTRODUCTION in A. paniculata leaves were andrographolide, neo-

Swietenia mahagoni (Linn.) Jacq. mainly grows in tropical areas
of Asia such as India, Malaysia, Indonesia and southern mainland
of China. It is also known as “Tunjuk Langit” by the local people.
Its seeds have been applied as folk medicine for treatment of
hypertension, diabetes and malaria, while the decoction of its bark
has been used as a febrifuge1. The biologically active ingredients,
tetranortriterpenoid and fatty acids are considered to be
responsible for these therapeutic effects2. The seed of S. mahagoni
has been reported for its anti-inflammatory, antimutagenecity, and
antitumour activities 3. The plant extracts have been accounted to
possess antibacterial and antifungal activities. Limnoid obtained
from S. mahagoni has antifungal activity and diabetes therapy4.
Andrographis paniculata, locally known as Hempedu Bumi
and commonly called the King of Bitter grows widely in tropical
area of South East Asia, India, and China with plant height of 30-
70 cm. In Malaysia and Indonesia, this plant has been extensively
used for traditional medicine and help against fever, dysentery,
diarrhoea, inflammation, and sore throat5. Furthermore, it is a
promising new way for the treatment of several diseases,
including HIV, AIDS, and numerous symptoms associated with
immune disorders6. The three main diterpenoid lactones identified
andrographolide and deoxyandrographolide7-8.The objective of
this work is to determine antioxidant activity and total phenolic
content of methanol extract of the S. mahagoni and A. paniculata.
2.0 EXPERIMENTAL
2.1 Raw Material and Sample Preparation
The S.mahagoni seeds were collected from Indonesia. Then, the
seeds were rinsed with water to remove any foreign particles and
dirt prior to drying. Then, the cleaned seeds were cut into small
pieces and dried using oven at 50°C for one week to remove
moisture. The seeds were grinded into powder by using blender
(Panasonic). A. paniculata sample was purchased from Malaysia,
where it was grinded into powder form.
2.2 Soxhlet Extraction (SE)
A conventional method of soxhlet extraction was performed. 5 g
of S.mahagoni and A. paniculata powder were weight
respectively and were placed in the thimble. 150 ml solvent of

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52 Liza Md Salleh et al. / Jurnal Teknologi (Sciences & Engineering) 69:4 (2014), 51–53

70% methanol was used for each plant. The extraction processes In this study, A. paniculata was showing higher yield compared
were done for 6 hours at temperature 65ºC. Then, the extraction than S. mahagoni due to the bioactive compound in A. paniculata
yields were put in the rotary evaporator at 50°C to remove the more polar than S. mahagoni where its seed contained more non-
solvent. polar compound 13.The used of methanol, ethanol or mixture of
these solvents with water usually give high yield of the extracts.
% yield = [𝑚1 ⁄𝑚0 ] × 100 (1) This theory was proven by one study where isolation of phenolic
compound from cherry liqueur pomace using 70% solvent-
Where m0 = mass of sample (g) mixture was more effective than using pure solvent only14.
m1 = mass of the extract (g)

2.3 The 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Free Radical

Scavenging

The free radical scavenging activity was measured using 2,2-

diphenyl-1-picrylhydrazyl (DPPH) assay. This assay was carried
out according to the method with a slight modification 9. Extract
solution were prepared by dissolving 0.025 g of dry extract in 10
ml of methanol to give final concentration at 2.5 mg/ml. Then, 77
μL of the extract solution were mixed with 3 ml of 6 x 10-5 M
methanolic solution of DPPH. After that, the mixtures were
placed in the dark for 30 minutes at room temperature and the
decreases in the absorption were measured at 517 nm using
spectrophotometer. The DPPH radical concentration was
calculated by using the following equation:

A Control−A Sample
DPPH radical concentration (%)= x 100 (2)
A Control

Where A Control is the absorbance value of the control reaction and Figure 1 Comparison of percentage yield of S. mahagoniand
A Sample is the absorbance value with the presence of the tested A.paniculataby using Soxhlet extraction
extracts in the sample.
3.2 DPPH Free Radical Scavenging Activity of S. Mahagoni
2.4 Total Phenolic Content (TPC)
and A. Paniculata
The TPC was determined according to the Folin-Ciocalteau
The antioxidant activity assay was carried out to examine the
method with slight modification10. The reaction mixture was
ability of S. mahagoni and A. paniculata to scavenge free radicals
composed by 1 mL of the extract (concentration of 0.01 g/mL), 5
in vitro by the improved of scavenging activity percentage. Figure
mL of Folin-Ciocalteu reagent and 4 mL of sodium carbonate (75
g/L) and was incubated for 1 hour in the dark at room 2 shown that A. paniculata extracts gave the higher DPPH
temperature. The absorbance was measured at 765 nm against a scavenging activity (89.83) compared to S. mahagoni (60.77%).
These results may explained by the fact that these extract enriched
reagent blank (containing all test reagents except for sample). The
with phenolic compounds which always play an important role in
TPC was calculated according to a standard curve. The
concentration of total phenolic compounds in the extract was the antioxidant activity of the plant15.
expressed as milligrams of gallic acid equivalent per gram of dry
3.3 Total Phenolic Content (TPC) of S. Mahagoni and A.
weight (mg GAE/g) of extract.
Paniculata

Total phenolic content (TPC), as determined by the Folin-

3.0 RESULTS AND DISCUSSION Ciocalteu method, was reported as gallic acid equivalents (mg
GA/g sample). This analysis was used to investigate contribution
3.1 Percentage Yield of S. mahagoni and A. paniculata in antioxidant activity of the plant extracts. The total phenolic
content extract is shown in Figure 3.
The result in Figure 1 shows the difference between the
TPC in S. mahagoniextract was found given better content of
percentage of extraction yield for S.mahagoni and A. paniculata.
phenolic compound (55 mg GAE/g sample) compared to the
Generally, the extraction of A.paniculatagave higher percentage
A.paniculataand it is well-matched with the result previously
yield (19.94%) compared to S. mahagoni (11.86%). The selection
since it has higher percentage yield.
of suitable solvent to extract the desired compounds should be
Meanwhile, A.paniculata had lower phenolic content (7.78
considered thoroughly since the extracted compound will be
mg GAE/g sample) in the extracts. The methanol 70% extract
depended on the type of solvent used11. A polar solvent will
shows significant amount of phenolic compound in the
favour polar compound more and vice versa, thus the differences
S.mahagoni extract. This might be contributed by the
of solvents used will defer the extracts and their
antioxidative activities of this extract. Phenols and polyphenolic
composition.Based on the findings, the extract had the highest
compounds, such as the flavonoid, are widely found in food
percentage yield when 70% of methanol was used as solvent.
products derived from plant sources, and they have been proven to
Polar solvents could extract andrographolide at higher yield
have significant antioxidant activities10.
and methanol was found to be the best solvent for the extraction
Basically, both of the extract contain high amount of
of andrographolide12. S. mahagoni seeds are non-polar because
phenolic compound and it is useful for the prevention of oxidative
most of the yield extract from seeds contained fatty acid mostly.
activities of the plants’ extracts.
53 Liza Md Salleh et al. / Jurnal Teknologi (Sciences & Engineering) 69:4 (2014), 51–53
Figure 2 DPPH free radical scavenging activity of S. mahagoniand A.
paniculata
Figure 3 Total Phenolic Content of S. mahagoniand A. paniculata
4.0 CONCLUSION
The methanolic extracts (70%) of S. mahagoni seed and A.
paniculata leaves extract contained total phenolic compounds and
were capable of inhibiting, quenching free radicals to terminate
the free radical chain reaction, and acting as reducing agents.
Acknowledgement
The authors gratefully acknowledge the financial support by
Ministry of Agriculture and Agro-based Industry Malaysia
(MOA) and acknowledgement is also extended to MOSTI,
UniversitiTeknologi Malaysia (UTM) and Vote no (4H013) for
the use of laboratory instruments and research grant during this
study.
References
[1] Chen, Y. Y., X. N. Wang, C. Q. Fan, S. Yin and J. M. Yue. 2007.
Swiemahogins A and B, Two Novel Limnoids from Swietenia
mahogany. Tetrahedron Letters. 48: 7480–7484.
[2] Bacsal, K., L. Chavez, I. Diaz, S. Espina, J. Javillo, H. Manzanilla, J.
Motalban, C. Panganiban, A. Rodriguez, C. Sumpaico and B. Talip and
S. Yap. 1997. The Effect of Swietenia mahagoni (Mahogany) Seed
Extract on Indomethacin-induced Gastric Ulcers in Female Sprague-
dawley Rats. Acta Medica Philippina. 3: 127–139.
[3] Guevara, A. P., A. Apilado, H. Sakurai, M. Kozuka and H Tokuda. 1996.
Anti-Inflammatory, Antimutagenic And Antitumor Promoting Activities
of Mahogany Seeds, Swietenia macrophylla (Meliaceae). Philippine
Journal of Science. 12: 5271–278.
[4] Ardahe, S. S., M. A. Abdulla, S. A. Razak, F. A. Kadir and P.
Hassandarvish. 2010. Gastroprotective Activity of Swietenia Mahagoni
Seed Extract on Ethanol-Induced Gastric Mucosal Injury in Rats. World
Academy of Science, Engineering and Technology. 67.
[5] Wongkittipong, R., L. Prat, S. Damronglerd and C. Gourdon .2000.
Solid-liquid extraction of andrographolide from plants-experimental
study, kinetic reaction and model. Separation and Purification
Technology. 40: 147–154.
[6] Calabrese, C., S. H. Berman and J. G. Babish. 2000. A Phase I Trial of
Andrographolide in HIV Positive Patients and Normal Volunteers.
Phytotherapy Research. 14: 333–338.
[7] Choudhury, B. R., S. J. Haque and M. K. Poddar. 1987. In Vivo and in
Vitro Effects of Kalmegh (Andrographis paniculata) Extract and
Andrographolide on Hepatic Microsomal Drug Metabolizing Enzymes.
Planta Medica. 53: 135–140.
[8] Rajani, M., N. Shrivastava and M. N. Ravishankara. 2000. A Rapid
Method for Isolation of Andrographolide from Andrographis Paniculata
Nees (Kalmegh). Pharmaceutical Biology. 38: 204–209.
[9] Miliauskas, G., P. R. Venskutonis and T. A. van Beek. 2004. Screening
of Radical Scavenging Activity of some Medicinal and Aromatic Plant
Extracts. Food Chemistry. 85: 231–237.
[10] Sahgal, G., S. Ramanathan, S. Sasidharan, M. N. Mordi, S. Ismail and S.
M. Mansor. 2009. In Vitro Antioxidant and Xanthine Oxidase Inhibitory
Activities of Methanolic Swietenia mahagoni Seed Extracts. Molecules.
14: 4476–4485.
[11] Zarnowski, R. and Y. Suzuki. 2004. Expedient Soxhlet Extraction of
Resorcinolic Lipids from Wheat Grains. Journal of Food Composition
and Analysis. 17: 649–664.
[12] Avanigadda, S. and M. Vangalapati. 2010. Experimental and Modelling
Studies of Andrographolide Extraction from Andrographis Paniculata.
International Journal of Chemical, Environmental and Pharmaceutical
Research. 1(1): 32–36.
[13] Majid, M. A., I. M. M. Rahman, M. A. H. Shipar, M. Helal Uddin and R.
Chowdhury. 2004. Physico-Chemical Characterization, Antimicrobial
Activity and Toxicity Analysis of Swietenia Mahagoni Seed Oil.
International Journal of Agriculture and Biology. 6(2): 350–354.
[14] Rodtjer, A., L. H. Skibsted and M. L. Andersen. 2006. Antioxidative and
Prooxidative Effect of Extracts Made From Cherry Liqueur Pomace,
Food Chemistry. 99: 6–14.
[15] Pourmorad, F., S. J. Hosseinimehr and N. Shahabimajd. 2006.
Antioxidant Activity, Phenol and Flavonoid Contents of Some Selected
Iranian Medical Plants. African Journal of Bioechnology. 5(11): 1142–
1145.