www.ejlst.

com European Journal of Lipid Science and Technology

Research Article
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Characterization of Virgin Avocado Oil Obtained via Advanced Green Techniques†

Running title: Virgin avocado oil characteristics

Chin Xuan Tana, Gun Hean Chongb, Hazilawati Hamzahc, Hasanah Mohd Ghazalia,*
a
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia
c
Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
*Corresponding author. E-mail: hasanah@upm.edu.my, Tel: +603-89468345,

Fax: +603-12 89423552

†This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences between
this version and the Version of Record. Please cite this article as doi: [10.1002/ejlt.201800170].

© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: April 4, 2018 / Revised: July 26, 2018 / Accepted: July 31, 2018

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Abstract
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The quality characteristics, bioactive phytochemicals, volatile compounds and antioxidant

capacities of virgin avocado oil extracted using a couple of green methods, namely, subcritical

CO2 extraction (SCO2) and ultrasound-assisted aqueous extraction (UAAE), were compared

with the oil extracted using the conventional solvent extraction. Results indicated the quality

properties of avocado oil were unaffected by extraction methods. The total phenolic content of

avocado oil was in the range of 111.27-130.17 mg GAE/100 g and the major phytosterol was

β-sitosterol (1.91-2.47 g/kg). Avocado oil extracted using SCO2 exhibited two to four times

greater levels of α- and γ-tocopherols than solvent extraction and UAAE. The volatile

components associated with nutty and grassy flavors were only detected in avocado oil

extracted under low-temperature extraction conditions such as SCO2 and UAAE. Based on the

antioxidant capacity tests, avocado oil obtained by SCO2 exhibited the strongest antioxidant

capacity compared with solvent extraction and UAAE.

Practical Applications

Virgin avocado oil extracted using subcritical CO2 method exhibits superior levels of

tocopherols content and antioxidant capacity than ultrasound-assisted aqueous and

conventional solvent methods. Subcritical CO2 is a promising oil extraction technique to obtain

solvent-free avocado oil with potential uses in functional foods and health care products.

Keywords: Avocado oil, Antioxidant capacity, Bioactive phytochemical, Subcritical CO2

extraction, Ultrasound-assisted aqueous extraction

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1. Introduction
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Avocado (Persea americana Mill) is an evergreen fruit crop belonging to the Lauraceae family

that grows in tropical or subtropical climates countries such as Australia, Mexico, Malaysia

and Indonesia. In 2016, the largest avocado producing country in the world was Mexico (1889

thousand tons), followed by Dominican Republic (601 thousand tons) and Peru (455 thousand

tons) [1]. The edible pulp constitutes the biggest portion of the fruit (65%) whereas the seed

(20%) and the peel (15%) constituted the rest [2]. Avocado fruit is also known as ‘butter fruit’

or ‘vegetable butter’ as the fleshy pulp is rich in lipid content. Avocado oil is a functional oil

extracted from the fleshy pulp, which has been widely used in food supplement, cosmetic and

healthcare products.

Plant oils are sources of phytosterols, tocols and unsaturated fatty acids. These components

are able to modulate the metabolic processes, thereby reducing the risk of certain chronic

diseases such as arthritis, diabetes, obesity, hypercholesterolemia, cancer and cardiovascular

disease [3–5]. Numerous studies on the health benefits of avocado oil consumption have been

conducted. Up to now, avocado oil has been scientifically proven for its effectiveness on

osteoarthritis treatment [4], wound healing [6] and anti-diabetic [3]. The hypocholesterolemic

and hepatoprotective properties of avocado oil have been reported in our recent study [7]. These

associated health benefits are attributable to the properties of avocado oil where it does not

contain cholesterol and trans-fatty acids, while rich in unsaturated fatty acids (oleic and

linolenic acids), tocopherols and bioactive phytochemicals such as phytosterols, carotenoids

and polyphenols.

Extractions of avocado oil using organic solvents [8–10], centrifugation force [11,12],

supercritical fluid [9,13] and cold-pressing [2,14] have been studied extensively in the past.

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However, chemical properties of the oil obtained following different extraction methods,
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particularly the qualities, bioactive phytochemicals, volatile components and antioxidant

capacities, have not been investigated. Solvent extraction using a Soxhlet extractor is the

standard technique and the main reference for evaluating the performance of the alternatives

[15]. Utilization of organic solvents in extracting edible oil has triggered safety concerns

among consumers due to trace amounts of solvent residues in the oil. The presence of hexane,

benzene or toluene residues in commercial plant oils has been reported by Ligor and Buszewski

[16]. These solvent residues may induce adverse health effects after long-term consumption.

In this context, green extraction techniques are currently being explored to replace the

conventional methods for edible oil extraction.

Recently, subcritical CO2 extraction (SCO2) and ultrasound-assisted aqueous extraction

(UAAE) have emerged as promising green techniques in edible oil extraction [17]. Both

techniques utilize green solvents (CO2 for SCO2 and water for UAAE) to extract the oils from

plant sources. Virgin avocado oil is a specialty oil extracted via natural or mechanical

approaches at low temperatures (<50 ºC) and without undergoing chemical refining [14,18].

The extraction conditions used in SCO2 and UAAE fulfilled the stated requirements and hence,

can also be classified as virgin avocado oil.

Previous studies indicated that solvents, temperatures and time used in oil manufacturing

process can significantly affect the qualities, bioactive phytochemicals, volatile components

and antioxidant capacities of plant oils [19–22]. There is an increased interest in the

development of oil extraction technologies that preserve the nutritional characteristics of virgin

avocado oil. Green extraction methods like SCO2 and UAAE are recently proposed as new

alternatives for conventional solvent method and suitable for edible oils production with no

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need for additional refining [17]. The attention has been directed to the use of such methods
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for the extraction of virgin avocado oil for their feasibility in pharmaceutical and food

industries. Consolidation of virgin avocado oil extraction sustainable technologies may

enhance the utilization of this functional oil. As the qualities, bioactive phytochemicals, volatile

components and antioxidant capacities of oils are greatly influenced by extraction conditions,

a study was conducted to analyze the extent of these parameters affecting avocado oil. This

was a prosecution of our previous work and conventional solvent extraction using a Soxhlet

extractor served as the control method.

2. Materials and Methods

2.1 Sample Preparation

Avocado fruits were harvested at commercial maturity from Universiti Putra Malaysia campus,

Malaysia in February 2016. Fruits were ripened at room temperature (25 ± 5ºC) until changes

in peel color (from green to red) and soft texture (where the fruits yielded slightly under the

pressure of a finger) occurred. The fruits were cut into halves and the pulp was manually

segregated from the seed and the peel. The pulp was dehydrated in an oven (Memmert UFB

400, Schwabach, Germany) at 35 ºC to constant weight. The dehydrated avocado pulp was

ground into powder using a home blender (Panasonic MS801S, Petaling Jaya, Malaysia) and

sieved through a stainless-steel sieve (360 µm).

2.2 Chemicals and Reagents

Analytical reagent grade of hexane, ethanol, petroleum ether, ethyl acetate and chloroform and

HPLC grade of methanol were purchased from Fisher Scientific (Pittsburgh, US). Potassium

hydroxide, sodium chloride, pyrogallol, 2’,7’-dichlorofluorescein, campesterol, Folin-

Ciocalteu reagent, sodium carbonate, gallic acid, β-carotene (97%), sodium sulfate, 5α-

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cholestane, Tri-Sil reagent, linoleic acid, tween 20, 2, 2’-azino-bis-3-ethylbenzothiazoline-6-

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sulphonic acid (ABTS), potassium peroxodisulfate, trolox, 2, 4, 6-Tris (2-pyridyl)-s-triazine

(TPTZ), sodium acetate and ferric chloride were purchased from Sigma-Aldrich (St. Louis,

US). Acetic acid and hydrochloric acid (37%) were purchased from J.T. Baker (New Jersey,

US).

2.3 Oil Extractions

2.3.1 Solvent Extraction

Solvent extraction was carried out using a Soxhlet extractor based on AOAC 920.39 method

[23]. Approximately 10 g of avocado powder was measured into a cellulose paper cone and

extracted with 200 mL of hexane for 8 hours at 70 ºC. The hexane was removed using a rotary

evaporator (Eyela N-1001S-W, Tokyo, Japan) at 70 ºC. To remove residual hexane, the oil was

dried in an oven at 70°C for 15 min. Oil extraction was repeated six times. The oil was stored

in sealed dark bottle at -20 ºC until use.

2.3.2 Subcritical CO2 Extraction (SCO2)

Subcritical CO2 extraction (SCO2) was carried out using a SCO2 instrument (FeyeCon

Development, Weesp, Netherland) consisted of three units, namely, extractor, reboiler and

condenser. This instrument operated at the fixed temperature (27 ºC) and pressure (68 bar).

Preliminary work indicated the highest yield was obtained at 7.5 hours when the 1 mL extractor

unit was loaded with 300 g of avocado powder [17]. The carrier solvent was 99.99% carbon

dioxide with the flow rate of 1.2 kg/min. A complete cycle extraction (approximately 3 mins)

was achieved when the carbon dioxide gas from the reboiler unit flowed back into the

condenser unit and condensed into liquid carbon dioxide. The oil was collected via the valve.

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Oil extraction was repeated three times. The oil was stored in sealed dark bottle at -20 ºC until
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use.

2.3.3 Ultrasound-Assisted Aqueous Extraction (UAAE)

Ultrasound-assisted aqueous extraction (UAAE) was carried out using the method developed

by Tan et al. [24]. Approximately 10 g of avocado powder was mixed with 60 mL of distilled

water and sonicated in an ultrasonic water bath (Thermo-10D, Waltham, US) at 35 ºC for 30

mins. The ultrasonic water bath was operated at a constant frequency (40 kHz) and output

power (240W). Subsequently, the mixture was pressed against a laboratory scale screw press

to obtain an aqueous-oil mixture and centrifuged at 1500 ×g for 20 mins at room temperature

to obtain the top oil layer. Oil extraction was repeated six times. The oil was stored in sealed

dark bottle at -20 ºC until use.

2.4 Determination of Quality Characteristics

Peroxide value (Cd 8-53) and p-anisidine value (Cd 18-90) were measured using the AOCS

official methods [25]. Total oxidation (Totox) value was calculated using the equation of Totox

= (2 × peroxide value) + p-anisidine value [26]

2.5 Determination of Tocol Content

The tocol content was determined using the method described by Shammugasamy et al. [27]

where 1 g of oil sample was added to a test tube containing 1 mL of 60% (w/v) potassium

hydroxide, 1 mL of 95% (w/v) ethanol, 1 mL of 1% (w/v) sodium chloride and 2.5 mL of 6%

(w/v) ethanolic pyrogallol. After incubation at 70 ºC for 45 min, 7 mL of 1% (w/v) sodium

chloride was added. The mixture was extracted twice with 10 mL of hexane-ethyl acetate (9:1,

v/v) using a separating funnel. The organic phases were pooled and washed with excess 1%

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(w/v) sodium chloride. A rotary evaporator (Eyela N-1001S-W, Tokyo, Japan) operated at 50
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ºC was used to remove the organic phase before addition of 8 mL of methanol and filtered

through a 0.2 µm nylon syringe filter (Sartorius, Gottingen, Germany).

Chromatographic analysis was performed on a high-performance liquid chromatography

(Agilent 1200, Santa Clara, US) equipped with a fluorescence detector (Agilent 1260, Santa

Clara, US) on a Kinetex pentafluorophenyl phase column (250 × 4.6 mm, with a particle size

of 5 µm; Phenomenex, Torrance, US). The injection volume was 20 µL and the column

temperature was 30 ºC. The mobile phases used were methanol and deionized water eluted in

gradient mode whereby at 0-15 min, the methanol (85%) increased linearly to 95% and held

isocratic for 3 min before returning to 85% within 2 min at constant flow rate of 1 mL/min.

The fluorescence detector was performed at an excitation wavelength of 290 nm and an

emission wavelength of 330 nm. The tocol content of samples was identified by retention time

and quantified using calibration curves of tocopherol (α, β, γ, and δ) and tocotrienol (α, β, γ,

and δ) standards.

2.6 Determination of Total Phenolic Content

The total phenolic content was carried out using the method of Singleton and Rossi [28] with

some modification described by Marathe et al. [29]. Initially, 0.25 g of oil was mixed with 5

mL of methanol and vigorously vortex for 1 min. The mixture was then centrifuged at room

temperature at 750 ×g for 10 min. The top layer (600 µL) was transferred to a test tube

containing 300 µL of 1 N Folin-Ciocalteu reagent and 600 µL of 2% (w/v) sodium carbonate

solution. The test tube was placed in the dark for 30 min at room temperature prior to measuring

the absorbance at 750 nm using a spectrophotometer (Thermo Scientific Genesys 10S,

Waltham, US) against a blank (80% methanol). A calibration curve was constructed using

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gallic acid as the standard and the total phenolic content of samples was expressed as
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milligrams of gallic acid equivalents per 100 g of oil (mg GAE/100 g oil).

2.7 Determination of Total Carotenoid Content

The total carotenoid content was determined using the method of Nehdi et al. [30]. Initially, β-

carotene was dissolved in hexane to construct a calibration curve in the range of 0-2.5 µg/mL.

Subsequently, the oil sample was diluted with hexane to an absorbance within the range of the

calibration curve. The absorbance was read at 440 nm against a blank (hexane). The total

carotenoid content of samples was expressed as milligrams of β-carotene per kg of oil (mg β-

carotene/kg oil).

2.8 Determination of Phytosterol Content

The phytosterol content was analysed using the method of Caligiani et al. [31]. Briefly, 2.5 g

of oil sample was saponified with 50 mL of 0.5 M ethanolic potassium hydroxide at 70 ºC for

an hour. Next, 50 mL of distilled water was added and the mixture was transferred to a

separating funnel and extracted three times with 25 mL of petroleum ether. The three ether

phases were pooled into a separating funnel and washed six times with 80 mL of distilled water.

To obtain the unsaponifiable fraction, the ether phase was dried over anhydrous sodium sulfate

and the solvent was removed using a rotary evaporator at 60 ºC. The unsaponifiable fraction

was then dissolved with 800 µL of hexane and spotted on a thin-layer chromatography (TLC)

silica gel 60 F254 plate (Merck, Darmstadt, Germany). To correctly identify the sterol band, a

reference sterol (campesterol) was spotted on the plate. The TLC plate was developed with

hexane-diethyl ether (1:1, v/v). After spraying with 2’,7’-dichlorofluorescein and viewed under

ultraviolet light, the sterol band was identified (on the basis of the reference spot). The sterol

band was scrapped off from the plate and extracted two times with 10 mL of hexane-ethyl

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acetate (1:1, v/v) for 30 mins under magnetic stirring. The solvent phases were pooled into a
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round bottom flask and evaporated to dryness using a rotary evaporator at 60 ºC. Then, 100 µL

of 5α-cholestane solution (internal standard) was added to the flask before derivatization with

130 µL Tri-Sil reagent at 60 ºC for an hour.

The GC-MS analysis was performed on a Shimadzu gas chromatography 2010 Plus system

equipped with a QP2010 Ultra mass selective detector (Shimadzu, Kyoto, Japan). The injection

volume was 1 µL and split injection mode (1:50) was applied. A BPX5 capillary column (30

m × 0.25 mm, with a film thickness of 0.25 µm; SGE, Melbourne, Australia) was used. The

carrier gas was 99.99% helium with an inlet pressure of 37.1 kPa at a column flow of 0.8

mL/min. The column temperature was initially 50 ºC, held for 2 mins and then increased at the

rate of 3 ºC/min to 300 ºC and held for 10 mins. Both the injection and ionization temperatures

were set at 250 ºC and the scan mode for the mass selective detector was 40-700 m/z. The

chromatogram was processed using GCMS solution software version 2.6 (Shimadzu, Kyoto,

Japan). The phytosterol components were identified based on mass spectra comparison with

the Wiley 299 library (John Wiley & Sons, New York, US). The concentration of each

phytosterol component in the oil was calculated using the equation as below:

Peak area of phytosterol × milligram of internal standard

Phytosterol (mg/100g) = × 100
Peak area of internal standard × gram of oil

2.9 Profiling of Volatile Compounds

The volatile content was analysed using the method described by Ligor and Buszewski [16]

with modifications. About 0.5 g of oil sample was placed in a 22 mL headspace vial (Sigma-

Aldrich, St. Louis, US), sealed with a hand crimper (Sigma-Aldrich, St. Louis, US), and

equilibrated at 95 ºC for 15 mins in a headspace sampler (Shimadzu 2010 Plus, Kyoto, Japan)

connected to a GC-MS (Shimadzu, Kyoto, Japan). A sampling needle (Shimadzu AOC-20i,

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Kyoto, Japan) was injected through the vial septum and left in the headspace at 95 ºC for 25
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mins to collect the vapour. The injection mode was splitless. A BPX5 capillary column (30 m

× 0.25 mm, with a film thickness of 0.25 µm; SGE, Melbourne, Australia) was used. The

column temperature was initially 40 ºC, held for 8 mins and then increased at the rate of 5

ºC/min to 200 ºC and held for 10 mins. The carrier gas was 99.99% helium with an inlet

pressure of 49.5 kPa at a column flow of 1 mL/min. Both injection and ionization temperatures

were set at 250 ºC and the scan mode for the mass selective detector was 40-450 m/z. The

chromatogram was processed using GCMS solution software version 2.6 (Shimadzu, Kyoto,

Japan). The volatile components were identified by mass spectra comparison with the Wiley

299 library (John Wiley & Sons, New York, US).

2.10 Determination of Antioxidant Capacities

2.10.1 Sample Extraction

About 1 g of oil sample was mixed with 3 mL of methanol and vigorously vortex for 1 min.

The mixture was then centrifuged at 750 ×g for 10 min at room temperature to obtain the

supernatant layer (methanol extract). The residue was re-extracted twice following the same

procedure. The three methanol extracts were pooled and the final volume was brought to 10

mL using methanol.

2.10.2 Determination of β-Carotene Bleaching Assay

The β-carotene bleaching assay was determined using the method of Sarikurkcu et al. [32] with

modifications. Initially, 1000 µL of β–carotene solution (0.5 mg/mL chloroform), 25 µL of

linoleic acid and 250 µL of tween 20 were mixed in a round bottle flask. The chloroform in the

mixture was removed using a rotary evaporator (Eyela N-1001S-W, Tokyo, Japan) at 40 ºC.

The residue was then added with 100 mL of distilled water and shaken vigorously to form an

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emulsion. Exactly 2000 µL of the emulsion was pipetted into a test tube containing 100 µL of
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the sample extract or control (methanol). As soon as the emulsion was added to the test tube,

the absorbance at the initial time (t = 0 min) was measured against a blank (distilled water) at

490 nm. The test tube was then incubated at 50 ºC and subsequent absorbance readings were

measured at the time intervals of 20, 40, 60, 80, 100 and 120 min. Positive control (α-tocopherol)

of the same concentration as the samples was used for comparison. The antioxidant activity

(AA) with respect to the successful bleaching of β-carotene at t = 120 min was calculated using

the following equation:

AA (%) = (DR of control – DR of sample)/ DR of control × 100

Where degradation rate (DR) of control and sample are calculated using the following equation:

DR = log (absorbance at t = 0 min/ absorbance at t = 120 min) × (1/120)

2.10.3 Determination of Trolox Equivalent Antioxidant Capacity (TEAC)

The trolox equivalent antioxidant capacity (TEAC) was carried out using the method of Re et

al. [33]. The ABTS radical cation (ABTS•+) stock solution was generated by mixing 7 mM of

ABTS powder with 2.55 mM of potassium peroxodisulfate in 10 ml of deionized water and

incubated in the dark for 16 hours at room temperature. The working solution was prepared by

diluting the ABTS•+ stock solution with absolute ethanol to an absorbance of 0.70 ± 0.05 at

734 nm. Exactly 100 µL of the sample extract was mixed with 1000 µL of diluted ABTS•+

solution. After incubating in the dark for 6 min at room temperature, the absorbance of the

mixture was measured against a blank (ethanol) at 734 nm. Positive control (α-tocopherol) of

the same concentration as the samples was used for comparison. A calibration curve was

prepared using trolox as standard and the TEAC of samples was expressed as millimolar of

trolox equivalent per kg of oil (mM TE/kg oil).

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2.10.4 Determination of Ferric-Reducing Antioxidant Power (FRAP)

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The ferric-reducing antioxidant power (FRAP) was determined according to the method of Tan,

Prasad and Ismail [34]. The FRAP reagent was freshly prepared by mixing 2.5 mL of 10 mM

TPTZ in 40 mM hydrochloric acid with 25 mL of 300 mM acetate buffer (pH 3.6) and 2.5 mL

of 20 mM ferric chloride solution. The FRAP reagent (1500 µL) was pipetted into a test tube

containing 100 µL of the sample extract and 150 µL of distilled water. After incubation in the

dark for 4 min at room temperature, the absorbance of the mixture was measured against a

blank (distilled water) at 593 nm. A calibration curve was prepared using trolox as standard

and the FRAP of samples was expressed as millimolar of trolox equivalent per kg of oil (mM

TE/kg oil).

2.11 Statistical Analysis

Statistical analysis was carried out using SPSS version 20 software (IBM Corp., Armonk, New

York). One-way analysis of variance (ANOVA) accompanied with Tukey’s post hoc were used

to determine the differences among the mean values. Mean values refer to three analytical

replicates and extraction replicate samples were pooled before analytical determination.

Pearson correlation test was used to evaluate the association between the antioxidant capacities

and bioactive phytochemicals. The level of significance was set at p<0.05.

3. Results and Discussion

3.1 Quality Characteristics

Peroxide, p-anisidine and total oxidation (Totox) values, commonly known as oxidation

indices, are used to measure the quality of edible oil [26]. Oil oxidation is a complex process

initiated by free radical reactions at the double bonds of unsaturated fatty acids. Avocado oil is

prone to oxidation due to its high degree of unsaturation (>65%) [24]. The recommended

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indices of oxidation markers for peroxide, p-anisidine and Totox values are less than 5 meq/kg,
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less than 20 and less than 26, respectively [26]. Current study indicated the peroxide (2.73-2.82

meq/kg), p-anisidine (2.82-3.03) and Totox (8.28-8.66) values (Table 1) of avocado oil were

unaffected by extraction methods and corresponds at the recommended indices of oxidation

ranges. These observations oppose to Samaram et al. [35], who reported that the oxidation

indices of highly unsaturated-papaya seed oil were affected by extraction methods. Prescha et

al. [36] reported higher peroxide (4.42-15.74 meq/kg) and p-anisidine (5.83-8.90) values in

cold-pressed avocado oil as compared to the present study.

3.2 Tocol, Phenolic and Carotenoid Contents

The tocols of plant oils exist in the form of tocopherols and tocotrienols. Both play an important

role as natural antioxidants to prevent the formation of free radicals and to delay the process of

autocatalytic lipid peroxidation. In the food industry, the stability and shelf life of food products

can be enhanced by the use of tocols. The tocol composition of avocado oil extracted using

solvent, SCO2 and UAAE is shown in Table 2. Within the experimental conditions, no

tocotrienols were detected. A similar observation was reported by Wong et al. [14]. Current

study detected two types of tocopherol components in avocado oil, namely, α-tocopherol and

γ-tocopherol. In contrast, Wong et al. [14] detected trace amounts (<10 mg/kg) of β- and δ-

tocopherols in cold-pressed avocado oil. The difference may have arisen from the analytical

methods used in determining the tocol components. Results indicated the tocol composition of

avocado oil was significantly affected (p<0.05) by extraction methods, whereby the contents

of α- and γ-tocopherols of SCO2-extracted oil were two to four times greater than solvent- and

UAAE-extracted oils, respectively. The superiority of SCO2 in extracting the tocol components

from plant materials was reported by Chia et al. [20], in which their study demonstrated the

tocols content of SCO2-extracted rice bran oil was ten times greater than solvent-extracted rice

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bran oil. Low level of tocopherols content in UAAE-extracted oil may be the poor solubility
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of non-polar tocopherols in a polar solvent (e.g. water). Compared to other plant oils, the total

tocol content of avocado oil (83.34-256.31 mg/kg) was comparable to extra-virgin olive oil

(130-190 mg/kg), but lower than supercritical CO2-extracted grape seed oil (355-559 mg/kg)

[37,38].

The stability and nutritional quality of plant oils are influenced by the levels of phenolic

content as phenolic compounds can act as antioxidants and prevent lipids peroxidation. The

total phenolic content (TPC) as determined using Folin-Ciocalteu colorimetric method

indicated that the UAAE-extracted oil (130.17 mg GAE/100 g) contained the highest TPC,

followed by solvent-extracted oil (128.17 mg GAE/100 g) and SCO2-extracted oil (111.27 mg

GAE/100 g) (Table 2). Although UAAE-extracted oil had the highest value, no significant

difference was observed between the TPC of solvent- and UAAE-extracted oils. This

observation partially agrees with Kozłowska et al. [39], who stated that the use of a polar

solvent during oil extraction able extract a greater amount of phenolic content from the plant

materials. Previously, the TPC of mechanically-pressed avocado oil and commercial avocado

oil were reported to be 46.07-77.36 mg GAE/100 g and 4.26-5.69 mg GAE/100 g, respectively

[10,40]. These values were much lower than the present study. The differences may be

explained by the extraction solvents used in the TPC analysis. Methanol is a preferred solvent

for TPC extraction compared with 50% acetone or 70-80% ethanol owing to its better

solubilization of lipophilic phenolic antioxidants [41]. It should be noted that some lipid

components (e.g. phospholipids) can react with the Folin-Ciocalteu reagent, resulting in the

overestimation of phenolic contents [42].

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Carotenoids are natural pigment present in the plant oils. It constitutes the orange, red and
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yellow colors of the oil. Apart from being vitamin A precursors, carotenoids also serve as

antioxidants to prevent free radical chain reactions. In Table 2, the total carotenoid content

(TCC) of solvent-extracted oil (5.24 mg β-carotene/kg) was significantly higher (p<0.05) than

SCO2- and UAAE-extracted oils (4.27 and 3.08 mg β-carotene/kg, respectively). A similar

observation was reported by Kiralan et al. [21], whereby the TCC of solvent-extracted black

cumin seed oil was two folds greater than cold-pressed black cumin seed oil. This can be

attributed to the good solubility of carotenoids in organic solvents such as hexane, petroleum

ether and ethyl acetate [43]. Krumreich et al. [10] reported very much higher of TCC (75.00-

104.62 mg β-carotene/kg) in mechanically-pressed avocado oil than the present study. In

contrast, Wong et al. [14] reported the TCC of cold-pressed avocado oil was 1.0-3.5 mg/kg.

Generally, the concentration of carotenoids in the plant oils is heavily influenced by extraction

methods, plant cultivar and climatic conditions [30].

3.3 Phytosterol Composition

Phytosterols are cholesterol-like components found in plant origin foods. It plays an important

role in inhibiting the intestinal cholesterol absorption, inclusive of circulating endogenous

biliary cholesterol, a key step of cholesterol elimination [44]. Piironen et al. [45] examined the

phytosterol content of several local fruits in Finland. Their study showed the phytosterol

content of avocado pulp (0.75 g/kg fresh fruit weight) was superior to the edible parts of apple,

banana, grape, kiwi, orange and plum, with a range value of 0.12-0.23 g/kg fresh fruit weight.

Phytosterols are lipid-soluble phytochemicals and it represents a major portion of the

unsaponifiable fraction of plant oils [46]. The phytosterol content of plant oils is stable during

storage and its concentration is slightly affected by cultivation conditions [46]. Table 3 shows

the phytosterol composition of avocado oil extracted using solvent, SCO2 and UAAE.

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Regardless of the extraction methods, β-sitosterol (1.91-2.47 g/kg) was the main sterol in the
Accept e d Article
avocado oil, which comprised about 66.39-73.75% of the total phytosterol content. This is in

accordance with Wong et al. [14], who reported the major phytosterol present in cold-pressed

avocado oil was β-sitosterol (2.23-4.48 g/kg). Compared to other plant sterols, the

physiological benefits of β-sitosterol on human health have been intensively investigated. For

instance, β-sitosterol has been scientifically proven for its effectiveness in reducing cholesterol

and antidiabetic properties [44].

Berasategi et al. [47] reported the total phytosterol content of commercial avocado oil to be

3.39 g/kg, which is in good agreement with the present study (2.59-3.60 g/kg). The total

phytosterol content of UAAE-extracted oil was significantly lower (p<0.05) than solvent- and

SCO2-extracted oils (Table 3). Previous study reported the total phytosterols of Araucaria

angustifolia seed oil obtained by subcritical propane extraction (8.78-9.33 g/kg) were greater

than solvent extraction (5.99 g/kg) [22]. However, a similar trend was not observed in the

current study. The difference can be attributed to the extracting solvents used. Generally, the

use of CO2 in food processing is preferable because of its non-toxic, inert and low-cost

properties.

3.4 Volatile Composition

A direct static headspace approach was used to determine the volatile composition of avocado

oil and the identified compounds are listed in Table 4. The desirable volatiles associated with

the nutty and grassy flavors were only detected in avocado oil extracted under low-temperature

conditions such as SCO2 and UAAE. Conversely, the use of high temperature in solvent

extraction leads to the degradation of these volatile components. As the quantitative analysis

was not carried out, it is not possible to assess whether the detected compounds were able to

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confer the corresponding flavours to the oil. In Figure 1, residual solvent peaks were detected
Accept e d Article
in the solvent-extracted oil despite using both rotary-evaporation and oven drying to remove

the solvent (hexane) used. This observation suggests the necessity of solvent-extracted oil to

undergo a refining process before consumption. Deodorization is a stripping stage of oil

refining, where the volatile odoriferous components (e.g. residual solvents) are removed.

However, the use of high temperatures (>200 ºC) in deodorization can drastically reduce the

heat-labile bioactive phytochemicals such as phenolics, carotenoids and phytosterols [48]. As

consumers become more concern over the safety of using organic solvents in food processing,

it is thus, important to use green solvents (e.g. CO2 and water) to extract edible oils.

By using solid-phase microextraction coupled with GC-MS, López et al. [49] detected six

volatile components (acetic acid, etanol, 3-methyl-butanol, pentanol, 3-hydroxy-2-butanone

and (E)-2-heptenal) in fresh avocado pulp while Haiyan et al. [50] detected nine volatile

components (acetic acid, pentanal, hexanal, (E)-2-hexenal, 1-hexanol, α-piene, β-piene, 3-

carene and nonanal) in cold-pressed avocado oil. In contrast, greater amounts of volatile

components detected in the current study may be due to the different analytical methods and

conditions used to determine the volatile components as well as the varietal difference of

avocados and the oil manufacturing conditions (e.g. extracting solvents, time and temperature).

3.5 Antioxidant Capacities

3.5.1 β-Carotene Bleaching Assay (BBA)

In this assay, free radicals are generated through the removal of hydrogen atom from the

diallylic methylene groups of linolenic acid at 50 ºC [51]. The liberated free radicals will

rapidly oxidize the β-carotene and the presence of antioxidants in the sample able to prevent or

reduce the extent of β-carotene oxidation. Figure 2 shows the changes in absorbance value of

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avocado oil extracted using different methods, control (methanol) and α-tocopherol (positive
Accept e d Article
control) using β-carotene bleaching assay (BBA). The reduction of absorbance over time is due

to the degradation of β-carotene in the assay. In Figure 2, the absorbance value of the control

reduced drastically throughout the experiments, indicating the degradation rate of β-carotene

was substantial and fast in the absence of antioxidants. Meanwhile, the presence of antioxidant

components in the oil samples is able to prevent or reduce the degradation rate of β-carotene.

The above observations are related by their respective antioxidant activity shown in Table

5, in which the percentage of antioxidant activity decreased in the order of α-tocopherol >

SCO2-extracted oil > solvent-extracted oil > UAAE-extracted oil. Regardless of the extraction

methods, the antioxidant activity of avocado oil was significantly lower (p<0.05) than α-

tocopherol. SCO2-extracted oil had the highest antioxidant activity as compared to solvent- and

UAAE-extracted oils, indicating a greater ability to neutralize the free radicals. Literature

comparison indicates the antioxidant activity of virgin avocado oil extracted by SCO2 (75.88%)

was greater than virgin coconut oil (71%) [52].

3.5.2 Trolox Equivalent Antioxidant Capacity (TEAC)

Trolox equivalent antioxidant capacity (TEAC) examines the capability of antioxidants to

inhibit the ABTS radical cation (ABTS•+) induced by potassium peroxodisulfate [34]. A

sample with higher antioxidant contents has a greater TEAC value [53]. In the present study,

avocado oil extracted using SCO2 (4.52 mM TE/kg) exhibited a highest TEAC value, followed

by solvent (2.99 mM TE/kg) and UAAE (1.71 mM TE/kg). There was a significant difference

(p<0.05) between the TEAC values and extraction methods. Literature comparison indicates

avocado oil (1.71-4.52 mM TE/kg) exhibits greater TEAC values than commercial corn (1.29

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mM TE/kg oil), sunflower (1.17 mM TE/kg oil) and olive (0.63 mM TE/kg oil) oils that are
Accept e d Article
reported by Pellegrini et al. [53].

3.5.3 Ferric-Reducing Antioxidant Power (FRAP)

This method involves the binding of TPTZ with ferric ion and forming a blue colored Fe (III)-

TPTZ complex at pH 3.6. The presence of antioxidants causes the reduction of Fe (III)-TPTZ

complex to dark blue colored Fe (II)-TPTZ. Theoretically, FRAP assay treats the antioxidants

as a reductant in a redox-linked colorimetric reaction [34]. Hence, the higher of antioxidants

within a sample, the greater the ferric ion is reduced to the ferrous form and a more intense of

dark blue color can be monitored. In Table 5, the FRAP value of SCO2-extracted oil (13.09

mM TE/kg) was significantly higher (p<0.05) than solvent- and UAAE-extracted oils (11.55

mM TE/kg and 6.59 mM TE/kg, respectively). Likewise, Shi et al. [54] reported the FRAP

value of roasted sesame seed oil obtained via subcritical butane extraction (2.37 mM TE/kg)

was greater than solvent extraction (1.64 mM TE/kg).

3.6 Correlations Between Bioactive Phytochemicals and Antioxidant Capacities

Correlations between bioactive phytochemicals and antioxidant capacities are statistically

analyzed and depicted in Table 6. Strong positive correlations (p<0.01) were found between

tocopherols (α and γ) and antioxidant capacities determined by BBA, TEAC and FRAP (r was

ranged from 0.835 to 0.979). Shi et al. [54] found that antioxidant capacities measured by

TEAC and FRAP were positively correlated with tocopherol of sesame oil. Another study by

Pellegrini et al. [53] reported that the high TEAC of soybean oil was due to its high tocopherols.

These findings, taking together, suggest that tocopherols are a major contributor to antioxidant

capacity in plant oils. Compared to solvent- and UAAE-extracted oils, greater antioxidant

activity observed in SCO2-extracted oil (Table 5) could have been its high tocopherols (Table

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2). Meanwhile, negative correlations were found between TPC and antioxidant capacities
Accept e d Article
determined by BBA, TEAC and FRAP (r was ranged from -0.709 to -0.945), probably the

interference of phospholipids. Previously, phospholipids were found to interfere the TPC of

nut oils, resulting in the overestimation of phenolic contents [42]. On the other hand, FRAP

showed a positive correlation with TCC (r = 0.759, p<0.05), campesterol (r = 0.797, p<0.05),

β-sitosterol (r = 0.913, p<0.01) and ∆5-avenasterol (r = 0.798, p<0.01), which agreed with the

previous study [54].

4. Conclusion

Utilization of green technologies (e.g. SCO2 and UAAE) in extracting edible oil is highly

recommended as trace amounts of solvent residues may leave in the extracted oil when using

the conventional solvent method. The quality properties of avocado oil were unaffected by

extraction methods and correspond at the recommended indices of oxidation ranges. Oil

extracted using SCO2 contained superior level of tocopherols content, but lower level of

carotenoids content compared with solvent method. Meanwhile, oil extracted using SCO2 and

solvent methods contained similar levels of phytosterols content and oil extracted using UAAE

and solvent methods exhibited similar levels of phenolics content. Volatile compounds

associated with the nutty and grassy flavors were detected in virgin avocado oil extracted using

green technologies. Based on the antioxidant capacity tests (BBA, TEAC and FRAP), avocado

oil obtained by SCO2 method exhibited the strongest antioxidant capacity compared with

solvent and UAAE methods. Strong positive correlations were found between tocopherols and

antioxidant capacities determined by BBA, TEAC and FRAP. It can be concluded that SCO2

method is a better choice for the extraction of avocado oil as compared to UAAE and solvent

methods.

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Acknowledgment
Accept e d Article
This work was financially supported by Universiti Putra Malaysia under grant number GP-IPS

9535600.

Conflict of Interest

The authors declare no conflict of interest.

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Table 1. Quality characteristics of avocado oil extracted using different methods

Accept e d Article
Parameters Extraction methods
Solvent SCO2 UAAE
a a
Peroxide value (meq/kg) 2.79 ± 0.14 2.73 ± 0.07 2.82 ± 0.15a
P-anisidine value 2.96 ± 0.10a 2.82 ± 0.13a 3.03 ± 0.07a
Totox value 8.55 ± 0.26a 8.28 ± 0.10a 8.66 ± 0.34a
Mean values in the same row with different letters are significantly different at p < 0.05.

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Table 2. Tocol, polyphenol and carotenoid contents of avocado oil

Accept e d Article
Component Solvent SCO2 UAAE
α-Tocopherol (mg/kg oil) 115.20 ± 0.62a 226.69 ± 0.93b 69.18 ± 0.82c
γ-Tocopherol (mg/kg oil) 18.07 ± 0.16a 29.62 ± 0.36b 14.16 ± 0.35c
Total tocols (mg/kg oil) 133.26 ± 0.74a 256.31 ± 1.30b 83.34 ± 0.93c
Total phenolic content (mg GAE/100 g 128.17 ± 0.04a 111.27 ± 0.03b 130.17 ± 0.04a
oil)
Total carotenoid content (mg β- 5.24 ± 0.36a 4.27 ± 0.18b 3.08 ± 0.22c
carotene/kg oil)
Mean values in the same row with different letters are significantly different at p<0.05.

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Table 3. Phytosterol composition of avocado oil

Accept e d Article
Component (g/kg oil) Solvent SCO2 UAAE
Campesterol 0.42 ± 0.02a 0.37 ± 0.07a 0.28 ± 0.01a
Stigmasterol 0.25 ± 0.01a 0.21 ± 0.14a 0.19 ± 0.06a
β-Sitosterol 2.39 ± 0.48a 2.47 ± 0.08a 1.91 ± 0.04b
∆5-Avenasterol 0.53± 0.02a 0.38 ± 0.08a 0.21 ± 0.01b
Total phytosterols 3.60 ± 0.54a 3.43 ± 0.23a 2.59 ± 0.01b
Mean values in the same row with different letters are significantly different at p<0.05.

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Table 4. Volatile composition of avocado oil extracted by different methods

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Peak Compounds Flavor Solvent SCO2 UAAE
No.
1 Bicyclo [2.2.2] octane-1- - + + +
carboxylic acid
2 2-Methylpentanal Fruity1 +
3 Acetic acid Vinegar1 + +
4 Hexane Petroleum3 +
5 Cyclopentane Petroleum3 +
6 Butanal Pungent2 +
7 Acetoin Buttery and creamy4 +
8 1, 2-Propanediol Odorless1 +
9 Pentanal Slightly fruity and nut-like1 +
10 2,3-Butanediol Odorless2 +
11 3-Hexanone Wine-like1 +
12 2-Pentanol Fusel oil4 +
13 3-Hexanol Medicinal1 +
14 Hexanal Fatty, green and grassy1 + +
15 2-Hexanol - +
16 1-Acetoxy-2-propanol - + +
17 Heptanal Fatty, green and nut-like4 + +
18 1-Methoxy-2-propyl acetate Sweet ether-like3 +
19 3-Hexyl hydroperoxide - +
20 (E)-Hept-2-enal Fatty and almond-like4 + +
21 2-Hexyl hydroperoxide - +
22 Octanal Strong fruity2 + +
23 Limonene Lemon-like3 +
24 Nonanal Fatty, floral and grassy4 + +
25 (E)-2-decenal Fatty1 + +
26 (E)-Caryophyllene Cloves and turpentine-like3 + + +
27 α-Cubebene Mild woody balsamic5 + + +
28 (E)-Nerolidol Woody-floral and waxy5 + +
29 Farnesol Floral4 + +
1
Burdock (2016); Larrañaga
2
et al. (2016); CAMEO 3
Chemicals
4
(https://cameochemicals.noaa.gov/); Flavor Extract Manufacturers Association
(https://www.femaflavor.org/); 5Galvao et al. (2016)

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4
A
Accept e d Article
5

1 12
11 13 21
15 19 23 26 27 28
6 16 29

B
3

10

1
8
17
7 14 16 20 22 24 25 26 27 29

14
18 20 24 26
23 25 27 28
9 17 22

Figure 1. Volatile profiles of avocado oils extracted by A: Solvent, B: SCO2 and C: UAAE.
Components are 1: bicyclo [2.2.2] octane-1-carboxylic acid; 2: 2-methylpentanal; 3: acetic acid; 4:
hexane; 5: cyclopentane; 6: butanal; 7: acetoin; 8: 1, 2-propanediol; 9: pentanal; 10: 2,3-butanediol; 11:
3-hexanone; 12: 2-pentanol; 13: 3-hexanol; 14: hexanal; 15: 2-hexanol; 16: 1-acetoxy-2-propanol; 17:
heptanal; 18: 1-methoxy-2-propyl acetate; 19: 3-hexyl hydroperoxide; 20: (E)-hept-2-enal; 21: 2-hexyl
hydroperoxide; 22: octanal; 23: limonene; 24: nonanal; 25: (E)-2-decenal; 26: (E)-caryophyllene; 27:
α-cubebene; 28: (E)-nerolidol; 29: farnesol

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Table 5. Antioxidant capacity of avocado oil

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Antioxidant capacity Solvent SCO2 UAAE α-tocopherol
BBA (%) 67.42 ± 1.24a 75.88 ± 1.66b 64.40 ± 1.11a 98.58 ± 0.64c
TEAC (mM TE/kg oil) 2.99 ± 0.15a 4.52 ± 0.14b 1.71 ± 0.04c 8.16 ± 0.01d
FRAP (mM TE/kg oil) 11.55 ± 0.34a 13.09 ± 0.15b 6.59 ± 0.08c -
Mean values in the same row with different letters are significantly different at p<0.05. BBA:

β-carotene bleaching assay; TEAC: Trolox equivalent antioxidant capacity; FRAP: Ferric-

reducing antioxidant power.

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Accept e d Article

Figure 2. Changes in absorbance values of different methods extracted avocado oil, control

and α-tocopherol using β-carotene bleaching assay.

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Accept e d Article
1 Table 6. Correlations between bioactive phytochemicals and antioxidant capacities

α- γ- TPC TCC Campesterol Stigmasterol β-Sitosterol ∆5- BBA TEAC FRAP

Tocopherol Tocopherol Avenasterol
α-Tocopherol 1 0.999** -0.924** 0.326 0.467 -0.127 0.641 0.390 0.973** 0.979** 0.860**
γ-Tocopherol 1 -0.926** 0.284 0.438 -0.169 0.610 0.347 0.969** 0.970** 0.835**
TPC 1 -0.144 -0.305 0.129 -0.544 -0.211 -0.945** -0.876** -0.709*
TCC 1 0.891** 0.440 0.885** 0.958** 0.299 0.503 0.759*
Campesterol 1 0.037 0.853** 0.827** 0.378 0.619 0.797*
Stigmasterol 1 0.328 0.558 0.022 -0.050 0.170
β-Sitosterol 1 0.883** 0.646 0.770* 0.913**
∆5-Avenasterol 1 0.386 0.544 0.798**
BBA 1 0.949** 0.828**
TEAC 1 0.939**
FRAP 1
2 *Significant at p<0.05
3 **Significant at p<0.01
4

34