Food Reviews International

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Virgin Coconut Oil: Extraction, Physicochemical

Properties, Biological Activities and Its
Authentication Analysis

Abdul Rohman, Irnawati, Yuny Erwanto, Endang Lukitaningsih, Muhamad

Rafi, Nurrulhidayah A. Fadzilah, Anjar Windarsih, Ainin Sulaiman & Zalina
Zakaria

To cite this article: Abdul Rohman, Irnawati, Yuny Erwanto, Endang Lukitaningsih, Muhamad Rafi,
Nurrulhidayah A. Fadzilah, Anjar Windarsih, Ainin Sulaiman & Zalina Zakaria (2021) Virgin Coconut
Oil: Extraction, Physicochemical Properties, Biological Activities and Its Authentication Analysis,
Food Reviews International, 37:1, 46-66, DOI: 10.1080/87559129.2019.1687515

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FOOD REVIEWS INTERNATIONAL
2021, VOL. 37, NO. 1, 46–66
https://doi.org/10.1080/87559129.2019.1687515

Virgin Coconut Oil: Extraction, Physicochemical Properties,

Biological Activities and Its Authentication Analysis
Abdul Rohmana,b, Irnawatia, Yuny Erwantoc, Endang Lukitaningsiha, Muhamad Rafic,
Nurrulhidayah A. Fadzilahd, Anjar Windarsihe, Ainin Sulaimanb, and Zalina Zakariab
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta,
Indonesia; bUniversity of Malaya Halal Research Centre (UMHRC), Universiti Malaya, Kuala Lumpur, Malaysia;
c
Institute for Halal Industry and Systems, Universitas Gadjah Mada, Yogyakarta, Indonesia; dInternational
Institute for Halal Research and Training, Gombak, Malaysia; eResearch Division for Natural Product
Technology, Indonesian Institute of Sciences (LIPI), Yogyakarta, Indonesia

ABSTRACT KEYWORDS
Virgin coconut oil (VCO) has emerged as functional food oil due to its Virgin coconut oil;
capability to provide some biological activities which are beneficial to authentication analysis;
human health. This is due to the fact that some minor components like functional oil;
tocopherols and phenolics compounds are retained. VCO is prepared physicochemical properties;
health benefits
from fresh, mature kernel of the coconuts by mechanical or natural
means, with or without the use of heat, but specifically without any
chemical refining, bleaching and deodorizing (RBD). As a consequence,
VCO has slight difference in terms of some physico-chemical properties
with RBD coconut oil. Due to phenolics compounds contained, VCO
exhibited several pharmacological activities including antioxidant, anti-
inflammatory and immunomodulatory, anti-hyperlipidemia, anticancer,
antidiabetic, anti-bacterial and neuroprotective activities. VCO has com-
manded high price value in the fats and oils industry, hence, VCO can be
target of adulteration with low priced oils. Fourier transform infrared
(FTIR) spectroscopy and chromatographic techniques combined with
multivariate analysis has been successfully reported for analysis of
adulteration practice involving the substitution or replacement VCO
with other oils. This review highlights some techniques for VCO extrac-
tion, physicochemical (characterization), biological activities and
authentication analysis of VCO.

Introduction
Coconut oil, known as minyak kelapa in Indonesia and Malaysia, is one of edible oils
obtained from the extraction of coconut kernel in the mature stage using either mechanical
or thermal processing. Because of the high saturated fatty acid and fat contents, coconut oil
is resistant to oxidative modifications, which make them suitable for cooking.[1] Coconut is
a widespread plantation and is grown in more than 80 countries. The world production of
coconut is estimated at around 55 million tons annually. Coconut oil has significant use in
the toiletry, food and various industrial applications.[2]
Some different types of oils prepared from coconut include coconut testa oil (CTO),
virgin coconut oil (VCO) and copra oil (CO).[3] Data from Research and Market reported

CONTACT Abdul Rohman [email protected] Department of Pharmaceutical Chemistry, Faculty of

Pharmacy, Universitas Gadjah Mada, Yogyakarta
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lfri.
© 2019 Taylor & Francis
 FOOD REVIEWS INTERNATIONAL 47

that the global market of VCO in the market is USD 2.7 billion in 2018 and is forecasted
that by 2024 the VCO market grow over 9% to reach USD 4.7 billion.[2] The difference in
coconut oil’s preparation itself causes changes in the physicochemical properties and the
biological activities.[3] CO is an oil collected with mechanical milling from copra (the
coconut kernel dried with direct sunlight or with the oven to reduce the contents of
water). CTO is the emerging form of coconut oil obtained by extracting the coconut testa
using isopropyl alcohol. Zhang et al.[4] reported that the optimum yield of CTO having up
to 63–76% was extracted using the extraction condition of temperature of 60°C, period of
extraction of 3 h and the ratio of substrate: isopropyl alcohol (1:4). Due to the involvement
of organic solvents during CTO extraction, this oil has not been widely applied yet for the
edible purposes.
VCO is obtained by extracting the fresh coconut kernel using natural means and does
not undergo any kinds of chemical treatments such as refining, bleaching and deodorizing
to produce refined-bleached-deodorized (RBD) oil.[5] In other words, VCO is produced
through wet method, namely via coconut milk.[6] Philippine National Standard together
with Bureau of Agriculture and Fisheries Product Standards[7] and Srivastava et al.[8] has
defined VCO as the oil obtained from fresh, mature kernel of the coconuts by mechanical
or natural means, with or without the use of heat, but specifically without chemical RBD
which does not lead to the alteration of the natural content of the oil. Since VCO is
produced differently from RBD coconut oil, the oil obtained is slightly different in terms
of its sensory characteristics. VCO is nearly colorless, with a slightly detectable acid aroma,
sweet and salty taste, and is perceptible nutty aroma and flavor. On other hand, RBD
coconut oil is distinctively yellow, slightly salty, and has no perceptible aroma and
flavor.[9]
VCO is considered as saturated fat because the contents of saturated fatty acids are
more than 90%. From epidemiological study, the consumption of high amounts of
cholesterol and saturated fat contributed to high blood cholesterol, as a consequence,
the perception on coconut oil is bad.[10] In the past few years, however, the pre-clinical
and clinical trials have been carried out on oil-derived coconut, and the reported results
showed that VCO exhibited positive outcomes to human health which might counter
those arguments.[6] The main fatty acids composed TG are considered as medium chain
triglyceride (MCT) which are more easily hydrolyzed and more absorbable than long
chain TG by some lipases in the gastrointestinal tract of humans. VCO is rich in lauric
acid, therefore, the hydrolysis of MCT yields mono-lauryl glycerol such as monolaurin
which is considered as pharmacologically active compound.[11] Having rich in MCT, the
consumption of VCO is associated with the increased levels of serum TG, but the lipid
profile may be improved due to the incorporation of structured lipid. Prior et al.[12]
reported that Polynesian populations consumed VCO regularly are not associated with
coronary heart diseases. Therefore, the campaign that VCO and other coconut oils
contributed to the bad cholesterol is a myth. VCO may have more beneficial effects
than CO, since it retains most of the unsaponifiable components. VCO exhibits some
important biological activities such as antiviral, antifungal, antiparasitic, antibacterial,
cardioprotective, hepatoprotective, antidiabetic, hypolipidemic, and antioxidants which
are beneficial to human health.[13] These effects may be attributed to the large amount
of short-chain fatty acids like caproic, caprylic, and capric acids.[9] Nevin and
Rajamohan[14] reported that VCO can lower triacylglycerols, phospholipids, total
48 A. ROHMAN ET AL.

cholesterol, low density lipoprotein (LDL), and very low-density lipoprotein (VLDL)
cholesterol levels. VCO also increase high density lipoprotein (HDL) in tissues and
serum. Phenolics contents of VCO were able to prevent LDL oxidation in vitro with the
formation of reduced level of carbonyl compounds.
The objective of this review was to discuss some extraction techniques to obtain VCO along
with its physicochemical characteristics and its biological activities. VCO commands high price
value which can be subjected to the adulteration with low quality edible oils, hence, the
authentication analysis of VCO was also addressed. During performing this review, we explored
several databases such as Science Citation index, PubMed, Medline, Scopus, and Google Scholar
to identify and to download the abstracts, reports, review articles, and research papers related to
the extraction, physicochemical properties, biological activities of VCO. The keywords used
during searching of information were: extraction + physicochemical + antioxidant (or anti-
inflammation, anti-hyperlipidemia, anti-bacteria) activities + authentication + VCO.

The extraction of VCO

The terms of “virgin” in VCO could be understood that VCO was prepared without any
RBD process, as a consequence, there was any alteration in the nature of oil.[6] VCO can
be extracted in a straightforward manner from coconut under ambient temperature;
therefore, the loss of minor components like pro-vitamin A, tocopherol, and phenolics
compounds due to solar UV irradiation during coconut drying can be avoided.[15] Based
on the mode of preparation, several types of VCO are available, namely cold extraction,
hot extraction, fermentation technique, and enzymatic extraction.
Cold extraction (CE) or aqueous extraction method is used to extract VCO directly from
coconut milk without the aid of heat. This method involves the chilling of coconut milk at
temperature of 2–8°C overnight and the separated oil is collected by centrifugation, filtered,
and stored. This is a simpler and cheapest method available for VCO preparation. CE method
eliminates the use of solvents because of the absence of RBD processes, therefore this method
is low cost and is environmentally friendly.[8] The disadvantage of CE method is that the oil
yield is relatively low which limits the application in commercial industry.[6] CE is rather
difficult due to the involvement of the emulsion breaking having high stability in coconut
milk. Onsaard et al.[16] have proposed three stages to break the emulsion (creaming, floccula-
tion, and coalescence). The creaming stage occurred through gravitational force which
resulted into two phases, namely aqueous phase with higher specific gravity (in the down
phase) and oil phase with lower specific gravity (the top phase). The flocculation stage
involved the oil phase moves as a form of group which does not involve the rupture of the
interfacial film, consequently the original globule does not change. Finally, the coalescence
step, the most critical destabilization phase of emulsion. In this step, the interfacial area is
broken and the globules are joined together thus reducing the interfacial area. The emulsion of
coconut milk can be also broken by pH adjustment at pH 3.0–5.6, and then inoculated with
bacteria cultures.[17] The coconut cream can be destabilized using acetic acid treatment. Che
Man et al.[18] have reported that acetic acid 25% vol/vol at different levels (0.1%, 0.2%, 0.3%,
and 0.4% vol/vol) in coconut cream for reaction time of 10–14 h at ambient temperature could
improve the yield of VCO extracted, with oil recovery up to 60%. Some techniques have been
also reported to break the coconut milk emulsion including heat and salts treatment, action of
 FOOD REVIEWS INTERNATIONAL 49

enzymes, refrigeration, and the use of short waves.[19] All techniques were possible because
milk proteins are easily precipitated at acidic pH (below 4).
Hamid et al.[20] have made innovation of VCO preparation through integrated wet process.
In this study, the meat of fresh coconut was pressed using mechanical pressing to obtain
coconut milk. In order to break emulsion, the coconut milk was chilled at temperature of 10°
C, so that the water and coconut butter are separated. The coconut butter was transferred and
then heated to 45°C and subjected to centrifugation for separating oil (VCO) from non-oil
fraction. Finally, VCO was filtered for removing any solid materials. VCO produced is
colorless retaining fresh aroma and sweet taste. The authors reported that this process can
maximize yield of approximately of 30–40%, which is 10–20% higher than conventional
technique with minimum cost, time, and energy.
Hot extraction (HE) which involved heat treatment is another technique used for VCO
extraction from coconut milk. This technique was traditionally used in Southern India and
VCO obtained was conventionally used in the Ayurvedic medicinal system for treatments
of children’s skin ailments. In HE system, the coconut milk is subjected to temperature of
100°C for 60 min or more until the oil was separated completely from the milk. Finally,
the oil formed is collected using filtration. It is reported that the use of heat can help
increasing the oil yield and releasing the bound phenolic acids.[1]
The fermentation technique for VCO extraction involves the uses of bacterial activity to
generate VCO. There are two types of fermentation, namely natural fermentation and induced
fermentation. In natural fermentation technique, the fresh-grated coconut kernel is extracted
using its water to collect the coconut milk and then is allowed at room temperature (or until
45°C) for 24–48 h to allow fermentation and separation of oil layer. The oil obtained is then
scooped out, filtered, and stored.[14] In induced fermentation technique, some bacteria are
used to extract VCO from coconut milk. The induced fermentation method is less popular
than natural fermentation. The induced fermentation using bacteria of Saccharomyces cerevi-
siae, Lactobacillus plantarum (strain 1041 IAM), and Lactobacillus delbrueckii has been
reported for the extraction of VCO from coconut milk.[21] Previously, Che Man et al.[22]
have also used induced fermentation using pure culture of Lactobacillus plantarum strain 1041
IAM to extract coconut oil and reported that this technique able to extract as much as 95%
of VCO.
VCO can be also obtained from enzymatic technique in aqueous extraction system. In this
technique, a mixture of enzymes is used to release the oil portion from the coconut milk. The
enzymes consisted of α-amylase to produce simpler carbohydrates from starch, protease to
remove plant proteins as well as polygalacturonase and cellulase to remove cell wall
components.[3] These enzymes are needed because plant cell wall consists of complex carbohy-
drate molecules (hemicellulose, cellulose, arabinogalactans, mannans, galactomannans, and
pectin) and protein.[6] Che Man et al.[18] have successfully extracted coconut oil with 1% enzyme
mixture of cellulase, alfa-amylase, polygalacturonase, and protease with an oil yield of 74%.

Physicochemical properties of VCO

Physicochemical properties of VCO were evaluated by determining some constants related
to edible fats and oils such as acid number, saponification number, etc., analysis of fatty
acid and triglyceride compositions, and identification and quantitative analysis of minor
components like tocopherols and phenolics contents. Marina et al.[6] have reported that
50 A. ROHMAN ET AL.

Table 1. Some physicochemical properties of virgin coconut oil extracted using different techniques.
Extraction techniques[23]
b
Fresh- Indonesian
a
Analysis Chilling Fermentation dry Enzyme APCC[7] Standard[24]
Iodine value 4.13 4.3 4.18 4.26 4.10–11.00 4.10–11.00
Free fatty acid Saponification 0.31 0.29 0.46 0.35 Maximal 0.5 Maximal 0.2
value
Moisture content (% wt) 258.23 256.73 258.42 262.72 Min. Not available
250–260
Viscosity (Pa.s) 0.11 0.06 0.04 0.11 0.1–0.5 Maximal 0.2
48.93 48.73 50.93 48.93 Not available Not available
a
APCC = Asian and Pacific Coconut Community; bStandard Nasional Indonesia (National Standard of Indonesia) SNI 7381:
2008.[22]

VCO had iodine value of 4.47–8.55 g I2/100 g, Saponification value of 252.45–260.67 mg

KOH/g, acid value of 0.13–0.27 mg KOH/g, peroxide value of 0.21–0.63 mEq/kg and
anisidine value of 0.16–0.49. Mansor et al.[23] have compared the physicochemical proper-
ties of VCO prepared from fresh-dry (grated coconut route), chilling and thawing,
enzymatic and fermentation method and the results showed that the physicochemical
parameters of studied VCOs were in accordance with those established by Codex
Alimentarius Commission and the Asian and Pacific Coconut Community (APCC), as
shown in Table 1. However, the moisture contents of produced VCO exceed the max-
imum limit set by Indonesian Standard SNI 7381 (2008).[24]
Dayrit et al.[25] have compared the physicochemical properties of VCO and RBD
coconut oil (RBD-CO). VCO had higher moisture, volatile matter, and free fatty acids
(FFAs) and lower peroxide value than RBD-CO, but the value range overlapped and there
was no single parameter could be used for differentiation of VCO from RBD-CO. In
addition, VCO and RBD-CO could be distinguished by the total amount of diglycerides
(DGs) in which VCO had average DGs content of 1.55% wt/wt, whereas RBD-CO gave
DGs of 4.10% wt/wt. Edible fats and oils are basically composed from TG, an ester of
glycerol with three fatty acids. Each edible oil has different fatty acid compositions in
terms of types and amount, therefore fatty acids can be used to characterize edible fats and
oils. Fatty acid (FA) compositions of VCO from several standards namely Indonesian
Standard, Codex Alimentarius Standard, Asian and Pacific Coconut Community (APCC)
along with those reported by some authors are presented in Table 2. Basically, the
composition of fatty acids in different types of coconut oil (VCO, RBD oil) is not

Table 2. Fatty acid composition of virgin coconut oil (VCO) and refined, bleached and deodorized (RDB)
coconut oil from various sources.
c d
Fatty acid composition Indonesia MS for Marina
a
(%) Codex[26] b
APCC[7] Standard[24] VCO[27] et al.[28] Dia et al.[29]
C6 (caproic acid) nd-0.70 0.40–0.60 ND-0.7 0.80–0.95 0.52–0.69 nd-0.60
C8 (caprylic acid0 4.60–10.0 5.00–10.00 4.6–10.0 8.00–9.00 7.19–8.81 5.98–10.44
C10 (capric acid) 5.0–8.0 4.50–8.00 5.0–8.0 5.00–7.00 5.65–6.59 5.37–6.60
C12 (lauric acid) 45.10–53.20 43.00–53.00 45.1–53.2 47.00–50.00 46.89–48.03 47.63–52.55
C14 (myristic acid) 16.80–21.00 16.00–21.00 16.8–21.0 17.00–18.50 16.23–18.90 16.79–20.08
C16 (palmitic acid) 7.50–10.20 7.50–10.00 7.5–10.2 7.50–9.50 7.41–9.55 6.38–10.17
C18:0 (stearic acid) 2.00–4.00 2.00–4.00 2.0–4.0 2.50–3.50 2.81–3.57 7.45–10.73
C18:1 (oleic acid) 5.00–10.00 5.00–10.00 5.0–10.0 4.50–6.00 5.72–6.72 5.15–6.03
C18:2 (linoleic acid) 1.00–2.50 1.00–2.50 1.0–2.5 0.70–1.50 0.90–1.60 nd-0.12
C18:3 (linolenic acid) nd-0.20 <0.5 Nd-0.2 nd nd nd
a
standard for RDB coconut oil; bAPCC = Asian and Pacific Coconut Community; cIndonesian Standard No. 7381 (2008);
d
MS = Malaysian Standard.
 FOOD REVIEWS INTERNATIONAL 51

Table 3. Triglyceride (TG) composition in VCO obtained from different method

calculated using normalization technique of peak area of liquid chromatogram.[23].
Extraction method†‡
TG composition Chilling Enzyme Fermentation Fresh-dry
CClCl – 0.31 0 0.44
CpCpLa 0.54 0.79 0.41 0.95
CpCLa 4.11 3.86 3.81 4.07
CCLa 13.55 13.36 13.82 12.71
CLaLa 17.05 17.34 18.9 21.1
LaLaLa 22.62 23.94 22.21 21.63
LaLaM 19.83 17.94 19.51 18.89
LaLaO 1.84 1.88 1.72 1.88
LaMM 11.07 10.8 12.17 10.77
LaMO 1.07 1.24 1.04 1
LaMP 6.62 5.94 5.01 4.72
LaOO 0.61 0.74 0.57 0.58
LaPP 1.19 1.64 0.83 1.08
MOO 0.27 0.22 0 0.19
Cp, caproic; Cl, caprylic; C, capric; La, lauric; M, myristic; P, palmitic; O, oleic.

significantly different. But, the main difference of VCO and RBD coconut oil is related to
the composition of active compounds such as tocopherol and phenolics which are retained
in VCO.[6] The main fatty acids composed VCO is lauric (C12:0), myristic (C14:0), and
palmitic (C16:0) acids which are classified as medium chain fatty acids. Lauric acid (C12:0)
was the most dominant of fatty acids present in VCO. In addition, VCO contains less
mono- and polyunsaturated FAs.
Another chemical property used for the characterization of VCO is triglyceride (TG) com-
positions, usually determined by liquid chromatography equipped with general detectors like
refractive index, evaporative light scattering detector, and mass spectrometer detector.[30] Marina
et al.[28] have reported that the major TG in VCO samples consisted of LaLaLa accounting of
22–25%, CCLa of 14–16%, CLaLa of 19–21%, LaLaM of 13–15%, and LaMM of 7–9%. The
authors also compared TG in VCO obtained from Malaysia and Indonesia, and the results
showed that Malaysian VCO had relatively higher contents of CpCpLa, CpCCpCLa, and LaOO
while Indonesian VCO had more of LaMP (Cp = caproic; La = lauric, C = capric, M = myristic,
P = palmitic, O = oleic acids). Mansor et al.[23] have evaluated TG of VCO prepared from fresh-
dry, chilling and thawing, enzymatic and fermentation methods, calculated using normalization
technique of peak area of liquid chromatogram, as shown in Table 3.

Pharmacological activities of VCO

Several pharmacological effects have been reported in VCO which include antioxidants,
anti-inflammation, anti-hyperlipidemia and antibacterial activities.[1] VCO prepared by
cold and fermentation techniques were extensively studied due to the active compounds
such as phenolics and tocopherols contained. Different researchers also studied the
pharmacological effects of VCO prepared from different extraction methods.

Antioxidant activities of VCO

Phenolics compounds are group of bioactive compounds present in edible oils capable of
exerting the antioxidant activities through several mechanisms, mainly hydrogen transfer
52 A. ROHMAN ET AL.

and reducing power. Several epidemiological studies showed that there is relationship
between antioxidant activity and diet containing phenolics compounds. The clinical
studies also revealed that VCO rich in polyphenol exhibit beneficial effects against
cardiovascular disease in recent randomized control trials.[31,32] Illam et al.[33] reported
that coconut oils contained some phenolic compounds namely ferulic acid, p-coumaric
acid, caffeic acid, quercetin, and catechins, and the these phenolics acids are found to be
higher in VCO compared to RBD-coconut oil (RBD-CO). The common phenolics com-
pounds present in VCO and commercial coconut oil are compiled in Table 4.
Marina et al.[35] have evaluated the antioxidant activities of VCO produced by chilling
and fermentation and compared its activity with RBD coconut oil using three in vitro
antioxidant methods, namely 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging,
beta-carotene-linoleate bleaching, and reducing power. In general, VCO revealed higher
antioxidant capacities than those of RBD-CO. This is based on the fact that some minor
components such as tocopherols and phenolics components are retained in VCO.[14] The
phenolic acids in VCO which correlated with these antioxidant activities were ferulic acid
and p-coumaric acid. These phenolics compounds were highly correlated with DPPH
radical scavenging activity, reducing power, and beta-carotene bleaching with coefficient
of correlation (r) values of 0.91, 0.96, and 0.83, respectively. The study confirmed that
phenolics compounds present in coconut oil contributed significantly to the antioxidant
capacities.[36]
VCO extracted from fresh coconut meat at temperature of 50°C was evaluated for
in vivo antioxidant activities through the measurement of antioxidant enzymes namely
superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx), Glutathione
reductase (GR), Glutathione content (GSH), and lipid peroxidation levels in male
Sprague–Dawley rats, compared to CO and groundnut oil (GO) as control for 45 days.
The results showed that polyphenol fractions in VCO increased the antioxidant enzymes
and reduced the lipid peroxide content.[37] In addition, VCO polyphenols (tentatively
identified as flavanones or dihydroflavonols like compounds) able to inhibit lipid perox-
idation than polyphenols from CO and GO. VCO extracted from fresh coconut meat
having high content of active components is superior in antioxidant property than coco-
nut oil extracted by dry process.[14] Rahim et al.[38] also reported that male Wistar rats
administrated with VCO could increase 8% SOD activity, 54% CAT activity, 20% GSH
content, and 12% GPx activity than those in control rats. In separate study, Illam et al.[33]
reported that VCO increased the levels of intracellular GSH which involved actively
during phase II detoxification system through GSH conjugation either in cell culture or
in animal models and in diabetic rats.[1] Furthermore, male inbred BALB/c mice treated

Table 4. Phenolic present in commercial coconut oil and in virgin coconut oil.
Commercial coconut oil Virgin coconut oil
Phenolic acid (mg/Kg) (mean ± standard error)[34] (mean ± standard deviation)[35]
Total polyphenols 91 ± 11 250.9 ± 22.6
Catechin 0.87 ± 0.1 not reported
Vanillic acid not reported 2.08
Syringic acid not reported 0.45 ± 0.3
p-Coumaric acid 0.34 ± 0.01 0.75 ± 0.1
Caffeic acid 0.13 ± 0.06 0.12 ± 0.1
Ferulic acid 0.31 ± 0.20 5.09 ± 2.3
 FOOD REVIEWS INTERNATIONAL 53

with VCO exhibited higher levels of brain antioxidants than mice control. VCO could
reduce lipid peroxidation and increase SOD activity in the serum subjected to the forced
swim test.[39,40]
Arunima and Rajamohan[41] compared the endogenous antioxidant status in vivo of
VCO CO, olive oil (OO), and sunflower oil (SFO) in Male Sprague-Dawley rats. The
dietary VCO improved the antioxidant status compared to CO, OO, and SFO (P < .05), as
indicated by the increased levels of antioxidant enzymes. Concentration of reduced
glutathione was also found to be increased significantly in liver, heart, and kidney due
to administration of VCO compared to those given by CO, OO, and SFO (P < .05). VCO
administration could prevent the oxidative stress, which is indicated by the decreased
formation of lipid peroxidation such as hydroperoxides, malondialdehyde, conjugated
dienes and by decreased protein oxidation products including protein carbonyls in
serum and tissues compared to rats fed with other oils (P < .05). The authors concluded
that VCO has a beneficial role in improving antioxidant status and hence preventing lipid
oxidation.

Anti-inflammatory and immunomodulatory effects of VCO

Several studies on the anti-inflammatory and immunomodulatory effects of VCO have been
reported in various experimental designs. Intahphuak et al.[42] have evaluated cold-extracted
VCO in acute and chronic inflammation models using rats as animal model. VCO exhibited
the protective effect toward granuloma formation in ear and paw oedemic models induced by
ethyl phenylpropionate, carrageenan, and arachidonic acid. VCO also showed the inhibitory
activity on chronic inflammation by reducing granuloma formation, the transudative weight,
and serum alkaline phosphatase. VCO prepared by fermentation technique has been reported
to reduce arthritis in rats induced by adjuvant, through the mechanism of downregulating the
expressions of cyclooxygenase, an enzyme responsible for inflammatory action, inducible by
nitric oxide synthase and tumor necrosis factor (TNF-α).[43] Zakaria et al.[44] reported that
fermentation-prepared VCO could reduce acute inflammation efficiently, but it revealed less
effective inflammation in chronic models.
The immunomodulatory effect of VCO has been investigated on skin inflammation
induced by lipopolysaccharide (LPS). LPS could alter the immune system when it enters
the body. LPS induced the stimulation of proinflammatory cytokine expression.
Administration of VCO showed beneficial effect to suppress proinflammatory cytokine
in monocytic leukemia both in protein and gene expression level.[45] VCO could suppress
the levels of proinflammatory cytokines of TNF-α and interleukins (IL) in protein and
gene expression responsible for inflammation, tissue damage, fever, and cell death. These
studies suggested that anti-inflammatory effects of VCO were achieved by suppressing the
inflammatory markers which include TNF-α, IL, and interferon (IFN-γ).
VCO showed the immunomodulatory effect in mice administered with diet containing
high refined carbohydrate.[46] High refined carbohydrate induced inflammatory disorder
in mice as well as altered the immune system by stimulating the high expression of
proinflammatory cytokines and leukocytes. Administration of VCO showed beneficial
effects on suppressing proinflammatory cytokines. VCO decreased several proinflamma-
tory cytokines concentration including TNF-α and IL-6. VCO also reduced the number of
leukocytes as well as mononuclear and polynuclear circulating cells.
54 A. ROHMAN ET AL.

The phenolic fraction of VCO showed immunomodulatory effects on human periph-

eral blood mononuclear induced by oxidized low-density lipoprotein (LDL). The oxidized
LDL could have several negative impacts on human body including stimulation of
inflammation signaling pathway through NF-kB, stimulation of toll like receptor (TLR),
and stimulation of proinflammatory cytokines such as IL-6 and TNF-α. Phenolic fraction
of VCO inhibited either the expression or translocation of NF-kB, therefore the inflam-
matory signal is inhibited. Moreover, phenolic fraction of VCO decreased the proinflam-
matory cytokines expression of TNF-α and IL-6 as well as decreased the production of
inflammatory mediators, especially prostaglandin (PGE2) and nitric oxide (NO).[47]

Anticancer and amelioration of toxicity induced by chemotherapy

Lauric acid (LA) present dominantly in VCO has been studied for anticancer activities. LA
is known to exhibit anticancer activity by reducing glutathione (GSH) level in cells. GSH is
believed to protect cancer cells from the increased oxidative stress inducing apoptosis.[48]
LA has been reported to induce apoptotic changes in numerous colorectal cancer cells
mediated by several reactive oxygen species and nitrogen reactive species (ROS/NOS).[49]
LA also could induce the cell cycle arrest in G0/G1 phase and G2/M phase and to activate
Rho associated kinase-mediated pathway and p21-dependent apoptosis.[50]
Craig-Schmidt et al.[51] reported that mice treated with the combination of coconut oil rich
in LC and menhaden oil exhibited the significant reduction of mammary tumorigenesis
induced by carcinogenic of dimethylbenzanthracene (DMBA). In another study, the diet
rich in VCO could reduce efficiently the incidence of colon cancer model induced by
azoxymethane-dextran sodium sulfate by increasing the levels of intestinal protein Mucin 2.
This protein involved in the proper maintenance of intestinal barrier integrity.[52] VCO
exhibited the cytotoxicity effects on human hepatocarcinoma cells (HepG2) by stimulating
hydroxyl radicals (•OH) to damage cancer cells promoting cell death.[53]
Due to its activity as antioxidant, the supplementation of VCO has been reported to
have hepatoprotective activity toward hepatotoxicity and oxidative damage which corre-
lated to the amelioration of toxicity-induced chemotherapy agents.[54] VCO supplementa-
tion could attenuate chemotherapy using anticancer drug of methotrexate through
inhibition of oxidative stress in rats. VCO supplementation could enhance the resistance
of biochemical alterations in rats induced by methotrexate.[55] VCO also demonstrated
hepatoprotective and chemotherapy using antibiotics of sulfamethoxazole-
trimethoprim[56] and anticancer drug of cyclophosphamide.[57]

Anti-diabetic activities
Diabetes mellitus is a metabolic disorder characterized by the increased levels of blood
glucose. Several studies have been carried out to evaluate the antidiabetic activities of VCO.
Maidin and Ahmad[58] have reported that VCO prepared from fermentation (F-VCO)
method could reduce the glucose levels of diabetic rats induced by alloxan. The similar
study on antidiabetic activity of VCO prepared from cold extraction method was also
conducted by Iranloye et al.[59] in which rats were treated with alloxan to induce diabetic.
The results showed that VCO had hypoglycemic action, enhance the insulin secretion and
also ameliorate the oxidative stress induced in type I diabetic male rats. El-Shemy[60] also
 FOOD REVIEWS INTERNATIONAL 55

found that VCO prepared from cold extraction method and administered orally at 10 mL/kg
body weight of rats, daily for 4 weeks was very effective against deleterious hyperlipidemic
on rats induced by alloxan. Besides, VCO also revealed the similar results of antidiabetic
activity on rats treated with streptozotocin.[61]
Siddalingaswamy et al.[62] have compared the antidiabetic activities of VCO extracted
from hot method (H-VCO) and cold-extracted VCO (C-VCO) on diabetic rats induced by
streptozotocin. The results showed that H-VCO had higher hypoglycemic activity and
insulin-sensitizing agent than C-VCO. These effects may be attributed to increased
polyphenolic and other antioxidants compounds present in H-VCO. F-VCO also effi-
ciently prevents the development of insulin resistance in high fructose-fed rats[3] and
exerts the protective effects on renal dysfunction in diabetic rats. It is believed that the
phenolic compounds play an important role in its antidiabetic activity and protective
effects on renal dysfunction by inhibiting reactive oxygen species such as hydroxyl radicals
responsible for the death of the beta cells.[62,63,64]

Anti-hyperlipidemia activities of VCO

VCO contain high levels of saturated fatty acids which are correlated with high levels of blood
cholesterols;[65] however, several studies revealed that VCO had the positive outcomes to lipid
profiles, mainly due to the presence of medium chain triacylglycerol (MCT) contained in
VCO. Srivastava et al.[66] have compared the hypolipidemic effects of VCO prepared by cold
extraction (CE), hot extraction (HE) and that commercially available (C-VCO) in India using
male Wistar rats. The cholesterols and LDL of in blood plasma of rats treated with VCOs have
been decreased significantly by on average of 25% and 40%, respectively, while high-density
lipoprotein (HDL) was significantly increased (p < .05) by 21% compared to control rats.
The effects of cold-prepared VCO on some lipid parameters in comparison with CO have
been evaluated by Nevin and Rajamohan.[67] The results revealed that VCO had lower triglycer-
ides in serum and tissues than those in CO and control animals. HDL cholesterol in rats treated
with VCO was increased, while LDL cholesterol level was decreased significantly compared to
CO. This finding also suggested that polyphenol fractions extracted from VCO was found to be
more effective than those extracted from CO, as indicated by preventing the copper-induced
oxidation of LDL as indicated by the low thiobarbituric acid reactive substance (TBARS) and
reduced carbonyl formation. In the subsequent study, the same authors[14,15] also reported that
the inhibitory effects on microsomal lipid peroxidation were increased in rats treated with VCO,
as indicated by reduced malondialdehyde (MDA) and conjugate diene content in the tissues. The
levels of lipid peroxide were also significantly decreased in the heart, liver and kidney of VCO fed
rats compared to CO. These findings indicated the potential benefits of VCO in maintaining lipid
metabolism.[5]
The clinical studies on the effects of daily VCO on plasma lipoproteins levels involving
35 healthy Thai volunteers using randomized, controlled, crossover trial approach have
been performed by Chinwong et al.[68] The results showed that daily VCO intake
significantly (p < .001) increased HDL cholesterol by 5.72 mg/dL compared to the control
regimen (2% carboxymethylcellulose solution). The daily consumption of 30 mL VCO in
young healthy adults significantly increased HDL cholesterol. There were no safety issues
reported during the daily consumption of VCO for 8 weeks.
56 A. ROHMAN ET AL.

Antibacterial activities
VCO has been also reported to have antibacterial activity. Ogbolu et al.[69] reported the
antibacterial activities of fermentation-prepared VCO on Candida species. They found
that the anticandidal effects of VCO diluted 1:4 were comparable with those of fluconazole
diluted 1:2. VCO also revealed the antibacterial activity toward Staphylococcus aureus.[70]
VCO obtained from the Philippines could reduce the mortality of Nile Tilapia fish
(Oreochromis niloticus) subjected to Streptococcus iniae infection.[71]
VCO could reduce plaque-related gingivitis, an oral disease induced by bacterial
infection.[72] Lauric acid, a major fatty acid present in VCO, is reported to the possible
compound responsible to antimicrobial activities.[73] In addition, Manohar et al.[74] found
that monolaurin compounds, major metabolite of VCO, contributed to antimicrobial
activities of VCO activity.

Neuroprotective effects of VCO

The studies on the effect of VCO with respect to neuroprotective activities have been
carried out. Nafar and Mearow[75] reported the possible beneficial effects of cold pressed
VCO in the prevention of neurodegenerative disorders. The treatment of VCO on cortical
neuronal cells could improve the cell survival by reducing the mitochondrial alterations.
VCO has several protective effects on cortical neuron disturbed by β-amyloid by enhan-
cing the signaling pathway in cortical neurons and inhibiting the oxidative reactions of
some oxidative markers of cellular stress, namely Caspase3 and reactive oxygen species
(ROS). The neuroprotective effect of VCO is also shown by activation of Akt and Erk
signaling pathways.[76] Fernando et al.[77] reported the possibilities of VCO as a potential
therapeutic agent against the neurological disorders including Alzheimer’s disease.
Rahim et al.[38] have reported that VCO could enhance Wistar rats memory. The
improvement in memory function is associated with cholinergic activity by enhancing
the cognitive functions of brain. VCO inhibits acetylcholinesterase activity, as a result of
the increased levels of acetylcholine, an important neurotransmitter for the cognitive
functions in brain. VCO has good neuroprotective effect by stimulating the expression
of p-tyrosine hydrolase and nerve growth factors. The neuroprotective effect of VCO also
correlated with the antioxidant activity of VCO through the inhibition of lipid peroxida-
tion reactions. Furthermore, the improvement on nerve system was also increased as
indicated by the presence of intracellular signaling molecules to mesenteric lymph node
and thymus.[78] VCO showed activity on normalizing NLRP3 (NOD-like receptor family
pyrin domain) in rats treated with β-amyloid and high fat diet. Either β-amyloid or high
fat diet significantly enhanced NLRP3 causing the impairment of memory and learning.
Administration of VCO at concentration of 8% and 10% exerted good effect on normal-
izing NLRP3 levels, as a consequence, the memory and learning function are repaired.[79]

Authentication analysis of VCO

VCO has emerged as functional oil in the fats and oils industry due to its capability to
provide the positive outcomes from the evaluations of biological activity in vitro and
in vivo. As a consequence, VCO has higher price value than common vegetable oils like
 FOOD REVIEWS INTERNATIONAL 57

palm, corn, and soybean oils. Unethical players may take advantage by replacing or
diluting VCO with low priced oils to get economical profits. Thus, the adulteration
issue may be raised due to the price difference, and analytical methods based on physi-
cochemical analysis have been developed, validated and used for detection of edible fats
and oils adulteration including VCO’ such as spectroscopy, differential scanning calori-
metry, chromatography, and electronic nose.[80,81] Table 5 compiled the analytical meth-
ods used for authentication analysis of VCO from other edible oils such as palm oil and
animal fats such as lard.
Fourier transform infrared (FTIR spectroscopy combined with the chemometrics of
multivariate calibration for quantitative analysis and discriminant analysis (DA) for
classification between VCO and VCO adulterated with other oils and animal fats appeared
as the most reported method for the authentication analysis of VCO. This is based on the
fact that FTIR spectra of fats and oils are fingerprint in nature, therefore, it is convenient
to search the specific peaks which are characteristics to VCO and VCO’s adulterant.[92]
The general procedure for authentication analysis of edible oils using FTIR spectroscopy
combined with chemometrics is as follows: (1) preparation of calibration and validation
samples (standards) or well characterized samples, (2) the acquisition of FTIR spectra
using certain FTIR spectral condition, (3) optimization of FTIR spectra condition capable
of providing the desired results which included spectral pre-processing and selection of
wavenumbers, (4) selection of calibration and validation sets, (5) calibration modeling
from calibration datasets, (5) validation the calibration models, and (6) evaluation of the
developed models in terms of its validation features namely accuracy, precision, sensitiv-
ity, and its application to predict unknown samples.[93]
Currently, Rohman et al.[94] have developed FTIR spectroscopy combined with multivariate
calibration of partial least square calibration (PLSR) and discriminant analysis (DA) for the
authentication of VCO from grape seed oil (GSO) and soybean oil (SO). FTIR spectra of VCO,
GSO, SO and its binary mixture of VCO-SO, and VCO-GSO were scanned at mid infrared
region (4000–650 cm−1) using attenuated total reflectance (ATR) technique and subjected to
FTIR spectral treatments. For quantitative analysis purpose, the wavenumbers (1/λ) was selected
based on its capability to provide the best prediction models in terms of highest R2 and lowest
root mean square error for calibration (RMSEC) and root mean square error for prediction/
validation (RMSEP). For the classification, 1/λ was selected based on its capability to classify
authentic VCO and adulterated VCO. Fig. 1 showed FTIR spectra of VCO, GSO, and SO at mid
infrared region (4000–650 cm−1). Each bands/peaks and shoulders are characteristics for FTIR
spectra of triglyceride (TG). There is a bit difference in terms of bands and shoulders intensity
between VCO and two other oils, mainly at 1/λ of about 3007 and 1654 cm−1. Bands at 1/λ of
3007 and 1654 cm−1 were absent in FTIR spectrum of VCO. These bands, corresponding to
stretching vibration of unsaturation degree (= CH vinyl and C = C), were observed in FTIR
spectra of GSO and SO. The difference was also observed at 1/λ of 1120–1095 cm−1, correspond-
ing to ether (C–O) vibration. VCO showed one peak at 1117 cm−1, while GSO and SO revealed
two peaks at 1117 and 1097 cm−1, respectively. These differences were used as basis for the
authentication analysis of VCO.
PLSR using absorbance values at combined 1/λ of 1200–900 and 3027–2985 cm−1 revealed
reliable method for the quantification of GSO in VCO, as indicated by high value of R2 (>0.99)
and low value of RMSEC (0.007% vol/vol) and RMSEP (1.32% vol/vol). In addition, PLSR using
FTIR spectra at the combined 1/λ of 1200–1000 and 3025–2995 cm−1 was preferred for
 58

Table 5. The different analytical techniques used for authentication of virgin coconut oil reported by authors.
Methods Oil adulterant Analytical results References
[82]
FTIR-ATR spectroscopy Palm kernel oil (PKO) Using whole mid IR at wavenumbers (1/λ) 4000–650 cm−1 combined with PLSR using 10 Principle
components, PKO at 1% could be detected. Discriminant analysis could classify VCO and VCO mixed with
other vegetable oils (walnut, extra virgin olive, soybean, sunflower, grapeseed, sesame, canola and corn
oils).
[83]
FTIR-ATR spectroscopy Palm oil (PO) PLSR at combined 1/λ of 3010–3000, 1660–1650 and 1120–1105 cm−1 exhibited a good relationship
between actual and FTIR-predicted values of PO with coefficient of determination (R2) of 0.999 and
standard error of calibration of 0.533. Discriminant analysis using 7 factors could classify pure VCO and that
A. ROHMAN ET AL.

adulterated with PO.

[84]
FTIR-ATR spectroscopy Palm oil (PO) in ternary PLSR at combined wavenumbers of 1120–1105 and 965–960 cm−1 is successfully applied for quantification
systems with VCO and olive of VCO adulterated with PO with high R2 (0.9996) and low RMSEC (0.494)
oil
[85]
FTIR-ATR spectroscopy Corn oil (CO) and sunflower PLSR using variable of absorbance values at combined wavenumbers of 858–705, 943–863, 1392–983, and
−1
oil (SFO) 3027–2983 cm was successfully used for prediction the levels of CO in VCO, and at 1685–686, 2946–1887,
and 3027–2983 cm−1 was used for quantification of SFO in VCO. The R2 > 0.999 for both CO and SFO. The
RMSEC values of CO
and SFO in VCO were 0.866% and 0.374% (v/v), respectively.
[86]
FTIR-ATR spectroscopy Lard (LD) The combined 1/λ of 3020–3000 cm−1 and 1120–1000 cm−1 using PLSR able to predict LD contents in VCO
with R2 value of 0.9990 for the relation between actual value of LD and FTIR predicted value.
RMSEC = 0.722%; RMSECV = 1.54%. DA at the same 1/λ could classify VCO and VCO adulterated with LD.
[87]
Nuclear magnetic resonance Refined coconut oil (RCO) Phosphorus-containing dioxaphospholane derivatives of monolgycerides (MGs), diglycerides (DGs), sterols
31
spectroscopy ( P NMR) and free fatty acids (FFA) were analyzed by 31P NMR. VCO had 40% higher of 1-MG content than RCO and
lower DG content (1.5%) than RCO (4.1%). VCO had sterol contents of 0.096%, lower than RCO (0.032%).
VCO had FFA contents 8 times higher than RCO.
[88]
Differential scanning calorimetry Soybean oil (SO), The heating curves of VCO adulterated with SFO and SO exhibited that adulteration peak appeared at the
(DSC) sunflower oil (SFO) and lower temperature region from 10% adulteration level. SMLR was used to predict the percent adulterant
palm kernel oil (PKO) with R2 of 0.9390 for SFO and 0.9490 for SO. For PKO adulteration, no adulteration peak was observed, but
there is good relationship between peak height of PKO and adulteration levels.
[89]
DSC Lard (LD) DSC provides unique thermal profiling for VCO and VCO adulterated with LD. In the heating thermogram,
one major endothermic peak (called with peak A) gradually smoothed out to the major peak with the
increased levels of LD. In the cooling thermogram, there are two major exothermic peaks, peak C which
increased as LD% increased and peak D which decreased in size as the LD% increased. SMLR able to predict
LD% adulteration in VCO with R2 (adjusted) of 95.82.
[90]
Electronic nose- SAW detector Palm kernel oil (PKO) Electronic nose using zNose equipped with surface acoustic wave (SAW) detector has been used for
detection of VCO adulteration with PKO. PCA using adulterant peaks was applied successfully to classify
VCO and VCO adulterated with PKO with 74% and 17% of the variations accounted for PC1 and PC2,
respectively.
[86]
Electronic nose-SAW detector Lard (LD) Binary admixtures of LD in VCO in various percentage concentrations ranging from 1% to 50% (v/v) have
been successfully assayed using electronic nose-SAW system. Ten different chromatogram peaks were
identified as the adulterant peaks. One peak (peak J) was found to have the best relationship, with R2 value
of 0.9344.
[91]
Two-dimensional gas Animal fats of lard (LD), The Changes in the cholesterol levels due to the addition of animal fats with VCO were used for detection
chromatography coupled with chicken fat (CF), beef tallow of VCO adulteration. The increased cholesterol levels in VCO are valid parameter that could be used to
time of flight mass (BF), and mutton tallow detect the adulteration of VCO with animal fats at a level as low as 0.25%.
spectrometry (GC x GC-TOF-MS) (MT)
 FOOD REVIEWS INTERNATIONAL 59

Figure 1. FTIR spectra of virgin coconut oil, grape seed oil, and soybean oil scanned at wavenumbers of
4000–650 cm−1 using attenuated total reflectance as sampling technique. Taken from Rohman et al.[94]
with permission from publisher.

quantitative analysis of SO in VCO. Discriminant analysis, one of the supervised pattern

recognitions, was also successfully used for the discrimination between VCO and VCO added
with adulterants of GSO and SO using the same wavenumbers used for quantitative analysis, as
shown in Fig. 2.

Figure 2. The Coomans plot of virgin coconut oil (VCO) and adulterants: (□) VCO; (Δ) VCO containing
adulterants of canola oil (A); grape seed oil (C); and Soybean oil (F). Taken from Rohman et al.[94] with
permission from publisher.
60 A. ROHMAN ET AL.

Che Man and Rohman[95] have applied FTIR spectroscopy combined with PLSR and DA
for quantification and classification of VCO adulteration with canola oil (CaO). The studied
oils (pure VCO, pure Ca-O, and the mixture of VCO-CaO) were scanned using FTIR-
spectrophotometer at wavenumbers of 4000–650 cm−1 using sampling technique of hor-
izontal-attenuated total reflectance (HATR). The authors have applied several pre-processing
techniques including derivative spectra to obtain the best prediction model. FTIR normal
spectra at combined 1/λ of 1200–900 and 3027–2985 cm−1 were suitable for the quantitative
analysis of CaO due to their capabilities to provide the high R2 values and low RMSEC and
RMSEP values. DA using the same wavenumbers used for quantitative analysis was able to
discriminate VCO and VCO adulterated with CaO without any misclassification reported.

Conclusion
VCO was prepared from wet methods without any RBD treatment, hence, the active
components such as phenolics and tocopherols are retained in VCO. These compounds
are responsible for biological activities including antioxidant, anti-inflammatory and
immunomodulatory, anti-hyperlipidemia, anticancer, antidiabetic, antibacterial and neu-
roprotective as proved from in vitro and in vivo studies. VCO commanded high price in
fats and oils industry which can be target of adulteration, therefore reliable analytical
techniques such as FTIR spectroscopy and chromatography has been successfully applied
for the authentication analysis. From this review, VCO is considered as functional food
oils which are potential to be used as component in food products.

Acknowledgments
The authors acknowledged the Ministry of Research and Higher Education, the Republic of
Indonesia for financial support during preparing this review article through the scheme “World
Class Research 2019” with contract number of 1973/UN1.DITLIT/DIT-LIT/LT/2019 awarded to
Prof. Dr Abdul Rohman and the program development of Twin center Universitas Gadjah Mada,
awarded to Dr Yuny Erwanto.

Funding
This work was supported by The ministry of Research and higher education [1973/UN1.DITLIT/
DIT-LIT/ LT/2019] through the scheme of World Class Research.

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