Ozone: Science and Engineering

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Study of Three Systems of Ozonized Coconut Oil

Maritza F. Díaz , Nayibi Núñez , Daraisy Quincose , Wilfredo Díaz & Frank
Hernández

To cite this article: Maritza F. Díaz , Nayibi Núñez , Daraisy Quincose , Wilfredo Díaz & Frank
Hernández (2005) Study of Three Systems of Ozonized Coconut Oil, Ozone: Science and
Engineering, 27:2, 153-157, DOI: 10.1080/01919510590925275

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Ozone: Science and Engineering, 27: 153–157
ISSN: 0191-9512 print / 1547–6545 online
Copyright # 2005 International Ozone Association
DOI: 10.1080/01919510590925275
Study of Three Systems of Ozonized Coconut Oil
Maritza F. Dı́az, Nayibi Núñez, Daraisy Quincose, Wilfredo Dı́az, and Frank Hernández
Ozone Research Center, National Center for Scientific Research, Havana, Cuba
In the present study, the products from the ozonation of
three systems (ozonized coconut oil, ozonized coconut oil
with water and ozonized coconut oil with ethanol) are
characterized and compared statistically. Peroxide, acidity
and viscosity indexes were determined. The reaction pro-
ducts were identified by 1H NMR and their antimicrobial
activity was evaluated. The ozonized coconut oil with etha-
nol showed the highest peroxide and acidity indexes. This
result suggests that in presence of ethanol a greater perox-
ide decomposition occurs leading to greater acid formation.
The variation coefficients obtained in the analysis methods
were smaller than 10%. Reaction products were identified
as ozonides and aldehydes compounds. The highest action
spectrum of antimicrobial activity by Staphylococcus aur-
eus was obtained with the ozonized coconut oil with water
and ethanol systems.
Keywords Ozone, Ozonides, 1H NMR, Coconut Oil, Anti-
microbial Activity
INTRODUCTION
Vegetable oils characterization of has been the subject
of academic study or commercial interest for many years.
In the last century, an additional stimulus has been the
growth of biochemistry and chemistry and the desire to
obtain a variety of natural products with different func-
tions. The vegetable oils show a wide variation in fatty
acids composition, which is associated with characteris-
tics and properties of the oils (Bailey, 1961).
Coconut oil is historically very popular in the food,
drugs, and cosmetic industry. For hundreds of years the
Philippine women have used coconut oil as liniment for
skin stimulation (DeNavarre, 1962). The coconut palm
grows along the West Coast of Central America and the
Indian Ocean. The tree grows to a height of 24 m and the
main producer is the Philippines. The copra is the source
Address correspondence to Maritza F. Dı́az, Department of
Ozonized Substances, Ozone Research Center, National Center for
Scientific Research, P.O. Box 6412, Havana, Cuba.
E-mail: ozono@infomed.sld.cu
Ozone: Science and Engineering
of coconut oil and contains 65–68% oil and 4–7% moist-
ure (Gunstone et al., 1986). The oil is characterized by the
presence of short chain fatty acids (6:0 to 12:0), and by its
low level of unsaturated fatty acids (Harwood and
Aparicio, 2000).
Oxidation reactions are very important in fatty acid
chemistry (Pryor and Wu, 1992; Pryor et al., 1995;
Rebrovic, 1992), since their reaction products are involved
in important microbiological effects (Dı́az et al., 2001).
The reaction of ozone with vegetable oil occurs almost
exclusively with the carbon-carbon double bonds present
in unsaturated fatty acids (Bailey, 1978). This reaction
produces several products such as Criegee ozonide, hydro-
peroxides and aldehydes (Dı́az et al., 1998) and these
compounds might be related to biological activity of
some vegetable oils (Lezcano et al., 1996). The concentra-
tion of these reaction products depends upon the solvent
used to initiate the reaction (Squadrito et al., 1992).
Analytical methods as peroxide index, acidity index,
and viscosity are used for determining the quality of ozo-
nized vegetable oils. These methods must be precise and
reproducible because these parameters are involved in the
quality of the ozonized vegetable oils (Thielmann, 1973).
The aim of the present paper is to determine the best
system for coconut oil ozonation in order to obtain a
product with adequate biological activity. Three different
systems (ozonized coconut oil, ozonized coconut oil with
water, and ozonized coconut oil with ethanol) are com-
pared using different analytical techniques, 1H NMR
spectroscopy, and antimicrobial activity.
MATERIALS AND METHODS
General Ozonation Procedure
Coconut oil (150 g) was introduced into a bubbling
reactor at room temperature. The reaction with ozone
was continued for 74.4 min. The same ozonation reac-
tions were obtained using a mixture of coconut oil (150 g)
with 15 mL of water or ethanol. The ozonized coconut oil
was stored at -10 ˚ C before further analysis.
April 2005 153
Ozone Generation
Ozone was generated by passing oxygen through a
Trailigaz Labo model 12–02 ozonator (Trailigaz, Paris,
France) at a voltage of 100 V, and a constant flow rate of
54 L/h. The ozone concentration obtained was 37.5 mg/L.
The Anseros Ozomat (Anseros, Tübingen, Germany),
equipment determined the ozone concentration.Total
dosage was 16.74 mg of ozone per gram of oil.
Analysis Technique
Peroxide Index. The peroxide index is the number
that expresses, in milliequivalents of active oxygen, the
quantity of peroxide contained in 1 000 g of the substance
(BP, 1998, 2000; Panreac, 1992).
Acidity Index. Acidity is frequently expressed as the
acidity index, which is the number of mg of potassium
hydroxide required to neutralize the free acids in 1.0 g of
the substance (BP, 1998, 2000; Panreac, 1992).
Viscosity. The viscosity was determined by the
Mettler RM 180 Rheomat rotational viscometer
(Mettler Toledo, Greifensee, Switzerland). Its open, con-
centric measurement system allows measurement by
immersion. The viscosity (Z) is calculated in Pascal
seconds (Pa s) (BP, 1998, 2000; Mettler, 1993).
The values of peroxides indexes, acidity, and viscosity
of the three studied systems were determined. Ten experi-
ences the same days (Repeatability) and three experiences
during five days (Intermedial Precision) were realized. All
data are expressed as means ± standard deviation.
Variation coefficients were calculated from each system.
Measurement of 1H NMR Spectra. The 1H NMR
spectra were obtained with a Bruker AC-250 F spectro-
meter (Bruker Optics, Ettlingen, Germany) with CDCl3
as solvent, and tetramethylsilane (TMS) as internal refer-
ence. 5 KHz spectral width, 60-degree pulse width (5 ms),
8 scans, and 32 Kbites of memory were used to obtain the
spectra. It was not necessary to use an internal standard
to measure the integrals of the signal areas because the
methylic signal of the compounds can be used as
TABLE 1. Peroxide Indexes Obtained in the Three Systems of Ozonized Coconut Oil
Number of experiences Mean ± Standard deviation Variation coefficients (%)
Ozonized Coconut Oil
Repeatability 10
Intermedial Precision 15
Ozonized Coconut Oil with Water
Repeatability 10
Intermedial Precision 15
Ozonized Coconut Oil with Ethanol
Repeatability 10
Intermedial Precision 15
154 Ozone: Science and Engineering
reference value for the relationship of the intensity values
of each spectrum.
Microbiological Studies
The antimicrobial activities from the ozonized oils
obtained by the three systems were determined by agar
diffusion sensitivity test (NCCLS, 1993). Oil aliquots
were poured in the central hole of an agar culture plaque,
which was inoculated with Staphylococcus aureus ATCC
25923, or Pseudomona aeruginosa ATCC 27853. The dis-
tance in millimeters of inhibition halo measured bacterial
growth inhibition. Each experiment was repeated at least
ten times. All data are expressed as means ± standard
deviations. Parallel experiments were performed in order
to determine the inhibitory growth of microorganisms
under the same conditions but with different antibiotic.
RESULTS AND DISCUSSION
Characterization and comparison of three systems
(ozonized coconut oil, ozonized coconut oil with water,
and ozonized coconut oil with ethanol) by different ana-
lysis methods.
Table 1 shows peroxide indexes values of the three
systems studied. Variation coefficients were less than
10% leading to good precision. There was also a remark-
able decrease in variation coefficients associated with
ozonized coconut oil with ethanol, which suggested a
better precision.
The coconut oil contains a low level of unsaturated
fatty acids as oleic acid (11%) and linoleic acid (2%)
(Gunstone et al., 1986). The reaction of ozone with coco-
nut oil occurs almost exclusively with the carbon-carbon
double bonds present in unsaturated fatty acids. This
reaction produces ozonized coconut oil with (260–470
meq O2/kg) peroxide indexes. On the other hand, the
ozone reaction with other vegetable oils, which have
high level of unsaturated fatty acids, led to rapid increase
of peroxide indexes. Olive contains a high proportion of
Peroxide indexes (meq O2/kg)
263.7 ± 13.66 5.18
231.0 ± 9.36 4.05
325.2 ± 14.09 4.33
243.4 ± 15.25 4.44
463.2 ± 10.22 2.21
402.5 ± 7.95 1.97
April 2005
TABLE 2. Acidity Indexes Obtained in the Three Systems of Ozonized Coconut Oil

Acidity Indexes (unit)

Number of experiences Mean ± Standard deviation Variation coefficients (%)

Ozonized Coconut Oil
Repeatability 10 5.26 ± 0.201 3.82
Intermedial Precision 15 6.31 ± 0.243 3.85
Ozonized Coconut Oil with Water
Repeatability 10 6.01 ± 0.172 2.86
Intermedial Precision 15 7.48 ± 0.285 3.81
Ozonized Coconut Oil with Ethanol
Repeatability 10 7.05 ± 0.158 2.24
Intermedial Precision 15 7.72 ± 0.220 2.85

oleic acid (65–85%), moreover, sunflower oil is rich in
linoleic acid (60%) and oleic acid (28%) (Capella, 1993;
Firestone and Reina, 1996). These ozonized oils should
have a higher proportion of peroxide indexes than ozo-
nized coconut oil.
The ozonized coconut oil with ethanol has a marked
increase in peroxide index value (463 meq O2/kg), (Table 1)
when compared to other systems. The results confirm that
the yield of ozonation products from unsaturated fatty
acids depends on the type of medium where the reactions
take place (Dı́az et al., 1998).
Table 2 shows acidity indexes values of the three stu-
died systems. All variation coefficients were less than 4%,
which leads to good method. Ozonized coconut oil with
ethanol showed the lowest variation coefficients and sug-
gested a slightly better precision.
An increase in acidity indexes was also observed in
ozonized coconut oil with ethanol possibly caused by
the decomposition of the hydroperoxides in acids. These
results agree with the highest peroxide index observed for
this system (Table 1).
The viscosity of the three systems studied is shown in
Table 3. Variation coefficients were less than 10% and are
representative of good precision. The system of ozonized
coconut oil with ethanol showed lower viscosity. This
behavior might be due to the solubility of oil with the
ethanol. When water is used in the systems the viscosity
increases because the emulsion appears due to the poor
solubility of oil with water.
Characterization and comparison by 1H NMR of the
three systems studied
Figure 1 shows 1H NMR spectrum from virgin coconut
oil, presenting singlete peak in d = 7.26 ppm, which
belongs to the chloroform-d, and multiplet peaks between
d =5.4 and d = 5.3 ppm, which belong to olefinic signals
from the fatty acid. Other present signals are d = 5.25
ppm (multiplet belong to glycerol methynic proton); d =
4.1 to d = 4.4 ppm (doblets belonging to glycerol protons);
d = 2.3 ppm (triplet from methylenic groups in a position
respect to carbonylic group); d = 2.0 ppm (multiplet from
methylenic group in both sides of olefinic protons); d =
1.62 ppm (multiplet from methylenic group in b position
with regards to carbonylic group); d = 1.3 ppm (signal
from methylenic groups in fatty acid chain); and d = 0.89
ppm (triplet from terminal methyl group).
1
H NMR spectrum from ozonized coconut oil with
water is displayed in Figure 2. This spectrum has the

TABLE 3. Viscosity Values Obtained in the Three Systems of Ozonized Coconut Oil

Viscosity (mPa.s.)

Number of experiences Mean ± Standard deviation Variation coefficients (%)

Ozonized Coconut Oil
Repeatability 10 62.7 ± 0.516 0.82
Intermedial Precision 15 63.3 ± 3.190 5.03
Ozonized Coconut Oil with Water
Repeatability 10 66.5 ± 0.547 0.82
Intermedial Precision 15 64.9 ± 2.870 4.42
Ozonized Coconut Oil with Ethanol
Repeatability 10 57.3 ± 0.516 0.90
Intermedial Precision 15 58.1 ± 2.060 3.54

Ozone: Science and Engineering April 2005 155


FIGURE 1. 250 MHz 1H NMR spectrum of virgin coconut oil.
same observed signals in virgin coconut oil (Figure 1) and
additionally other two signals at d = 9.75 ppm (triplet
from aldehydic protons), d = 5.2 to d = 5.1 ppm (multi-
plet from ozonides). As it can be seen, the comparison
with the virgin coconut oil spectrum (Figure 1) is the
non-appearance of olefinic signals from the fatty acid
between d = 5.4 and d = 5.3 ppm as the ozonation
reaction consumed all bond doubles.
In the three ozonized systems 1H NMR spectra,
showed the same signals. Measurements of intensity
values of new signals were different from each spectrum
(Table 4). The maximal formation of ozonides was
achieved in the system ozonized coconut oil with water
(intensity relationship 1.2) while in the system ozonized
coconut oil with ethanol there was a minor conversion
(intensity relationship 0.9). This result is important
because the ozonide has been considered as one of the
active principles of ozonized sunflower oil with antimi-
crobial activity (Dı́az et al., 2001). The aldehyde protons
were observed weak intensity signals. Volatile compound
formation is higher in the system with ethanol (0.62) than
in water (0.32). This fact confirms that ethanol is a better
medium for aldehyde formation (Dı́az et al., 1997).

Microbiological Analysis
Data for antimicrobial spectra of three systems of ozo-
nized coconut oil and antibiotic are presented in table 5. The
FIGURE 2. 250 MHz 1H NMR spectrum of ozonized coconut oil
with water.
tests for three systems were antimicrobial activity. An
increase of the action spectrum of ozonized coconut oil

TABLE 4. Signal Intensity Values of Reaction Product in Three Systems of Ozonized Coconut Oil

Intensity values by 1H NMR

Ozonides of methynic protons Aldehydic protons

Ozonized coconut oil 0.4 0.28
Ozonized coconut oil with water 1.2 0.32
Ozonized coconut oil with ethanol 0.9 0.62

TABLE 5. Antimicrobial Effect by Agar Diffusion Test of Three Systems of Ozonized Coconut Oil

Staphylococcus aureus Pseudomonas aeuriginosa

ATCC 25923 ATCC 27853

Means ± SD (mm) Means ± SD (mm)
Coconut Oil 0 0
Ozonized Coconut Oil 21.2 ± 0.5 17.5 ± 0.69
Ozonized Coconut Oil with water 28.0 ± 0.42 24.0 ± 0.81
Ozonized Coconut Oil with ethanol 28.5 ± 0.41 25.2 ± 0.75
Gentamicin 18.0 ± 0,48 19.0 ± 0.58
Chloramphenicol 20.0 ± 0.77 21.2 ± 0.34
Kanamycin 20.0 ± 0.38 17.4 ± 0.25
Erythromycin 17.0 ± 0.66 18.6 ± 0.34
Means ± SD (mm): zone of growth inhibition.

156 Ozone: Science and Engineering April 2005

with ethanol was observed. The ATCC strains
Staphylococcus aureus in the systems ethanol and water
were more sensitive than the antibiotics. This result is the
most important because Staphylococcus aureus is a very
resistant gram-positive bacterium to different antibiotic
(Linuma et al., 1994). Coconut oil alone showed no antimi-
crobial activity.
CONCLUSIONS
Higher peroxide decomposition occurs when ethanol is
added to the reaction of coconut oil with ozone. This
favors a higher acids and aldehyde formation. When
water is present there is a higher ozonides formation.
All these oxygen compounds formed in ethanol or water
presence are the effectors of antimicrobial activity of
ozonized coconut oil. It was demonstrated that 1H
NMR technique is an appropriate spectroscopic method
for the detection of specific oxygen compounds from
ozonized coconut oil.
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