988 Eur. J. Lipid Sci. Technol.

2015, 117, 988–998

Research Article
Correlation of structural characterization and viscosity
measurements with total unsaturation: An effective method
for controlling ozonation in the preparation of ozonated grape
seed and sunflower oils

Pamela Guerra-Blanco1, Tatyana Poznyak1, Isaac Chairez2 and Marco Brito-Arias2

1
Escuela Superior de Ingeniería Química e Industrias Extractivas (ESIQIE)-IPN, México, D.F., Mexico
2
Unidad Profesional Interdisciplinaria de Biotecnología (UPIBI)-IPN, México, D.F., Mexico

Ozonated oils have demonstrated promising results for clinical applications. The reaction of ozone with the
unsaturated compounds of oils produces by-products such as ozonides and poly peroxides. A deeper
knowledge of the dynamics of by-product formation is helpful in determining the required ozonation degree
to obtain therapeutic effects. The aim of this paper is to show the relationship between ozonation degree and
structural and viscosity changes during the ozonation of grape seed (GS) and sunflower (SF) oils. Structural
characterization was done by Fourier transform infrared (FT-IR) and hydrogen-1 nuclear magnetic
resonance (1H NMR) spectroscopy, with iso-ozonides being identified. Viscosity showed a significant
increase during ozonation, a fact associated with poly peroxide formation. We have made use of the total
unsaturation (TU) method to measure the ozonation degree. The TU of non-ozonated GS oil was found to
be higher than for SF oil (5.94 and 4.49 mmol per g of oil, respectively), and their by-product distributions
were also found to differ. In GS oil, three reaction steps were observed for double-bond conversion into iso-
ozonides and poly peroxides, while the ozonides and poly peroxides were formed in parallel in SF oil. The
studies we implemented characterized the differences in the reactivities of these oils with ozone.

Practical applications In this work, we propose using the TU method to measure the ozonation
degree of ozonated oils. TU experimental determination is based on the ozonation of the sample, and the
ozone-oxidizable substrate is quantified. Despite GS and SF oils having similar fatty acid compositions,
they contain other unsaturated compounds specific to their vegetal sources. These compounds are also
reactive with ozone, and are also quantified by the TU method. The differences in distributions of by-
products among ozonated oils from different sources could explain why the ozonation degree need not be
the same for different oils. Studies like this represent a feasibility foundation for controlling the
therapeutic application of ozonated oils and correctly interpreting their well-known clinical effects.

Keywords: FT-IR spectroscopy / 1H NMR spectroscopy / Ozonated grape seed oil / Ozonated sunflower oil / Ozone /
Total Unsaturation (TU)
Received: June 18, 2014 / Revised: November 12, 2014 / Accepted: November 28, 2014
DOI: 10.1002/ejlt.201400292

1 Introduction in medicine. In fact, in some European countries, such as

Ozonated vegetable oils have many interesting applications in
the food, cosmetic, and pharmaceutical industries, as well as
Correspondence: Dr. Pamela Guerra Blanco, Edif. 8, 3er piso (SEPI-
ESIQIE), UPALM, C.P. 07738, México, D.F México
E-mail: pamelaguerrablanco@gmail.com
Fax: þ52 (55) 5729 6000 EXT. 52600
Abbreviations: TU, Total Unsaturation; GS, Grape seed; SF, Sunflower;
PCNA, Proliferating cell nuclear antigen; NFkB, Nuclear factor kappa B;
VEGF, Vascular endothelial growth factor; 4-HNE, 4-Hydroxynonenal
Germany, they can be obtained on prescription from
pharmacies [1]. Countries such as Cuba have developed
commercial ozonated oils, and they have been successfully
tested for the treatment of many diseases [2–4].
The therapeutic effects achieved by ozone’s medical
applications depend on administration pathway and dose
[5]. In this sense, ozonated oils provide stable ozonation by-
products (mainly iso-ozonides, hydroperoxides, and poly
peroxides) with therapeutic potential. The medical appli-
cations of ozonated oils have been extensively studied.
Several papers have demonstrated their bactericidal and

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

Eur. J. Lipid Sci. Technol. 2015, 117, 988–998
antifungal effects, as well as their role as wound-healing
modulators [6–9].
Due to the lack of control methods for ozonated oil
production, the required ozonation degree to achieve the
desired therapeutic effects has not been well established.
In vitro tests have shown an increase in bactericidal and
antifungal effects with increasing ozonation degree [10–12].
In vivo tests have shown that, depending on the disease and
the nature of the oil, the ozonation degree must be lower or
higher [1, 13, 14]. In fact, when ozonation degree is not
controlled, the positive effects of ozonated oils may be
reversed.
The application of ozonated sesame oil for wound healing
in mice has shown that this oil must be ozonated until a
“medium” degree of peroxidation (PV ¼ 1631  64 mEq/kg)
is achieved. Lower and higher oxidation levels have been
shown to be not as effective [13]. On the other hand, the
periodic application of completely ozonated olive oil (iodine
value ¼ 0) to the skin caused hair loss at the site and contact
hypersensitivity. The authors have found that ozonated olive
oil produces both swelling-suppressive and proinflammatory
effects. However, its repeated application markedly induces
the swelling reaction [1]. These results reveal the need to
develop control methods for ozonated oil production.
The therapeutic effects of ozonated oils are strongly
related to the accumulation of ozone reaction products. The
ozonation of unsaturated compounds, including fatty acids,
is well characterized by the Criegee mechanism [15–17].
Ozone reacts mainly with the double bonds of unsaturated
fatty acids (free and as esters in triglycerides) of vegetable oils.
As a consequence of this reaction, oxidized products such as
iso-ozonides, hydroperoxides, and poly peroxides, are
formed (depending on reaction conditions).
Scheme 1 shows the Criegee mechanism for the reaction
of ozone with the double bonds of vegetable oils (in the
absence of polar solvents). In this scheme, the primary
product of the reaction is so-called molozonide (I), which is
unstable and decomposes rapidly to zwitterion (II) and a
carbonyl compound (aldehyde or ketone, III). These
Scheme1. Criegee mechanism for the reaction of ozone with >C –
– C< of vegetable oils.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Structural characterization of ozonated oils 989
intermediate compounds can combine again and form a
normal ozonide (iso-ozonide, IV–VI). The reaction products
usually consist of a mixture of the normal ozonide (IV),
the crossed ozonides (V) and (VI), and poly peroxides
(oligomeric ozonides, VII) [15–17].
By-product identification has already been reported [18–
23]; however, the formation/decomposition dynamics of
these by-products during ozonation has not received much
attention. The differences in by-product distributions among
ozonated oils from different sources could explain why the
ozonation degree need not be the same for different oils.
In order to elucidate the ozone reaction pathway with
vegetable oils, several techniques have been used to character-
ize vegetable oils as well as their ozone reaction products.
Spectroscopic methods are adequate, because they make it
possible to observe changes derived from the ozone’s reaction
with specific functional groups. Fourier transform infrared
(FT-IR) and hydrogen-1 nuclear magnetic resonance
(1H NMR) spectroscopy have been extensively used to study
these reactions, and the signals corresponding to reactant and
product groups are well identified [19, 20, 24–26].
Even though the information obtained with these
analytical techniques is highly reliable, the required infra-
structure makes them impractical for routine control
methods. In this sense, some simple-determination analytical
techniques, such as peroxide value, iodine value, and
viscosity determination [18, 22], as well as calorimetry
[27], have been attempted to correlate them with the
information obtained from spectroscopic analyses, in order
to establish them as control methods of ozonation degree.
In the second half of the last century, Russian researchers
developed the Total Unsaturation (TU) technique [28–30].
With this technique, it is possible to determine the oxidizable
substrate by ozone in a short time (1–3 min) and with high
precision (1%). The TU determination is made by
measuring the ozone necessary to react with the sample
and comparing it with the ozone consumed in a secondary
reaction with a standard sample of known chemical structure
and concentration. Ozone reacts with all the >C –– C< with
www.ejlst.com
990 P. Guerra-Blanco et al.
similar reaction-rate constants, regardless of the chemical
structure of the compounds containing them [31], so TU
accurately represents the >C –– C< concentration in analyzed
samples (in mol of >C –– C< per g of oil). The fundamentals of
this method can be reviewed in [29, 30, 32].
Since TU provides a direct measure of the total substrate
that is oxidizable by ozone, the ozonation degree corre-
sponding to all substrates that react with ozone is also
measured. The correlation between ozonation degree and
reaction products accumulation, as well as therapeutic
effects, makes it possible to control ozonated oil production
based on kinetic fundamentals.
This work studies the ozonation of two oils: grape seed
(GS) and sunflower (SF) oil. GS and SF oils were selected
because of their similar composition [33], in order to
compare reaction product distributions in the ozonation of
two oils with similar fatty acid composition but different
sources. Identification of by-products was done by FT-IR
and 1H NMR spectroscopy. Double-bond decomposition
was monitored during ozonation, as were ozonation by-
product dynamics and viscosity changes. All these variables
were compared with ozonation degree as measured by TU, in
order to correlate the latter with reaction product accumu-
lation. In this sense, TU may be used as an easy, reliable, and
accessible method for controlling ozonated oil production.
2 Materials and methods
The experiments were performed in triplicate; where
appropriate, mean values and SD were calculated and
displayed.
2.1 Oils
Commercial extra-virgin edible GS and SF oils were bought
in local markets. These were used in order to provide a study
close to conditions applying to commercial products.
2.2 Ozonation
Ozone was generated from extra-dry oxygen (99.9%). Nine
grams of each oil were ozonated in a semi-batch reactor using
an AZCO ozone generator with an ozone concentration of
30 mg/L and gas flow of 0.5 L/min. Ozone in gas phase in the
reactor outlet was measured with a BMT 930 ozone analyzer
connected to a computer. The ozone monitoring was used to
measure the ozone consumption. These results are repre-
sented by the characteristic plot of ozone concentration
versus time, called an ozonogram. The complete exper-
imental design can be reviewed in a previous work [34].
The ozonation degree of vegetable oils was measured and
characterized by spectroscopic (FT-IR, 1H NMR) and
physicochemical (viscosity measurement) techniques, as well
as by their TU.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2015, 117, 988–998
2.3 FT-IR spectroscopy
Oil samples were diluted with CHCl3 (0.1 g/mL), and a thin
layer was then formed in a potassium bromide (KBr)
window. The solvent was evaporated, and FT-IR spectra in
the 4000–400 cm1 region were obtained with a Thermo
Nicolet Nexus 470 spectrometer.
1
2.4 H NMR spectroscopy
1
H NMR spectra were obtained with a Varian Mercury
300 MHz spectrometer. All the samples were diluted with
CDCl3.
2.5 Viscosity
The oils’ kinematic viscosities were measured with Cannon–
Fenske routine viscometers (calibrated, size 200–400) in a
viscosity bath with temperature regulated at 37 °C (0.1 °C).
2.6 Total unsaturation (TU)
Oil samples (ozonated and non-ozonated) were weighed and
dissolved in CHCl3; then 10 mL of this solution was injected
into a reactor containing CCl4 with an ozone gas flow of
0.1 L/min. The ozonogram’s area (ozone reacted with oil)
was measured and compared with a standard area (ozone
reacted with stilbene, 5  105 mol/mL). Based on the area,
the known concentration and >C –– C< per mol in the
standard, the >C –– C< concentration in the new sample can
be calculated and expressed as mol of >C –– C< per gram of oil
by comparing the two areas [30]. The mathematical
expression for the TU is:
 
CST V ST SS V SOL mol>C¼C<
TU ¼ ½¼
SST V S W S goil
where, CST is the concentration of the standard solution
(mol/mL), VST, and VS are the volumes of the standard
sample and the analyzed sample, respectively (mL), SST and
SS are the ozonogram areas for the standard and analyzed
samples (conditional units [30]), VSOL is the solution volume
of the analyzed sample (mL), and WS is the weight of the oil
sample (g).
3 Results and discussion
3.1 Oil compositions
The main components of vegetable oils are triglycerides; their
fatty acid ester distributions differ according to seed
characteristics. The compositions of GS and SF oils are
shown in Table 1. As can be seen, GS and SF oils have similar
oleic acid (C18:1) and linoleic acid (C18:2) compositions,
www.ejlst.com
Eur. J. Lipid Sci. Technol. 2015, 117, 988–998
Table 1. Lipid composition of oils
Oil Saturated fats Mono-unsaturated fats
(%)a) (%)a)
Grape seed 10 21
Sunflower 10 28
a)
Nutritional product information.
b)
CODEX-STAN 210, 1999 [33].
these being their major components. Hereafter, “fatty acids”
refers to free fatty acids and their esters in triglycerides.
3.2 Oil ozonation
GS and SF oils were ozonated until the ozone reaction was
complete, meaning when the outlet ozone’s concentration
was equal to the inlet concentration (5.0 h for the
experimental conditions). The latter was controlled by the
ozonogram, with the plot representing ozone that was not
consumed during the ozonation process. Comparing the
ozonograms of GS and SF oils in Fig. 1, it can be seen that
GS oil consumes more ozone than SF oil, indicating that GS
oil contains more oxidizable substrate. In fact, some
differences in the formation and decomposition rates of
by-products from GS and SF ozonation were found,
although it was expected that their behaviors would be
similar, taking into account their similarities in fatty acid
composition.
3.3 Ozonated oil characterization
3.3.1 FT-IR and 1H NMR spectroscopic analysis
Based on FT-IR spectra, the conversion from double bonds to
ozonides was observed. The latter are the principal ozonation
Figure 1. Ozonograms of vegetable oils.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Structural characterization of ozonated oils 991
Poly-unsaturated fats Oleic acid Linoleic acid
(%)a) (%)b) (%)b)
69 12.0–28.0 58.0–78.0
62 14.0–39.4 48.3–74.0
by-products produced in the absence of protic solvents [25].
The expected decreases in characteristic wave numbers of
C –– C double-bond stretching (1654 cm1) and –– C–H stretch-
ing (3009 cm1) were observed [35], as well as the increase on
ozonide C–O stretching (1105 cm1) [22, 24].
More specific information was obtained by 1H NMR
spectroscopy in order to differentiate the chemical structures
modified during ozonation. Four different types of signals
were observed in the GS and SF oil spectral data, based on
their behavior under ozonation. We identified these types as
conserved, decreasing, increasing, and increasing/decreasing
signals. The 1H NMR spectra of GS oil (before and after 5 h
of ozonation) are shown in Fig. 2. Changes observed in GS
oil were also observed in SF oil.
The conserved signals, for which the non-exchangeable
protons were not in the proximity of the double bonds, did
not suffer any modification upon ozone treatment. These
signals correspond to the a- and b-protons of carbonyl groups
at 2.32, 1.61, 1.22–1.25, and 0.87 ppm for the methylene
and methyl protons and at 4.16, 4.28, and 5.22 ppm for
the glycerol moiety.
The decreasing signals were assigned to sp2 protons
located at 5.33 ppm corresponding to the protons of the
double bonds for the oleic-linoleic acids present as a mixture
(CH –– CH), and to vinyl protons at 2.02 ppm (CH2–C ––
C–CH2 and CH2–C –– C–C–C –– C–CH2) and 2.75 ppm (C ––
C–CH2–CC), with multiplicity patterns of doublet of triplet
and triplet, respectively.
The increasing signals corresponding to the newly formed
protons surrounding the ozonides showed a complex signal
pattern due the mixture of the two ozonides for the linoleic
acid and the single ozonide for the oleic as cis/trans isomers
[19]. These protons appeared at 1.64 ppm for the methylene
attached to the ozonide, at 2.04–2.18 ppm for the methylene
bonded to the ozonides, and a multiplet signal system
at 5.12–5.19 ppm, 5.22–5.33 ppm, and 5.42–5.47 ppm
assigned to the ozonide protons. These data are in agreement
with reports in the literature [26]. Finally, the presence of
aldehyde was not observed for any of the GS and SF oils
under the reaction conditions (i.e., during a period of 5.0 h).
The principal new chemical shifts observed in GS and SF
oils with their respective assignations are listed in Table 2.
Both spectroscopic analyses showed a correspondence
between double-bond elimination and ozonide formation,
whose dynamics are presented in Figs. 3 and 4. In order to
www.ejlst.com
992 P. Guerra-Blanco et al.
Figure 2. 1H NMR spectra of GS oil before and after 5 h of ozonation.
observe the trends, the ozonation by-product distributions
are presented as normalized values. For the FT-IR results,
the intensities of the moieties were normalized by the C–H
stretch (2930 cm1) intensity, which was not affected by
ozonation. For the 1H RMN spectra, the normalization was
done in a similar way relative to the proton signals at
d ¼ 0.84 ppm (CH3). In both cases, all values were addi-
tionally normalized relative to their corresponding maximum
values.
The FT-IR data showed that the double-bond decom-
position for SF oil was faster than for GS oil, and that their
corresponding ozonide formation dynamics followed this
behavior as well (Fig. 3). This behavior was expected in view
of the oils’ ozonograms (Fig. 1), which show SF oil ozonation
ending before GS oil ozonation. On the other hand, the
1
H NMR data did not show significant differences in double-
bond decomposition dynamics between the two oils;
however, ozonide formation was faster for GS oil than for
SF oil (Fig. 4). The probable explanation for these differ-
ences relates to the variety of unsaturated compounds
contained in vegetable oils. Any extraction or sample
treatment was done before the spectroscopic analysis,
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2015, 117, 988–998
following which the reaction of ozone with all the unsaturated
compounds contained in oils was observed by FT-IR
spectroscopy. This approach is justified because the FT-IR
technique is less specific than 1H NMR when small
differences in chemical structure are present.
1
H NMR data showed the reaction of ozone with only the
>C –– C< corresponding to fatty acids. The chemical shifts
observed in this technique were barely influenced by
neighboring moieties. Comparing the spectra for the oils
and the isolated fatty acids (oleic and linoleic), some signals
in the region around d ¼ 5.33 ppm (sp2 protons) were present
in oils, but were absent in isolated fatty acids (data not
shown). It is assumed that these signals correspond to the
other unsaturated compounds besides fatty acids. For data
treatment, these signals were deliberately omitted and only
those corresponding to fatty acids were considered.
It is worth mentioning that, under the ozonation
conditions employed, the 1H NMR signals corresponding
to vinyl protons at 2.02 and 2.75 ppm and to double-bond
protons signals at 5.33 ppm disappeared after 2.5 h (for both
oils), while the ozonide proton signals emerged at 20 min.
The final signal type, corresponding to the increasing/
www.ejlst.com
Eur. J. Lipid Sci. Technol. 2015, 117, 988–998 Structural characterization of ozonated oils 993

Table 2. 1H NMR chemical shifts of ozonated oils [19, 26]

d [ppm] Associated protons Key

1.4 (bp) OZ1

1.7 (m) OZ2

5.1 (m) OZT

Figure 3. Double-bond elimination and ozonide formation dynam-

ics obtained by FT-IR spectroscopy.

unsaturated compounds (other than fatty acids), so that it

needs more time to react fully with ozone. It has been
2.5 (m) OZ5 demonstrated that GS extracts are potent scavengers of
oxygen free radicals [37, 38], which may explain why GS oil
contains more unsaturated compounds than SF oil besides
fatty acids. As can be seen in Fig. 4, after 2.5 h practically all
the fatty acids in both oils had reacted with ozone. At this
2.1 (m) OZ6
time, all the >C –– C< in SF oil had been reacted, while in GS
oil their reaction continued until 4.0 h (Fig. 3).

3.3.2 Viscosity measurement

5.4–5.5 (m) OZ7
We investigated viscosity as a complementary study to
confirm the oils’ ozonation pathways. Figure 5 shows the
viscosity dynamics of both oils. The results of this study are in

5.5 (m) OZ8

bp, broad peak; m, multiplet.

decreasing pattern located at 5.50 ppm, 5.35–5.28 ppm, and

2.42–2.35 ppm, began to be present from 20 min as a
multiplet and disappeared completely by 3.0 h. This behavior
can be attributed to the formation of unstable or reactive
species such as hydroperoxides, which are further decom-
posed during the ozonation process [36].
In this sense, Figs. 3 and 4 compare the ozone’s reaction
with all unsaturated compounds in oils and only with fatty
acids, respectively. As expected, the reaction with fatty acids
was very similar in the two oils (Fig. 4), while the reaction
with all >C –– C< in oils was faster for SF than for GS oil Figure 4. Double-bond elimination and ozonide formation dynam-
(Fig. 3). This fact indicates that GS oil contains more ics obtained by 1H NMR spectroscopy.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

994 P. Guerra-Blanco et al.
Figure 5. Viscosity profiles during GS and SF oil ozonation.
agreement with results presented by other authors, who have
found that viscosity increases as the ozonation degree
increases. This observation is commonly explained by a
decrease in the flexibility of ester chains because the total
number of unsaturations has decreased, and by the formation
of chemical species with higher molecular mass, which are
assumed to be oligomers [21, 22, 26].
In the present study, the viscosity dynamics were different
for GS and SF oils. As can be seen in Fig. 5, in the first hour of
ozonation, the viscosity of both oils increased more than
twofold. This increase continued as an approximately linear
trend in SF oil until 3.0 h, while in GS oil an increase in
viscosity slope was observed. After 5.0 h, the viscosities had
increased 29 and 10 times for GS and SF oils, respectively. In
fact, the final viscosity of GS oil was 2.5 times higher than the
viscosity of SF oil. The differences in viscosity dynamics
evidenced the formation of poly peroxides (oligomeric
ozonides) in higher proportions in GS oil than in SF oil.
3.4 Control method for oil ozonation: Measurement
of ozonation degree by TU
The characterization of ozonated oils by spectroscopic and
physicochemical analysis enables an understanding of their
ozonation pathways. However, all these techniques are
mainly qualitative, and quantitative methods are still scarce.
Some authors have reported the 1H NMR quantification of
fatty acids from vegetable oils [39, 40], but oils contain other
unsaturated compounds with different chemical natures
(specifically, unsaponifiable matter), such as sterols and
polyphenols, as well as characteristic compounds of their
source (such as antioxidants); even as minor components,
they also react with ozone. Therefore, although their
influence on therapeutic effects has not yet been studied;
these additional unsaturated compounds must be quantified
as part of the total oxidizable substrate by ozone.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2015, 117, 988–998
In fact, not much variety in control methods exists for oil
ozonation capable of quantifying the overall substrate that
reacts with ozone directly. Some of the reported control
methods apply to the main ozonation by-products (i.e.,
ozonides), rather than directly to the oxidizable substrate.
For example, calorimetric techniques [27] are based on the
decomposition enthalpy of ozonides, or peroxide value [22];
that is based on the content of oxygen as peroxide in the
sample.
A recent study [14] has revealed that the peroxidation levels
and viscosity of ozonated oils are not the only factors that seem
to be involved in the wound-healing process. The testing of
three ozonated oils of different natures but with similar
peroxide values and viscosities resulted in different wound-
healing processes, measured by wound closure rate, PCNA
(proliferating cell nuclear antigen) expression, NFkB (nuclear
factor kappa B) activation, and VEGF (Vascular endothelial
growth factor) and 4-HNE (4-hydroxynonenal) levels, even
though they were expected to have similar effects. This study
shows the importance of the oil’s properties in clinical effects.
In fact, its authors attribute the differing effects on wound
healing to the different compositions of polyunsaturated fatty
acids and probably to rheological behaviors.
In the present study, the TU method is proposed to
control the oil ozonation. The main advantage of TU over
other methods is that it provides a direct measure of the total
substrate oxidizable by ozone through its reaction with
ozone. In this sense, it is possible to measure the ozonation
degree of an oil through this method.
An additional advantage of this technique is the infra-
structure and knowledge needed to develop it. The TU
method is based on ozonation of the sample, and people
dedicated to producing ozonated oils are familiar with
ozonation methods and have access to the necessary
infrastructure. Not all these producers have access to
spectroscopic analysis, but they all have ozone generators.
In this sense, few modifications would be needed to measure
TU. These modifications include flow control and an
ozone gas sensor with enough sensitivity to detect low ozone
concentration changes precisely.
The TU of each oil was measured before and after ozona-
tion, and the observations displayed in ozonograms (Fig. 1)
were confirmed and quantified. GS oil contains more oxi-
dizable substrate by ozone, because its TU is higher than that of
SF oil (5.94 and 4.49 mmol per g of oil, respectively). Figure 6
shows the TU decrease during ozonation of GS and SF oils.
The slope of TU decrease is very similar for both oils, as ozone
reacts with all the >C¼C< with similar reaction rate constants,
about 2  105 L/mol  s [31], regardless of the chemical
structures of the compounds containing double bonds.
Figure 7 shows the comparison between TU and
spectroscopic (FT-IR and 1H NMR) methods for determin-
ing the oxidizable substrate elimination in GS oil. All values
are presented in normalized form in order to compare their
dynamics. Two facts are corroborated in Fig. 7. First, the
www.ejlst.com
Eur. J. Lipid Sci. Technol. 2015, 117, 988–998 Structural characterization of ozonated oils 995

be a more potent scavenger of oxygen free radicals than other

common antioxidants such as vitamins C and E [37, 38]. The
presence of this kind of compound should be related to the
higher TU of GS oil.

3.5 Relationship between the characterizations of

ozonated oils and their TU

Figures 8 and 9 show the relationship between the TU

decrease and the observed by-product dynamics during GS
and SF oils ozonation. Two 1H NMR signals corresponding
to ozonides are shown, OZT (total ozonides formed) and
OZ6 (ozonides formed from the second and/or third double
bonds of linoleic and linolenic acids; see chemical structures
in Table 1). The viscosity dynamic represents poly peroxide
formation. All quantities are presented in a normalized form.

Figure 6. TU profiles of GS and SF oils during ozonation.

FT-IR profile provides information on overall >C –– C<

elimination. Its dynamic is virtually overlapped with the TU
dynamic, both being slower than the 1H NMR profile, which
represents only the elimination of >C –– C< corresponding to
fatty acids. Second, oils contain compounds in addition to
fatty acids, that simultaneously react with ozone and may be
involved in therapeutic effects.
Grapes are a well-known natural source of antioxidants.
Although grape seeds make up a small percentage of the mass
of grapes, they contain two-thirds of the extractable phenols
[41]. The seeds may contain up to 5–8% phenols by mass
[42] and these are essentially flavonoids. The polyphenolic
structure of flavonoids makes them suitable substrates for
ozone oxidation. Grape seed extracts have also been shown to
Figure 8. TU and by-product dynamics in GS oil ozonation.

Figure 7. Comparison of methods used to determine >C –

– C<
elimination during ozonation. Figure 9. TU and by-product dynamics in SF oil ozonation.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

996 P. Guerra-Blanco et al. Eur. J. Lipid Sci. Technol. 2015, 117, 988–998

The TU decomposition dynamic is similar in both oils,

but their by-product distributions are different. Comparing
the by-product slopes of the two oils, ozonide and poly
peroxide formation rates are faster in GS oil than SF oil. In
both oils, the OZ6 accumulation dynamic appears to be
related to the viscosity increase, and by association to poly
peroxide accumulation.
In GS oil, three reaction steps were observed. First, the
double bonds were converted mainly into iso-ozonides (up
to an ozonation degree of 40%). Then, between 40% and
80% degree of ozonation, there is a competition between
ozonide and poly peroxide formation. Finally, as the last 20%
of double bonds are converted into poly peroxides, the total
ozonide accumulation is not significant, although viscosity
increases.
The reaction products of SF oil follow a different
behavior: initially (up to an ozonation degree of 40%), the
slope for total ozonide formation is significantly higher than Figure 11. Relationship between TU and viscosity.
the slope for viscosity. After that, both slopes are very similar,
indicating that ozonides and poly peroxides are formed in
parallel in SF oil ozonation.
Figures 10 and 11 show the relationship between TU and determined via the comparison of by-products of >C –– C<
the representative variables of ozonation reaction, such as decomposition from different sources (fatty acids and the
>C –– C< from fatty acid elimination and viscosity increase. A other moieties of vegetable oils).
linear relationship between TU and the integral signal Figure 12 shows the relationship between >C –– C<
(d ¼ 5.3 ppm) decrement was observed in the initial exper- elimination (measured by 1H NMR) and viscosity. The
imental points (Fig. 10). However, after 40% of TU behavior of double-bond elimination observed by 1H NMR
elimination (or ozonation degree) the slope changes for (integral signal at d ¼ 5.3 ppm) versus viscosity is very similar
both oils. The corresponding changes were also observed in to that observed in Fig. 10, confirming the relationship
Fig. 11, more noticeably in GS oil. between TU and these variables. Figure 12 confirms
The observed dynamics confirmed the competition of that the first reaction step favors iso-ozonide formation,
reaction with ozone between >C¼C< from fatty acids and and consequently that the viscosity does not greatly
from other moieties. In this sense, the “optimal” ozonation increase. Once 50% of >C –– C< (from fatty acids) have
degree, according to the intended application, may be been eliminated, the slope significantly changes, indicating

Figure 10. Relationship between TU and >C¼C< elimination Figure 12. Relationship between viscosity and >C –
– C< elimina-
(measured by 1H NMR spectroscopy). tion (measured by 1H NMR).

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

Eur. J. Lipid Sci. Technol. 2015, 117, 988–998
that poly peroxide formation and consequently viscosity
increase.
4 Conclusions
The FT-IR and 1H NMR spectroscopic analyses and
viscosity results were useful in a qualitative way, in enabling
the construction of a feasible explanation of the main reaction
occurring during the ozonation of the GS and SF oils. These
results were complemented by the oils’ TU measurements,
making it possible to relate the structural changes in the oils
to their corresponding ozonation degrees.
This work has shown that the relationship between
ozonation degree and structural and viscosity changes during
ozonation is different for GS and SF oils. Moreover, ozonides
and poly peroxides were formed at different rates for the two
oils, although it was expected that these rates would be
similar, given that the fatty acid compositions of the oils were
very similar.
In the early reaction stage, the major ozonation by-
products accumulated were iso-ozonides. In GS oil, three
reaction steps were observed: first, the double bonds were
converted mainly into iso-ozonides (up to an ozonation
degree of 40%). Then, between 40 and 80% degree of
ozonation, there was a competition between ozonide and
poly peroxide formation. Finally, the last 20% of double
bonds were converted into poly peroxides. The reaction
products of SF oil followed a different behavior, with the
ozonides and poly peroxides formed in parallel.
The differences in by-product distribution between
ozonated oils from different sources could explain why the
ozonation degree need not be the same for different oils.
Further, accurate knowledge of formation/decomposition
dynamics of ozonation by-products could help in selecting
the required ozonation degree of a vegetable oil for the
intended application.
All studies were successfully implemented to character-
ize the differences in reactivity of double bonds from oils
with ozone, which is essential for improving the clinical
applications of ozonated oils. The TU method presented in
this study can be considered as a simple alternative for
controlling the ozonation degree in the preparation of
ozonated oils.
The authors thank the Department of Graduate Study and
Investigation of the Instituto Politécnico Nacional of Mexico
(Project 20130453) and the Consejo Nacional de Ciencia y
Tecnología of Mexico-CONACyT (Projects 83593, 83275, and
153356). P. Guerra thanks CONACyT for the scholarship
support.
The authors declare that there is no conflict of interest regarding
the publication of this article.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Structural characterization of ozonated oils 997
References
[1] Kataoka, H., Semma, M., Sakazaki, H., Nakamuro, K.,
et al., Proinflammatory event of ozonized olive oil in mice.
Ozone Sci. Eng. 2009, 31, 238–246.
[2] Menéndez, S., Falcón, L., Simón, D. R., Landa, N., Efficacy
of ozonized sunflower oil in the treatment of Tinea Pedis.
Mycoses 2002, 45, 329–332.
[3] Hernández, F., Hernández, D., Zamora, Z., Díaz, M.,
et al., Effects of an ozonized sunflower oil product
(Oleozon1) on in vitro trophozoites. Exp. Parasitol. 2009,
121, 208–212.
[4] Menéndez, S., Falcón, L., Maqueira, Y., Therapeutic
efficacy of topical OLEOZONé in patients suffering from
onychomycosis. Mycoses 2011, 54, e272–e277.
[5] Viebahn-Hansler, R., The use of ozone in medicine. ODREI-,
Publishers, Germany 2002.
[6] Valacchi, G., Fortino, V., Bocci, V., The dual action of ozone
on the skin. Br. J. Dermatol. 2005, 153, 1096–1100.
[7] Travagli, V., Zanardi, I., Valacchi, G., Bocci, V., Ozone and
ozonated oils in skin diseases: A review. Mediators Inflamm.
2010, 2010, 9.
[8] Sánchez, G. M., Re, L., Perez-Davison, G., Delaporte, R.
H., Las aplicaciones mádicas de los aceites ozonizados,
actualizacién. Rev. Esp Ozonoterapia 2012, 2, 121–139.
[9] Campanati, A., De Blasio, S., Giuliano, A., Ganzetti, G.,
et al., Topical ozonated oil versus hyaluronic gel for the
treatment of partial- to full-thickness second-degree burns: A
prospective, comparative, single-blind, non-randomised,
controlled clinical trial. Burns J. Int. Soc Burn Inj. 2013,
39, 1178–1183.
[10] Díaz, M., Lezcano, I., Molerio, J., Hernández, F., Spectro-
scopic characterization of ozonides with biological activity.
Ozone Sci. Eng. 2001, 23, 35–40.
[11] Díaz, M. F., Hernández, R., Martínez, G., Vidal, G., et al.,
Comparative study of ozonized olive oil and ozonized
sunflower oil. J. Braz. Chem. Soc. 2006, 17, 403–407.
[12] Skalska, K., Ledakowicz, S., Perkowski, J., Sencio, B.,
Germicidal properties of ozonated sunflower oil. Ozone Sci.
Eng. 2009, 31, 232–237.
[13] Valacchi, G., Lim, Y., Belmonte, G., Miracco, C., et al.,
Ozonated sesame oil enhances cutaneous wound healing in
SKH1 mice. Wound Repair Regen. Off. Publ. Wound Heal.
Soc. Eur. Tissue Repair Soc. 2011, 19, 107–115.
[14] Valacchi, G., Zanardi, I., Lim, Y., Belmonte, G., et al.,
Ozonated oils as functional dermatological matrices: effects
on the wound healing process using SKH1 mice. Int. J.
Pharm. 2013, 458, 65–73.
[15] Criegee, R., Mechanismus der ozonolyse. Angew. Chem.
1975, 87, 765–771.
[16] Bailey, P. S., Ozonation in Organic Chemistry, Academic
Press, New York 1978.
[17] Razumovskiıc, S., Zaikov, G., Kinetics and mechanism of
the reaction of ozone with double bonds. Russ Chem Rev.
1980, 49, 1163–1180.
[18] Hernández-Tápanes, R., Martínez-Tellez, G., Díaz-
Gómez, M. F., Aspectos Quámico-Fásicos del aceite de
girasol ozonizado. Rev. CENIC Cienc. Quám. 2004, 35,
159–162.
www.ejlst.com
998 P. Guerra-Blanco et al.
[19] Díaz, M. F., Gavín, J. A., Characterization by NMR of
ozonized methyl linoleate. J. Braz. Chem. Soc. 2007, 18,
513–518.
[20] Soriano, N. U., Jr, Migo, V. P., Matsumura, M., Ozonation
of sunflower oil: Spectroscopic monitoring of the degree of
unsaturation. J. Am. Oil Chem. Soc. 2003, 80, 997–1001.
[21] Sadowska, J., Johansson, B., Johannessen, E., Friman, R.,
et al., Characterization of ozonated vegetable oils by
spectroscopic and chromatographic methods. Chem. Phys.
Lipids 2008, 151, 85–91.
[22] Zanardi, I., Travagli, V., Gabbrielli, A., Chiasserini, L.,
Bocci, V., Physico-chemical characterization of sesame oil
derivatives. Lipids 2008, 43, 877–886.
[23] Jardines, D., Correa, T., Ledea, O., Zamora, Z., et al., Gas
chromatography–mass spectrometry profile of urinary
organic acids of Wistar rats orally treated with ozonized
unsaturated triglycerides and ozonized sunflower oil. J.
Chromatogr. B 2003, 783, 517–525.
[24] Soriano, N. U., Jr, Migo, V. P., Matsumura, M., Functional
group analysis during ozonation of sunflower oil methyl
esters by FT-IR and NMR. Chem. Phys. Lipids 2003, 126,
133–140.
[25] Díaz, M. F., Gavín Sazatornil, J. A., Ledea, O., Hernández,
F., et al., Spectroscopic characterization of ozonated sun-
flower oil. Ozone Sci. Eng. 2005, 27, 247–253.
[26] Sega, A., Zanardi, I., Chiasserini, L., Gabbrielli, A., et al.,
Properties of sesame oil by detailed 1H and 13 C NMR
assignments before and after ozonation and their correlation
with iodine value, peroxide value, and viscosity measure-
ments. Chem. Phys. Lipids 2010, 163, 148–156.
[27] Cataldo, F., Chemical and thermochemical aspects of the
ozonolysis of ethyl oleate: Decomposition enthalpy of ethyl
oleate ozonide. Chem. Phys. Lipids 2013, 175-176, 41–49.
[28] Sergeev, V., Burkin, I., Levin, A., Poznyak, T., URSS Patent
1500943 (1989).
[29] Poznyak, T. I., Kiseleva, E. V., Turkina, T. I., Distribution
of the total unsaturation in lipid components of plasma as a
new differential diagnostic method in clinical analysis. J.
Chromatogr. A 1997, 777, 47–50.
[30] Poznyak, T., García, A., Kiseleva, E., Ozone application for
the people health state monitoring by the total unsaturation
index determination—Medigraphic. com. Rev. Ozonoterapia
2008, 1, 15–23.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2015, 117, 988–998
[31] Razumovskiıc, S. D., Zaikov, G. E., Ozone and Its Reactions
with Organic Compounds; Studies in organic chemistry;
Elsevier, 1984.
[32] Brajnikov, E., Lisitsyn, D., Poznyak, T., URSS patent
882307 (1979).
[33] CODEX STAN 210, 1999. Codex standard for named
vegetable oils. Current official standards (Amended 2003,
2005). FAO/WHO Food standards. Codex Alimentarius.
http://www.codexalimentarius.net.
[34] Guerra, P., Amacosta, J., Poznyak, T., Siles, S., et al., in
Chamy, R. (Ed.), Aerobic Biodegradation Coupled to Prelimi-
nary Ozonation for the Treatment of Model and Real Residual
Water, Biodegradation—Engineering and Technology;
InTech, Rijeka, Croatia 2013, p. 365–387.
[35] Vlachos, N., Skopelitis, Y., Psaroudaki, M., Konstantinidou,
V., et al., Applications of Fourier transform-infrared
spectroscopy to edible oils. Anal. Chim. Acta 2006, 573–
574, 459–465.
[36] Guillén, M. D., Goicoechea, E., Detection of primary and
secondary oxidation products by Fourier transform infrared
spectroscopy (FTIR) and 1H nuclear magnetic resonance
(NMR) in sunflower oil during storage. J. Agric. Food Chem.
2007, 55, 10729–10736.
[37] Balu, M., Sangeetha, P., Haripriya, D., Panneerselvam, C.,
Rejuvenation of antioxidant system in central nervous system
of aged rats by grape seed extract. Neurosci. Lett. 2005, 383,
295–300.
[38] Bagchi, D., Garg, A., Krohn, R. L., Bagchi, M., et al.,
Oxygen free radical scavenging abilities of vitamins C and E,
and a grape seed proanthocyanidin extract in vitro. Res.
Commun. Mol. Pathol. Pharmacol. 1997, 95, 179–189.
[39] Miyake, Y., Yokomizo, K., Matsuzaki, N., Determination of
unsaturated fatty acid composition by high-resolution
nuclear magnetic resonance spectroscopy. J. Am. Oil Chem.
Soc. 1998, 75, 1091–1094.
[40] Guillén, M. D., Ruiz, A., 1H nuclear magnetic resonance as
a fast tool for determining the composition of acyl chains in
acylglycerol mixtures. Eur. J. Lipid Sci. Technol. 2003, 105,
502–507.
[41] Tsai Su, C., Singleton, V. L., Identification of three flavan-3-
Ols from grapes. Phytochemistry 1969, 8, 1553–1558.
[42] Amerine, M. A., Joslyn, M. A., Table Wines: The Technology of
Their Production, University of California Press, California 1970.
www.ejlst.com