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Research Article
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Identification and Quantitative Analysis of β-sitosterol Oxides in Oil of 2 Macrotermitinae Varieties from Congo
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V. N`Goka,
D. Julien-David
and
E. Marchioni
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ABSTRACT
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Termites are one of the edible insects in the Congo, but very little scientific data are available on this food. Sitosterol oxides are present in vegetable food. However, as cholesterol oxides, biological utility and food safety of some sitosterol oxides are controversial and not clarified. In a chemical study, sitosterol oxides were identified and quantitatively determined in oil of Bellicositermes natalensis Haviland from two regions of the Congo by gas chromatography (GC) and Mass spectrometry (MS) analysis. The following β-sitosterol oxides have been found in oil of termites in various amount: 5,6β-epoxysitosterol, 7α-hydroxysitosterol, 7β-hydroxysitosterol, 5α,6β-dihydroxysitosterol, 5,6α-epoxysitosterol and 7-ketositosterol. Total sitosterol oxides amount found was 29.1 μg g-1 for termites dried by sun exposure and 39.1 μg g-1 for smoked termites. These results showed also that oil of termites contained more sitosterol oxides than edible vegetable oils previously reported. On the other hand this study demonstrated for the first time that phytosterol oxides may be present in some animal oils.
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Received: November 15, 2012;
Accepted: January 01, 2013;
Published: February 01, 2013
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INTRODUCTION
β-sitosterols are reported to be the main component in phytosterols fraction
of unsaponifiables in vegetable oils. Many researchers are interesting on phytosterols
because of their biological activities and their use in some food, as they are
useful for health (Ling and Jone, 1995; Moghadasian,
2000; Pioronen et al., 2000; Zhang
et al., 2005a, b, 2006a;
Mortuza, 2006; Fernandez-Cuesta
et al., 2012). It has been shown, in vitro, that some cholesterol
oxides may be cytotoxic, mutagenic, atherogenic and carcinogenic (Schroepfer
Jr., 2000; Guardiola et al., 2002). It is also
well known that, molecular structures of cholesterol and phytosterol are highly
similar. Thats how, many and various studies were undertaken on phytosterols
and their oxides. The main objectives of these researches were: identification,
quantitative determination and biological evaluation of their activities (Guardiola
et al., 2002, 2004; Lea
et al., 2004; Roussi et al., 2005;
Liang et al., 2011; Garcia-Liatas
and Rodriguez-Estrada, 2011). For example, some biological studies have
been done on phytosterol oxides concentrate from which, genotoxicity and subchronic
toxicity were not demonstrated (Lea et al., 2004).
Abramsson-Zetterberg et al. (2007) reported
a study on phytosterol oxides from a mixture of sitosterol and campesterol,
therein, no genotoxic effect was observed in vivo in mice. Recently,
a study of sitosterol oxides, namely 7-ketositosterol, 7β-hydroxysitosterol,
7α-hydroxysitosterol, a mixture of 6β-hydroxy-3-ketositosterol and
6α-hydroxy-3-ketositosterol has been reported. In this work, no mutagenic
activity, towards Salmonella Typhimurium strains, was observed (Koschutnig
et al., 2010).
Although, the above mentioned literatures are favourable for no toxicity from
sitosterol oxides, some controversial biological effects have been reported.
Koschutnig et al. (2009) reported cytotoxic
and apoptotic activities of some β-sitosterol oxides. Differences were
observed by comparing of activities of 7-ketositosterol, 7β-hydroxysitosterol,
7α-hydroxysitosterol and some mixtures of others β-sitosterol oxides.
In this study, 7-ketositosterol and 7α-hydroxysitosterol were found to
be more active than the others. On the other hand, Liang
et al. (2011) working with male hamsters, reported effects of β-sitosterol,
stigmasterol and their corresponding oxides in lipoprotein profile and aortic
function. These authors found favorable effects for β-sitosterol and stigmasterol
but not favorable for both corresponding oxidation products.
Zhang et al. (2006b) reported that sitosterol
oxides in oil content can change under sunlight exposure, from 9.6 to 1007 μg
g-1 in sunflower oil, from 0.8 to 895 μg g-1 in soybean
oil, from 0.1 to 676 μg g-1 in olive oil and 1.9 to 2421 μg
g-1 in rapeseed oil. The same oil showed variation of total sitosterol
oxides from 9.6 to 354 μg g-1 for sunflower oil, 0.8 to 331
μg g-1 for soybean oil, 0.1 to 294 μg g-1 for
olive oil and 1.9 to 1083 μg g-1 for rapeseed oil, under UV
light exposure.
Having said that, further researches are required for a better understanding,
of the influence of plant sterols and their oxides on consumers health.
In a previous study, composition of oil, from termites from Congo, has been
reported (NGoka and Julien-David, 2012). This
animal oil was comparable to vegetable olive oil taking into account their monounsaturated
components. These findings encouraged us to generate complementary information
for this oil. The present study reports identification and quantification of
β-sitosterol oxides in oil of Bellicositermes natalensis Haviland
from two regions of the Congo.
MATERIALS AND METHODS Samples: The termites are the same species of Bellicositermes natalenis Haviland obtained from two origins: Sangha and Plateaux-Cuvette, respectively named termites S and termites PC. A sample was bought at the market in Brazzaville Congo on 2005 September. Termites were transported to the laboratory where they were conserved at -18°C.
Extraction of Bellicositermes natalenis oil: Three individual
10 g samples of dry termites of each origin were refluxed with 300 mL of chloroform
in weighed flasks using a Soxhlet apparatus according to AACC
(1987) method. The fat matter of the termites has been extracted with the
help of a Soxhlet equipped with extraction cartridges in cellulose. The termites
ground, with the help of a homogenizer to knives, have been submitted to an
extraction under ebullition of chloroform during 9 h. The oils were recovered
by distilling the solvent in a rotary evaporator at 45°C, then dried to
constant weight in a vacuum oven at 90°C for 1 h and weighed.
Extraction of phytosterol oxides: Extraction of phytosterol oxides was
carried out as previously described (Zhang et al.,
2005b), taking advantage of the previously synthesized standards of phytosterol
oxides (Zhang et al., 2005a). Briefly, a 200
mg oil sample was spiked with a 19-hydroxycholesterol solution (20 μL,
1 mg mL-1 in ethyl acetate) as an internal standard. After removal
of the solvent with a gentle flow of nitrogen, the sample was dissolved in a
mixture of ethanol (9 mL) and a saturated aqueous KOH solution (0.5 mL) before
it was submitted to an overnight (15 h) soft saponification at room temperature.
The unsaponifiable fraction was extracted with diethyl ether and the oxides
were separated from the matrix using a Solid-phase Extraction (SPE) silica gel
cartridge and converted to Trimethylsilyl (TMS) ethers with pyridine (50 μL)
and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), 40 μL. Extraction
steps were done in brown bottles and at room temperature. Analysis of 1 μL
samples was carried out using a GC-MS chromatograph as described below.
Gas chromatography-mass spectrometry (GC-MS) analysis: GC-MC analysis
was carried out as described by Zhang et al. (2005b).
GC-MS analyses were performed on a Varian Star 3400 GC instrument equipped with
an on-column SPI injector coupled to a Varian Saturn 2000 mass sensitive detector
(Varian, France) operating in the Electron Impact (EI) ionization mode at 70
eV and monitored on the full-scan range (m/z 40-600). Data acquisition and processing
and instrumental control were performed by Varian Saturn WS software. Analytes
were separated in a VF-5 ms capillary column (phase stationary: 5% phenyl-95%
dimethyl polysiloxane, thickness of 0.1 μm, 60 mx0.25 mm, Varian, France).
The column temperature gradient was programmed from 105°C (hold for 2 min)
to 170°C at 20°C min-1 and then, to 320°C at 7°C
min-1 (hold for 15 min). The injector operating conditions were as
follows: injection volume 1 μL; initial injector temperature of 105°C
was increased to 300°C at 100°C min-1 (hold for 40 min).
Helium (purity 99.9995%) was used as a carrier gas with a flow rate of 1 mL
min-1.
Quantitative data were carried out using Selected Ion Monitoring (SIM) analysis.
The analytes were quantified as previously reported (Zhang
et al., 2005b).
RESULTS AND DISCUSSION
Identification and quantitative determination of β-sitosterol oxides were
carried out in oil of Bellicositermes natalensis Haviland from two regions
of the Congo. The total amount of their sitosterol oxides varied from 29.1 to
39.1 μg g-1 of oil from these edible insects. This animal oil
has proved to contain phytosterol oxides which are generally present in vegetable
matter. β-sitosterol oxides composition of the oil and their amount in
μg g-1 are presented in Table 1. Sitosterol
oxides in oil of termites found were: 5,6β-epoxysitosterol, 10.3 and 12.5
μg g-1; followed by 7α-hydroxysitosterol, 6.4 and 10.0
μg g-1; 7β-hydroxysitosterol, 5.2 and 9.3 μg g-1;
5α,6β-dihydroxysitosterol, 3.2 and 4.3 μg g-1; 5,6α-epoxysitosterol,
2.0 and 4.0 μg g-1 and 7-ketositosterol, 2.0 and 3.3 μg
g-1, respectively for termites PC and termites S.
Table 1: |
Concentration of β-sitosterol oxides in oil of termites
from two origins |
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Values are Mean±SD |
This finding was already observed in edible vegetable oils by Zhang
et al. (2005b, 2006b). Therein the authors
showed that β-sitosterol oxides were present and total amount was reported
to be 9.6 μg g-1 for sunflower oil, 0.8 μg g-1
for soybean oil, 0.1 μg g-1 for olive oil and 1.9 μg g-1
for rapeseed oil. In the animal material, the subject of this study, the total
amount of β-sitosterol oxides was 29.1 μg g-1 for termites
PC and 39.1 μg g-1 for termites S. Termites S contained more
oxidized phytosterol then termites PC. This observation suggested that smoking
procedure occurred with more oxidation than sun drying. Previous studies of
experimental oxidation of edible oils have been reported. Zhang
et al. (2005b) have been shown that temperature could increase β-sitosterol
oxides in oils. More oxidizing compounds were found under sunlight exposure
than under UV light exposure, Zhang et al. (2006b).
The present study showed that termites oil contained more β-sitosterol
oxides (29.1 to 39.1 μg g-1) than edible vegetable oils previously
reported. The reason of variation between the chemical compositions of oils
of termites PC and S may depend on climatic, geographic conditions, harvest
drying procedure.
In our knowledge, sitosterol oxides, therefore phytosterols, are reported for the first time in oil of animal. The high amount of these oxides and the high importance of their biological properties showed that, oil of termites must be chemically characterized and biologically evaluated. Further study must be done to determine phytosterol and phytosterol oxides in fresh termites for a comprehensive study. CONCLUSION
5,6β-epoxysitosterol, 7α-hydroxysitosterol, 7β-hydroxysitosterol,
5α,6β-dihydroxysitosterol, 5,6α-epoxysitosterol and 7-ketositosterol
were identified and quantitatively determined in oil of Bellicositermes natalensis
Haviland. This study demonstrated for the first time that phytosterol oxides
may be present in some animal oils. Under normal conditions of production and
conservation, it has been shown that termites contained more sitosterol oxides
than edible vegetable oils previously reported. Large amount of these compounds
in termites suggested that much attention must be done for evaluation of risk
because biological effects and safety aspects of phytosterol oxides are still
unclear.
ACKNOWLEDGMENTS We are grateful to the «Laboratoire dInnovation Thérapeutique and Laboratoire de Chimie Analytique des Molécules Bioactives; Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, 67400 Illkirch France» for reception of Victor NGOKA.
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