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Total antioxidant activity of selected vegetable oils and their influence on total antioxidant values in vivo: A photochemiluminescence based analysis
Accepted Manuscript Short communication Total antioxidant activity of selected vegetable oils and their influence on total antioxidant values in vivo: A photochemiluminescence based analysis Dhavamani Sugasini, Yalagala Poorna Chandra Rao, Belur R Lokesh PII: DOI: Reference:
S0308-8146(14)00770-5 http://dx.doi.org/10.1016/j.foodchem.2014.05.064 FOCH 15847
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 May 2013 11 April 2014 13 May 2014
Please cite this article as: Sugasini, D., Poorna Chandra Rao, Y., Lokesh, B.R., Total antioxidant activity of selected vegetable oils and their influence on total antioxidant values in vivo: A photochemiluminescence based analysis, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.05.064
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1
Total antioxidant activity of selected vegetable oils and their influence on
2
total antioxidant values in vivo: A photochemiluminescence based analysis
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Dhavamani Sugasini, Yalagala Poorna Chandra Rao and Belur R Lokesh*
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Department of Lipid Science and Traditional Foods,
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CSIR-Central Food Technological Research Institute,
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Mysore- 570 020, Karnataka, India
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*Corresponding Author
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Dr. B. R. Lokesh,
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Chief Scientist,
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Department of Lipid Science and Traditional Foods,
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CSIR-Central Food Technological Research Institute,
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Mysore -570 020, India
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Tel:91-821-2514153
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Fax: 91-821-2517233
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E-mail: [email protected]
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33 34
Abstract This
study
evaluated
the
antioxidant
activity
of
vegetable
oils
using
35
photochemiluminescence based assay. The following oils were selected for the study - palm
36
oil (PO); olive oil (OLO); sunflower oil (SNO); rice bran oil (RBO); sesame oil (SESO) and
37
linseed oil (LSO). The antioxidant activity of oils was reduced significantly when
38
unsaponifiable matter was removed from the oils. The rats fed unsaponifiable matter removed
39
vegetable oils showed significantly reduced antioxidant activity but no change in overall fatty
40
acid composition in serum lipids. Therefore the minor constituents in unsaponifiable matter
41
influences antioxidant activity exhibited by vegetable oils.
42 43
Keywords: Photochemiluminescence assay, vegetable oils, total antioxidant activity,
44
antioxidant status of serum.
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2
66
1. Introduction
67
Antioxidants play an important role in providing the stability to vegetable oils. As a
68
dietary component, they also play a vital role in reducing the oxidative stress in vivo. Dietary
69
polyunsaturated fatty acids play a significant role in maintaining serum lipids at desirable
70
levels. The polyunsaturated fatty acids such as linoleic acid lower serum total and LDL
71
cholesterol. But excess intake of linoleic acid increases the vulnerability of LDL lipids to
72
peroxidation which initiates cascading events leading to foam cell formation and ultimately to
73
atherosclerotic plaques (Reena & Lokesh, 2011; Calder, 2012). Therefore the intake of
74
antioxidants along with PUFA rich diet is recommended to overcome the oxidative stress.
75
Minor constituents in unsaponifiable fractions of edible oils have drawn considerable
76
interest for their antioxidant and health-promoting effects. Rice bran oil contains oryzanol.
77
Sesame oil contains lignans such as sesamin and sesamolin. Palm oil contains β- carotene and
78
tocotrienols. Olive oil contains polyphenols. Most of the oils have tocopherol at different
79
levels. The contributions of these minor constituents in the oils on the antioxidant value in
80
biological systems has not been thoroughly studied. In the present study, the commercially
81
available oils and the oils whose minor constituents were removed by column
82
chromatography were fed to rats and its impact on antioxidant value in serum lipid were
83
measured in a chemiluminescense based assay using an Photochem (from Analytik Jena
84
AG, Germany) (Popov & Lewin, 1996; Barba et al, 2013; Sielicka, Malecka & Purlan,
85
2014).This system has high sensitivity and requires less time (< 3minutes/sample) for
86
analysis. Each analysis requires few microliters of the sample and the antioxidant value can
87
be quantified in nanomolar range.
88 89
2.0 Material & Methods
90
2.1 Materials
91
Different vegetable oils such as palm oil (PO), olive oil (OLO), sunflower oil (SNO),
92
rice bran oil (RBO), sesame oil (SESO), linseed oil (LSO) were procured from a local super
93
market in Mysore, India. Kits for determination of Antioxidant capacity of lipid-soluble
94
substances (ACL) by photochemiluminescence (PCL) assay were purchased from Analytik
95
Jena AG (Jena, Germany). Oryzanol (>99% purity) was a gift from Amohusu Chemical
96
Industries Inc, Tokyo, Japan. Sesamolin was prepared in the laboratory according to the
97
method of Hemalatha & Ghaforunissa (2004). Tertiary butylated hydroxyl quinone (TBHQ),
98
butylated hydroxyl toluene (BHT), butylated hydroxyl anisole (BHA), α-tocopherol, 3
99
tocotrienol, BF3 in methanol, gallic acid, β-carotene, thiobarbituric acid, 1,1,3,3,-tetraethoxy
100
propane, sesamol, sesamin and fatty acids were procured from Sigma Chemical Co. (St.
101
Louis, MO, U.S.A.). All other chemicals and solvents (analytical grade) were purchased from
102
Sisco Research Laboratories Ltd (Mumbai, India).
103 104
2.2 Methods
105
2.2.1 Evaluation of total antioxidant value of commercial edible oils
106
Selected oils were evaluated for total antioxidant value by photochemiluminescence
107
(PCL) assay using Photochem (Analytica Jena AG, Germany) (Sielicka, Malecka & Purlan,
108
2014). The antioxidant values of the samples were determined as per the instructions
109
provided by the manufacturer and results are expressed in terms of trolox equivalents.
110 111
2.2.2 Fatty acid composition
112
Fatty acid composition of oils and serum lipids were analyzed by Gas
113
chromatography (Fisons, fitted with a flame ionization detector [FID]) using the method
114
described by Morrison & Smith (1964). The fatty acid methyl esters prepared using 14% BF3
115
in methanol were separated on a fused silica capillary column (25 m × 0.25 mm, Parma bond
116
FFAP-DF-0.25, Machery Negal GmbH Co., Duren, Germany). Individual fatty acid was
117
identified by comparing with the retention times of standards (Reena and Lokesh, 2011).
118 119 120 121
2.2.3 Unsaponifiable matter of oils The unsaponifiable matter in oils was measured according to the AOCS official method (1998).
122 123
2.2.4 Estimation of minor constituents in oils
124
Tocopherol and tocotrienols in oils were determined according to the method of
125
Rogers et al. (1993). Shimadzu LC-10A (Shimadzu Corporation, Tokyo Japan) HPLC fitted
126
with C18 column (250mm 4.6 mm length, 5 µm Supleco, USA) and fluorescence detector
127
was used for separating isomers of tocopherols and tocotrienols. The mobile phase consisted
128
of acetonitrile /methanol/isopropanol/ water (48:45:5:2) and run in isocratic condition at a
129
flow rate of 1.0 ml/min. Individual tocopherols were identified and quantified with respective
130
standard tocopherols. Tocotrienols were identified by the elution pattern of palm oil
131
tocotrienols and quantified according to AOCS method (AOCS, 1998). 4
132
The oryzanol content in RBO was determined by HPLC method. Shimadzu LC 20A
133
system with Phenomenex develosil column (250mm4.6mm, 5µm i.d) and photodiode array
134
detector was used for analysis. Following conditions were used for the separation of oryzanol
135
components (Wavelength 325nm: mobile phase acetonitrile:methanol:isopropylalcohol
136
(10:9:1 v/v/v): flow rate 1mL/min (Gopala Krishna et al, 2001). -oryzanol (>99% purity)
137
obtained as gift from Amohusu Chemical Industries Inc, Tokyo, Japan was used as standard
138
for quantification.
139 140
Sesamol, sesamin and sesamolin in oils was estimated by HPLC using Phenomenex
141
C18 column of 250 × 4.60 mm, 5 micron particle size. The mobile phase was 70:30 (v/v)
142
methanol: water and individual peaks were analysed at 295nm using UV detector
143
(Amarowicz, Shahidi, & Pegg, 2001).
144 145
The phenolic compounds were extracted from oils using liquid–liquid extraction
146
according to Taga, Miller & Pratt (1984). The concentration of total phenols in the
147
methanolic extract was estimated with Folin-Ciocalteau reagent. Gallic acid (0.05- 0.4
148
mg/ml) was used as reference standard.
149 150
β-carotene in oil samples were quantified using an HPLC system (LC- 10 A,
151
Shimadzu, Kyoto, Japan) equipped with photodiode array (PDA) detector (SPD- M 20A,
152
Shimadzu). β- carotene was separated on a C-30 (ODS) column (250mm× 4.6 mm i.d., 5-μm
153
particle size; Princeton, SPHE Germany) by isocratically eluting with acetonitrile–
154
dichloromethane–methanol (60:20:20, v/v/v) containing 0.1% ammonium acetate as a mobile
155
phase (Raju et al., 2007). Carotenoids were quantified using the standard curve generated
156
with reference standards of β-carotene (2-10 µg).
157 158
2.2.5 Preparation of minor constituents removed oils
159
The minor components in unsaponifiable fractions of PO, OLO, SNO, RBO, SESO
160
and LSO were removed according to the method of Cardenia et al (2011). The oils that were
161
stripped of minor constituents was aliquoted into brown glass bottles, flushed with nitrogen,
162
and stored at -20C until use.
163 164 5
165
2.2.6 Total antioxidant value of oils
166
10 mg of oil was dissolved in 3ml of hexane. 30 μl of aliquots were used for analysis.
167
The antioxidant values of the samples were determined by PCL as per the instructions
168
provided by the manufacturers of the instrument (Photochem®, Analytik Jena, Germany) and
169
results are expressed in terms of trolox equivalents (Sielicka, Malecka & Purlan, 2014).
170 171
. 2.2.7 Animal experiments
172
Male Wistar rats [OUBT-Wistar, IND-cft (2c)] (Rattus norvegicus) weighing 200 g
173
were grouped (six rats in each group) by random distribution and housed in individual cages,
174
under a 12 h light/dark cycle, in an approved animal house facility at the Central Food
175
Technological Research Institute in Mysore, India. The rats were fed standard rodent pellets
176
for 15 days (Sai Durga Feeds, Bangalore). The rats had free access to food and water
177
throughout the study. In addition, the rats were administered 1 mL of native or unsaponifiable
178
fractions removed oils (PO, OLO, SNO, RBO, SESO and LSO) by gavage once a day at 10
179
AM for 15 days. After 15 days of feeding, rats were fasted overnight and sacrificed by ether
180
anesthesia. Blood was drawn by cardiac puncture and serum was separated by centrifugation
181
at 1,100 g for 30 min. The experimental protocol for these studies was approved by the
182
Institutional Animal Ethics Committee recognized by the Government of India.
183 184
2.2.8 Total antioxidant activity in serum
185
Total lipid was extracted from serum using the method of Bligh & Dyer (1959). Total
186
lipid present in serum was in the range of 1.9 to 2.1mg/mL of serum. Serum lipid was
187
dissolved with 200 µl of hexane and 30μl aliquots were taken for measuring antioxidant
188
activity. The antioxidant values of the samples were determined as per the instructions
189
provided by the manufacturers of the instrument (Photochem®, Analytik Jena, Germany) and
190
results are expressed in terms of trolox equivalents.
191 192
2.2.9 Serum lipid peroxides in rats given native or minor constituents removed oils
193
Lipid peroxides in the serum was measured as thiobarbituric acid reactive substance
194
following the method described by Ledwozyw et al (1986). The serum lipid peroxides were
195
expressed as nmol malondialdehyde/dL.
196 197 6
198
2.2.10 Statistical analysis
199
Results are represented as means ± standard deviation for each group. The data was
200
analyzed by one way ANOVA followed by a post hoc Tukey test to compare the control and
201
treatment groups; p-values less than 0.01 were considered as statistically significant. All
202
statistical analysis was performed using SPSS statistical software package version 17.0.
203 204
3. Results & Discussion
205
3.1. Total antioxidant activity of synthetic and natural antioxidants
206
A typical response of photochemiluminescence in the presence of different
207
concentrations of trolox standard is shown in fig 1a. As the concentration of trolox was
208
increased there is an increase in lag phase, and a change in the rate and extent of quenching
209
of signals was observed. A standard curve was generated from this and used for measuring
210
trolox equivalents of experimental samples (Fig 1b). The duration of the lag phase is
211
determined by the inflection point and subsequently, the slope (tangent) at the inflection point
212
was calculated. The intersection point of the tangent through the inflection point with the x-
213
axis defines the duration of the lag time. The difference in the lag time of the sample and the
214
lag time of the blank values was calculated (Barba et al, 2013; Sielicka, Malecka & Purlan,
215
2014).
216
The total antioxidant activity measured by this system and expressed in μg of trolox
217
equivalent/mg were as follows: TBHQ (1227); BHT (728); BHA (790); α-tocopherol (348);
218
tocotrienol (282); β-carotene (369), -oryzanol(195), sesamin (128) and sesamolin (119).
219 220
3.2 Total antioxidant activity of vegetable oils
221
The total antioxidant value of vegetable oils before and after the removal of minor
222
constituents was evaluated. The total antioxidant value of PO, OLO, SNO, RBO, SESO and
223
LSO expressed in terms of µg of trolox equivalent/mg oil were 79, 111, 34, 130, 122 and 61
224
respectively. When the minor constituents were removed from these oils and tested for
225
antioxidant activity, the values observed (µg trolox equivalent/mg oil) for PO, OLO, SNO,
226
RBO, SESO and LSO were 10.4, 12.7, 9.2, 13.2, 14.3 and 10.3 respectively. Thus oils lost
227
much of their antioxidant activity when unsaponifiable matter was removed.
228
We quantitated some of the minor constituents present in unsaponifiable fractions of
229
oils which are associated with the antioxidant activities. RBO contained oryzanol comprising
230
of (in mg/100g oil) methyl ferulate (228), cycloartenyl ferulate (105), 24 methylene 7
231
cycloartenyl ferulate (492), Campesteryl ferulate (375) and β-sitosteryl ferulate (186). The
232
lignans present in SESO comprised of (in mg/100g oil) sesamin (91.8), sesamolin (147.6) and
233
sesamol (0.82). β-carotene was present to an extent of 41.8mg/100g oil in PO (Table 1).
234
Isomers of tocopherols and tocotrienols present in vegetable oils understudy is shown in
235
Table 1. The content of unsaponifiable matter in PO, SNO, OLO, LSO, SESO and RBO were
236
0.9%, 0.7%, 1.3%, 1.2%, 2.1% and 4.5% respectively. The residual unsaponifiable matter
237
remaining in the oils (PO, SNO, OLO, LSO, SESO and RBO) after passing it through the
238
columns (as described in methods) were 0.1%, 0.1%, 0.2%, 0.2%, 0.3% and 0.3%
239
respectively. More than 90-95% of unsaponifiable fraction was thus removed from the oil
240
which also stripped minor constituents of oils. However removal of unsaponifiable fraction
241
did not alter the fatty acid composition of oils (data not shown).
242 243
3.3. Total antioxidant activity of serum lipid in rats given native or minor constituent
244
removed oils
245
There was no significant difference in the total lipid observed in serum of rats given
246
native oil or the minor constituent removed oil (1.8-2.1mg/mL). We observed that the
247
removal of unsaponifiable fractions from oils resulted in diminished antioxidant activity
248
exhibited by the oil. In order to evaluate whether removal of minor constituents present in
249
unsaponifiable fractions oils has any impact on the antioxidant activity in vivo, we fed the
250
rats with native and minor constituents removed oils for 15 days. The antioxidant status of
251
rats given minor constituent removed oils was significantly lowered. The total antioxidant
252
activity of serum lipid was reduced by 30% and by 71% in rats given SNO and LSO which
253
were stripped of minor constituents. The total antioxidant activity of serum lipid was reduced
254
by 83 -87% when PO, OLO, RBO or SESO which were stripped of minor constituents were
255
given to rats (Table 2). Thus minor constituents present in PO, OLO, RBO or SESO has
256
greater influence on total antioxidant activity in serum lipid. This also reflected on lipid
257
peroxide levels in serum. When rats were given minor constituents removed oils the lipid
258
peroxide levels were increased by 17- 61% as compared to those given respective native oils
259
(Table 2). However the fatty acid composition of serum lipids remained same in rats given
260
individual native or minor constituents removed oils (Table3). These studies thus indicate
261
that minor constituents in oils had significant influence on antioxidant status but not on the
262
overall fatty acid composition of serum lipids. Interestingly, we also observed that the rats
263
given LSO which was stripped of unsaponifiable matter consistently showed a small but a 8
264
significant increase in long chain omega-3 fatty acids 20:5 n-3 and 22:6 n-3 PUFA as
265
compared to those given LSO containing minor constituents (Table 3). This needs to be
266
further evaluated.
267 268
The dietary lipids are known to have a significant effect on the fatty acid composition
269
of serum lipids, tissue lipids and that of red blood cells. However it is not known whether the
270
endogenous antioxidants/minor constituents associated with oils have any influence on the
271
antioxidant status as well as fatty acid composition of serum and tissue lipids. Our present
272
studies indicated that the components present in unsaponifiable fractions of oils have a
273
significant effect on total antioxidant status in vivo. However, the fatty acid composition of
274
serum lipids were not different when oils with or without the presence of unsaponifiable
275
components were fed to rats.
276 277
In summary, photochemiluminescence based assay is a rapid and sensitive method to
278
study the effect of minor constituents in oils on antioxidant status in vivo. The antioxidant
279
values in serum lipid were significantly lowered when unsaponifiable fractions removed oils
280
were fed to rats as compared to those given native oils. However, removal of the
281
unsaponifiable fraction components from oils had no effect on the fatty acid composition of
282
serum lipids. Therefore while studying the effect of dietary lipids on antioxidant status in
283
experimental systems; one should also consider the contributions of endogenous compounds
284
present in unsaponifiable fractions of oils.
285 286
Acknowledgements
287
The authors thank Director of Central Food Technological Research Institute, Mysore for
288
his encouragement and support for this work. D. Sugasini and Y. Poorna Chandra Rao
289
acknowledge Senior Research Fellowship granted by Indian Council of Medical Research,
290
New Delhi, India.
291 292
Conflict of interest
293
There is no conflict of interest
294 295 296 9
297
References
298
Amarowicz, R., Shahidi, F., & Pegg, R. B. (2001). Application of semi preparative RP-18
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HPLC for the purification of sesamin and sesamolin. Journal of Food Lipids, 8, 85- 94. AOCS. (1998). Unsaponifiable matter. Official methods and recommended practices of the American Oil Chemists Society. 5th edn, AOCS Press Champaign, Illinois
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Barba, F. J., Esteve, M. J., Tedeschi, P., Brandolini, V., & Frígola, A. (2013). A comparative
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study of the analysis of antioxidant activities of liquid foods employing
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spectrophotometric, fluorometric, and chemiluminescent methods. Food Analytical
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Methods, 6, 317-327.
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Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911- 917.
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Calder, P.C. (2012). The role of marine omega-3 (n-3) fatty acids in inflammatory process,
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atherosclerosis and plaque stability. Molecular Nutrition and Food Research, 56, 1073-
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Cardenia, V., Waraho, T., Rodriguez-Estrada, M. T., McClements, D. J., & Decker, E. A.
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(2011). Antioxidant and pro oxidant activity behaviour of phospholipids in stripped
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soybean oil in water emulsions. Journal of American Oil Chemists Society, 88, 1409-
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GopalaKrishna, A.G., Khatoon, S., Sheila, P.M., Saramandal, C.V., & Indira.T.N. (2001).
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Effect of refining of crude rice bran oil on the retention of oryzanol in the refined oil.
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Journal of American Oil Chemists Society, 78, 127-131.
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Hemalatha, S., & Ghafoorunissa. (2004). Lignans and tocopherols in Indian sesame cultivars. Journal of American Oil Chemists Society, 81, 467- 470.
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plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during
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human atherosclerosis. Clinica Chimica Acta, 155, 275- 285.
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Morrison, W. R., & Smith, L. M. (1964). Preparation of fatty acid methyl esters and
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dimethylacetals from lipids with boron trifluoridemethanol. Journal of Lipid Research,
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Popov, I. N., & Lewin, G. (1996). Photochemiluminescent detection of antiradical activity;
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Raju, M., Varakumar, S., Lakshminarayana, S., Krishnakantha, T.P., & Baskaran.V. (2007).
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Carotenoid composition and vitamin A activity of medicinally important green leafy
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vegetables. Food Chemistry, 101, 1598-1605.
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Reena, M. B., & Lokesh, B. R. (2011). Effect of feeding blended and interesterified vegetable
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11
Table 1. Minor components present in vegetable oils Minor components
Oils PO
OLO
SNO
RBO
SESO
LSO
α
22.9±1.2 c
13.3±1.0 b
36.9±2.7 d
13.2±0.4 b
1.1±0.2 a
23.4±2.3 c
β+
nd
1.1±0.2 a
2.93±0.3 b
23.3±1.7 d
65.7±4.2 e
12.1±0.8 c
nd
0.8±0.1 a
0.67±0.1 a
2.1±0.2 b
3.8±0.3 c
0.6±0.1 a
α
27.5±2.1 b
nd
nd
16.5±0.9 a
nd
nd
β+
1.1±0.2 a
nd
nd
54.2±3.7 b
nd
nd
nd
nd
0.91±0.1
a
nd
nd
Tocopherols(T) (mg/100g of oil)
Tocotrienols (T3)(mg/100g of oil)
b
12.2±0.6
Total (T+ T3) (mg/100g of oil)
63.7± 4.1 d
15.2± 1.3 a
40.5± 3.1 c
110.2± 2.3 f
70.6± 4.7 e
36.1± 3.2 b
Methyl ferulate
nd
nd
nd
228 ± 10.6
nd
nd
Cycloartenyl ferulate
nd
nd
nd
105 ± 9.8
nd
nd
24-Methylene cycloartenyl ferulate
nd
nd
nd
492 ± 18.6
nd
nd
Campesteryl ferulate
nd
nd
nd
375 ± 16.3
nd
nd
β-sitosteryl ferulate
nd
nd
nd
186 ± 11.8
nd
nd
Sesamin
nd
nd
nd
nd
91.8±8.6
nd
Sesamolin
nd
nd
nd
nd
147.6±12
nd
Sesamol
nd
nd
nd
nd
0.82±0.1
nd
nd
nd
nd
32 ± 0.8 a
0.3±0.1a
0.7±0.1b
0.6±0.1b
0.8±0.2b
Oryzanol (mg/100g of oil)
Lignans(mg/100g of oil)
Polyphenols (mg/100g of oil)
nd
38.2 ±3.6
β-carotene (mg/100g of oil)
41.8±4.9 d
2.4±0.3c
b
Values are mean ± SD, n = 4; Mean in a row with different superscript differ significantly at P<0.01, nd: not detected
1
Table 2. Total antioxidant activity and lipid peroxides in serum of rats fed native (N) or minor constituents removed (MCR) oils Total antioxidant activity Oils fed to rats
Lipid Peroxides
N
MCR
N
MCR
(µg of Trolox
(µg of Trolox equivalent
(nmoles MDA/dL)
(nmoles MDA/dL)
equivalent /dL)
/dL)
PO
22.8±0.9 b
3.5±0.4 a
24.8±0.8 a
30.8±1.4 b
OLO
26.3±1.5b
4.5±0.3 a
21.3±1.2 a
27.2±0.9 b
SNO
9.2±0.4 b
2.8±0.2 a
37.2±2.7 a
43.6±1.0 b
RBO
35.2±1.4 b
4.7±0.2 a
17.8±1.4 a
28.6±1.2 b
SESO
29.7±1.7 b
3.6±0.5 a
22.7±0.9 a
31.4±0.5 b
LSO
20.6±1.1b
3.9±0.2 a
34.3±1.0 a
46.9±0.4 b
Values are mean ± SD, n = 4; MDA: Malondialdehyde, Mean with different superscript for individual oils in native or MCR forms differ significantly at P <0.01 for parameters measured,
2
Table 3. Fatty acid composition (%) of serum lipids in rats given native (N) and minor constituent removed (MCR) oils Fatty acid
PO
OLO
SNO
RBO
SESO
LSO
14:0
N 0.9 0.3 a
MCR 0.6 0.2 a
N nd
MCR nd
N nd
MCR nd
N 1.3 0.5 a
MCR 1.8 0.4 a
N 2.7 0.5 a
MCR 2.4 0.7 a
N nd
MCR nd
16:0
28.7 1.9 a
29.2 2.7 a
23.0 2.0 a
24.8 1.2 a
24.7 2.4a
26.1 1.3a
24.0 2.3a
26.7 1.8a
22.1 2.1 a
24.2 1.1 a
23.0 1.2 a
25.1 1.4 a
16:1
3.9 0.6 a
4.2 0.4a
2.6 0.5 a
2.0 0.2a
2.4 0.2a
2.4 0.12 a
1.8 0.2 a
1.9 0.3 a
1.7 0.2 a
1.3 0.08 a
2.8 0.7 a
2.0 0.38 a
18:0
12.2 0.9 a
16.0 1.0 b
7.1 0.7 a
7.9 0.5 a
6.2 0.5 a
7.3 0.7 a
10.9 1.1 a
12.4 1.6 a
10.2 0.7a
11.4 0.3a
10.3 0.4 a
10.4 0.43a
18:1
24.2 2.9 a
24.1 2.4 a
32.2 1.0 a
34.1 1.7 a
15.7 1.7 a
17.3 0.6a
29.3 1.6 a
31.0 2.6 a
27.4 2.4 a
28.1 1.8a
27.4 1.0a
29.1 0.64 a
18:2
25.2 0.3a
24.5 0.5a
22.9 0.6 a
21.2 0.9a
33.8 2.0a
30.1 1.3a
19.4 0.9 a
16.1 2.9 a
22.0 1.5a
20.2 1.2a
21.7 0.9a
21.1 0.59a
18:3
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
2.6 0.3 b
1.7 0.12 a
20:4
14.9 1.6 a
13.4 0.4 a
12.2 1.7 a
10.0 0.5 a
17.2 0.9 a
16.8 0.6 a
13.3 1.8a
10.1 0.6 a
13.9 1.8 a
12.4 0.6 a
10.8 0.9 a
9.0 0.35 a
20:5 (n3)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.3 0.1 a
0.6 0.05 b
22:5(n3)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.2 0.01b
0.1 0.05 a
22:6(n3)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.3 0.06 a
0.9 0.08 b
Values are mean ± SD, n = 4; Mean with different superscript for individual oils in native or MCR forms differ significantly at P <0.01, nd: not detected.
3
Fig. 1a. Photochemical luminescence responses at different concentrations of Trolox (S1- 0.5 nmoles/mL, S1- 1.0 nmoles/mL, S1- 2.0 nmoles/mL and B- Blank)
Fig 1b. Standard curve for total antioxidant activity generated using different concentrations of trolox in photochemical luminescence assay..
Highlights
Photochemiluminescence assay was used for measuring total antioxidant activity of oils
Minor constituent removed oils significantly reduced antioxidant activity in rat serum
Minor constituent removed oils did not affect overall fatty acid composition of serum lipids
S0308-8146(14)00770-5 http://dx.doi.org/10.1016/j.foodchem.2014.05.064 FOCH 15847
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 May 2013 11 April 2014 13 May 2014
Please cite this article as: Sugasini, D., Poorna Chandra Rao, Y., Lokesh, B.R., Total antioxidant activity of selected vegetable oils and their influence on total antioxidant values in vivo: A photochemiluminescence based analysis, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.05.064
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1
Total antioxidant activity of selected vegetable oils and their influence on
2
total antioxidant values in vivo: A photochemiluminescence based analysis
3 4
Dhavamani Sugasini, Yalagala Poorna Chandra Rao and Belur R Lokesh*
5
Department of Lipid Science and Traditional Foods,
6
CSIR-Central Food Technological Research Institute,
7
Mysore- 570 020, Karnataka, India
8 9 10 11 12 13 14 15 16
*Corresponding Author
17
Dr. B. R. Lokesh,
18
Chief Scientist,
19
Department of Lipid Science and Traditional Foods,
20
CSIR-Central Food Technological Research Institute,
21
Mysore -570 020, India
22
Tel:91-821-2514153
23
Fax: 91-821-2517233
24
E-mail: [email protected]
25 26 27 28 29 30 31 32 1
33 34
Abstract This
study
evaluated
the
antioxidant
activity
of
vegetable
oils
using
35
photochemiluminescence based assay. The following oils were selected for the study - palm
36
oil (PO); olive oil (OLO); sunflower oil (SNO); rice bran oil (RBO); sesame oil (SESO) and
37
linseed oil (LSO). The antioxidant activity of oils was reduced significantly when
38
unsaponifiable matter was removed from the oils. The rats fed unsaponifiable matter removed
39
vegetable oils showed significantly reduced antioxidant activity but no change in overall fatty
40
acid composition in serum lipids. Therefore the minor constituents in unsaponifiable matter
41
influences antioxidant activity exhibited by vegetable oils.
42 43
Keywords: Photochemiluminescence assay, vegetable oils, total antioxidant activity,
44
antioxidant status of serum.
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2
66
1. Introduction
67
Antioxidants play an important role in providing the stability to vegetable oils. As a
68
dietary component, they also play a vital role in reducing the oxidative stress in vivo. Dietary
69
polyunsaturated fatty acids play a significant role in maintaining serum lipids at desirable
70
levels. The polyunsaturated fatty acids such as linoleic acid lower serum total and LDL
71
cholesterol. But excess intake of linoleic acid increases the vulnerability of LDL lipids to
72
peroxidation which initiates cascading events leading to foam cell formation and ultimately to
73
atherosclerotic plaques (Reena & Lokesh, 2011; Calder, 2012). Therefore the intake of
74
antioxidants along with PUFA rich diet is recommended to overcome the oxidative stress.
75
Minor constituents in unsaponifiable fractions of edible oils have drawn considerable
76
interest for their antioxidant and health-promoting effects. Rice bran oil contains oryzanol.
77
Sesame oil contains lignans such as sesamin and sesamolin. Palm oil contains β- carotene and
78
tocotrienols. Olive oil contains polyphenols. Most of the oils have tocopherol at different
79
levels. The contributions of these minor constituents in the oils on the antioxidant value in
80
biological systems has not been thoroughly studied. In the present study, the commercially
81
available oils and the oils whose minor constituents were removed by column
82
chromatography were fed to rats and its impact on antioxidant value in serum lipid were
83
measured in a chemiluminescense based assay using an Photochem (from Analytik Jena
84
AG, Germany) (Popov & Lewin, 1996; Barba et al, 2013; Sielicka, Malecka & Purlan,
85
2014).This system has high sensitivity and requires less time (< 3minutes/sample) for
86
analysis. Each analysis requires few microliters of the sample and the antioxidant value can
87
be quantified in nanomolar range.
88 89
2.0 Material & Methods
90
2.1 Materials
91
Different vegetable oils such as palm oil (PO), olive oil (OLO), sunflower oil (SNO),
92
rice bran oil (RBO), sesame oil (SESO), linseed oil (LSO) were procured from a local super
93
market in Mysore, India. Kits for determination of Antioxidant capacity of lipid-soluble
94
substances (ACL) by photochemiluminescence (PCL) assay were purchased from Analytik
95
Jena AG (Jena, Germany). Oryzanol (>99% purity) was a gift from Amohusu Chemical
96
Industries Inc, Tokyo, Japan. Sesamolin was prepared in the laboratory according to the
97
method of Hemalatha & Ghaforunissa (2004). Tertiary butylated hydroxyl quinone (TBHQ),
98
butylated hydroxyl toluene (BHT), butylated hydroxyl anisole (BHA), α-tocopherol, 3
99
tocotrienol, BF3 in methanol, gallic acid, β-carotene, thiobarbituric acid, 1,1,3,3,-tetraethoxy
100
propane, sesamol, sesamin and fatty acids were procured from Sigma Chemical Co. (St.
101
Louis, MO, U.S.A.). All other chemicals and solvents (analytical grade) were purchased from
102
Sisco Research Laboratories Ltd (Mumbai, India).
103 104
2.2 Methods
105
2.2.1 Evaluation of total antioxidant value of commercial edible oils
106
Selected oils were evaluated for total antioxidant value by photochemiluminescence
107
(PCL) assay using Photochem (Analytica Jena AG, Germany) (Sielicka, Malecka & Purlan,
108
2014). The antioxidant values of the samples were determined as per the instructions
109
provided by the manufacturer and results are expressed in terms of trolox equivalents.
110 111
2.2.2 Fatty acid composition
112
Fatty acid composition of oils and serum lipids were analyzed by Gas
113
chromatography (Fisons, fitted with a flame ionization detector [FID]) using the method
114
described by Morrison & Smith (1964). The fatty acid methyl esters prepared using 14% BF3
115
in methanol were separated on a fused silica capillary column (25 m × 0.25 mm, Parma bond
116
FFAP-DF-0.25, Machery Negal GmbH Co., Duren, Germany). Individual fatty acid was
117
identified by comparing with the retention times of standards (Reena and Lokesh, 2011).
118 119 120 121
2.2.3 Unsaponifiable matter of oils The unsaponifiable matter in oils was measured according to the AOCS official method (1998).
122 123
2.2.4 Estimation of minor constituents in oils
124
Tocopherol and tocotrienols in oils were determined according to the method of
125
Rogers et al. (1993). Shimadzu LC-10A (Shimadzu Corporation, Tokyo Japan) HPLC fitted
126
with C18 column (250mm 4.6 mm length, 5 µm Supleco, USA) and fluorescence detector
127
was used for separating isomers of tocopherols and tocotrienols. The mobile phase consisted
128
of acetonitrile /methanol/isopropanol/ water (48:45:5:2) and run in isocratic condition at a
129
flow rate of 1.0 ml/min. Individual tocopherols were identified and quantified with respective
130
standard tocopherols. Tocotrienols were identified by the elution pattern of palm oil
131
tocotrienols and quantified according to AOCS method (AOCS, 1998). 4
132
The oryzanol content in RBO was determined by HPLC method. Shimadzu LC 20A
133
system with Phenomenex develosil column (250mm4.6mm, 5µm i.d) and photodiode array
134
detector was used for analysis. Following conditions were used for the separation of oryzanol
135
components (Wavelength 325nm: mobile phase acetonitrile:methanol:isopropylalcohol
136
(10:9:1 v/v/v): flow rate 1mL/min (Gopala Krishna et al, 2001). -oryzanol (>99% purity)
137
obtained as gift from Amohusu Chemical Industries Inc, Tokyo, Japan was used as standard
138
for quantification.
139 140
Sesamol, sesamin and sesamolin in oils was estimated by HPLC using Phenomenex
141
C18 column of 250 × 4.60 mm, 5 micron particle size. The mobile phase was 70:30 (v/v)
142
methanol: water and individual peaks were analysed at 295nm using UV detector
143
(Amarowicz, Shahidi, & Pegg, 2001).
144 145
The phenolic compounds were extracted from oils using liquid–liquid extraction
146
according to Taga, Miller & Pratt (1984). The concentration of total phenols in the
147
methanolic extract was estimated with Folin-Ciocalteau reagent. Gallic acid (0.05- 0.4
148
mg/ml) was used as reference standard.
149 150
β-carotene in oil samples were quantified using an HPLC system (LC- 10 A,
151
Shimadzu, Kyoto, Japan) equipped with photodiode array (PDA) detector (SPD- M 20A,
152
Shimadzu). β- carotene was separated on a C-30 (ODS) column (250mm× 4.6 mm i.d., 5-μm
153
particle size; Princeton, SPHE Germany) by isocratically eluting with acetonitrile–
154
dichloromethane–methanol (60:20:20, v/v/v) containing 0.1% ammonium acetate as a mobile
155
phase (Raju et al., 2007). Carotenoids were quantified using the standard curve generated
156
with reference standards of β-carotene (2-10 µg).
157 158
2.2.5 Preparation of minor constituents removed oils
159
The minor components in unsaponifiable fractions of PO, OLO, SNO, RBO, SESO
160
and LSO were removed according to the method of Cardenia et al (2011). The oils that were
161
stripped of minor constituents was aliquoted into brown glass bottles, flushed with nitrogen,
162
and stored at -20C until use.
163 164 5
165
2.2.6 Total antioxidant value of oils
166
10 mg of oil was dissolved in 3ml of hexane. 30 μl of aliquots were used for analysis.
167
The antioxidant values of the samples were determined by PCL as per the instructions
168
provided by the manufacturers of the instrument (Photochem®, Analytik Jena, Germany) and
169
results are expressed in terms of trolox equivalents (Sielicka, Malecka & Purlan, 2014).
170 171
. 2.2.7 Animal experiments
172
Male Wistar rats [OUBT-Wistar, IND-cft (2c)] (Rattus norvegicus) weighing 200 g
173
were grouped (six rats in each group) by random distribution and housed in individual cages,
174
under a 12 h light/dark cycle, in an approved animal house facility at the Central Food
175
Technological Research Institute in Mysore, India. The rats were fed standard rodent pellets
176
for 15 days (Sai Durga Feeds, Bangalore). The rats had free access to food and water
177
throughout the study. In addition, the rats were administered 1 mL of native or unsaponifiable
178
fractions removed oils (PO, OLO, SNO, RBO, SESO and LSO) by gavage once a day at 10
179
AM for 15 days. After 15 days of feeding, rats were fasted overnight and sacrificed by ether
180
anesthesia. Blood was drawn by cardiac puncture and serum was separated by centrifugation
181
at 1,100 g for 30 min. The experimental protocol for these studies was approved by the
182
Institutional Animal Ethics Committee recognized by the Government of India.
183 184
2.2.8 Total antioxidant activity in serum
185
Total lipid was extracted from serum using the method of Bligh & Dyer (1959). Total
186
lipid present in serum was in the range of 1.9 to 2.1mg/mL of serum. Serum lipid was
187
dissolved with 200 µl of hexane and 30μl aliquots were taken for measuring antioxidant
188
activity. The antioxidant values of the samples were determined as per the instructions
189
provided by the manufacturers of the instrument (Photochem®, Analytik Jena, Germany) and
190
results are expressed in terms of trolox equivalents.
191 192
2.2.9 Serum lipid peroxides in rats given native or minor constituents removed oils
193
Lipid peroxides in the serum was measured as thiobarbituric acid reactive substance
194
following the method described by Ledwozyw et al (1986). The serum lipid peroxides were
195
expressed as nmol malondialdehyde/dL.
196 197 6
198
2.2.10 Statistical analysis
199
Results are represented as means ± standard deviation for each group. The data was
200
analyzed by one way ANOVA followed by a post hoc Tukey test to compare the control and
201
treatment groups; p-values less than 0.01 were considered as statistically significant. All
202
statistical analysis was performed using SPSS statistical software package version 17.0.
203 204
3. Results & Discussion
205
3.1. Total antioxidant activity of synthetic and natural antioxidants
206
A typical response of photochemiluminescence in the presence of different
207
concentrations of trolox standard is shown in fig 1a. As the concentration of trolox was
208
increased there is an increase in lag phase, and a change in the rate and extent of quenching
209
of signals was observed. A standard curve was generated from this and used for measuring
210
trolox equivalents of experimental samples (Fig 1b). The duration of the lag phase is
211
determined by the inflection point and subsequently, the slope (tangent) at the inflection point
212
was calculated. The intersection point of the tangent through the inflection point with the x-
213
axis defines the duration of the lag time. The difference in the lag time of the sample and the
214
lag time of the blank values was calculated (Barba et al, 2013; Sielicka, Malecka & Purlan,
215
2014).
216
The total antioxidant activity measured by this system and expressed in μg of trolox
217
equivalent/mg were as follows: TBHQ (1227); BHT (728); BHA (790); α-tocopherol (348);
218
tocotrienol (282); β-carotene (369), -oryzanol(195), sesamin (128) and sesamolin (119).
219 220
3.2 Total antioxidant activity of vegetable oils
221
The total antioxidant value of vegetable oils before and after the removal of minor
222
constituents was evaluated. The total antioxidant value of PO, OLO, SNO, RBO, SESO and
223
LSO expressed in terms of µg of trolox equivalent/mg oil were 79, 111, 34, 130, 122 and 61
224
respectively. When the minor constituents were removed from these oils and tested for
225
antioxidant activity, the values observed (µg trolox equivalent/mg oil) for PO, OLO, SNO,
226
RBO, SESO and LSO were 10.4, 12.7, 9.2, 13.2, 14.3 and 10.3 respectively. Thus oils lost
227
much of their antioxidant activity when unsaponifiable matter was removed.
228
We quantitated some of the minor constituents present in unsaponifiable fractions of
229
oils which are associated with the antioxidant activities. RBO contained oryzanol comprising
230
of (in mg/100g oil) methyl ferulate (228), cycloartenyl ferulate (105), 24 methylene 7
231
cycloartenyl ferulate (492), Campesteryl ferulate (375) and β-sitosteryl ferulate (186). The
232
lignans present in SESO comprised of (in mg/100g oil) sesamin (91.8), sesamolin (147.6) and
233
sesamol (0.82). β-carotene was present to an extent of 41.8mg/100g oil in PO (Table 1).
234
Isomers of tocopherols and tocotrienols present in vegetable oils understudy is shown in
235
Table 1. The content of unsaponifiable matter in PO, SNO, OLO, LSO, SESO and RBO were
236
0.9%, 0.7%, 1.3%, 1.2%, 2.1% and 4.5% respectively. The residual unsaponifiable matter
237
remaining in the oils (PO, SNO, OLO, LSO, SESO and RBO) after passing it through the
238
columns (as described in methods) were 0.1%, 0.1%, 0.2%, 0.2%, 0.3% and 0.3%
239
respectively. More than 90-95% of unsaponifiable fraction was thus removed from the oil
240
which also stripped minor constituents of oils. However removal of unsaponifiable fraction
241
did not alter the fatty acid composition of oils (data not shown).
242 243
3.3. Total antioxidant activity of serum lipid in rats given native or minor constituent
244
removed oils
245
There was no significant difference in the total lipid observed in serum of rats given
246
native oil or the minor constituent removed oil (1.8-2.1mg/mL). We observed that the
247
removal of unsaponifiable fractions from oils resulted in diminished antioxidant activity
248
exhibited by the oil. In order to evaluate whether removal of minor constituents present in
249
unsaponifiable fractions oils has any impact on the antioxidant activity in vivo, we fed the
250
rats with native and minor constituents removed oils for 15 days. The antioxidant status of
251
rats given minor constituent removed oils was significantly lowered. The total antioxidant
252
activity of serum lipid was reduced by 30% and by 71% in rats given SNO and LSO which
253
were stripped of minor constituents. The total antioxidant activity of serum lipid was reduced
254
by 83 -87% when PO, OLO, RBO or SESO which were stripped of minor constituents were
255
given to rats (Table 2). Thus minor constituents present in PO, OLO, RBO or SESO has
256
greater influence on total antioxidant activity in serum lipid. This also reflected on lipid
257
peroxide levels in serum. When rats were given minor constituents removed oils the lipid
258
peroxide levels were increased by 17- 61% as compared to those given respective native oils
259
(Table 2). However the fatty acid composition of serum lipids remained same in rats given
260
individual native or minor constituents removed oils (Table3). These studies thus indicate
261
that minor constituents in oils had significant influence on antioxidant status but not on the
262
overall fatty acid composition of serum lipids. Interestingly, we also observed that the rats
263
given LSO which was stripped of unsaponifiable matter consistently showed a small but a 8
264
significant increase in long chain omega-3 fatty acids 20:5 n-3 and 22:6 n-3 PUFA as
265
compared to those given LSO containing minor constituents (Table 3). This needs to be
266
further evaluated.
267 268
The dietary lipids are known to have a significant effect on the fatty acid composition
269
of serum lipids, tissue lipids and that of red blood cells. However it is not known whether the
270
endogenous antioxidants/minor constituents associated with oils have any influence on the
271
antioxidant status as well as fatty acid composition of serum and tissue lipids. Our present
272
studies indicated that the components present in unsaponifiable fractions of oils have a
273
significant effect on total antioxidant status in vivo. However, the fatty acid composition of
274
serum lipids were not different when oils with or without the presence of unsaponifiable
275
components were fed to rats.
276 277
In summary, photochemiluminescence based assay is a rapid and sensitive method to
278
study the effect of minor constituents in oils on antioxidant status in vivo. The antioxidant
279
values in serum lipid were significantly lowered when unsaponifiable fractions removed oils
280
were fed to rats as compared to those given native oils. However, removal of the
281
unsaponifiable fraction components from oils had no effect on the fatty acid composition of
282
serum lipids. Therefore while studying the effect of dietary lipids on antioxidant status in
283
experimental systems; one should also consider the contributions of endogenous compounds
284
present in unsaponifiable fractions of oils.
285 286
Acknowledgements
287
The authors thank Director of Central Food Technological Research Institute, Mysore for
288
his encouragement and support for this work. D. Sugasini and Y. Poorna Chandra Rao
289
acknowledge Senior Research Fellowship granted by Indian Council of Medical Research,
290
New Delhi, India.
291 292
Conflict of interest
293
There is no conflict of interest
294 295 296 9
297
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298
Amarowicz, R., Shahidi, F., & Pegg, R. B. (2001). Application of semi preparative RP-18
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HPLC for the purification of sesamin and sesamolin. Journal of Food Lipids, 8, 85- 94. AOCS. (1998). Unsaponifiable matter. Official methods and recommended practices of the American Oil Chemists Society. 5th edn, AOCS Press Champaign, Illinois
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Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911- 917.
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(2011). Antioxidant and pro oxidant activity behaviour of phospholipids in stripped
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Effect of refining of crude rice bran oil on the retention of oryzanol in the refined oil.
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Journal of American Oil Chemists Society, 78, 127-131.
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Hemalatha, S., & Ghafoorunissa. (2004). Lignans and tocopherols in Indian sesame cultivars. Journal of American Oil Chemists Society, 81, 467- 470.
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plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during
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human atherosclerosis. Clinica Chimica Acta, 155, 275- 285.
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11
Table 1. Minor components present in vegetable oils Minor components
Oils PO
OLO
SNO
RBO
SESO
LSO
α
22.9±1.2 c
13.3±1.0 b
36.9±2.7 d
13.2±0.4 b
1.1±0.2 a
23.4±2.3 c
β+
nd
1.1±0.2 a
2.93±0.3 b
23.3±1.7 d
65.7±4.2 e
12.1±0.8 c
nd
0.8±0.1 a
0.67±0.1 a
2.1±0.2 b
3.8±0.3 c
0.6±0.1 a
α
27.5±2.1 b
nd
nd
16.5±0.9 a
nd
nd
β+
1.1±0.2 a
nd
nd
54.2±3.7 b
nd
nd
nd
nd
0.91±0.1
a
nd
nd
Tocopherols(T) (mg/100g of oil)
Tocotrienols (T3)(mg/100g of oil)
b
12.2±0.6
Total (T+ T3) (mg/100g of oil)
63.7± 4.1 d
15.2± 1.3 a
40.5± 3.1 c
110.2± 2.3 f
70.6± 4.7 e
36.1± 3.2 b
Methyl ferulate
nd
nd
nd
228 ± 10.6
nd
nd
Cycloartenyl ferulate
nd
nd
nd
105 ± 9.8
nd
nd
24-Methylene cycloartenyl ferulate
nd
nd
nd
492 ± 18.6
nd
nd
Campesteryl ferulate
nd
nd
nd
375 ± 16.3
nd
nd
β-sitosteryl ferulate
nd
nd
nd
186 ± 11.8
nd
nd
Sesamin
nd
nd
nd
nd
91.8±8.6
nd
Sesamolin
nd
nd
nd
nd
147.6±12
nd
Sesamol
nd
nd
nd
nd
0.82±0.1
nd
nd
nd
nd
32 ± 0.8 a
0.3±0.1a
0.7±0.1b
0.6±0.1b
0.8±0.2b
Oryzanol (mg/100g of oil)
Lignans(mg/100g of oil)
Polyphenols (mg/100g of oil)
nd
38.2 ±3.6
β-carotene (mg/100g of oil)
41.8±4.9 d
2.4±0.3c
b
Values are mean ± SD, n = 4; Mean in a row with different superscript differ significantly at P<0.01, nd: not detected
1
Table 2. Total antioxidant activity and lipid peroxides in serum of rats fed native (N) or minor constituents removed (MCR) oils Total antioxidant activity Oils fed to rats
Lipid Peroxides
N
MCR
N
MCR
(µg of Trolox
(µg of Trolox equivalent
(nmoles MDA/dL)
(nmoles MDA/dL)
equivalent /dL)
/dL)
PO
22.8±0.9 b
3.5±0.4 a
24.8±0.8 a
30.8±1.4 b
OLO
26.3±1.5b
4.5±0.3 a
21.3±1.2 a
27.2±0.9 b
SNO
9.2±0.4 b
2.8±0.2 a
37.2±2.7 a
43.6±1.0 b
RBO
35.2±1.4 b
4.7±0.2 a
17.8±1.4 a
28.6±1.2 b
SESO
29.7±1.7 b
3.6±0.5 a
22.7±0.9 a
31.4±0.5 b
LSO
20.6±1.1b
3.9±0.2 a
34.3±1.0 a
46.9±0.4 b
Values are mean ± SD, n = 4; MDA: Malondialdehyde, Mean with different superscript for individual oils in native or MCR forms differ significantly at P <0.01 for parameters measured,
2
Table 3. Fatty acid composition (%) of serum lipids in rats given native (N) and minor constituent removed (MCR) oils Fatty acid
PO
OLO
SNO
RBO
SESO
LSO
14:0
N 0.9 0.3 a
MCR 0.6 0.2 a
N nd
MCR nd
N nd
MCR nd
N 1.3 0.5 a
MCR 1.8 0.4 a
N 2.7 0.5 a
MCR 2.4 0.7 a
N nd
MCR nd
16:0
28.7 1.9 a
29.2 2.7 a
23.0 2.0 a
24.8 1.2 a
24.7 2.4a
26.1 1.3a
24.0 2.3a
26.7 1.8a
22.1 2.1 a
24.2 1.1 a
23.0 1.2 a
25.1 1.4 a
16:1
3.9 0.6 a
4.2 0.4a
2.6 0.5 a
2.0 0.2a
2.4 0.2a
2.4 0.12 a
1.8 0.2 a
1.9 0.3 a
1.7 0.2 a
1.3 0.08 a
2.8 0.7 a
2.0 0.38 a
18:0
12.2 0.9 a
16.0 1.0 b
7.1 0.7 a
7.9 0.5 a
6.2 0.5 a
7.3 0.7 a
10.9 1.1 a
12.4 1.6 a
10.2 0.7a
11.4 0.3a
10.3 0.4 a
10.4 0.43a
18:1
24.2 2.9 a
24.1 2.4 a
32.2 1.0 a
34.1 1.7 a
15.7 1.7 a
17.3 0.6a
29.3 1.6 a
31.0 2.6 a
27.4 2.4 a
28.1 1.8a
27.4 1.0a
29.1 0.64 a
18:2
25.2 0.3a
24.5 0.5a
22.9 0.6 a
21.2 0.9a
33.8 2.0a
30.1 1.3a
19.4 0.9 a
16.1 2.9 a
22.0 1.5a
20.2 1.2a
21.7 0.9a
21.1 0.59a
18:3
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
2.6 0.3 b
1.7 0.12 a
20:4
14.9 1.6 a
13.4 0.4 a
12.2 1.7 a
10.0 0.5 a
17.2 0.9 a
16.8 0.6 a
13.3 1.8a
10.1 0.6 a
13.9 1.8 a
12.4 0.6 a
10.8 0.9 a
9.0 0.35 a
20:5 (n3)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.3 0.1 a
0.6 0.05 b
22:5(n3)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.2 0.01b
0.1 0.05 a
22:6(n3)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.3 0.06 a
0.9 0.08 b
Values are mean ± SD, n = 4; Mean with different superscript for individual oils in native or MCR forms differ significantly at P <0.01, nd: not detected.
3
Fig. 1a. Photochemical luminescence responses at different concentrations of Trolox (S1- 0.5 nmoles/mL, S1- 1.0 nmoles/mL, S1- 2.0 nmoles/mL and B- Blank)
Fig 1b. Standard curve for total antioxidant activity generated using different concentrations of trolox in photochemical luminescence assay..
Highlights
Photochemiluminescence assay was used for measuring total antioxidant activity of oils
Minor constituent removed oils significantly reduced antioxidant activity in rat serum
Minor constituent removed oils did not affect overall fatty acid composition of serum lipids