Dietary effects of diacylglycerol rich mustard oil on lipid profile of normocholesterolemic and hypercholesterolemic rats

Abstract

Several recent studies have established that diacylglycerol (DAG) rich oils significantly reduce the body weight. The present study was conducted to evaluate the dietary effects of DAG-rich mustard oil on normal and hypercholesterolemic rats. DAG-rich mustard oil (45.5% DAG) was prepared in the laboratory by enzymatic glycerolysis process. For the feeding experiment, 32 rats were taken and divided into four groups (average body weight 130 g) and body weight gain, food efficiency ratio, lipid profile of plasma, liver, mesentery and erythrocytes membrane (EM), HMG Co-A reductase activity and plasma leptin content were measured and compared with the normal TAG-rich diet. The dietary DAG rich mustard oil significantly decreased body weight and FER compared to TAG rich mustard oil both in normal and hypercholester-olemic rats. The total cholesterol content was decreased with significant increase in HDL-cholesterol by feeding DAG rich diet. Total lipid and TAG content of both liver and mesentery were significantly decreased in DAG diet group compared to control group. Liver HMG CoA: mevalonate ratio was also found to be significantly decreased in the DAG group. Blood leptin level signifi-cantly reduced with DAG rich diet compared to the TAG rich dietary groups.

Full text

ORIGINAL ARTICLE
Dietary effects of diacylglycerol rich mustard oil
on lipid profile of normocholesterolemic
and hypercholesterolemic rats
Rupali Dhara & Pubali Dhar & Mahua Ghosh
Revised: 7 April 2011 /Accepted: 19 April 2011
#
Association of Food Scientists & Technologists (India) 2011
Abstract Several recent studies have established that
diacylglycerol (DAG) rich oils significantly reduce the
body weight. The present study was conducted to evaluate
the dietary effects of DAG- rich mustard oil on normal and
hypercholesterolemic rats. DAG- rich mustard oil (45.5%
DAG) w as prepared in the laboratory by enzymatic
glycerolysis process. For the feeding experiment, 32 rats
were taken and divided into four groups (average body
weight 130 g) and body weight gain, food efficiency ratio,
lipid profile of plasma, liver, mesentery and erythrocytes
membrane (EM), HMG Co-A reductase activity and plasma
leptin content were measured and compared with the
normal TAG-rich diet. The dieta ry DAG rich mustard oil
significantly decreased body weight and FER compared to
TAG rich mustard oil both in normal and hypercholester-
olemic rats. The total cholesterol content was decreased
with significant increase in HDL- cholesterol by feeding
DAG rich diet . Total lipid and TAG content of both liver
and mesentery were significantly decreased in DAG diet
group compared to control group. Liver HMG CoA:
mevalonate ratio was also found to be significantly
decreased in the DAG group. Blood leptin level signi fi-
cantly reduced with DAG rich diet c ompared to the TAG
rich dietary groups.
Keywords Diacylglycerol
.
Lipid profile
.
Triacylglycerol
.
Cholesterol
.
Leptin
.
HMG-CoA reductase
Introduction
Numerous scientific reports have shown the effectiveness
of diacylglycerol (DAG) oil in preventing body fat
accumulation and obesity related disorders. (Nagao et al.
2000; Yamamoto et al. 2001; Hibi et al. 2011). At the
current growth rate of obese population throughout the
world, the necessity for oils with health beneficial effects
will be high and it can be expected that the global market
demand for DAG oil will increase in the future. The long-
term ingestion of dietary DAG composed mainly of 1, 3-
DAG decreased both body weight and visceral fat mass in
humans, in comparison with ingestion of TAG (Rudkowska
et al. 2005). Moreover, several studies with humans have
demonstrated that DAG ingestion reduced postprandial
hypertriglyceridemia compared with TAG ingestion (Saito
et al. 2010) It has been found that energy values of the TAG
oil and DAG oil, when measured in bomb calorimeter, were
almost si milar and being having more or less equal
digestibility also, the reduced fat accumulation by dietary
DAG is only caused by differential metabolic fates of both
the lipids after absorption in the GI tract (Hibi et al. 2009;
Matsuzawa et al. 1995; Pi-Sunyer 1991). A vast population
of East Asia use mustard oil as the primary cooking oil.
Mustard oil (Brassica juncea) contains 8–9% of saturated
fatty acid and 88–91% of unsaturated fatty acid in which
R. Dhara
:
M. Ghosh
Dept. of Chemical Technology, University of Calcutta,
Kolkata, India
P. Dhar
Dept. of Home Science, University of Calcutta,
Kolkata, India
M. Ghosh (*)
Oil Technology Division, Department of Chemical Technology,
University of Calcutta,
92, Acharyya Prafulla Chandra Road,
Kolkata 700009, India
e-mail: mahuag@gmail.com
M. Ghosh
e-mail: mgchemtech@caluniv.ac.in
J Food Sci Technol
DOI 10.1007/s13197-011-0388-y

48–50% is erucic acid (C22:1). According to various
studies, erucic acid takes time to digest in human system
which leads to less deposition of lipid in different organs
(Kannel et al. 1991). More even mustard oil contains two
essential fatty acids (EFA) in appreciable a mount and
natural antioxidant, tocopherol, in significant amount.
Therefore it could be a potential raw material to produce
a low calorie, healthful edible oil. In the present study
mustard oil (Brassica juncea) was converted into DAG rich
mustard oil by enzymatic glycerolysis (Dhara and Ghosh
2009). The dietary effects of DAG rich mustard oil in
comparison with TAG rich mustard oil on the tissue lipid
profile of normal and hypercholeste rolemic rats was
studied.
Materials an d methods
Materials
Mustard oil was extracted from brown mustard seeds at
laboratory by solvent extraction process and bleached with
bleaching earth and activated carbon and finally physically
refined to remove free fatty acid (FFA) and allyl isothiocya-
nate so that enzymatic esterification can be done properly
(John 1976). The 1, 3 specific immobilized lipase Thermo-
myces lanuginosus (TLIM) was a gift of M/s Novozymes
India Ltd., Bangalore, India. According to the literature
supplied by the manufacturer it is produced by submerged
fermentation of genetically modified Aspergillus oryzae and
possesses activity of 250 IU/g. The product complies with
the recommendation purity specifications for food-grade
enzymes given by the Joint FAO/WHO. According to
literature this liapse is suitable for hydrolysis and esterifica-
tion (Fernandes et al. 2004). Glycerol and all other reagents
used were of analytical grade and were of procured from
Merck India Ltd. Mumbai, India. All the enzyme kits used
for the measurement of various blood parameters were
procured from Merck India Ltd., Mumbai, India.
Methods
Glycerolysis reaction
Physically refined mustard oil and glycerol were taken in a
molar ratio of 2:1 in round bottom flask and 10% (w/w)
enzymes (on the basis of total substrate weight) was added
to it. The reaction was carried out at 60 °C with constant
stirring of 200 rpm under vacuum for 26 h (Dhara and
Ghosh 2009) for optimum DAG production. Final product
was filte red to remo ve enzyme, excess glycerol was
separated out by gravity separation and water washing.
The product that is DAG rich mustard oil was then vacuum
dried, checked for FFA content and stored at refrigerator for
feeding experiments.
Estimation of amount of DAG and MAG present in dietary
oils
HPTLC analysis was done to estimate MAG and DAG (%
w/w) present in the dietary oils (Macala et al. 1983). The
extracted lipids were separated by TLC using a Silica gel
60-precoated high-performance TLC (HPTLC) plate
(Merck, Germany) and hex ane/diethyl ether/acetic acid
(80:20:1, by vol) as the development solvent. For quanti-
fication of DAG, HPTLC plates were sprayed with 40%
sulfuric acid, immediately heated to 180 °C to visualize the
lipids, and used for densitometry. The integrated optical
density (IOD) of the lipids was measured using a
WINCATS-3 software program, CAMAG- HPTLC Scanner
3″ (Scanner 3_ 130214″ S/N 130214). Standards for sn-1,2-
DAG (99% pure, Sigma) and 1,3-DAG (98% pure, Sigma)
and 1 and 2- MAG (prepared in the laboratory) were
applied to the plate, and t he calibration c urv es were
constructed by plotting the IOD vs the amount of lipid
loaded. The standard curve was linear and the value of the
IOD of the lipid was interpolated on the corresponding
calibration curve.
Gas Liquid Chromatography (GLC)
Gas–liquid chromatography technique was employed for
the determination of fatty acid composition of different
dietary oils after convert ing them into methyl esters by
Litchfield’s method (Litchfield 1972).
Feeding experiment
The animal experiments were performed with the approval
of the Ethics Committee for experimental animals of the
Department of Chemical Technology (University of Cal-
cutta). The animal experiment was designed on the basis of
earlier reports published from this laboratory. Male albino
rats (average body weight per gr-170 g) of Charles Foster
strain (selected for the authenticity of the strain) were
chosen as animal system for feeding experiment. Rats were
housed individually in stainless steel cages (27×21×
14 cm3) with mesh floors in a room maintained under
constant temperature (25
–30 °C) and a 10 h light/14 h dark
cycle (appropriate light and dark cycle is responsible to
maintain the biological rhythm which maintains hormonal
cycles that in turn controls the lipid metabolism). Following
a 7 days adaptation during which the animals were
maintained on a standard diet and water ad libitum. Each
group of rats received different experimental oil while the
other dietary components were same. The rats were fed
J Food Sci Technol

balanced diet (Jones and Foster 1942) having the following
composition: fat free casein-18% (protein source), fat-20%,
starch-55% (carbohydrate source), Salt mix ture 4% [com-
position of salt mixture No. 12 (wt in gm): NaCl-292.5;
KH
2
PO
4
-816.6; MgSO
4
-120.3; CaCO
3
-800.8; FeSO
4
,
7H
2
O-56.6; KCl-1.66; MnSO
4
,2H
2
O-9.35; ZnCL
2
-
0.5452; CuSO
4
,5H
2
O-0.9988; CoCl
2
-, 6H
2
O-0.0476];
cellulose-3%; and one multiv itamin capsule (Vitamin A I.
P. 10,000 units, thiamine mononitrate I.P.5 mg, vitamin B.I.
P. 5 mg, calcium pantothenate USP 5 mg, niacinamide I.P.
50 mg, ascorbic acid I.P. 400 units, cholecalciferol USP 15
units, menadione I.P. 9.1 mg, folic acid I.P. 1 mg, and
vitamin E USP 0.1 mg) per kg of diet. The diet was
adequate in all nutrients. The animals were divided into 4
groups (average body weight 170 g), each consisting of
eight animals, naming MO, DAG, MOCh and DAGCh.
MO group received normal mustard oil as dietary lipid,
DAG group was fed with DAG rich mustard oil as dietary
fat. Two groups, MOCh and DAGCh were made hyper-
cholesterolemic by addition of cholesterol (1% of total
dietary fat). MOCh group received normal mustard oil and
DAGCh group received DAG rich mustard oil respectively.
Table 1 describes the details of feeding groups.
The amount of daily diet consumed by each rat and
weekly body weight gain were noted. The Food Efficiency
ratio (FER) of each rat was calculated by the following
equation:-
FER ¼ Body weight gain=Food consumed:
At the end of 28 days of experimental period rats were
fasted over-night for 12 h and then sacrificed under
anesthesia using chloroform. The abdomen was opened,
blood samples was collected from hepatic vein into clear
heparinised centrifuge tube and centrifuge at low speed
(3,000 g, 4 °C) for 10 min to isolate the plasma. Plasma
was separated as the supernatant layer and was collected
carefully without disturbing the rest lower part. The liver,
heart and mesentery were immediately excised, cleaned by
washing with saline (0.98% NaCl sol), blotted, weighed
and stored at deep freeze temperature (− 40 °C) for
subsequent extraction of tissue lipid.
Lipid analysis
According to the standard methods, the lipid components
such as total cholesterol (Allain et al. 1974), and high-
density lipoprotein (HDL)-cholesterol (Warnick et al.
1985) and triacylglycerol (TAG) (Bucolo and David
1973) of plasma were analyzed using enzymatic kits
Ecoline CHOD PAP method, supp lied by Mer ck India
Ltd., Mumbai, India.
Preparation of EM ghost
After plasma separation, the red blood cells (RBC) were
washed three times by centrifugation at 3,000 g, for 10 min
with three volumes of a cooled isotonic solution containing
0.15 M NaCl and 10
−5
M EDTA. RBC was haemolysed
using hypotonic solution and centrifuged at 20,000 ⋅ g for
40 min in a cold centrifuge at 4 °C. The supernatant was
removed carefully with a pasteur pipette. The process was
repeated two more times. After the last wash step, the
supernatant was removed as much as possible and the
loosely packed milky-looking membrane pellet was re-
suspended at the bottom of the tube using 0.89% NaCl
solution. Concentrated membrane solution was taken in
2 ml screw cap vial and stored at −40 °C (Rose and
Oklander 1965).
Plasma lipid peroxidation
Plasma lipid peroxidation was measured by the assay of
thiobarbituric acid-reactive substances (TBARS) according
to the stan dard method (Wills 1987). The amount of
malonedialdehyde formed was calculated by taking the
extinction coefficient of malonedialdehyde to be 1.56×
105 M
−1
cm
−1
.
Liver & mesentery tissue lipid extraction
Total lipids were extracted from an aliquot o f tissue
homogenate by the method of Bligh and Dyer (Bligh and
Dyer 1959).
Table 1 Food composition of different dietary groups
Animal Groups Composition of diet Dose Duration
Male Albino Rat Control (MO) Balanced diet+20% physically refined mustard oil
(Charles foster strain) Treated (DAG) Balanced diet+20% DAG rich mustard oil
Control (MOCh) Balanced diet+20% physically refined mustard oil+Cholesterol
(1% of dietary oil)
10 g of food/rat/day for
each group
28 days
Treated (DAGCh) Balanced diet+20% DAG rich mustard oil+Cholesterol (1% of
dietary oil)
J Food Sci Technol

Estimation of total protein
Total protein was estimated by the method of Lowry et al.
(Lowry et al. 1951).
Estimation of phospholipid
Phospholipid content in tissue lipid was determined by
estimating phosphorous according to the method of Chen et
al. (Chen et al. 1956).
Measurement of HMG CoA: mevalonate ratio
To measure the HMG Co A reductase activity an indirect
method was used (Rao and Ramakrishnan 1975). 1 g of
fresh liver tissue was homogenized in 10 ml saline-arsenate
solution and equal volume of diluted perchloric acid was
added and kept for 5 min. After centrifugation at 2,000 rpm
for 10 min, 1 ml of the filtrate was utilized for HMG-Co A
analysis with 0.5 ml alkaline hydroxylamine (pH 5.5) and
1.5 ml ferric chloride and 1 ml of the filtrate was utilized
for measuring the mevalonate with 0.5 ml acidic hydrox-
ylamine (pH 2.1) and ferric chloride. The absorbance was
measured at 540 nm.
Measurement of leptin
Leptin content was measured using ELISA Kit procured
from LINCO Research, MO, USA [Cat. No. E6083-K] by
the method provided with the kit.
Statistical analysis
The data was expressed as mean±standard error of mean
(SEM). One-dway analysis of variance (ANOVA) was also
used for statistical analysis between groups. F ratio of one-
way ANOVA is significant when p value<0.05. Tukey’s
multiple range method (Scheffe 1961) was used for
comparison. For statistical analysis Origin 7 softwar e was
used to calculate the results.
Results and discussions
The physioco-chemical properties of TAG rich mustard oil
and DAG rich mustard oil used as dietary oils for the
feeding experi ment are depicted in Table 1. The fatty acid
composition of TAG rich mustard oil was similar with that
of DAG rich mustard oil and so both the oils similar in
respect to fatty acid composition. The amount of TAG,
DAG, MAG, FFA and unsapponifiable matter in original
(physically refined) mustard oil and DAG rich mustard oil
prepared by enzymatic glycerolysis process are given in
Table 2. There was an increase in DAG percentage from
1.18% to 45.5% and decrease in the percentage of TAG
from 95.37% to 50.21% in DAG rich mustard oil. There
was no significant change observed in amount of MAG,
unsapponifiable matter and FFA in both the oils. Fatty acid
composition of different dietary DAG (1, 3 DAG and 1,2/
2,3 DAG) produced by enzyme catalysed glyce rolysis
reaction of mustard oil is shown in Table 2. Mean body
weight gain observed in the experimental subjects is given
in Table 3 and the food efficiency ratio (FER) calculated
from body weight gain and food intake of each rat per week
is represented by Fig. 1. The FER of DAG group was
significantly decreased with respect to the other three
dietary groups in all the 4 weeks. In 3rd week the group
DAGCh showed a significant lower FER compared to the
group MOCh. Consistent with previous reports of Murase
et al. (Murase et al. 2002) feeding with the high DAG diet
for 1 month resulted in significant decreases in body weight
and food efficiency ratio compared with rats fed with TAG
rich diet. Taguchi et al. reported that the apparent
digestibility of diacylglycerol and TAG oil was identical
(96.3%) in rats, and the energy content measured in a bomb
calorimeter was similar (38.9 and 39.6 kJ/kg for DAG and
Table 2 Amount of DAG, MAG, TAG, FFA and unsap matter in original mustard oil and DAG rich mustard oil
Oils TAG
b
(% w/w)
DAG
c
(% w/w) MAG
d
(% w/w)
FFA
e
(% w/w)
Unsap
f
(%w/w)
Total 1,3 (% of total) 1,2 (% of total)
Mustard oil (Physically refined) 95.37±0.56 1.18±0.015 68.12±2.42 30.76±0.75 1.02±0.02 0.33±0.07 1.98±0.25
DAG rich mustard oil
a
50.21±0.25 45.50±0.35 66.42±0.35 33.58±0.08 2.21±0.02 0.25±0.03 1.83±0.14
a
Mole ratio between Mustard oil: Glycerol=2:1 and 10% enzyme TLIM was used at 60 °C, 200 r.p.m for 26 h. (all values are mean±SD, n=3)
b
TAG- Triacylglycerol
c
DAG- Diacylglycerol
d
MAG- Monoacylglycerol
e
FFA- Free fatty acid
f
unsap- Unsaponifiable matter
J Food Sci Technol

TAG respectively) (Taguchi et al. 2001). The plasma lipid
profile, LDL and plasma peroxidation of four di etary
groups of rats are given in Table 4. The total cholesterol
decreased in DAG group in comparison with the other three
dietary groups. DAGCh group showed significantly low-
ered total cholesterol than MOCh group. Plasma HDL
cholesterol levels of rats fed with high DAG diet was
significantly higher than those of rats fed the high TAG diet
and the plasma non-HDL cholesterol and TAG concen-
trations were significantly lowered in rats fed with high
DAG in comparison with the high TAG diet (MO Vs DAG
and MOCh Vs DAGCh). The plasma lipid profile indicates
that DAG rich mustard oil reduced total cholesterol, TAG,
non-HDL cholesterol and raised HDL cholesterol even
when cholesterol was supplemented in diet. High DAG diet
has shown a reduced plasma peroxidation and LDL
peroxidation decreased in the hypercholesterolemic
DAGCh compared to the MOCh diet. To further examine
the local accumulation of fat, we determined the liver and
mesentery lipid profiles that are represented in Table 5. The
total lipid accumulation in liver and mesentery were
significantly lower in high DAG group (DAG) compared
to the high TAG group (MO). Similarly, in 1% cholesterol
fed groups the mesentery lipid was high in TAG rich group
(MOCh) compared to the DAG rich group (DAGCh) group.
The total liver cholesterol content significantly decreased in
DAGCh group in comparison with MOCh group and the
TAG of liver was decreased with high DAG group than
high TAG group (MO). The liver phospholipid content was
increased in the DAG group than the MO group. In the
mesentery, the total cholesterol and TAG was reduced in
DAG groups (DAG and DAGCh) than high TAG groups
(MO and MOCh). Measurement of plasma leptin was an
excellent index of obesity. Table 6 shows the liver HMG-
CoA: Mevalonate ratio and plasma leptin content of
different dietary groups. Plasma leptin content was signif-
icantly lowered in high DAG fed groups compared to that
of high TAG fed groups both without and with cholesterol.
During cholesterol biosynthesis 3 hydroxy 3-methyl glu-
teryl CoA (HMG CoA) is converted into mevalonate by
HMG CoA reductase, a rate limiting enzyme for cholesterol
synthesis. DAGCh group consuming DAG along with
cholesterol showed increased HM G-CoA: Mevalonate ratio
than TAG fed MOCh group which in turn illustrates
decreased activity of HMG-CoA reductase that indicated
decreased synthesis of liver cholesterol in DAG Ch group.
Lipid profile of erythrocyte membrane (EM) of different
dietary groups is presented in Table 7. TAG content
decreased in DAG fed group compared to the TAG fed
group.
In the present study, we examined the effects of dietary
DAG rich mustard oil in normal and hypercholesterolemic
rat model. The results of this study indicate that structural
differences between DAG and TAG, did not affect the fatty
acid composition, has markedly affect nutritional behavior
of lipids including body fat accumulation, serum lipid
profile and lipid profile of the liver and mesentery and the
antiobesity hormone leptin . Many studies have been
conducted to determine the preventive or therapeutic effects
of various dietary oils on obesity. The decrease in FER and
Table 3 Mean body weight gain of rats of different dietary groups
Week Weight gain in gm
MO DAG MOCh DAGCh
I 15.55±0.04 8.28±0.04 17.68±0.70 17.0±0.32
II 12.3±0.31 10.26±0.16 14.36±0.10 16.35±0.06
III 10.32±0.26 9.56±0.21 18.35±0.13 15.21±0.13
IV 12.60.14 10.48±0.09 15.55±0.18 14.62±0.15
All values are means±SEM of 8 rats/diet
MO Mustard oil control group; DAG DAG rich mustard oil; MOCh
mustard oil with 1% cholesterol; and DAGCh DAG rich mustard oil
with 1% cholesterol
0
0.05
0. 1
0.15
0. 2
0.25
0. 3
0.35
0. 4
0.45
1234
Week s
Food Efficiency Ratio (FER)
MO
DAG
MOCh
DAG Ch
Fig. 1 Food efficiency ratio of
different dietary groups of rats.
MO: mustard oil control group;
DAG: DAG rich mustard oil;
MOCh: mustard oil with 1%
cholesterol and DAGCh: DAG
rich mustard oil with 1% choles-
terol. All values are means±SEM
of 8 rats/diet
J Food Sci Technol

body weight gain in DAG rich oil group suggests that there
is reduced accumulation of energy derived from dietary
DAG. In control feeding studies diacylglycerol prevented
the accumulation of body weight and fat associated with the
high fat and high sucrose diet in obesity prone mice
(Murase et al. 2001). These effects do not appear du e to the
poor digestibility or reduced energy content of DAG. The
apparent digestibility of DAG and TAG oil was identical
(96.3%) in rats, and the energy content was similar
(Taguchi et al. 2001). Therefore, decreased body weight is
due to energy expenditure and food or both (Maki et al.
2002). Watanabe et al. (Watanabe and Tokimitsu 2004)
found that in compa rison with a triacylglycerol control,
oxygen consumption in rats increased 1 ml.kg-1 min − 1
during the 90 min after DAG administration, suggesting a
short term increase in energy expenditure. Studies also
suggested that, fatty acids released from dietary 1,3
diacylglycerol (DAG) oil are not effectively incorporated
into chylomicron after absorption from the intestinal lumen,
resulting in greater fatty acid oxidation in the small
intestine (Murase et al. 2002) and liver (Mori et al. 2005;
Murase et al. 2001; Murata et al. 1997; Nagao et al. 2000)
which leads to lower postprandial plasma triglyceride
(PPTAG) level (Bauer et al. 2006 ; Umeda et al. 2006) and
lower body weight with DAG enriched diets. Lower
postprandial TAG has also been observed (Tada et al.
2001; Taguchi et al. 2000 ) people with type –II diabetes
mellitus. Consumption of DAG oil also reduces glycosy-
lated hemoglobin (HbA1
C
) in diabetic patient (Yamamoto
et al. 2001). Another important difference between DAG
and TAG metabolism is the substrate specificity of the
diacylglycerol acyltransferase (DGAT) enzyme, DGAT-1
and DGAT-2 in the small intestines (Cases et al. 1998)
DGAT has low substrate specificity toward 1, 3 DAG and
therefore does not significantly convert 1,3 DAG to TAG
(Lehner and Kuksis 1993). There are three possible causes
for the slightly lower recovery of fatty acids originated from
1,3 DAG in lymph. First, a part of the fatty acids from
DAG might be transferred to the portal vein preferentially
for β oxidation in liver (Watanabe et al. 1997). Second,
Table 4 Plasma Lipid Profile, Lipid peroxidation and Lipoprotein peroxidation of Rats in different dietary groups
Groups Total cholesterol
(mg/dL)
HDL-cholesterol
(mg/dL)
Non-HDL
cholesterol (mg/dL)
Triacylglycerol
(mg/dL)
Plasma lipid peroxidation
(n mole of MDA/ml of plasma)
LDL-peroxidation
(n mole of MDA/mg of
non-HDL cholesterol)
MO 85.54±3.06 22.86 ±1.22
a
62.68±2.07
b
97.74±1.33
t
5.46±0.40 14.76±1.66
DAG 83.18±1.94 28.71±1.31
a
54.17±2.74
b
86.27±2.49
t
3.34±0.30 12.98±2.54
MOCh 114.16±1.61** 22.48 ±0.47 60.24 ±1.60
bb
126.72±2.64
tt
4.11±0.34
aa
22.9±1.06
DAGCh 103.50±1.73** 20.84±0.76 81.64±1.66
bb
92.5 ±2.44
tt
3.34±0.34
aa
17.51±1.83
MO Mustard oil control group; DAG DAG rich mustard oil; MOCh mustard oil with 1% cholesterol; and DAGCh DAG rich mustard oil with 1%
cholesterol
All values are means±SEM of 8 rats/diet, **MOCh vs. DAGCh,
a
MO vs DAG,
b
MO vs DAG,
bb
MOCh Vs DAGCh, MO vs DAG,
tt
MOCh
vs DAGCh,
aa
MOCh vs. DAGCh
Significant F ratios for total cholesterol (**p=0.0492) HDL-cholesterol (
a
p=0.0091), non-HDL-cholesterol (
b
p=0.0082,
bb
p=0.0279) and
triacylglycerol (
t
p=0.02418,
tt
p=0.0360) total LDL peroxidation (
aa
p=0.023)
Table 5 Liver and mesentery lipid profile of rats of different dietary groups
Groups Total lipid (mg/g of tissue) Total Cholesterol (mg/g of tissue) TAG (mg/g of tissue) Phospholipid (mg/g of tissue)
Liver Mesentery Liver Mesentery Liver Mesentery Liver Mesentery
MO
b
51.52±0.64
$
231.75±1.25 3.81±0.86
M
37.33±2.42
a
32.52±2.00
@
174.44±4.57
p
15.16±1.47 17.95±1.13
DAG
b
45.2±0.80
$
196.62±2.26 2.88 ±0.27
M
31.92±1.09
a
24.25±1.26
@
151.0±1.30
p
22.01±1.55 14.24±0.41
MOCh 65.3 ±1.84 * 88.25±1.18 **4.17±0.55
MM
55.21±3.21 37.27±1.01
bb
206.62±1.25 25.23±1.52 26.81±0.92
DAGCh 63.36±2.30 *262.27±1.42 **2.36±1.01
MM
46.79±4.43 34.0±1.05
bb
188.37±2.04 24.10±2.53 24.05±0.52
MO Mustard oil control group; DAG DAG rich mustard oil; MOCh Mustard oil with 1% cholesterol; and DAGCh DAG rich mustard oil with 1%
cholesterol
All values are mean±SEM of 8 rats/diet
b
MO vs DAG, *MOCh vs. DAGCh, **MOCh vs. DAGCh
a
MO vs DAG,
p
MO vs DAG,
$
MO vs. DAG,
M
MO vs. DAG,
MM
MOCh vs.
DAGCh,
@
MO vs.DAG,
bb
MOCh vs.DAGCh,. Significant F ratios for total lipid content (
b
p=0.04917) total cholesterol (**p=0.01396,
MM
p=
0.00001) and triacylglycerol (
a
p=0.024) total liver phospholipid, (
p
p=0.02) for total mesentery lipid content (
$
p=0.037), triacylglycerol (
@
p=
0.0001,
bb
p=0.020),cholesterol (
M
p=0.001,
MM
p=0.002)
J Food Sci Technol

there is a possibility that a part of the fatty acids from DAG
was oxidized in intestinal cells. Murase et al. (Murase et al.
2002) reported that when DAG was fed to mice for 10 days
mRNA expression of all β oxidation enzymes like acyl
CoA oxidase, medium chain acyl coA dehydrogenase and
uncoupling protein −2 in intestine were increased.
Dietary high DAG significantly changed the plasma lipid
profiles. The decrease in plasma TAG with DA G rich oil as
observed in the present study may be due to distinct
metabolic pathways of the ingested TAG and DAG. 1, 3
DAG is digested in the metabolic tract to 1-MAG or 3-
MAG, those a re poorly re-esterifi ed into TAG in the
intestinal mucosa (Osaki et al. 2005; Tomonobu et al.
2006). For 1-MAG to follow this pathway, it must be
hydrolysed to glycerol, which releases a free fatty acid
(FFA) that could be available for TAG re-synthesis or
potentially for energy utilization. As a result, postprandial
elevations in TAG-rich lipoproteins were lowered signifi-
cantly after consumption of DAG rich oil (Tada et al. 2001;
Taguchi et al. 2000). Experimental studies in animals and
humans showed that diacylglycerol (mainly 1, 3 DAG)
decreases postprandial triglyceridemia in comparison with a
triacylglycerol control (Taguchi et al. 2000; Wang et al.
2010). It was reported that dietary DAG had anti-obesity
activity and prevented postprandial hypertriacylglycerole-
mia in experimental animals and humans (Ikeda and
Yanagita 2004; Murase et al. 2002, 2001). Ikeda and
Yanagita (Ikeda and Yanagita 2004) suggested that delayed
absorption of DAG compared to TAG may be the important
determinant in preventing body fat accumulation. A major
contributor to the clearance of TAG rich lipoproteins from
plasma is lipoprotein lipase (Ikeda and Yanagita 2004;
Masui et al. 2001; Kokie et al. 2001). Murata et al. showed
that in vitro, DAG emulsions were better substrates for
lipoprotein lipase mediated lipolysis. Therefore, efficient
hydrolysis of DAG by lipoprotein lipase may attribute to
the decreased plasma triacylglycerol level. DAG feeding
may be utilized preferentially for fatty acid oxidation rather
than for body fat accumulation (Murata et al. 1997). A
report also revealed that the ingestion of DAG significantly
decreased the respiratory quotient (RQ), which is calculated
from consumed oxygen and expired carbon dioxide, and
increased lipid oxidation compared with that of TAG in a
human clinical study (Kamphuis et al. 2003) and in animals
(Kimuru et al. 2003). A decrease in RQ from the baseline
indicates an increament in fat utilisation as an energy
source. In our study we observed that the DAG rich
mustard oil is capable of reducing arteriosclerotic factors
like tota l cholesterol and non-LDL cholesterol and increase
antiatheroscleotic factor such as HDL cholesterol. The
results are consistent with the previous report of Masui et
al. and Koike et al. (Kokie et al. 2001; Masui et al. 2001).
Our DAG rich mustard oil contains about 45% of DAG
which reduced the liver total cholesterol in hyperlipidemic
rats reducing the HMG-CoA reductase activity (Frayn
2002), with net uptake of fatty acids in the postprandial
state and net release between meals.
Leptin is a protein hormone a product of ob gene
containing 167 amino acids with important effects in
regulating body weight, metabolism and reproductive
function. It is secreted from white adipose tissues and
decreased hunger and food i ntak e by inhibi ting gene
expression of neuropeptide Y (NPY). Leptin is a product
of ob gene with important effects in regulating body
Table 6 Liver HMG CoA-Mevalonate ratio and Leptin content (ng/mL)
in different dietary groups
Groups HMG CoA-Mevalonate ratio Leptin content (ng/mL)
MO 2.29±0.33*
a
1.45±0.149
DAG 2.86±0.86*
a
0.87±0.03
MOCh 2.17±0.29**
b
2.5±0.085
DAGCh 3.75±0.86**
b
1.61±0.065
MO: mustard oil control group; DAG: DAG rich mustard oil; MOCh:
mustard oil with 1% cholesterol;
and DAGCh: DAG rich mustard oil with 1% cholesterol
All values are means±SEM, n=8., **MOCh vs DAGCh,
a
MO vs
DAG,
b
MOCh vs DAGCh Significant F ratios for liver HMG CoA-
Mevalonate ratio (**p=0.02456), total plasma leptin content (
a
p=
0.003,
b
p=0.007)
Table 7 Lipid profile of erythrocyte membrane (EM) ghost of different dietary groups
Groups TG (mg/mg of protein) Cholesterol (mg/mg of protein) Phospholipid (mg/mg of protein)
MO 0.24
a
±0.01 0.13±.07 0.60±0.03
DAG 0.15
a
±0.08 0.20±0.07 0.64±0.06
MOCh 0.22**±0.07 0.33
$
±0.02 0.56*±0.01
DAGCh 0.19**±0.09 0.24
$
±0.08 0.44*±0.06
MO Mustard oil control group; DAG DAG rich mustard oil; MOCh mustard oil with 1% cholesterol; and DAGCh DAG rich mustard oil with 1%
cholesterol
All values are means±SEM, n=8,
a
MO vs. DAG, **MOCh vs. DAGCh,
$
MOCh vs. DAGCh, *MOCh vs. DAGCh; Significant F ratios for EM
TAG (
a
p=0.025, **p=0.0002), cholesterol (
$
p=0.0038), phospholipids (*p=0.0025)
J Food Sci Technol

weight, metabolism and reproductive function. Leptin’s
effects on body weight are mediated through effects on
hypothalamic centres that control feeding behaviour and
hunger, body temperature and energy expenditure. The
body weight reduction in the DAG dietary groups possibly
due to the reduced leptin synthesis by the adipose tissues
compared to TAG rich dietary oil fed rats. We can propose
another mechanism in prevention of body fat accumulation
of DAG. Mustard oil contains 45–50% of erucic acid
(22:1). After a meal the increase in blood glucose is
followed by insulin secretion. When TAG is consumed with
meal both blood glucose and post prandial TAG level are
increased.
When glucose is exhausted insulin level decreased and
due to slow absorption of high molecular weight fatty acid
like erucic acid, preferentially used for fatty acid oxidation
for energy expenditure rather than to be stored as
triglyceride. Oxidation of fatty acids leads to satiety and
the food intake between meals is inhibited. Therefore, the
amount of chylomicron triglycerides entering the blood
stream after mustard oil DAG consumption is less than that
for TAG because lymphatic transport of triglyceride is
delayed after feeding of DAG.
Conclusions
Thus the present study demonstrates that the DAG rich
mustard oil prepared from normal mustard oil reduces body
weight as evidenced by food efficiency ratio, lowering of
plasma leptin content and reduced atherosclerotic factors
such as plasma TAG, Non-LDL cholesterol.
Acknowledgement The work was funded by University Grants
Commission, Govt. of India. Help and suggestions obtained from
Prof. D.K. Bhattacharyya and Dr. Santinath Ghosh are gratefully
acknowledged.
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