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Research Article Open Access
Antioxidant activity and phenolic contents of Ajwa date and
their effect on lipo-protein profile
Sumaira Khalid1,2, Asif Ahmad1, Muhammad Kaleem1
1Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi,
Shamsabad, Muree Road, Rawalpindi, Pakistan; 2Department of Food Science and Nutrition,
GCUF-layyah Campus, Rawalpindi, Pakistan
Corresponding author: Sumaira Khalid, PhD, Department of Food Technology, PMAS-Arid
Agriculture University, Rawalpindi, 46300, Pakistan
Submission date: February 28th, 2017, Acceptance Date: June 27th, 2017, Publication Date: June
30th, 2017
Citation: Khalid S., Ahmad A., Kaleem M., Antioxidant activity and phenolic contents of Ajawa
date and their effect on lipo-protein profile. Functional Foods in Health and Disease 2017; 7(6);
396-410
ABSTRACT
Background:!Dates (Phoenix dactylifera L.) are well known as both a food and economic crop
for many years worldwide due to its substantial nutritional, health, and economic benefits besides
its appeal and environmental value.!However, although Date pits are rich in phenolic and
antioxidant contents, they are generally neglected and treated as a waste product. Ethnobotanical
records indicate the potential of Ajwa dates pits and flesh having phenolics, antioxidants, and some
other nutrients.
Objective: The purpose of the study was to extract the phenolic contents and to determine the
antioxidant scavenging activity of Ajwa date flesh and pits in comparison to local date varieties in
three different solvents with three different concentrations, and to observe their effect on the high
lipo-protein profile of albino mice.
Materials and Methods: Three solvents viz. Methanol, Ethanol, and Acetone were used at
concentrations of 70%, 80%, and 90% to make nutraceutical extracts. These extracts were
characterized for numerous nutritional parameters. Correlation of flavonoid with phenolic, DPPH,
ABTS, and other parameters were studied. The above extracted Ajwa flesh and pits extracts were
then administrated to high cholesterol induced albino mice under three different treatments (10%,
20% and 30%) in four different groups where one group was studied as control
Results: Pits appeared to be the richest source for total phenols and total flavonoid contents. Such
extracts from pits were high in DPPH and ABTS activity compared to that of the flesh in all date
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cultivars, which were the highest in Ajwa pits (3932.3 mg GAE/100g, 2956.2 mg QEC/100g,
96.3% and 86.2% respectively). In the comparative analysis of solvents, 80% ethanol extracted the
highest antioxidant activity for both DPPH and ABTS assay in all date varieties and their parts.
Behaviour of solvent for polyphenol (Total phenols and total flavonoids) extraction was highly
variable so that a single solvent cannot be recommended for extraction. Highly significant
correlation of flavonoids with DPPH was observed in pits part. Phenolic contents positively
correlated with flavonoids, DPPH, and ABTS. DPPH and ABTS also revealed a strongly positive
correlation. In PCA analysis phenolic contents, flavonoid contents and antioxidant activity showed
a strong relation among pits, while demonstrating a weak relation in flesh parts. Furthermore,
results also depicted that 30% extracts of Ajwa pits have a higher effect on the percent reduction
of total protein, total triglycerides, total cholesterol, and LDL-C levels, in addition to a significant
percent increase in HDL-C levels in treated albino mice
Conclusion: The work identifies variability among the flesh and pits of Ajwa date and local date
varieties for their polyphenols contents, antioxidant activity, and their health beneficial effect
against a high Lipo-protein profile that can be used as an active ingredient against different
maladies in food products and supplements.
Keywords: PCA, Ajwa, lipo-protein, phenolics
INTRODUCTION
Dates (Phoenix dactylifera) are the oldest cultivated plant in the world and renown for its
substantial nutritional, health, and economic benefits, in addition to its aesthetic and biological
importance.!In addition to their nutritional properties, dates are believed to have medicinal
properties due to the presence of different bioactive compounds. The presence of natural
antioxidants and phenolics in dates make them suitable against different maladies like infectious
and bacterial diseases, diabetes, hyperlipidaemia, and cancer. However, these phenolic and
antioxidant components may differ in date varieties and their parts [1; 2]. The differences in the
quantity of these antioxidants depend on many factors including, solvents, variety, and parts of
date fruit. Among these factors, the solvent type is important to quantify the phenolic and
antioxidant contents, as different solvents with different concentrations may have an effect on the
quantity of these antioxidants [3]. Similarly, another important factor which can affect nutritional
and medicinal value is variety. Among all date varieties, the Ajwa date can be distinguished due
to the higher nutritional and medicinal properties it contains. In addition to the variety of dates,
different date parts within the same variety have different composition of nutrients and bioactive
components [4]. There are documented ethano-pharmacological records indicating the potential of
date flesh and pits against hyperlipidaemic problems and several other ailments. However, there
are less experimental results on the effects of Ajwa date parts against hyperlipidaemia. This
demands a detailed scientific study to explore total phenolics and the antioxidant activity of Ajwa
dates in comparison to other varieties. From a public health point of view, one of the major factors
contributing to the development of cardiovascular diseases is hyperlipidaemia [4].
Until recently, several studies have demonstrated that controlling hyperlipidaemia results in
significant reduction in death rate due to stroke and other heart diseases. With this scientific need
in mind, the purpose of the study was to extract phenolic contents of Ajwa date parts (flesh and
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pit) in different solvents and their concentrations, in addition to also determining their antioxidant
scavenging activity in comparison to local date varieties. Finally, the potential of the extract for
having the best profile for phenolic contents and antioxidant scavenging activity was evaluated to
study parameters of hyper lipo-protein profile in albino-mice..
MATERIALS AND METHODS
Preparation of Samples
The Ajwa date samples were purchased from renowned stores of Rawalpindi, Pakistan and were
botanically identified from Department of Botany, PMAS-Arid Agriculture University,
Rawalpindi while local date varieties (Aseel and Zaidy) were bought from Date palm research
centre, Jhang, Punjab, Pakistan. Fully developed fruits of uniform size, free of fungal infection and
any physical injury were selected, washed with distilled water, and then dried. Date pits were
grinded with a heav duty grinder and all samples were stored at ambient temperature until used for
further analysis.
Preparation of Extracts
Date flesh and pits samples were extracted separately using three solvents (methanol, ethanol,
and acetone) in three different concentrations (70%, 80%, and 90%) for each solvent as
explained in Figure 1.
Figure. 1. Schematic diagram for extraction in different solvents
Total Phenolic Contents (TPC)
The total phenolic content of both flesh and pits of all date samples were determined with the
Folin- Ciocalteiu reagent method of Benzie and Strain [5]. The concentration of total phenolic
contents in extracts was expressed as mg Gallic acid equivalent (GAE) per 100 g.
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Total Flavonoid Contents (TFC)
Total flavonoid contents in all date samples were determined by DPPH assay according to the
method of Kim et al [6]. The concentration of total flavonoid contents in extracts was expressed
as mg QEC equivalent (QEC) per 100 g.
DPPH Scavenging activity
DPPH solution was prepared for determination of antioxidant activity in date samples according
to the method of Brand-Williams et al. [7]. Stock solutions of DPPH were prepared by adding 0.2
g of DPPH in in 50 ml of respective solvents (methanol, ethanol, and acetone). Similarly, stock
samples in their respective solvent were prepared containing 1mg extract/ml of solvent. 1 ml from
these respective stock solutions were added to the respective test tubes labelled with their name. 2
ml from respective DPPH stock solution was added to each test tube. In the same way, control
solutions were also prepared containing 2 ml of DPPH and 1ml of Gallic acid solution. All test
tubes were incubated at room temperature in the dark for 30 minutes. The decrease in absorption
of extract mixtures and control was recorded at 517 nm and was measured using
spectrophotometer. Percent inhibition of DPPH radicals by samples and control was calculated
using the following formula:
!""#$%&' ( )* + ),
)* -$,..
where Ao = absorbance of control and A1= absorbance of sample extract
ABTS Scavenging Activity
The ABTS assay is based on the capacity of a sample to scavenge the ABTS radical cation (ABTS)
compared to a standard antioxidant (Quercetin), by adopting the method of Arnao et al. [8]. The
ABTS solution was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium
persulfate (final concentration) for 12-16 h, in the dark, at room temperature. Prior to use, the
ABTS working solution was prepared by diluting the stock solution with EtOH to an absorbance
of 0.70 ± 0.02 at 734 nm. The samples and Quercetin standards (20 µl) were combined with the
ABTS working solution (170 µl, absorbance 0.70 ±0.02). After 6 min of incubation at 30°C, the
absorbance at 734 nm was measured. ABTS scavenging activity for all samples was expressed as:
)/01$1234567867%&' ( 9:;9
9: -$,.. Where, Ao = Absorbance of control and A= Absorbance of
sample
The extract of date parts studied with the best profile of phytochemicals and their antioxidant
activities were further studied for their efficacy against induced hypo cholesterol in animal models.
EFFECT ON LIPO-PROTEIN PROFILE
Hyper cholesterol induction to animals
In the second experiment, the effect of the date extract with the best profile of bioactive
components on the lipoprotein profile were studied in comparison with local date varieties. Higher
cholesterol levels were induced by feeding mice feed mixed with cholesterol and casein powder.
Serum samples of some mice were tested to ensure hyper cholesterol induction in the animals.
Animals and experimental protocol
During the study period, the experimental animals (albino mice) of uniform age (4 weeks old)
were given a diet mixed with date extracts and water ad libitum. Male and female animals were
kept in separate animal houses under same conditions (12 hrs of light/dark, 24 ± 20C temperature,
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and 65% humidity) and provided with treatments. The study was carried out on four groups of
mice (10 mice per group, 5 males and 5 females). One group (male and female) was studied as the
control group, while the other three groups of mice were studied under date pit extracts of the best
profile. After the end of the experiment, period blood samples from the animals in all groups was
taken under light ether anaesthesia with a fasting period of 18 hours. Blood samples from all
animals were taken for further analysis. Data of all parameters was presented as percent change in
comparison to control group data.
Experiments were performed as per ethics and permission of Pakistan veterinary medical
council.
Serum separation
Serum samples were prepared by collecting mice blood after sacrificing in Eppendorf tubes
containing 0.8 ul EDTA and were mixed gently, which was followed by centrifuging blood at 5000
rpm for 20 minutes and were stored at low temperature (-20◦ C) for further analysis.!!
!
The following analysis were conducted:
Determination of total proteins
Total proteins for all serum samples were assessed as per method of Gadder, 1996 [9]. Briefly,
three test tubes labelled as sample, standard, and blank were taken. In these tubes, 20 µl sample,
20 µl distilled water and 1000 µl reagent 1 were added. All solutions were mixed well and
incubated at 20-25oC for 5 min., before the absorbance (A1) was read at 540 nm against the blank.
Afterwards, 250 µl of reagent 2 was added to both tubes and incubated again at 20-25oC for 5 min.
Finally, absorbance (A2) was measured and change in absorbance calculated according to the
formula:
∆A= [(A2-A1)] sample – [(A2-A1)] blank
Then, two test tubes labelled as sample and blank were taken. In these test tubes, 20 µl sample
and 20 µl distilled water were added respectively, followed by the mixing of 1000 ml of mono-
reagent in both tubes. After mixing well, test tubes were nurtured at 37oC for 5 min and then
absorbance was measured at 540 nm. Total proteins were then calculated according to the
following formula:
∆A= A (sample) – A (blank)
0*<3=$>?*<586$7
@= (A) 13B>=5
A)$ 1<36@3?@ -$2*62$*C$D<36@3?@
Determination of total triglycerides
Total triglycerides were determined according to the procedure of Natio, 2003 [10]. Briefly,
1000µl of reagent and 10µl of sample was taken and well mixed followed by incubation for 10
minutes at 20oC ± 3oC for 5 minutes. Afterwards, absorbance of sample (As) resp. of the standard
(Ast) was measured against the reagent blank. The absorbance remains stable for 1 hour. Total
triglycerides were calculated as:
Triglycerides concentration= )DE$FGHIJ$$GK$LMNHONPO
9$LM
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Determination of total cholesterol
Total cholesterol was determined by the method discussed by Natio, 2003 [10]. Three test tubes
were taken labelled as sample, standard, and blank. In each test tube, 10 µl of the sample, standard,
and distilled water was added, followed by the addition of 1000 µl of reaction solution to each test
tube, and then incubation for 10 minutes at 20 oC- 25 oC. Absorbance of sample (As) and standard
(Ast) was measured against the reagent blank at 500 nm. The absorbance remains stable for 45
minutes (Natio, 2003). Concentration of total cholesterol was calculated as:
Concentration of cholesterol= Q9$LNRSTU
Q9$VMO EW*62$1<@J XB7Y@=Z
Determination of HDL-C
High density lipoprotein-cholesterol for all serum samples were calculated by the following
method of Natio, 2003 [10]. Three test tubes labelled as blank, standard, and sample were taken.
In the blank test tube, 2.4 µl of distilled water, 2.4 µl of standard, and 2.4 µl of sample were added.
Afterwards, in all three test tubes, 240 µl of reagent R1 was added before being well mixed and
incubated at 37oC for 4 min. 40 sec. Absorbance of sample (A1) and standard (As1) were
measured, then added to 80 µl of reagent R2 in blank and sample test tubes. Next, we mixed them
well and incubated the test tubes again for 4 minutes and measured the absorbance of sample (A2)
and standard (As2). HDL was calculated as:
Concentration of HDL= 9[;9\$%LNRSTU'
9L\;9L[%LMNHONPO'EW*62$*C$D<36@3?@
Determination of LDL- C
Low density lipoprotein-cholesterol for all serum samples were calculated by following the
method of Sinha [11]. Same procedure was followed, taking three test tubes labelled as sample,
blank and standard. 30µl of sample, blank, and standard was followed by the addition of 300µl of
reaction solution added to each test tube. Test tubes were mixed well and then incubated at thirty-
seven degree centigrade for five minutes. The absorbance was measured as the following: first
absorbance of sample (A1) and standard (As1) were measured against blank at 600 nm; then 90 µl
of reagent B was added in all three test tubes and absorbance of standard (As2) and sample (As1)
was measured again; concentration of LDL was calculated by the formula:
Concentration of LDL= 9[;9\$%LNRSTU'
9L\;9L[%LMNHONPO'EW*62$*C$D<36@3?@
STATISTICAL ANALYSIS
Principal component analysis, correlation and Scatterplot analysis were used to observe
relationships within various parameters. For all statistical analysis, R studio and Minitab version
16 soft wares were used.
RESULTS AND DISCUSSION
Total phenolic contents
Phenolic contents of flesh and pit of different date varieties fluctuate widely in each type of solvent
concentration. Interaction between solvent, concentration, varieties, and part of date (flesh or pits)
have a significant effect on TPC (p>0.05). Results showed that among all of the date varieties,
Ajwa date had higher amounts of TPC, while pits of all date varieties have significantly higher
total phenolic contents compared to the flesh parts. Similarly, among all solvents and their
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concentrations, 90% ethanol was found to be the most effective to extract higher amounts of TPC
in all date varieties and their parts (Figure 2). Results further showed that among all date varieties
and their parts, Ajwa pits had a range of phenolic contents between 3932.3 mg GAE/100g (90%
ethanol) to 3154.7 mg GAE/100g (70% ethanol), while Zaidy pits had a lower range of total
phenols from 2432.2 mg GAE/100g (90% ethanol) to 2333.5 mg GAE/100g (70% ethanol).
Furthermore, variable trends for TPC extraction in different solvents and their concentrations is an
evident depiction that in some parts TPC can be extracted using 90% ethanol, while in other parts
TPC can be extracted through 80% ethanol, 70% ethanol, and 90% acetone. Results suggest that
solvent polarity plays a basic role in enhancing phenolic solubility [3] and that water-organic
solvent mixtures are more effective towards the extraction of total phenolic contents. Therefore, it
is difficult to develop a standard extraction procedure that will be appropriate for the extraction of
phenolic contents of all plants and their parts [3]. Furthermore, results suggest that pits have
remarkable potential for polyphenols compared to that of the flesh, so date pits can be a better
source of phenolic contents than flesh parts of date cultivars irrespective of variety and solvent
used. Differences in flesh and pits quantities may be due to their physiology and chemical
constituents. However, due to their higher potential of phenols, these can be used as potential
source of phenolics and antioxidants in food products [1; 12]. The results further suggest that more
work on the isolation of these phenols derived from date parts is required so that synthetic phenols
can be replaced in foods.
Figure 2. Total phenolic contents in date varieties and parts in different solvents and their different
concentration
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Total flavonoid contents (TFC)
Results showed that among all date varieties, Ajwa date had a higher amounts of TFC, whereas
pits of all date varieties have significantly higher total flavonoid contents compared to that found
within the flesh parts of the dates. Similarly, among all solvents and their concentrations, 90%
ethanol was found to be the most effective in extracting higher amounts of TFC in all date varieties
and their parts (Figure 3). However, the flesh and pits of date cultivars also revealed extensive
differences for flavonoids contents in all varieties (Figure 3). In contrast, Ajwa pits highest
quantity of TFC ranged from 1897.4 mg QEC/100g (70% acetone) to 2956.2 mg QEC/100g (80%
acetone), while zaidy pits have the least amount of flavonoids in a range between 1013.6 mg
QEC/100 g (90% methanol) - 1962.3 mg QEC/100g (80% actone). The significant effect of solvent
and its different concentrations is also evident on flavonoids. These differences may be due to
variable polarity of the solvents which plays a basic role in enhancing flavonoids solubility [3].
Results further suggest that 80% acetone was the best solvent to extract flavonoids from both parts
of all date cultivars, recommending it as the best solvent to extract flavonoid contents which may
be due to its higher capacity to dissolve polar flavonoids. Overall, pits of date varieties are better
sources of flavonoids than flesh. The variation in flavonoid contents of flesh and pits may be due
to their genetic makeup, solvent used, and solvent concentration, which suggests there should be
more studies on date pits to explore their flavonoids and to also make it possible to use them in
food industry to minimize their wastage. Further studies are also recommended for extraction of
non-flavonoids contents from date parts and cultivars.
Figure. 3. Total flavonoids contents (mg GAE/100g) in date varieties and parts in different
solvents and their different concentrations
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Total antioxidant activity
Different methods are employed for evaluation of antioxidant activity in fruits, of which ABTS
and DPPH are most common. In this study, the antioxidant activity of date flesh and pits were
evaluated by two methods viz. DPPH and ABTS. Moreover, antioxidant activity is affected by
solvent type, concentration, varieties, and their different parts. For DPPH assay, interaction
between solvent, concentrations, variety, and parts have significant effects on DPPH scavenging
activity of date parts (p>0.05) (Figure 4). Generally, among all date varieties, Ajwa dates have
higher amounts of DPPH scavenging activity, whereas pits of all date varieties have significantly
higher amounts of DPPH scavenging activity compared to the flesh parts of dates. Similarly,
among all solvents and their concentrations, 90% ethanol was found most effective in extracting
higher amounts of DPPH scavenging activity in all date varieties and their parts (Figure 4). Among
pits, Ajwa pits exhibited the highest DPPH scavenging activity range between 96.3% (80%
ethanol) to 67.3% (70% acetone), while Zaidy pits had the lowest DPPH scavenging activity
varying from 67.1% (70% ethanol) to 47.6% (90% methanol) in different solvents (Figure 4).
Similarly, in ABTS assay solvent, interaction between variety, concentrations of solvent, solvent
and parts had significant effects on ABTS scavenging activity(p> 0.05). Normally, date pits
significantly showed more ABTS scavenging activity in comparison to date flesh of all varieties
(Figure 5). Within parts, Ajwa pits have highest amount of ABTS scavenging activity in range of
86.2% to 69.3% followed by Zaidy pits which have ABTS scavenging activity varying from 65.9%
to 54.2% (Figure 5). Results suggest that the antioxidant activity of different extracts has strong
association with different concentrations of solvent used, mainly due to different scavenging
capacity of compounds with different polarities [14]. Therefore, it can be suggested that
antioxidants are more dissolved in polar solvents and that among them ethanol and its water
concentrations (80% and 70%) are the most effective solvent for extraction of date antioxidants,
due to a wide range of compounds that it can dissolve. Differences among pits and date flesh
recommend pits to be a greater source for antioxidants, which may be due to their higher phenolic
and flavonoid contents. However, further studies are recommended for the isolation of these
antioxidants.
Figure 4. DPPH scavenging activity (%) in date varieties and parts in different solvents and their
different concentrations
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Figure 5. ABTS scavenging activity (%) in date varieties and parts in different solvents and their
different concentrations
Relationship analysis
Irrespective of date parts and solvents, the histogram shows that most of date cultivars have
phenolic contents ranged between 200-1000 mg GAE/10 0g, flavonoids between 300-1200 mg
QEC/100 g and DPPH and ABTS contents between 55-65% (Figure 6). Phenolic contents have a
significantly higher positive correlation with flavonoids (0.96), DPPH (0.63), and ABTS (0.75).
Similarly, a strongly positive correlation was observed between flavonoids, DPPH (0.61), and
ABTS (0.74), while DPPH and ABTS (0.89) were also revealed to have a strong positive
increasing relationship, as evident in Figure 4. Results suggest that phenolic and flavonoid contents
have a strong effect on antioxidant activity of date fruits. So varieties could be nominated by
relying on one of the above stated attributes for these parameters while studying their antioxidant
properties.
Figure 6. Relationship between phenolic, flavonoids and antioxidant activity
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Principle component analysis
Significantly, new plant sources for natural antioxidant are being used to replace synthetic
antioxidants. Many literature reports designate studies of antioxidants from date palm and their
parts. To understand more about variables and variation between date parts, PCA was applied
based on their antioxidant, phenolic, and flavonoids contents. The scatter plot was used to classify
the number of principle components (Figure 7). Eigen value for first two components dropped
sharply while remaining stagnant for others with a cumulative variation of 96.8%, so we will take
the first two components for study. Bi-plot shows variability among three varieties on the basis of
their variables in two dimensions: V1 is highly apart from V2 and V3, while V2 and V3 are
overlapping (Figure 7). The leading PC1 explains variability of 82.3% and is highly contributed
by variables of V1 (Ajwa). while PC2 explains 14.6% variability for the variables of V2 and V3.
In particular, PC1 is highly correlated with phenolics, flavonoids, and DPPH and ABTS activity
of V1, while PC2 is positively correlated with phenolic, flavonoids, and DPPH and ABTS contents
of V2 (aseel) and V3 (zaidy). This technique serves as a a valuable tool for understanding much
more about the explanation of results from the research study and effects of date varieties on them.
Assessment of phenolics, flavonoids, and antioxidants in each variety can assist in the
classification of date cultivars.
Figure 7. PCA plot for data set of date varieties
Effect on lipoprotein
In this assay, the effect of Ajwa date pit extracts dissolved in 90% ethanol were evaluated against
higher lipoprotein profile in both male and female albino mice. The results revealed that among
the three different treatments of Ajwa pits extracts, 30% dose of extracts was most effective in
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reducing percent level of total cholesterol in both male (18.4%) and female (19.2%) mice
significantly, while 10% dose was least effective in both genders (16.8%; 14.1% respectively)
(Figure 8 A). The same trend was also observed for percent decrease in total protein contents,
where also 30% dose of the Ajwa pits extracts was most effective in both male (28.5%)and female
(25.7%) albino mice, while 10% dose was least effective in both genders (23.5%; 23.9%
respectively) (Figure 8B). Furthermore, results also revealed that 30% dose of Ajwa pits was most
effective for the percent decrease in total triglycerides levels in both male (35.1%)_and female
mice (44.5%), while 20% dose of Ajwa pits were least effective for the percent decrease in TTG
levels in male mice and 10% dose being the least effective in female mice (Figure 8C). The same
trend was also observed in percent decrease in LDL levels where 30% dose of Ajwa pit extracts
was most effective respectively in male and female mice (32.5%). Likewise, results also revealed
that in male mice 20% dose of ajwa pits was least effective on percent decrease in serum LDL in
both male (13.5%) and female (10.9%) groups (Figure 8D). Epidemiological studies suggest that
there is positive correlation between protein consumption and CHD (r=0.78) because these protein
levels are the primary source of cholesterol and saturated fats which increases risk of CHD (Figure
8). Our results suggest that Ajwa pit extracts can significantly reduce serum total protein
percentages in cholesterol fed male and female mice, which may be due to their high enrichment
in phenols and flavonoids. Several studies have revealed the protective effect of polyphenols on
coronary heart diseases [14] by reducing the oxidative changes in lipoprotein fractions. The
significantly higher effects of 30% dose may be due to the higher number of polyphenols than
other treatments. Moreover, the differences between genders can be due to their endocrinological
and physiological factors. A strong relationship exists between triglycerides levels and cardio-
vascular diseases. Triglycerides are the most problematic lipid, usually measured in the evaluation
of CVD risk, which is due to its strongly inverse relationship with HDL. It lowers levels of HDL
and LDL-C, which increases risk of CVD [15;16]. The results of this study demonstrated a
promising effect of 30% extracts of Ajwa pits in significantly reducing serum triglycerides level
in mice. While among genders female mice have higher percentages of reducing triglycerides,
which may be due to their ability to survive in environment provided and may also be due to the
presence of elevated levels of estrogenic hormone, which is linked with reduction of lipids and
cholesterol in blood. Similarly, results also showed that in both male (10.2%) and female (8.9%)
groups, 30% was more effective for percent increase in serum HDL levels while 20% dose was
least effective in both male (5.0%) and female (4.9%) groups (Figure 8D). Oxidative damages are
main reasons for atherosclerosis, which is mainly due to modification of LDL for scavenging
receptor uptake causing accumulation of cholesterol in cells. The antioxidants in Ajwa pits
inhibited peroxidation of LDL and reduced risk of atherosclerosis in hyperlipidaemic in both male
and female mice [17]. Furthermore, it is suggested that LDL is generally oxidized by metal ions
or muscle cells, endothelial cells, and macrophages which usually produce free radicals that causes
oxidation of LDL; thus, a defensive system against these free radicals is required that can prevent
LDL from oxidation [18; 19]. These results suggest that administrating 30% Ajwa date pits can
prevent LDL oxidation which can also protect against atherosclerotic progression. Recent studies
have suggested that HDL can promote the reverse cholesterol transport pathway. This pathway
includes that HDL can induce efflux of excessive accumulated cholesterol protecting the
production of oxidative modified LDL [19; 20]. The results stated above suggest that Ajwa pit
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extracts have positive effects on inhibition of LDL modification and also promote the excessive
efflux of accumulated cholesterol from cells by elevating serum HDL levels [21; 22]. The results
further suggest that 30% dose of Ajwa pit extracts have hypocholesterolaemia effect on both
genders on mice but on male mice the effect was significantly higher, which may be due to
endocrinology and physiological effect.
Figure 8: Effect of Ajwa pits treatments on Lipo-protein profile
CONCLUSIONS
Comparative analysis between date varieties and parts (flesh and pit) showed higher quantities of
total phenolic, total flavonoids, and antioxidant contents in date pits than flesh in 90% ethanol,
while Ajwa pits were found to be the richest source of all of the parameters described above in
both parts of all date varieties. In correlation analysis, phenolic contents have a significant positive
correlation with flavonoids and DPPH and ABTS. Similarly, a strongly positive correlation was
also observed between flavonoids and DPPH and ABTS, while DPPH and ABTS also revealed a
strongly positive increasing relationship. Based on PCA analysis, the data group was clearly
divided into two subsets. Pits were separated from flesh parts based on their phenolic contents,
flavonoid contents, and antioxidant activity, which will be helpful in the classification of date parts
and cultivars. Furthermore, Ajwa pits were evaluated for their biological effect against lipo-protein
profile, concluding that 30% extract of Ajwa pit was effective against percent decrease in total
Gender
Treatment(%)
MF
302010302010
20
10
0
% decrease in TC
G ender
Treatm ent(% )
MF
302010302010
30
15
0
% decrea se in TP
Gender
Treatment(%)
MF
302010302010
40
20
0
% decrea se in TTG
Gender
Treatment(%)
MF
302010302010
20
10
0
% decrea se in LDL
Gender
Treatment(%)
MF
302010302010
16
8
0
% i ncrease in HDL
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aa
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e
a
b
c
cd
d d
AB
E
CD
Functional Foods in Health and Disease 2017; 7(6); 396-410 Page 409 of 410
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protein, total cholesterol, total triglycerides, and LDL levels, and were also effective for percent
increase in HDL levels. Thus, higher phenolic and antioxidant components in Ajwa date pits
emphasize their use in food and pharmaceutical industry as functional and disease protective
ingredient in food products and supplements
List of abbreviations: Total phenolic contents(TPC), total flavonoid contents(TFC), 2,2-diphenyl-
1-picrylhydrazyl (DPPH), 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS), High
density lipo-protein(HDL), Low density lipoproteins (LDL), Total proteins (TP), Total
triglycerides(TTG), Total cholesterol(TC).
Competing interest: Authors have no conflict of interest
Author’s contribution: All authors have equally contributed in this work
Acknowledgements: We highly acknowledge the Higher education commission for providing
financial assistance in this project
REFERENCES
1. Ebtesam, S., Tawakif, M.S., Abu-Taroush, H. M. (2011). Phenolic contents and
antioxidant activity of various date palm (phoenix dactylifera l.) fruits from Saudi Arabia.
2(10),1134-1141.
2. Zhang, C. A., Aldosari, S. A., Vidyasagar, P. V., Nair, K. M. and Nair, M. G. (2015).
Antioxidant and anti-inflammatory assays confirm bioactive compounds in Ajwa date
fruit. Journal of Agriculture and Food Chemistry, 61, 5834-5840.
3. Naczk, M., and Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables:
Occurrence, extraction and analysis. Journal of Pharmacology and Biomedical Analysis,
41, 1523–1542.
4. Santas, J. Carbó, R. M., Gordon, H. and Almajano, M. P. (2008). Comparison of the
antioxidant activity of two Spanish onion varieties. Food Chemistry, 107, 1210–1216.
5. Benzie, I. F. F. & Strain, J. J. (1999). Ferric reducing/antioxidant power assay: Direct
measure of total antioxidant activity of biological fluids and modified version for
simultaneous measurement of total antioxidant power and ascorbic acid concentration.
Methods in Enzymology, 299:15–27.
6. Kim, J. K., Lee, S., Choi Suh, J. S., Chung, H. Y., Song, Y. O., Choi, W. C. (2003). The
first total synthesis of 2, 3, 6-tribromo-4, 5-dihydroxybenzyl methyl ether (TDB) and its
antioxidant activity. Bull Korean Chemistry Society, 23(5), 661-662.
7. Brand-Williams, W. M., Cuvelier , E. and C. Berset. (1995). Use of free radical method
to evaluate antioxidant activity. Lebensm Wiss Technology, 28, 25-30.
8. Arnao, M. B., Cano, A. and Acosta, M. (2001). The hydrophilic and lipophilic
contribution to total antioxidant activity. Journal of Food Chemistry, 73, 239–244.
9. Gudder, D. A., K. M. Fritz., L. Gray. 1996. The relationship of floods, drying, flow and
light to primary production and producer biomass in a prairie stream. Hydrobiol., 333:
151-159
Functional Foods in Health and Disease 2017; 7(6); 396-410 Page 410 of 410
!
!
10. Natio, M., S. Sakurna and A. Miyazaki. 2003. Intravenous injections of rabbit apo-
lipoprotein habits progression of artheosclorosis in cholesterol fed rabbits. Artherioscler.
Thromb. Vasc. Biol., 15: 1882-1885.!
11. Tietz, N. W. 1995. Clinical Guide to Laboratory Tests. 3rd edition. W.B. Saunders
Company, Philadelphia, USA.
12. Sinha, A. K. 1972. Colorimetric assay of catalase. Analytical Biochemistry, 47, 389-394.
13. Sharma, G. N., Dubey, S. K., Sati, N. and Sanadya, J. (2011). Anti-inflammatory activity
and total flavonoid content of Aegle marmelos seeds. International Journal of
Pharmacological Sciences and Drug Research, 3, 214-218. Sharma, G. N., Dubey, S. K.,
Sati, N. and Sanadya, J. (2011). Anti-inflammatory activity and total flavonoid content
of Aegle marmelos seeds. International Journal of Pharmacological Sciences and Drug
Research, 3, 214-218.
14. Khanavi, M., Saghari, Z. Mohammadirad, A., Khademi, R., Hadjiakhoondi, A. and
Abdollahi, M. (2008). Comparison of antioxidant activity and total phenols of some date
varieties. Daru. 17, 104-107.
15. Arnao, M. B., Cano, A. and Acosta, M. (2001). The hydrophilic and lipophilic
contribution to total antioxidant activity. Journal of Food Chemistry, 73, 239–244.
16. Mansouri, A., Embarek, G., Kokkalou, E. and Kefalas, P. (2005). Phenolic profile and
antioxidant activity of the Algerian ripe date palm fruit (Phoenix dactylifera L.) Food
Chemistry, 89: 411-420.
17. Comporti, M. (1985). Lipid peroxidation and cellular damage in toxic liver injury. Lab.
Investigation. Journal of Technical Methods in Pathology, 53(6): 599-623.
18. Al-Farsi, M., Alasalvar, C. A., Morris, M. B. and Shahidi, F. (2005a). Comparison of
antioxidant activity, anthocyanins, carotenoids, and phenolics of three native fresh and
sun-dried date (Phoenix dactylifera L.) varieties grown in Oman. Journal of Agriculture
and Food Chemistry, 53(19), 7592-7599.
19. Al-Farsi, M., Alasalvar, C. A., Morris, M. B. and Shahidi, F. (2005b). Compositional
and sensory characteristics of three native sun-dried date (Phoenix dactylifera L.)
varieties grown in Oman Journal of Agriculture and Food Chemistry, 53(19), 7586-
7591.
20. Al-Laith, A. A. (2009). Degradation kinetics of the antioxidant activity in date palm
(Phoenix dactylifera L.) fruit as affected by maturity stages. Arab Gulf Journal of
Scientific Research, 268, 16-25.
21. Hong, Y., Tomas-Barberan, J., Tomas-Barberan, F. A. and Mitchell, A. A. (2006). The
flavonoid glycosides and procyanidin composition of Deglet Noor dates (Phoenix
dactylifera). Journal of Agriculture Food Chemistry, 54(6), 2405-2411.
22. Saafi, E. B., Louedi, M. A., Elfeki, A., Zakhama, M. F., Najjar, M., and Achour, L.
(2011). Protective effect of date palm fruit extract (Phoenix dactylifera L.) on dimethoate
induced-oxidative stress in rat liver. Experimental Toxicology and Pathology, 63(5), 433-
441
23. Kawada, N., Kuroki, T. Kobayashi, Inoue, K. Nakatani, M. K. Kaneda, K. and Nagata,
K. (1996). Expression of heat-shock protein in mouse liver. Cell Tissue Research, 284,
341-346.
24. Vayalil, P. K. (2011). Date fruits (Phoenix Dactylifera Linn): An emerging medicinal
food. Critical Review in Food Science and Nutrition, 52, 24
