Utilization from leaves of olive and pomegranate as a source of bioactive components

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Journal of Advances in Biological and Basic Research 01[01] 2015
www.asdpub.com/index.php/jabbr e-ISSN-2454-6097
© ASD Publisher All rights reserved. 28
Original Article
Utilization from leaves of olive and pomegranate as a source of bioactive
components
Amany M. Basuny1, Hayam A. Elsawy 2 and Shaker M. Arafat*3
1,3Oils & Fats Research Department, Food Technology Research Institute, Agriculture Research Center, Giza. Egypt.
2 Special Food & Nutrition Department, Food Technology Research Institute, Agriculture Research Center, Giza.
Egypt.
*Corresponding Author
Prof. Dr. Shaker Arafat
Oils & Fats Research Department,
Food Technology Research Institute,
Agriculture Research Center, Giza. Egypt.
E-mail: dr_shakerarafat@yahoo.com
Keywords:
Olive,
Pomegranate,
Phenolic compounds,
Antioxidants,
Phytochemistry,
1. Introduction
Reactive oxygen species (Ros) readily attack and induce
oxidative damage to various biomolecules including proteins, lipids,
lipoproteins, and DNA. The oxidative damage is a crucial etiological
factor implicated in several chronic human diseases, namely
cardiovascular diseases, rheumatism, diabetes mellitus and
cancer[1]. Based on growing interest in free radical biology and the
lack of effective therapies for most chronic diseases, the usefulness of
antioxidants in protection against these diseases is warranted.
Antioxidants are chemical substances that reduce or prevent
oxidation. They have the ability to counteract the damaging effects of
free radicals in tissues and thus are believed to protect against
cancer, arteriosclerosis, heart disease and several other diseases [2].
Many studies have shown that phenolic compounds display
antioxidant activity as a result of their capacity to scavenge free
radicals[3]. Phenolic compounds can also act as antioxidants by
chelating metal ions, preventing radical formation and improving the
antioxidant endogenous system[4]. These compounds are known to
act as antioxidant not only because of their ability to donate
hydrogen or electrons but also because they are stable radical
intermediates.
Probably the most important natural phenolic are
flavonoids because of their broad spectrum of chemical and
biological activities, including antioxidant and free radical scavenging
properties [5]. In fact, flavonoids have been reported as antioxidants,
scavengers of a wide range of reactive oxygen species and inhibitors
of lipid peroxidation [6]. These compounds which are widely
distributed across the plant kingdom represent the most abundant
antioxidants in the diet and they have gained tremendous interest as
potential therapeutic agents against a wide variety of diseases, most
of which involve oxidant damage. The unusually wide
pharmacological spectrum of flavonoids was originally thought to
result from their antioxidant activity; however, recent studies
suggest that various flavonoids may use other protective mechanisms
are well.
Flavonoids have also been shown to be highly effective
scavenging of most types of oxidizing molecules, including singlet
oxygen and other various free radicals that are probably involved in
several diseases. On the other hand, numerous studies have shown
structure-activity relationships governing antioxidant capacities of
flavonoids [7,8]. Food chemists have proposed the replacement of
synthetic antioxidants such as butylhydroxyanisole (BHA) and
butylhydroxytoluene (BHT), with natural ones because the synthetic
antioxidants are suspected to be carcinogenic [9]. In summary,
natural antioxidants are useful in the food industry as preservatives
to increase the life of food products preventing the loss of their
sensory and nutritional value. Epidemiology studies have shown that
the traditional Mediterranean diet is associated with low incidence of
cardiovascular disease and certain cancers [10]. These beneficial
effects on human health have been attributed to the presence in the
Mediterranean diet of antioxidants such as phenolic compounds,
carotenoids, and tocopherols that play an important role in disease
prevention [11]. The olive leaf (Oleaceae) has been widely used in
folk medicine for several thousand years in European Mediterranean
islands and countries. Historically, olive leaf has been used as a
remedy for fever and other diseases such as malaria [12,13]. Olive
foods such as olive oil and olive leaf in the Mediterranean diet are the
primary source of phenolic compounds, which are also important
markers for evaluating the quality of olive-based food products. The
major active components in olive leaf are known to be oleuropein
and its derivatives such as hydroxytyrosol and tyrosol, as well as
caffeic acid, p-coumaric acid, vanillic acid, vanillin, luteolin,
diosmetin, rutin, luteolin-7-glucoside, apigenin-7-glucoside, and
diometin-7-glucoside [14-16]. Also, pomegranate is one of the
important and oldest fruits of tropical and subtropical regions, which
originated in the Middle East. It is widely reported that pomegranate
exhibits antivirus, antioxidant, anticancer, antiproliferative activities
[17,18]. For centuries, the barks, leave, flowers fruits and seeds of
this plant have been used to ameliorate some diseases [19]. The
leaves have been widely used by traditional medicine in America,
Abstract
Crude juices of olive and pomegranate leaves were obtained by hydraulic press.
The level of polyphenolic compounds in the (olive and pomegranate) juice were510.00
and 722.00ppm. Aliquots of the concentrated olive and pomegranate juice leaves,
represent 200, 400, 800 and 1600ppm and butylated hydroxy toluene (BHT, 200ppm)
were investigated by Rancimat method at 100ºC and 1, 1-diphenyl-2-picrylhydrazyl
(DPPH) free radical scavenging method. These compounds were administrated to rats
daily for 6 weeks by stomach tube. The liver (Aspartate aminotransferase, alanine
aminotransferase and alkaline phosphatase activities) and kidney (bilirubin, uric acid and
creatinine) function tests and serum contents (total lipids, total cholesterol and low and
high-density lipoproteins) were measured to assess the safety limits of the phenolic
compounds in the olive and pomegranate juice leaves. The data of the aforementioned
measurements indicates that the administration of olive and pomegranate juice leaves did
not cause any changes in liver and kidney functions. On the contrary, BHT at 200ppm
induced significant increases in the enzyme activities and the serum levels of total lipids,
uric acid and creatinine.

Amany M. Basuny et al / Utilization from leaves of olive and pomegranate as a source of bioactive components
© ASD Publisher All rights reserved. 29
Asia, Africa and Europe for the treatment of different types of
diseases [20]. The phytochemistry of pomegranate has been widely
studied by some researchers and this fruit is found to be a rich source
of polyphenolic compounds[19].
The present study was entailed on the direct use of juice
obtained by pressing olive and pomegranate leaves without recourse
to extraction and fractionation of the total polyphenols. Total
phenolic content, electron donating ability by DPPH and antioxidant
activity by Rancimat method. Also, identification of phenolic
compounds in juice (olive by HPLC and the study effect of crude leaf
juice (olive and pomegranate) on rat serum constituents.
2. Materials & Methods
2.1 Source of leaves
The ripe olive and pomegranate leaves were collected
during the year 2014 from the Horticulture Research Institute,
Agriculture Research Centre, Giza, Egypt.
2.2 Source of sunflower oil
Refined sunflower oil was obtained from Savola Oil .The oil
acid and peroxide values were 0.30 mg KoH g-1 and 0.60 meq./ kg-1,
respectively.
2.3 Solvents, reagents and kits
All solvents used throughout the whole work were
analytical grade and distilled before use. Caffeic acid (98%) and folin-
Ciocalteau reagent were purchased from Sigma-Aldrich (St. Louis,
MO, USA) and Gerbsaure Chemical Co. Ltd., Germany, respectively.
Alkaline phosphatase (AP), aspartate aminotransferase (AST),
alanine aminotransferase (ALT), bilirubin, creatinine, and uric acid
kits obtained from Borhringer Ingelheim GmbH, Ingelheim, Germany.
2.4 Experimental animals
Albino male rats, 50 days old with an average weight of 60-
70g were obtained from the Faculty of Veterinary Medicine, Cairo
University, Giza, Egypt.
2.5 Preparation of crude leaves juice
Leaves were cleaned and remove seeds them pressed by
hydraulic laboratory press. The resultant crude juice was
concentrated using freeze dryer (Labconco Corporation, Kansas, City,
Mo, USA) and kept in a brown bottle at 5ºC until use.
2.6 Determination of total polyphenolic
The levels of total polyphenols of fresh crude juice were
determined according to the method of Gutfinger (1981) [21]. Caffeic
acid was served as a standard compound for the preparation of the
calibration curve.
2.7 Phenolic fraction
Phenolic facrtion was isolated by solid phase extraction
and analyzed by reversed-phase HPLC using a diode array UV
detector. A Hewlett-Packard series 1,100 liquid chromatographic
system (Waldbronn, Germany) equipped with diode array detector
and a lichrosorb Rp18 column (4.00 mmid C250 mm, particle size
5mm, Merck, Darmstdt) used. Elution was performed at a flow rate of
1.00 ml/min with mobile phase of water/acetic acid (98:2 v/v,
solvent A) and methanol/ acetonitril (50:50, v/v, solvent B), starting
with 5% B then increase to levels of 30 % at 25 min, 40% at 35 min.,
52 % at 40 min.; 70% at 50 min., 100 % at 55 min., and kept at this
stage for 5 min. Quantification of phenolic compounds was carried
out at wave length of 280nm using P-hydroxybenzoic acid as an
internal standard.
2.8 Oxidation system
Different concentrations of phenolic compounds from juice
(200, 400, 800 and 1600ppm) and BHT (200 ppm) were individually
add to sunflower oil to study their antioxidant behavior. The
designation of an induction period, measured by using a Rancimat
Instrument, was taken as a tool to compare the effectiveness of the
phenolic compounds on sunflower oil stability.
2.9 Designation of induction period by Rancimat
Rancimat method was used to evaluate oxidative stability,
because it is fast and reliable [22]. Stability was expressed as the
oxidation induction time (hr) measured with the Rancimat 679
apparatus (Metrohm Co. Switzerland) using an oil sample of 5.0 g
warmed to 100 ± 2ºC, and an air flow of 20 l/hr. the time taken to
reach a fixed level of conductivity was measured.
2.10 DPPH free radical-scavenging activity
The DPPH free radical scavenging assay was carried out, as
previously reported by Cheel et al[23]., (2007) with some
modification. The crude juice from olive and pomegranate at various
concentrations (200, 400, 800 and 1600ppm) were added to a
0.06nm DPPH solution in ethanol and reaction mixture was shaken
vigorously. After incubation for 30 min at room temperature, the
absorbance at 517nm was recorded spectorphotometrically. Vitamin
E was used as a reference as the test compound. A control solution,
without the tested compound, was prepared in the same manner as
the assay mixture. All the analysis was done in triplicate. The degree
of discolorisation indicates the free radical scavenging efficiency of
the substances. The antioxidant activity was calculated as an
inhibitory effect (IE %) of the DPPH radical formation as follows:
IE% = 100 X (A517cntrol-A517sample /A517cntrol), and expressed ad IC50. The
IC50 value was defined as the concentration (in μg/ml) of the
compound required to scavenge the DPPH radical by 50%.
2.11 Nutrition experiments
A total of eighty albino male rats were raised in the animal
house of faculty of Agriculture, Cairo University, Giza, Egypt. The
animals were fed on a basal diet for 7 days as an adaptation period.
The basal diet was formulated according to A. O. A. C. (2005) method
and consisted of casein (15%), corn oil (10%), cellulose (5%), salt
mixture (4%), vitamin mixture (1%) and starch (65%). Water was
available ad libitum. Known volumes of the concentrated crude sidr
(fruit and leaf) juice were dissolved in a mixture of distilled water
and Tween 20 (12:1, V/V) to obtain polyphenols concentrations of
200, 400, 800 and 1600ppm. BHT solution (200ppm) was prepared
exactly as mentioned above for juice (olive and pomegrante). The
rats divided into ten groups and each group comprised eight rats. The
first group presents the control rats, second group was given BHT at
200ppm, third, fourth, fifths and sixth were given 200, 400, 800 and
1600 of polyphenols from olive leaf juice. Seventh, eighth, ninth and
tenth groups were given 200, 400, 800 and 1600ppm of polyphenols
from pomegrante leaf juice. Each rat group was stomach ingested by
gavage daily (1ml) from the crude juice (olive and pomegrante) and
BHT solution for 6 weeks.
2.12 Blood samples
Blood samples were taken at the start of the experiment
and after 1, 2, 3, 4, 5, and 6 weeks of the administration of the crude
sidr juice (olive and pomegrante) and BHT. The blood samples were
obtained from orbital plexus venous by means of fine capillary glass
tubes according to the method outlined by Schermer [24](1967). It
was not possible to collect 10 ml blood from a single rat, hence the
blood of eight rats in each group was pooled. The blood samples were
placed in dry and clean centrifuge tubes and allowed to clot for 1-2 h
at room temperature. Sera were then removed using a Pasteur
pipette and centrifuged for 20 min at 110 rpm. The clean supernatant
Sera were then kept frozen until analysis.
2.13 Sera analysis
Alanine aminotransferase ALT (E. C. 2. 6. 1. 2), Aspartate
aminotransferase AST (E. C. 2. 6. 1. 1) and AP (E. C. 3. 1. 3. 1) activities
were measured according to the methods described by Kachmar &
Moss [25] (1976), Bergmryer & Harder [26] (1986) and Varley et
al[27]., (1980), respectively. Types of bilirubin (total and direct), urea
and acid were determined according to the methods described by
Fawcett & Scott [28] (1960), Dounas et al[29]., (1987) and Barham &
Trinder [30](1972), respectively. The levels of serum cholesterol, low
and high density lipoproteins and total lipids were determined
according to the methods outlined by Roechlau et al[31]., (1974),
Assmann[32], (1979) & Frings & Dunn [33] (1979). Each
determination was carried out in triplicate and the mean values are
presented in the text.

Amany M. Basuny et al / Utilization from leaves of olive and pomegranate as a source of bioactive components
© ASD Publisher All rights reserved. 30
2.14 Statistical analysis
Data were collected and expressed and expressed as the
mean ± saturated deviation of three independent experiments and
analysis for statistical significant from control, using the Dunnett test
(SPSS 11.5 Statistics Software; SPSS, Chicago, IL).
3. Results & Discusssion
3.1 Polyphenols content
There is currently great interest world-wide in finding new
and safe antioxidant from natural sources to prevent oxidative
rancidity of foods and so the present study focused on extracts of
olive and pomegranate containing polyphenols, which do not have an
undesirable odor when inhaled through the nose or an undesirable
and tongue taste. Although, in this publication, the active principal is
assumed to be polyphenolic in origin the occurrence of other
unidentified active principles is not excluded. Thus though reference
is always made to the polyphenolic material as being antioxidant, the
case is that we have proved it unequivocally. The total polyphenols
contents obtained are shown in Table 1. The concentration of
polyphenols in the different plant is as follows: olive > pomegranate.
It can be seen from Table 1 that pomegranate contain as low total
polyphenols as olive.
3.2 Phenolic compounds
Identification of phenolic compounds by HPLC technique
was used to identify the major phenolic compounds in the leaf juice
(olive and pomegranate). The identification was based on
comparisons on the chromatographic retention time and UV
absorbance spectra of compounds in olive and pomegranate samples
with those of authentic standards. Data of the HPLC analysis of leaf
juice samples (olive and pomegranate) are given in Table 2. Data
show that the phenolic compounds of leaf juice (olive and
pomegranate) were made up of 13 compounds. The main phenolic
compounds of olive leaves are (tyrosol, hydroxytyrosol, P-
hydroxybenzoic acid and tannic acid), while the main phenolic
compounds of pomegranate are (tyrosol, hydroxytyrosol, P-
hydroxybezoic acid and vanillic acid).
3.3 The antioxidant activity of olive and pomegranate phenolic
compounds:
The antioxidant activities of phenolic compounds from the
leaf juice (olive and pomegranate) were assessed by the Rancimat
method. This method the induction period for the onset of oxidative
rancidity in sunflower oil at 100ºC. In the present study, simple
model systems comprising sunflower oil with phenolic compounds
were used to assess oxidation behavior. An experiment was
performed with sunflower and BHT (200ppm) to compare the
antioxidant efficiency of the phenolic compounds from olive and
pomegranate with the most commonly used synthetic antioxidant
material. It has been reported that synthetic antioxidants (BHT, BHA
and PG) are added at concentrations of 100- 400ppm to fats and oils
to suppress the development of peroxides during food storage[34].
Therefore, the phenolic compounds were added to sunflower oil at
concentrations of 200, 400, 800 and 1600ppm. Table 3 shows the
olive and pomegranate leaves on the oxidative rancidity of sunflower
oil. The results illustrate that all the polyphenols of olive and
pomegranate juice and added at various concentrations to the test
system, exhibited antioxidant activity. However, statistical analysis
showed that had no significant differences of phenolic compounds for
both plant olive and pomegranate leaves on sunflower oil stability.
3.4 Scavenging capacity of DPPH free radicals
The changes in scavenging capacity of the phenolic
compounds antioxidant measured with the hydrophobic DPPH free
radical is shown in Table 4. Four levels of the concentrations of leaf
juice phenolic compounds (olive and pomegranate) were used with a
very high scavenging capacity of 40.00 after only 10 min. In all cases
the scavenging capacity did not increase after the first 10 min of
incubation. The reaction of BHT with DPPH was similar to phenolic
compounds with DPPH; the scavenging capacities were similar.
3.5 Alanine aminotransferase (ALT), aspartate aminotransferase
(AST) and phosphatase activities (AP)
Table 5, 6 &7 show the activities of ALT, AST and AP for
control rats and the values were slightly increased during the whole
experiment (6 weeks). The administration of BHT at 200 ppm to
experimental rats induced significant increases in serum ALT and
AST activities after 3 weeks from the commencement and towards
the end of the experiment. In the mean time, BHT induced significant
increase in serum AP activity after only 1 week from the beginning of
the experiment. The administration of polyphenols in crude leaf juice
(olive and pomegranate) at 200, 400 800 and 1600 ppm did not
cause any significant changes in enzyme activities compared with the
control experiment. Baron (1987) mentioned that the rise in the
activities of ALS, AST AND AP in rat serum is a sign of hepatocelluor
damage. This case has been found in rats administered only BHT and
not with polyphenolic of leaf juice (olive and pomegranate).
3.6 Serum total lipids
The results (Table 8) show that there was no significant
increase in the total lipids for control rats throughout the whole
experiment. The administration of BHT at 200 ppm caused significant
and gradual increase in serum total lipids. The rise in total lipids at
the end of the experiment was approximately 1.1 greater than that at
the beginning of the experiment. The administration of polyphenolic
compounds at 200, 400, 800 and 1600 ppm present in crude leaf
juice (olive and pomegranate) induced non-significant rise in rat
serum total lipids. In other words, the several of phenolic compounds
possessed the same function as that noticed with control rats.
3.7 Serum total cholesterol and low-density lipoproteins
cholesterol
Tables (9 &10) show the levels of serum total cholesterol
and low-density lipoprotein cholesterol (LDL-C) of control rats. Rats
administered BHT (200 ppm) and phenolic compounds (200, 400,
800 and 1600ppm) of crude leaf juice (olive and pomegranate). The
results for the control rats and rats administered BHT (200ppm),
indicated that there were no significant increase in total cholesterol
and LDL-C levels during the entire experiment. Also, administration
of polyphenols of leaves (olive and pomegranate) juice at various
concentrations exhibited non-significant increases in the total
cholesterol and LDL-C.
3.8 Serum high-density lipoprotein cholesterol
The results (Table 11) for the rats of the control and BHT
group indicated that there was no significant rise in the levels of
high-density lipoprotein cholesterol (HDL-C) throughout the whole
experiment. Also, crude leaf juice (olive and pomegranate)
polyphenols at various concentrations induced non-significant rise in
rat serum HDL-C.
3.9 Serum total bilirubin
Table 12 shows the effect of leaf juice polyphenolic
compounds (olive and pomegranate) at various levels (200, 400, 800
and 1600 ppm) and BHT (200 ppm) on the levels of rat serum total
those administered various concentrations of phenolic compounds in
leaf juice (olive and pomegranate), did not show any changes in the
levels of total bilirubin. Conversely, BHT 200 ppm caused significant
and gradual increases in total bilirubin levels during the entire
experiment.
3.10 Serum uric acid
The data (Table 13) for the control rats and rats
administered various concentrations of crude leaf juice (olive and
pomegranate) phenolic compounds showed non-significant changes
in the levels of uric acid during the entire experimental period. On the
contrary, BHT at 200ppm exhibited gradual increases on the levels of
rat serum uric acid. It is worth noting that a significant increase in
uric acid levels occurred at the second week and towards the end of
experiment.
3.11 Serum creatinine
The data (Table 14) for control rat group and the
polyphenols rat groups indicate that there were no significant

Amany M. Basuny et al / Utilization from leaves of olive and pomegranate as a source of bioactive components
© ASD Publisher All rights reserved. 31
changes of serum creatinine levels during the entire experiment. In
contrast, BHT at 200ppm caused increases in the levels of rat serum
creatinine and the increase took place from the second week of the
experiment. In generals, liver and kidney functions of rats
administered crude leaf juice (olive and pomegranate) did not cause
any adverse effects in liver and kidney functions. Consequently, one
would suggest adding crude leaf juice (olive and pomegranate) to
increase their shelf-life of oils without any deleterious effect on
human health. In general, the data for biochemical measurements
demonstrate that the polyphenolic compounds of crude leaf juice
(olive and pomegranate) are quite safe for human health. Conversely,
the results made it clear that BHT as a synthetic antioxidant has to be
abandoned from use.
Table 1: Phenolic contents as caffeic acid of leaves
(Olive and pomegranate)
Phenolic contents (ppm)
Olive leaves
510.00 ± 10.90
Pomegranate leaves
722.00 ± 12.15
Mean value ± standard deviation (SD).
Table 2: Phenolic compounds (ppm) of leaves (olive and
pomegranate)
Leaves
Olive
Pomegranate
Quercetin
3.00±0.01
1.50±0.001
Caffeic acid
7.30±0.91
8.30±0.82
Gallic acid
5.20±0.52
2.10±0.02
Apigenin
2.50±0.01
5.30±0.46
Chlorogenic acid
6.20±0.63
1.42±0.01
Ferulic acid
5.77±0.60
6.50±0.56
Cinamic acid
10.32±1.01
9.22±0.98
P-coumaric acid
9.50±0.91
6.30±0.67
P- hydroxy benzoic acid
19.07±2.53
18.50±2.00
Tannic acid
12.11±1.55
10.30±1.30
Hydroxy tyrosol
22.30±3.00
25.60±4.31
Vanillic acid
9.30±0.95
16.70±1.91
Tyrosol
25.50±4.50
31.20±5.81
Mean value ± standard deviation (SD).
Table 3: Effect of polyphenols obtained from leaves (olive and
pomegranate) on sunflower oil oxidative stability.
System
Induction period (hr)
Sunflower oil (control, C)
7.50±2.00
C+BHT (200PPM)
11.30±3.15
Polyphenols of olive leaves(POL):
C+POL (200ppm)
10.50±2.95
C+POL (400ppm)
11.70±3.20
C+POL (800ppm)
14.30±5.13
C+POL (1600ppm)
18.50±7.50
Polyphenols of pomegranate leaves (PPL)
C+PPL (200ppm)
10.60±2.55
C+PPL (400ppm)
11.50±3.25
C+PPL (800ppm)
15.00±6.00
C+PPL (1600ppm)
18.70±7.19
Mean value ± standard deviation (SD).
Table 4: Scavenging capacity of DPPH free radicals by
polyphenols leaves (olive and pomegranate) and BHT
System
Scavenging capacity (%)
Control
40.00±2.30
BHT (200ppm)
60.00±3.50
POL (200ppm)
52.00±2.90
POL (400ppm)
63.00±3.70
POL (800ppm)
75.00±5.51
POL (1600ppm)
93.00±7.30
PPL (200ppm)
54.00±2.95
PPL (400ppm)
67.00±3.85
PPL (800ppm)
78.00±5.18
PPL (1600ppm)
95.00±7.63
Mean value ± standard deviation (SD).
Table 5: Influence of leaves (olive and pomegranate) juice and BHT on the activity of serum alanine aminotransferase of rats (IU L-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
11.40±0.30
11.50±0.11
11.60±0.31
10.411.65±
11.70±0.40
11.73±0.71
11.52±0.01
BHT (200ppm)
11.40±0.30
12.80±0.41
13.50±0.13
17.00±0.61
20.22±0.41
22.30±0.62
25.50±0.04
POL (200ppm)
11.40±0.30
11.60±0.10
11.65±0.11
11.72±0.03
11.84±0.43
11.90±0.57
12.01±0.31
POL (400ppm)
11.40±0.30
11.66±0.33
11.73±0.31
11.90±0.46
12.00±0.19
12.05±0.35
12.21±0.41
POL (800ppm)
11.40±0.30
11.56±0.16
11.80±0.01
11.85±0.12
11.93±0.32
12.00±0.22
12.05±0.25
POL(1600ppm)
11.40±0.30
11.55±0.22
11.70±0.09
11.90±0.17
12.01±0.23
12.15±0.22
12.25±0.51
PPL (200ppm)
11.40±0.30
11.61±0.29
11.62±0.16
11.73±0.03
11.89±0.13
11.95±0.46
12.01±0.39
PPL (400ppm)
11.40±0.30
11.50±0.16
11.69±0.23
11.81±0.25
11.92±0.31
12.00±0.97
12.19±0.51
PPL (800ppm)
11.40±0.30
11.53±0.19
11.71±0.03
11.80±0.17
11.95±0.37
12.03±0.42
12.20±0.54
PPL(1600ppm)
11.40±0.30
11.67±0.06
11.83±0.20
11.83±0.20
11.92±0.39
11.98±0.43
12.17±0.13
Mean value ± standard deviation (SD).
Table 6: Influence of leaves (olive and pomegranate) juice and BHT on the activity of serum aspartate aminotransferase of rats (IU L-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
11.61±0.34
11.71±0.29
11.95±0.11
12.00±0.11
12.11±0.50
12.20±0.31
12.30±0.21
BHT (200ppm)
11.61±0.34
11.90±0.61
12.93±0.17
13.85±0.21
15.93±0.26
22.13±0.31
23.15±0.19
POL (200ppm)
11.61±0.34
11.63±0.21
11.72±0.20
11.75±0.61
11.70±0.19
11.83±0.23
11.85±0.51
POL (400ppm)
11.61±0.34
11.65±0.32
11.70±0.63
11.82±0.09
11.91±0.21
11.99±0.22
12.01±0.57
POL (800ppm)
11.61±0.34
11.70±0.51
11.75±0.04
11.93±0.11
11.95±0.19
12.00±0.59
12.11±0.65
POL(1600ppm)
11.61±0.34
11.82±0.63
11.85±0.63
11.94±0.19
11.99±0.12
12.03±0.05
12.15±0.32
PPL (200ppm)
11.61±0.34
11.79±0.59
11.79±0.06
11.83±0.01
11.89±0.19
11.93±0.34
12.00±0.53
PPL (400ppm)
11.61±0.34
11.75±0.56
11.79±0.11
11.82±0.60
11.90±0.29
11.93±0.31
11.99±0.45
PPL (800ppm)
11.61±0.34
11.79±0.41
11.83±0.21
11.92±0.05
11.95±0.01
12.05±0.04
12.22±0.43
PPL(1600ppm)
11.61±0.34
11.75±0.71
11.83±0.21
11.90±0.19
11.94±0.21
11.99±0.01
12.15±0.32
Mean value ± standard deviation (SD).

Amany M. Basuny et al / Utilization from leaves of olive and pomegranate as a source of bioactive components
© ASD Publisher All rights reserved. 32
Table 7: Influence of leaves (olive and pomegranate) juice and BHT on the activity of alkaline phosphatase of rats (IU L-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
65.00±0.46
64.93±0.13
65.03±0.13
65.06±0.13
65.19±0.29
65.20±0.31
65.31±0.07
BHT (200ppm)
65.00±0.46
65.50±0.12
65.90±0.51
67.20±0.01
71.30±0.36
73.20±0.56
80.02±0.16
POL (200ppm)
65.00±0.46
65.03±0.30
65.12±0.39
65.19±0.15
65.31±0.23
65.23±0.66
65.41±0.23
POL (400ppm)
65.00±0.46
65.13±0.52
65.20±0.24
65.22±0.20
65.35±0.19
65.41±0.75
65.31±0.66
POL (800ppm)
65.00±0.46
65.00±0.01
65.09±0.19
65.14±0.52
65.29±0.64
65.25±0.18
65.46±0.31
POL(1600ppm)
65.00±0.46
65.04±0.50
65.19±0.35
65.22±0.01
65.49±0.19
65.31±0.70
65.33±0.39
PPL (200ppm)
65.00±0.46
65.13±0.09
65.25±0.61
65.29±0.61
65.31±0.25
65.36±0.01
65.29±0.41
PPL (400ppm)
65.00±0.46
65.05±0.19
65.13±0.72
65.17±0.19
65.16±0.39
65.22±0.49
65.25±0.53
PPL (800ppm)
65.00±0.46
65.13±0.22
65.16±0.64
65.10±0.34
65.31±0.46
65.22±0.05
65.36±0.41
PPL(1600ppm)
65.00±0.46
65.23±0.31
65.23±0.31
65.32±0.29
65.40±0.55
65.41±0.33
65.39±0.18
Mean value ± standard deviation (SD).
Table 8: Influence of leaves (olive and pomegranate) juice BHT on the levels of serum total lipids of rats (mg dl-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
288.00±0.83
288.50±0.15
288.61±0.31
288.71±0.90
288.90±.11
288.95±0.45
290.00±0.76
BHT (200ppm)
288.00±0.83
290.00±0.90
292.00±1.04
295.30±2.50
299.70±0.09
303.00±0.13
310.20±0.82
POL (200ppm)
288.00±0.83
288.10±1.03
288.30±2.03
288.51±0.80
288.90±0.42
288.85±1.09
290.15±2.00
POL (400ppm)
288.00±0.83
288.40±0.60
288.55±1.19
288.67±0.89
288.95±2.01
290.00±0.35
290.30±1.06
POL (800ppm)
288.00±0.83
288.20±0.19
288.35±1.11
288.61±0.95
288.75±0.46
288.95±0.56
290.10±0.66
POL(1600ppm)
288.00±0.83
288.29±0.49
288.50±2.30
288.78±1.22
288.30±0.67
288.50±0.19
288.11±0.76
PPL (200ppm)
288.00±0.83
288.00±0.57
288.30±1.01
288.60±0.97
288.13±0.95
288.90±0.89
288.30±0.73
PPL (400ppm)
288.00±0.83
288.19±1.03
288.35±2.00
288.45±2.11
288.52±0.81
288.55±0.59
288.90±1.01
PPL (800ppm)
288.00±0.83
288.11±0.77
288.49±1.00
288.56±0.66
288.11±0.89
288.90±1.17
290.00±0.90
PPL(1600ppm)
288.00±0.83
288.01±1.00
288.19±1.00
288.50±0.66
288.90±1.90
289.00±2.05
289.50±0.79
Mean value ± standard deviation (SD).
Table 9: Influence of leaves (olive and pomegranate) juice and BHT on the levels of serum total cholesterol of rats (mg dl-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
160.00±0.14
160.50±0.29
161.20±0.30
160.30±0.01
160.22±0.19
162.00±0.82
161.90±0.39
BHT (200ppm)
160.00±0.14
160.20±0.19
160.00±0.34
161.50±0.45
161.90±0.72
161.50±0.21
162.00±0.31
POL (200ppm)
160.00±0.14
160.30±0.01
160.80±0.42
161.20±0.41
161.90±0.81
162.03±0.61
162.00±0.13
POL (400ppm)
160.00±0.14
160.33±0.19
160.45±0.03
160.55±0.80
161.31±0.39
162.13±0.18
161.00±0.09
POL (800ppm)
160.00±0.14
160.55±0.23
160.90±0.75
161.00±0.42
161.20±0.31
161.33±0.01
161.70±0.61
POL(1600ppm)
160.00±0.14
160.00±0.21
160.31±0.19
161.00±0.22
161.50±0.69
161.72±0.39
161.49±0.11
PPL (200ppm)
160.00±0.14
160.09±0.59
161.00±0.30
161.50±0.19
161.00±0.09
161.31±0.41
161.50±0.40
PPL (400ppm)
160.00±0.14
160.15±0.19
160.22±0.39
160.52±0.82
160.75±0.13
160.90±0.31
160.99±0.12
PPL (800ppm)
160.00±0.14
160.29±0.22
160.56±0.39
160.75±0.45
160.90±0.56
161.00±0.61
161.20±0.65
PPL(1600ppm)
160.00±0.14
160.00±0.29
160.55±0.34
161.20±0.01
161.35±0.01
161.00±0.19
161.00±0.41
Mean value ± standard deviation (SD).
Table 10: Influence of leaves (olive and pomegranate) juice and BHT on the levels of serum HDL-cholesterol of rats (mg dl-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
40.13±0.52
40.33±0.22
40.50±0.35
41.00±0.09
41.31±0.14
41.60±0.67
42.00±0.31
BHT (200ppm)
40.13±0.52
40.00±0.60
40.50±0.05
40.39±0.35
41.22±0.19
41.50±0.01
41.00±0.30
POL (200ppm)
40.13±0.52
41.00±0.70
41.39±0.19
40.71±0.27
40.79±0.42
41.30±0.18
41.39±0.37
POL (400ppm)
40.13±0.52
40.59±0.64
40.75±0.55
41.03±0.67
41.90±0.01
42.00±0.63
42.10±0.13
POL (800ppm)
40.13±0.52
40.60±0.13
40.90±0.63
40.85±0.72
41.30±0.94
41.50±0.13
42.00±0.29
POL(1600ppm)
40.13±0.52
40.00±0.60
40.81±0.33
40.90±0.01
40.85±0.72
41.00±0.19
41.35±0.18
PPL (200ppm)
40.13±0.52
40.05±0.67
40.19±0.82
40.71±0.13
41.30±0.84
41.73±0.35
42.00±0.60
PPL (400ppm)
40.13±0.52
40.50±0.19
40.66±0.01
40.75±0.04
40.91±0.42
41.00±0.13
41.30±0.19
PPL (800ppm)
40.13±0.52
40.39±0.72
40.69±0.89
40.18±0.32
41.09±0.43
41.33±0.84
41.90±0.93
PPL(1600ppm)
40.13±0.52
40.80±0.61
41.00±0.22
41.35±0.81
41.69±0.11
41.69±0.59
42.00±0.19
Mean value ± standard deviation (SD).

Amany M. Basuny et al / Utilization from leaves of olive and pomegranate as a source of bioactive components
© ASD Publisher All rights reserved. 33
Table 11: Influence of leaves (olive and pomegranate) juice and BHT on the levels of serum LDL-cholesterol of rats (mg dl-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
100.60±1.06
100.71±0.83
100.39±0.29
100.81±2.04
101.00±0.91
101.20±0.19
101.50±0.56
BHT (200ppm)
100.60±1.06
101.00±0.78
101.30±0.35
101.34±0.49
100.90±0.85
101.50±0.95
101.66±1.09
POL (200ppm)
100.60±1.06
100.33±0.88
101.11±2.03
101.19±0.50
101.50±0.59
101.90±0.19
102.00±0.89
POL (400ppm)
100.60±1.06
100.90±0.67
101.00±0.52
101.50±0.64
101.90±0.66
101.95±0.23
102.01±0.85
POL (800ppm)
100.60±1.06
100.75±0.52
100.85±0.39
100.95±0.25
101.00±1.03
101.50±0.14
102.00±0.45
POL(1600ppm)
100.60±1.06
100.90±0.18
101.11±0.38
101.19±0.91
101.50±0.55
101.90±0.71
102.00±0.79
PPL (200ppm)
100.60±1.06
100.65±0.13
100.75±1.05
100.80±1.00
100.95±2.03
101.95±0.19
102.15±0.89
PPL (400ppm)
100.60±1.06
100.75±0.39
101.00±0.95
101.35±0.90
101.45±0.85
101.90±1.16
102.60±0.81
PPL (800ppm)
100.60±1.06
100.90±1.00
100.95±0.89
101.20±0.72
101.31±0.33
101.22±0.89
101.55±0.19
PPL(1600ppm)
100.60±1.06
100.63±0.90
100.63±0.90
100.83±0.41
101.09±0.42
101.29±0.30
101.39±0.42
Mean value ± standard deviation (SD).
Table 12: Influence of leaves (olive and pomegranate) juice and BHT on the levels of serum total bilirubin of rats (mg dl-1)
Concentration of phenolic
compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
1.00±0.01
1.01±0.01
1.01±0.02
1.02±0.01
1.01±0.03
1.00±0.01
1.02±0.01
BHT (200ppm)
1.00±0.01
1.01±0.01
1.07±0.003
1.23±0.01
1.33±0.01
1.39±0.02
1.43±0.02
POL (200ppm)
1.00±0.01
1.01±0.01
1.01±0.02
1.02±0.02
1.01±0.02
1.02±0.01
1.01±0.03
POL (400ppm)
1.00±0.01
1.01±0.03
1.00±0.01
1.01±0.04
1.01±0.01
1.02±0.02
1.02±0.01
POL (800ppm)
1.00±0.01
1.01±0.02
1.00±0.04
1.01±0.003
1.01±0.003
1.02±0.02
1.02±0.01
POL(1600ppm)
1.00±0.01
1.01±0.01
1.01±0.01
1.02±0.02
1.02±0.02
1.01±0.02
1.01±0.02
PPL (200ppm)
1.00±0.01
1.01±0.01
1.01±0.01
1.02±0.01
1.02±0.01
1.01±0.01
1.01±0.02
PPL (400ppm)
1.00±0.01
1.00±0.02
1.01±0.02
1.02±0.03
1.01±0.01
1.01±0.03
1.02±0.03
PPL (800ppm)
1.00±0.01
1.00±0.01
1.01±0.02
1.00±0.02
1.01±0.02
1.01±0.01
1.01±0.01
PPL(1600ppm)
1.00±0.01
1.01±0.01
1.02±0.01
1.02±0.01
1.01±0.01
1.01±0.02
1.02±0.02
Mean value ± standard deviation (SD).
Table 13: Influence of leaves (olive and pomegranate) juice and BHT on the levels of serum urea of rats (mg dl-1)
Concentration of
phenolic compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
30.00±0.30
30.19±0.42
30.18±0.13
30.11±0.15
30.00±0.17
29.19±0.17
29.50±0.43
BHT (200ppm)
30.00±0.30
33.85±0.10
39.00±0.31
45.00±0.13
52.00±0.04
62.00±0.31
69.50±0.31
POL (200ppm)
30.00±0.30
30.00±0.60
30.00±0.32
29.15±0.33
29.50±0.13
30.11±0.41
30.19±0.41
POL (400ppm)
30.00±0.30
30.00±0.90
30.01±0.04
29.50±0.10
29.90±0.41
29.85±0.69
30.00±0.13
POL (800ppm)
30.00±0.30
30.00±0.72
30.00±0.19
30.00±0.66
30.00±0.01
29.81±0.19
29.00±0.51
POL(1600ppm)
30.00±0.30
29.85±0.22
29.90±0.25
30.00±0.32
29.00±0.39
30.00±0.34
30.00±0.21
PPL (200ppm)
30.00±0.30
29.00±0.33
29.50±0.41
29.20±0.19
29.11±0.22
30.00±0.25
30.00±0.68
PPL (400ppm)
30.00±0.30
30.12±0.29
30.00±0.31
30.01±0.39
29.80±0.35
29.50±0.22
29.00±0.01
PPL (800ppm)
30.00±0.30
30.27±0.22
30.20±0.23
30.15±0.14
30.30±0.04
30.00±0.05
30.00±0.16
PPL(1600ppm)
30.00±0.30
29.00±0.33
29.00±0.69
29.50±0.01
29.55±0.10
29.00±0.42
29.00±0.42
Mean value ± standard deviation (SD).
Table 14: Influence of leaves (olive and pomegranate) juice and BHT on the levels of serum creatinine of rats (mgdl-1)
Concentration of
phenolic compounds
Blood withdrawal period (week)
0
1
2
3
4
5
6
Control rats
0.88±0.10
0.88±0.03
0.87±0.09
0.87±0.04
0.88±0.04
0.088±0.11
0.88±0.19
BHT (200ppm)
0.88±0.10
1.01±0.30
1.29±0.03
1.48±0.01
1.60±0.02
1.80±0.01
1.91±0.01
POL (200ppm)
0.88±0.10
0.87±0.10
0.86±0.41
0.86±0.08
0.85±0.07
0.85±0.13
0.85±0.14
POL (400ppm)
0.88±0.10
0.87±0.10
0.86±0.07
0.84±0.06
0.85±0.13
0.85±0.00
0.85±0.12
POL (800ppm)
0.88±0.10
0.88±0.19
0.88±0.09
0.86±0.01
0.83±0.03
0.80±0.10
0.80±0.10
POL (1600ppm)
0.88±0.10
0.85±0.20
0.85±0.22
0.83±0.09
0.83±0.09
0.82±0.07
0.80±0.02
PPL (200ppm)
0.88±0.10
0.88±0.02
0.87±0.10
0.85±0.08
0.85±0.08
0.85±0.07
0.85±0.10
PPL (400ppm)
0.88±0.10
0.85±0.10
0.85±0.30
0.84±0.14
0.84±0.12
0.84±0.10
0.84±0.12
PPL (800ppm)
0.88±0.10
0.86±0.09
0.85±0.42
0.82±0.09
0.82±0.12
0.81±0.11
0.80±0.11
PPL(1600ppm)
0.88±0.10
0.85±0.10
0.84±0.01
0.82±0.01
0.82±0.30
0.80±0.11
0.80±0.11
Mean value ± standard deviation (SD).

Amany M. Basuny et al / Utilization from leaves of olive and pomegranate as a source of bioactive components
© ASD Publisher All rights reserved. 34
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