Content uploaded by Ufuk Koca
Author content
All content in this area was uploaded by Ufuk Koca
Content may be subject to copyright.
For Peer Review
Journal of Medicinal Food: http://mc.manuscriptcentral.com/medicinalfood
Wound Repair Potential of Olea europaea L. Leaf Extracts
Revealed by in vivo Experimental Models and Comparative
Evaluation of the Extracts’ Antioxidant Activity
Journal:
Journal of Medicinal Food
Manuscript ID:
JMF-2010-0039.R2
Manuscript Type:
Original Article
Date Submitted by the
Author:
27-Apr-2010
Complete List of Authors:
Koca, Ufuk
Pesin Süntar, Ipek; Gazi University, Pharmacognosy
Kupeli Akkol, Esra; Gazi University, Pharmacognosy
Yılmazer, Demet
Alper, Murat
Keyword:
oleuropein
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
For Peer Review
1
Wound Repair Potential of Olea europaea L. Leaf Extracts Revealed by in
vivo Experimental Models and Comparative Evaluation of the Extracts’
Antioxidant Activity
Ufuk Koca1, Ipek Peşin Süntar1, Esra Küpeli Akkol1*, Demet Yılmazer2, Murat Alper2
1 Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkey
2 Department of Pathology, Dışkapı Yıldırım Beyazit Education and Research Hospital, Ankara, Turkey
RUNNING TITLE: Wound Healing Potential of Olea europaea
* Corresponding author: Tel: +90 312 2023185; Fax: +90 312 2235018.
E-mail address: esrak@gazi.edu.tr (E. Küpeli Akkol).
Page 1 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
2
ABSTRACT
Leaves and fruits of Olea europaea L. (Olive) have been used externally as emollient for skin
ulcers, and healing of inflammatory wounds. n-Hexane and aqueous extracts, prepared from the dried
leaves of olive, were evaluated for their wound healing activity by using in vivo wound models of linear
incision and circular excision in comparison with the reference ointment Madecassol®. The group of
animals treated with the aqueous extract demonstrated increased contraction (87.1%) on excision and a
significant increase in wound tensile strength (34.8%) on incision models as compared to other groups.
Moreover, antioxidant activity assay showed that aqueous extract has higher scavenging ability than the
n-hexane extract. According to the experimental data, the aqueous extract of O. europaea leaves
displayed wound healing activity. Secoiridoid oleuropein (4.6059%) was identified as the major active
compound according to HPLC analysis of the aqueous extract.
KEY WORDS: Excision, Incision, Oleuropein, Tensiometer, Wound healing
INTRODUCTION
Olea europaea L. (Oleaceae) is one of the most ancient tree of the Mediterranean region. The
cultivation process is believed to be through the selection of trees with large fruit size and/or high oil
content.1 Commercial olives belong to the species Olea europaea L. (Olive, Zeytoon, Zeytin), which is
a long-lived evergreen tree that is recognizable by the dense assembly of branches with thick, leathery
and oppositely arranged leaves.2
Olive oil is well known for its flavor and health benefits, while its leaves have been used
medicinally in various indications for ages. In west coast of Turkey, decoction of the leaves is used to
vanish the nodules and also against diabetes.3 A fixed oil is made with white wax that is melted in olive
oil can be applied onto wounds twice a day in Northwest part of Anatolia. Moreover, fruits or fixed oil
are pounded with onions and employed to treat muscular or rheumatic pain.4 The oil is also pounded
with salt and ground soap in order to make an ointment, which is applied onto cuts and wounds for
rapid healing. Olive and olive products have been used not just in Turkey but in various parts of the
world. Decoction, made out of olive fruits and leaves, has been recommended to be used with lemon
Page 2 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
3
juice as a laxative in chronic constipation and as an antidot for poisoned livestocks in Jordan.5 Fruits
and leaves are also used to treat hepatic troubles, infection, urinary system symptoms and hypertension
at the same region. Additionally, decoction prepared from the leaves are used as antihypertensive, and
vitamin; the oil, the raw fruits and the leaves are applied to soothe and to heal burns in Bulgaria, Italy
and Portugal.6 Verbal literature revealed that the leaves, olive oil and fruits are applied as an emollient
to heal the pain and inflammation in rheumatologic diseases; to reduce the blood glucose in diabetes and
to control body weight in obesity in Portugal.7
Besides their ethnopharmacological usage, olive leaf and the leaf extracts brought attention with
their bioassays supported antioxidant, antimicrobial and, in vivo proven blood pressure lowering,
hypocholesterolemic, antidiabetic and anti-inflammatory effects.8-12 Aside from olive oil content,
studies have also focused on the composition of olive leaf extracts due to the availability and low cost of
raw material in the origin.13, 14 The principle components of the leaf extract were shown to be
biophenolics, the secoiridoid oleuropein, and their biodegradation products.14 However, investigation of
the traditional usage of olive leaves on wound healing in a scientific platform have been neglected for
years. Inspite of number of recorded ethnopharmacological information on leaf extracts, our rational
was to provide a scientific base for the traditional usage of the leaves on wound healing. In order to do
that the aqueous and n-hexane extracts were prepared from the leaves of the plant and were tested in
vivo by applying on mice and rats using circular excision and linear incision wound models. Moreover,
antioxidant activity of the extracts was analyzed by using commonly accepted DPPH method. The
major compound was determined in the extracts by using chromtatographical methods.
MATERIALS AND METHODS
Plant material
Branches of Olea europaea var. europaea were collected from Bahcelievler region, near the
city center of Balıkesir, Turkey, in October 2008. The plant was authenticated and a voucher specimen
was deposited in the Herbarium of Faculty of Pharmacy, Gazi University (GUE-2621).
Extraction
Page 3 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
4
Following the collection of young branches of Olea europaea, they were shade dried. Leaves
were separated, graded into little pieces and 30 gram of leaves (for each extraction) were submitted to
successive n-hexane and water extractions at room temperature for a day (500 ml each solvent). This
was repeated in two trials. After filtration, the extracts were evaporated at 400C (Buchi, Switzerland) to
dryness in vacuo. Yields of each extracts were 18.23% for aqueous and 12.38% for n-hexane extracts.
Animals
Male, Sprague–Dawley rats (160–180 g body weight) and Swiss albino mice (20–25 g body
weight) were purchased from the animal breeding laboratories of Experimental Animal Research Center
of Gazi University (GÜDAM) (Ankara, Turkey). The animals were left for 3 days at room conditions
for acclimatization and maintained on standard pellet diet and water ad libitum throughout the
experiment. A minimum of six animals were used in each group. The study was permitted by the
Institutional Animal Ethics Committee and was performed according to the international rules
considering the animal experiments and biodiversity rights.
Wound Healing Activity
Excision and incision wound models were used to evaluate the wound healing activity. For the
in vivo wound models, samples were prepared in the ointment consists of glycol stearate: propylene
glycol: liquid paraffin (3:6:1) and applied topically onto the test animals. Extracts were prepared as 1%
in the ointment, and 0.5 g of each was applied on wounded area immediately after wound was created
artificially.
The animals in the vehicle group were treated with the ointment base only. Animals of the
negative control group were not treated with any material. Commercial Madecassol®, 0.5 g (Bayer,
00001199) was applied as a reference drug that contains 1% extract of Centella asiatica extract.
Circular excision wound model
With the purpose of monitoring wound contraction and wound closure time, this model was
applied to mice. Each group of animals (six-animal each) was anaesthetized by 0.01 ml Ketalar®. The
back hairs of the mice were depilated by using a shaving machine. A circular wound was created on the
Page 4 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
5
dorsal interscapular region of each animal by excising the skin carefully with a 5 mm biopsy punch;
circular wounded areas were left open till the end of the experiment.15 The extracts, the reference
material (Madecassol® Bayer) and the vehicle ointment were administered once a day until the wounds
were completely healed in one of the applied group. The progressive changes in wound area were
monitored by a camera (Fuji, S20 Pro, Japan) and wound area evaluated by using AutoCAD program
every other day. Wound contraction was calculated as percentage of the reduction in wounded area. At
the end of the experiment, a specimen sample of tissue was isolated from the healed skin of each group
of mice for the histopathological examination.16
Linear incision wound model
Firstly, the animals used in this experiment were anaesthetized with 0.15 ml Ketalar®. In order
to have clean surface, the back hair of the rats were shaved by using a shaving machine. Five cm long,
two linear-paravertebral incisions were made with a sterile blade through the full thickness of the skin at
the distance of 1.5 cm from the midline of each side of the vertebral column.17 The artificially created
wounds were stiched with three surgical interrupted sutures of 1 cm apart. Related materials were
topically applied once in a day through 9 days on animals in all groups excluding the negative control
group. All the sutures were removed on the 9th post wound day. On day 10, all the animals were killed
with ether anesthesia. One linear-paravertebral incised skin was measured for its tensile strength, using
tensiometer (Zwick/Roell Z0.5, Germany), the other incised skin was sent for histopathological
examination.18, 19
Histopathological Study
Sample tissues were fixed in 10% formalin and were embedded in paraffin wax. Serial
sections (5 micrometer thickness) of paraffin embedded tissues were cut. The tissues were stained by
haematoxylin and eosin and were examined by light microscope (Olympus BX51). Ulceration, necrosis
and epithelisation were evaluated. Also congestion, edema, PNL, mononuclear cells, fibroblasts and
vascularisation were quantified as -, +, ++, +++, from negative to the most observed.
Page 5 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
6
Statistical Analysis of the Data
The data on percentage wound healing was statistically analyzed using one-way analysis of
variance (ANOVA). The values of p ≤ 0.05 were considered statistically significant. Histopathologic
data were considered to be nonparametric; therefore, no statistical tests were performed.
Analysis of Oleuropein in the Extracts
The HPLC analysis was carried out using an Agilent Technologies 1200 series HPLC system
according to method validated by Ansari et al.20 The system consisted of a quaternary pump, in line
degasser, column thermostate and UV detector. For the data processing and acquisition, Agilent
Chemstation software was used. The chromatographic separation of the fractions was performed using a
LiChrospher® 100 RP C-18, 5 µm, 250-4.0 mm (Supelco Inc.) column with an isocratic elution of
HPLC grade water adjusted to pH 2.5 with ortophosphoric acid (o-H3PO4): acetonitrile (CH3CN)
(80:20). Detection wavelength was 280nm, flow rate was optimised at 1mL/min. The reference
compound oleuropein (Chromadex, CA, USA) was dissolved in HPLC grade water to prepare stock
solution (1 mg/mL), calibration curve was prepared by diluting the stock 6 times and the equation was
calculated. The dry aqueous extract was dissolved in water, whereas n-hexane extract was dissolved in
small amount of n-hexane, then diluted with isopropanol to the volume. The reference and the extracts
were filtered through a membrane filter (0.45 µm), reference, aqueous and n-hexane extracts were
injected for comparison of their oleuropein content.
Antioxidant Activity Assay
Antioxidant activity of the extracts were assayed on the basis of their scavenging effect on the
2,2-diphenyl-1-picrylhidrazyl (DPPH radical Sigma, St Louis, MO,USA) according to the method of
Blois (1958).21 The concentration of DPPH was 6 x 10-5 molL-1. The extracts were dissolved in 75%
ethanol and then 77 µl of them were added to 3ml DPPH solution, incubated for 15 min at room
temperature. Butylated hydroxytoluene (BHT, Sigma, St Louis, MO, USA) was used as a reference.
The decrease in absorbance of the extracts, and the reference were measured at 515 nm after the blank
reading. Scavenging ability was calculated from the equation as follows:
Page 6 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
7
Inhibition (%) = {(Ablank-Asample)/Ablank} x 100
RESULTS
The wound healing activity of the n-hexane and aqueous extracts prepared from the leaves of
Olea europaea were evaluated on mice and rats by circular excision and linear incision wound models
to confirm the claimed folkloric usage of the plant on a scientific base. The histopathological changes
formed by these extracts were also assessed. Additionally, antioxidant activities of the extracts were
compared with each other and then the major compound of the aqueous extract was determined by
HPLC.
The measurements of the progress of wound healing induced by the extracts, reference drug,
vehicle groups, and negative control in the excision wound model are shown in Table 1.
Animals treated with the aqueous extract revealed 80.1% (p<0.01) contraction on day tenth, and
the same extract demonstrated 87.1% (p<0.001) contraction on day twelfth, which was close to
contraction value of the reference drug Madecassol® (100%) on the same day. The other extract
demonstrated no significant result on the given days.
The results of the measurements of tensile strength are shown in Table 1. Tensile strength of the
animals treated with the aqueous extract of olive leaves demonstrated the highest value (34.8%, p<0.01)
on day 10. Topical application of the aqueous extract on incision wound model expressed a significant
increase in wound tensile strength as compared to and n-hexane treated group and the control groups.
On the other hand reference drug Madecassol® demonstrated 51.1% (p<0.001) tensile strength.
Overall the histopathological examinations showed that healing process in aqueous extract
treated group was comparably close to the reference drug treated group whereas no healing was
observed in the negative control group (untreated). Treatment of rats and mice with the aqueous extract
of leaves of O. europaea led to reduced congestion, edema and number/infiltration of
polymorphonuclear leucocytes. Enhanced epithelialization, establishment of collagen fibers,
development of fibroblasts and mature hair follicles were observed (Fig. 1). The histopathological
studies demonstrated a significant healing effect by the aqueous extract of the leaves compared to
untreated negative control group, which exhibited a wide area of ulceration containing mixed type
Page 7 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
8
inflammatory cells and congested vessels in dermis (Fig. 2). Re-epitelialization was not observed in the
negative control group. Complete re-epithelialization together with mature hair follicles and collagen
fibers were obviously noticed in the reference drug Madecassol® treated group (Fig. 3). Accordingly,
histopathological observations supported the outcome of in vivo wound models of this study.
Initially, the calibration curve and the equation were established by using oleuropein reference
with the purpose of determining that in the extracts. Retention time was recorded as 9.2±3 for the
oleuropein (Fig. 4-A) with the method used. The same retention time was obtained when the aqueous
extract was applied to the column (Fig. 4-B). The reference oleuropein and the aqueous extract were
injected together in order to verify whether the observed major peak is oleuropein. Increase in the area
of the same peak (area=2948.2) supported the idea that the major peak was oleuropein in the aqueous
extract. Comparison of the UV profiles of oleuropein and the major peak present in the aqueous extract
also concluded that the major peak was oleuropein. The n-hexane extract was also injected in parallel,
but the observed peaks were below the detection limits on the chromatogram (Fig. 4-C). Moreover, it
has also been determined that 1g of aqueous extract contains 46.06 ± 2.30 mg oleuropein. Previously in
a different study, the levels of oleuropein in different stage of flowers, leaves and fruits were analyzed
in detail in Arbequina olives and oleuropein level was found as 38.13 ± 1.81 mg g-1 and remained fairly
constant throughout the full expansion of the leaves, which is in range close to our findings.22
Free radical scavenging activity of the extracts were examined by DPPH assay compared to
reference BHT. DPPH is a molecule containing a stable free radical. In the presence of an radical
scavenger, which can donate an electron to DPPH, the purple color of free DPPH radical decays, and
the change can be measured by a spectrophotometry. Results of the assay demonstrated that scavenging
ability of the aqueous extract (63.59%) was somewhat close to the reference (72.23%) at different
concentrations, whereas the scavenging ability of the same extract was superior of the n-hexane extract
(29.17%) at all the concentrations (Table 2).
DISCUSSION
Wound healing entails a multifaceted series of interactions between, the extracellular matrix,
cytokine mediators, and different cell types. These interactions between different cell types occur in
Page 8 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
9
several phases like haemostasis, inflammation, proliferation, and remodeling which flow concurrently,
but independent from each other. Therefore we thought that the use of two different wound models
combined with histopathology and antioxidant activity might be more informative to assess the effect of
Olea europaea extracts on the various phases of wound healing. This study investigated wound healing
potency of the aqueous and the n-hexane extracts of olive leaves by using two in vivo models. Polar
(aqueous) extract demonstrated potent wound healing activity over nonpolar (n-hexane) extract that had
very similar content with the oil. In excision wound model, the aqueous extract showed faster healing
compared with the negative control group and also wound contraction rate was faster with the aqueous
extract than the n-hexane extract. The stronger tensile strength of aqueous extract treated wounds in this
model might be due to the increase in collagen concentration and stabilization of the fibers. The
beneficial role of antioxidants on tensile or wound breaking strength were experimentally proved
previously.23 Effect of radical scavengers from plant extracts on healing of wounds were shown to be
important by several studies. In this study, in vitro antioxidant activity of the olive leaf extracts was
searched and it was demonstrated that the aqueous extract has more free radical scavenging capacity,
that improved the speed of the wound healing in comparison to n-hexane extract.
Olive leaves might share possible health benefiting bioactive phytochemicals with olive oil,
although it has some superior futures over the oil. First of all, its natural material is easily available with
low cost. Studies demonstrated that the major polyphenolic constituent in olive fruits and leaves (Olea
europaea L.) is oleuropein glycoside. This compound is almost entirely absent from olive oil because of
its low solubility in oil and its widespread enzymatic degradation during olive oil production.22 In a
previous study, oleuropein, hydroxytyrosol, hydroxytyrosol acetate, luteolin, luteolin- 7-O-glucoside,
and luteolin 4’-O-glucoside were identified as well in the olive leaf extracts. These phytochemicals also
demonstrated considerable power of radical scavenging.24 Radical oxygen has a major role in the
pathogenesis and chronic wounds. Mass production of reactive oxygen species (ROS) results in
oxidative stress, which might cause cytotoxicity and furtheremore delayed wound healing.
Consequently, removal of ROS could be a vital strategy in therapy of chronic wounds. Thus, drugs that
are able to inhibit lipid peroxidation, most probably increase the strength of collagen fibres, prevent cell
damage, support novel DNA synthesis and rise the circulation in the healing tissue, which results in
Page 9 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
10
increased viability of collagen fibrils.25 Similarly, more potent in vitro antioxidant activity of the
aqueous leaf extract of O. europaea might explain the different potential of the O. europaea extracts on
in vivo wound healing.
Besides antioxidant activity, the antimicrobial effect of olive leaves might be an other
mechanism for their benefitial effect on wound healling. In addition to the whole leaf extract, the
phenolic compounds isolated from olive fruits and leaves were shown to inhibit the growth of
Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus.26 Oleuropein, a major compound
of the leaf extract, were shown to inhibit Bacillus cereus, and hydroxytyrosol, a derivative of
oleuropein, was reported to be active against clinical human pathogenic strains of Haemophilus
influenzae, Moraxella catarrhalis, Salmonella enterica subsp. enterica servar Typhi, Vibrio
parahaemolyticus and S. aureus.10, 12 As a wound healer, the olive extracts might aid the healing process
by providing an optimal healing environment via their effect against bacteria and/or virus, which results
in decreased rates of infection.
Investigation on olive leaf extratcs so far demostrated that the extract has variety of
phytochemicals; emphasizing the major one being oleouropein that is followed by flavonoids. Various
studies also showed that the olive leaf extract and the isolated compounds have major radical
scavenging and considerable antimicrobial, antiviral activity. Because of these features, olive leaf
extract is gaining a strong place in the world of new herbal drugs or functional foods. The results of this
study provided further evidence by demostrating that the aqueous extract has significant antioxidant
properties in association with the speed of the healing.
CONCLUSION
According to literature review this is the first study revealing wound healing activity of the
aqueous extract of Olea europea leaves. In addition to utilization of the extract as a food supplement
and in some cosmetical prepearations, the use of olive leaf extract as a component of wound healing
drugs/pomads due to its antioxidant and antimicrobial features might be advantageous, as these are
natural, easily obtained and demonstrate strong ‘in vivo’ wound healing activity.
Page 10 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
11
ACKNOWLEDGMENT
The authors would like to thank Associate Prof. Ugur Tamer and Sebnem Yilmaz (PhD.) from
Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry for their kind help during
HPLC analysis of the extracts.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
REFERENCES
1. Liphschitz N, Gophna R, Hartman M, Biger G: The beginning of olive (Olea europaea)
cultivation in the old world: a reassessment. J Archaeol Sci 1991;18:441-453.
2. Tan K: The Olea europaea complex. The Davis and Hedge Festschrift, Edinburgh University
Press, United Kingdom, 1989, pp. 287–299.
3. Tuzlacı E, Eryasar Aymaz P: Turkish folk medicinal plants, Part IV: Gonen/Balıkesir.
Fitoterapia, 2001;72:323-343.
4. Yesilada E, Sezik E, Honda G, Takaishi Y, Takeda Y, Tanaka T: Traditional medicine in
Turkey IX: Folk medicine in north-west Anatolia. J Ethnopharmacol 1999;64:195-210.
5. Aburjai T, Hudaib M, Tayyem R, Yousef M, Qishawi M: Ethnopharmacological survey of
medicinal herbs in Jordan, the Ajloun Heights region. J Ethnopharmacol 2007;110:294-304.
6. Leporatti ML Ivancheva S: Preliminary comparative analysis of medicinal plants used in the
traditional medicine of Bulgaria and Italy. J Ethnopharmacol 2003;87:123-142.
Page 11 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
12
7. Neves JM, Matosa C, Moutinho C, Queirozd G, Gomesa LR: Ethnopharmacological notes
about ancient uses of medicinal plants in Trás-os-Montes (northern of Portugal). J
Ethnopharmacol 2009;124(2):270-283.
8. Lasserre B, Kaiser R, Pham HC, Ifansyah N, Gleye J Moulis C: Effect on rats of aqueous
extracts of plants used in folk medicine as antihypertensive agents. Naturwissenschaften
1983;70:95-96.
9. De Pasquale R, Monforte MT, Trozzi A, Raccuia A, Tommasini S, Ragusa S: Effects of leaves
and shoots of Olea europaea and oleuropein on experimental hypercholesterolemia in rat.
Plantes Med Phytother 1991;25:134-140.
10. Tassou CC, Nychas GJE: Effect of phenolic compounds and oleuropein on the germination of
Bacillus cereus T spores. Biotechnol Appl Bioc 1991;13:231-237.
11. Bennani-Kabchi N, Fdhil H, Cherrah Y, Kehel L, El Bouayadi F, Amarti A, Saidi M, Marquie
G: Effects of Olea europaea var. oleaster leaves in hypercholesterolemic insulin-resistant sand
rats. Therapie1999;54:717–723.
12. Bisignano G, Tomaino A, LoCascio R, Crisafi G, Uccella N, Saija A: On the in-vitro activity of
oleuropein and hydroxytyrosol. J Pharm Pharmacol 1999;51:971–974.
13. Briante R, Patumi P, Terenziani S, Bismuto E, Febbraio F, Nucci R: Olea europaea L. leaf
extract and derivatives: Antioxidant properties. J Agr Food Chem 2002;50:4934-4940.
14. Meirinhos J, Silva BM, Valentao P, Seabra RM, Pereira JA, Dias A, Andrade PB, Ferreres F:
Analysis and quantification of flavonoidic compounds from Portuguese olive (Olea europaea
L.) leaf cultivars. Nat Prod Res 2005;19:189-195.
Page 12 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
13
15. Tramontina VA, Machado MA, Nogueira Filho Gda R, Kim SH, Vizzioli MR, Toledo S: Effect
of bismuth subgallate (local hemostatic agent) on wound healing in rats. Histological and
histometric findings. Braz Dent J 2002;13:11-16.
16. Sadaf F, Saleem R, Ahmed M, Ahmad SI, Navaid-ul Z: Healing potential of cream containing
extract of Sphaeranthus indicius on dermal wounds in Guinea pigs. J Ethnopharmacol
2006;107:161-163.
17. Ehrlich HP, Hunt TK: The effect of cortisone and anabolic steroids on the tensile strength of
healing wounds. Ann Surg 1969;170:203-206.
18. Suguna L, Singh S, Sivakumar P, Sampath P, Chandrakasan G: Influence of Terminalia
chebula on dermal wound healing in rats. Phytother Res 2002;16:227-231.
19. Lodhi S, Pawar RS, Jain AP, Singhai AK: Wound healing potential of Tephrosia purpurea
(Linn.) Pers. in rats. J Ethnopharmacol 2006;108:204-210.
20. Ansari M, Dianat S, Kazemipour M, Adib N: Simple HPLC-UV method for the determination
of oleuropein in Olea europaea L. leaves extract and pharmaceutical preparations. National
Biotechnology Conference. AAPS J 2006;8:002590.
21. Blois MS, Antioxidant determinations by the use of a stable free radical. Nature 1958;
181:1199–1200..
22. Malik NSA, Bradford JM: Changes in oleuropein levels during differentiation and development
of floral buds in ‘Arbequina’ olives. Sci Hortic 2006;110:274–278.
Page 13 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
14
23. White ML, Heckler FR: Wound healing-oxygen free radicals and wound healing. Clin Plast
Surg 1990;17:473-484.
24. Goulas V, Exarchou V, Troganis AN, Psomiadou E, Fotsis T, Briasoulis E, Gerothanassis IP:
Phytochemicals in olive-leaf extracts and their antiproliferative activity against cancer and
endothelial cells. Mol Nutr Food Res 2009;53(5):600-608.
25. Shetty S, Udupa S, Udupa L: Evaluation of Antioxidant and Wound Healing Effects of
Alcoholic and Aqueous Extract of Ocimum sanctum Linn in Rats. Evid-Based Compl Alt
2008;5(1):95-101.
26. Pereira A, Ferreira I, Marcelino F, Valentão P, Andrade PB, Seabra R, Estevinho L, Bento A,
Pereira JA: Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv.
Cobranc¸ osa) leaves. Molecules 2007;12:1153-1162.
LEGENDS
Table 1. Activities of the extracts from Olea europaea on circular excision and linear incision wound
model
Table 2. In vitro Antioxidant activity of the n-hexane and aqueous leaf extracts of Olea europaea
Figure 1. Microscopic view of the section of 10 days old wound tissue treated with aqueous extract. (a)
intact epidermis; (b) collagen fibres; (c) fibroblast and (d) hair follicle.
Figure 2. Microscopic view of the section of negative control group (untreated) (a) area of ulceration;
(b) congested vessel; (c) mixed type inflammatory cells and (d) edema
Figure 3. Microscopic view of the section of 10 days old wound tissue treated with Reference material
Madecassol® (a) intact epidermis; (b) hair follicle; (c) fibroblast; (d) blood vessel and (e) collagen
fibers.
Figure 4. Determination of oleuropein (A) in the aqueuous extract (B) and n-hexane extract (C).
Page 14 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
TABLE 1. ACTIVITIES OF THE EXTRACTS FROM OLEA EUROPAEA ON CIRCULAR EXCISION AND LINEAR INCISION WOUND MODELS
Wound area ± S.E.M. (Contraction %)
Material 0 day 2 day 4 day 6 day 8 day 10 day 12 day
Statistical Mean ± S.E.M.
(Tensile Strength%)
Vehicle 20.07 ± 2.03 16.83 ± 1.87
(17.5)
13.52 ± 1.11
(19.9)
11.28 ± 1.34
(17.8)
9.40±0.97
(6.1)
7.37 ± 0.41
(16.6)
4.56 ± 0.20
(12.5)
16.66 ± 1.24
(4.5)
Negative Control 21.32 ± 2.63 20.41 ± 1.79 16.89 ± 1.82 13.72 ± 1.06 10.01 ± 0.27 8.84 ± 0.54 5.21 ± 0.16 15.94 ± 0.57
Aqueous extract 19.80 ± 1.85 13.71 ± 1.49
(18.5)
7.22 ± 1.30
(46.6)*
6.61 ± 0.95
(41.4)*
3.28 ± 0.36
(65.1)**
1.47 ± 0.08
(80.1)**
0.59 ± 0.02
(87.1)***
22.46 ±1.39
(34.8)*
n-Hexane extract 20.14 ± 1.97 13.95 ± 1.35
(17.1)
12.29 ± 1.12
(9.1)
8.33 ± 1.81
(26.2)
6.18 ± 0.41
(34.3)
4.95 ± 0.16
(32.8)
2.77 ± 0.14
(39.3) 16.03 ± 1.55
Madecassol® 19.26 ± 1.66 12.53 ± 2.17
(25.5)
6.23 ± 1.43
(53.9)**
3.93 ± 1.50
(65.2)**
2.39 ± 0.99
(74.6)**
0.40 ± 0.02
(94.6)***
0.00 ± 0.00
(100.0)***
25.18 ± 1.01
(51.1)***
* : p < 0.05; ** : p < 0.01; *** : p < 0.001; S.E.M.: Standart error meaning
Percentage of contraction and tensile strength values: Vehicle group was compared to Negative control group; The extracts and reference material were
compared to vehicle group
Page 15 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
TABLE 2. IN VITRO ANTIOXIDANT ACTIVITY OF THE n-HEXANE AND AQUEOUS LEAF
EXTRACTS OF OLEA EUROPAEA
*Values are mean ± Standard error of mean (SEM) of three replicates; BHT: Butylated hydroxytoluene
Scavenging Ability (%)
*
Material 2000 µg/mL-1 1000 µg/mL-1 500 µg/mL-1 100 µg/mL-1
BHT 72.23 ± 0.36 69.05 ± 0.13 67.38 ± 0.22 54.01 ± 0.47
Aqueous extract 63.59 ± 0.51 56.09 ± 0.06 53.06 ± 0.11 48.18 ± 0.63
n-Hexane extract 29.17 ± 0.06 14.73 ± 0.58 13.63 ± 0.11 13.18 ± 0.63
Page 16 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
Microscopic view of the section of 10 days old wound tissue treated with aqueous extract. (a) intact
epidermis; (b) collagen fibres; (c) fibroblast and (d) hair follicle.
119x77mm (300 x 300 DPI)
Page 17 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
Microscopic view of the section of negative control group (untreated) (a) area of ulceration; (b)
congested vessel; (c) mixed type inflammatory cells and (d) edema
179x115mm (72 x 72 DPI)
Page 18 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
Microscopic view of the section of 10 days old wound tissue treated with Reference material
Madecassol® (a) intact epidermis; (b) hair follicle; (c) fibroblast; (d) blood vessel and (e) collagen
fibers.
99x78mm (300 x 300 DPI)
Page 19 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For Peer Review
Determination of oleuropein (A) in the aqueuous extract (B) and n-hexane extract (C).
545x381mm (300 x 300 DPI)
Page 20 of 20
Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801
Journal of Medicinal Food
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
