Investigation of effects of fucoidan polysaccharides extracted from

Investigation of effects of fucoidan polysaccharides extracted from two species of Padina on
wound healing process in the rat
Moazameh KORDJAZI,1*, Bahareh SHABANPOUR1, Ebrahim ZABIHI2, Mohammad Ali
FARAMARZI3, Hassan AHMADI GAVLIGHI4, Seyed Mohammad Amin FEGHHI5, Seyed
Abbas HOSSEINI1, Fatemeh FEIZI2
1
Department of Fisheries, College of Fisheries and Environmental Sciences, Gorgan University
of Agricultural Sciences and Natural Resources, Gorgan, Iran, P.O. Box 49138–15739, Fax:
+981732424155. Email: [email protected]
2
Cellular and Molecular Biology research Center, Babol University of Medical Sciences, Ganje-
Afrooz Road, Babol, Iran
3
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology
Research Center, Tehran University of Medical Sciences, Tehran, Iran
4
Faculty of Agriculture, Department of Food Science and Technology, Tarbiat Modares
University, Tehran, Iran
5
Persian Gulf Biotechnology Park, Persian Gulf Biotechnology Research Center, Sooza Road,
Qeshm Island, Iran
1
Abstract
Seaweeds contain different types of polysaccharides with the high abilities. The aim of this study
was fucoidan polysaccharides potential for wound healing properties. Fucoidans of two species
of Padina (P. tetrastromatica and P. boergesenii), collected from Persian Gulf northern coastal
area, were extracted and analyzed using high performance anion exchange chromatography
(HPAEC). Purified fucoidans were used to prepare 2% topical ointment. Under aseptic
conditions, two identical paravertebral full- thickness skin wounds (either by burn or excision)
were made in 40 anesthetized male rats allocated in 4 groups. Either fucoidan ointments or the
vehicle (ointment base) were applied on rats’ wounds twice daily for 14 days. In addition to
wound area reduction (days: 3, 7, 10, and 14) and histopathological assays (days: 7 and 10),
tissue specimens were taken to laboratory. Fucoidans obtained from both seaweeds induced
significant wound area reduction compared to the vehicle at some time points after treatment.
Histopathological examinations showed improvement in some healing signs including
angiogenesis, collagen fiber formation, and epidermis formation. In conclusion, both species of
Padina have significant wound healing effects and fucoidan polysaccharides are the active
constituents for this healing property. However, fucoidan obtained from P. boergesenii showed
superior effects in this regard.
Keywords: Fucoidan polysaccharides, burn wound, excisional wound, wound healing, rat
2
1. Introduction
Brown algae or phaeophyceae are widely distributed tropical marine seaweeds which their
chemical constituents include: fucoidans, alginate and some other types of polysaccharides with
interesting biological activities (1, 2). Fucoidan are sulphated polysaccharides (composed of
fucose, glucose, xylose, galactose, etc.) existing in the brown seaweeds (3). They have molecular
structures similar to heparin and recently introduced as candidates for wound healing (4). Other
properties of these important seaweed constituents include: antioxidant, antithrombotic, antiinflammatory, antitumoral and antiviral effects (5, 6). Fucoidans obtained from different species
and geographical areas have different structures with potential diversity in their pharmacological
activities (7, 8). However, there is little information on their in vivo wound healing effects so far.
Wound healing, as a complex process, has been extensively subjected to numerous medical and
pharmacological studies in the recent decades. Three main phases in wound healing, after
primary cessation of bleeding (hemostasis) and clotting, are: inflammation, proliferation and
maturation (9). To improve wound healing process by a substance, at least one of these phases
must be enhanced. Skin burn and cut wounds are two common traumas which sometimes need
extensive care medicine at daily human life. The complexity of wound healing process in
addition to the shortage of effective medicines has initiated numerous investigations on new drug
candidates.
Marine products especially algae derivatives have shown many interesting activities and their
constituents have been investigated in medical studies since ancient times. Seaweeds have long
been recognized as rich and with valuable natural remedies because of their various biological
properties (10, 11). In the present study, fucoidan content of two main species of Padina (P.
tetrastromatica and P. boergesenii) collected from the northern coasts of Persian Gulf was
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extracted and purified which might have distinct pharmacological effects to be of benefit in
wound healing processes. Also, this study was designed to evaluate the histopathological
changes in rat’s burn and excisional skin wound models after topically administration of purified
fucoidans.
2. Materials and methods
2. 1. Raw material
P. tetrastromatica and P. boergesenii were collected from Kani (55º23 765 E and 26º34 344 N)
and Bahman (56º16 646 E and 26º57 645 N) ports at Qeshm island, Iran. Taxonomic
identification of the specimens was done in “Persian Gulf Biotechnology Park” (P.
tetrastromatica No. 66-20 p and P. boergesenii No. 44-14 p). After drying at room temperature,
the raw algae mass was powdered by a mechanical blender and later sieved to obtain uniform
powdered samples. Fresh samples were kept frozen at -20ºC for further analysis.
2. 2. Extraction of fucoidan polysaccharides
Fucoidan polysaccharides were extracted from powdered algae masses according to the
procedure of Yang et al. (12). Briefly, the dried samples (20 g) were extracted twice with
distilled water (400 mL) at 65ºC while stirring for 2 h. The obtained extracts were combined and
centrifuged at 13000 g for 10 min. The supernatant was mixed with 1% CaCl2 and kept at 4ºC
overnight to precipitate alginic acid. The solution was centrifuged at 13000 g for 10 min and
EtOH (96%) was added to the supernatant to reach the final concentration of 30%. The latter
solution was kept at room temperature for 4 h, and then centrifuged at 13000 g for 15 min. The
alcohol alcoholic concentration in the supernatant was increased to 70% and kept at 4ºC
overnight. Using a nylon membrane (0.45 µm pore size, Whatman International Ltd., UK), and
4
following washing with EtOH (96%) and acetone, the brown-yellowish creamy precipitates were
collected and dried at room temperature in a desiccator. The yield of fucoidan purification was
reported based on the primary dried samples weight.
2. 3. Sample preparation for high-performance anion-exchange chromatography with
pulsed amperometric detection (HPAEC-PAD)
Approximately 15-20 mg of purified fucoidans (200-500 µm particle size) was dissolved in
deionized water. Then, after adding 600 µL trifluoroacetic acid (TFA; 2 M), the apparatus was
heated at 121ºC in a drying oven for 2 h without stirring and allowed to cool off at room
temperature. Hydrolysates were lyophilized and kept at -20ºC until analysis. Prior to analysis by
HPAEC-PAD, the lyophilized sample hydrolysates were dissolved in 5 mL of double deionized
water. The prepared total fucoidans were filtered through 0.20 µm nylon syringe filters into the
sample vials used for the AS50 auto-sampling chromatography system.
2. 3. 1. Determination of carbohydrate composition
The HPAEC-PAD analysis of the samples fucoidans was performed as previously described by
Arnous and Meyer (13). The monosaccharide contents of the hydrolysates and standards were
determined using a BioLC system consisting of a GS50 gradient pump, an AS50 chromatograph,
an ED50 electrochemical detector coupled to an AS50 autosampler (Dionex Corp., Sunnyvale,
CA). The separation was performed using a CarboPacTM PA20 analytical column (3×150 mm)
(Dionex Corp., Sunnyvale, CA) and the eluent flow rate was 0.5 mL/min. The monosaccharides
were separated using a double eluents system consisting of deionised water and NaOH (500
mM). Neutral monosaccharides were eluted isocratically with 2.5 mM NaOH for 20 min
followed by a second isocratic elution for 10 min by higher NaOH concentration (500 mM) to
elute acidic monosaccharides (gulucronic). This high concentration of NaOH simultaneously was
5
used to wash the column. Before each injection (10 µL), a column re-equilibration program was
run for 5 min with 100 mM NaOH followed by another 5 min with 2.5 mM NaOH. Different
carbohydrate standards were mixed to resemble the matrix of the fucoidans lysates. Data
collection was performed using the following pulse potentials/durations for detections: E1¼0.1
V, t1 ¼400 ms; E2¼_2 V, t2¼ 20 ms; E3 ¼ 0.6 V, t3 ¼10 ms; E4¼ 0.1 V, t4 ¼70 ms, datacollection rate: 0.2 Hz (13).
2. 3. 2. Determination of fucoidans sulfate content
One of the important characteristics of fucoidans is the percentage of their sulfate contents which
has been determined by the following procedure. From 1.1 ml of the fucoidan aqueous solution
(0.05%), 1.2 ml of 8% trichloroacetic acid (TCA) was added and mixed well. Then 0.6 ml of the
appropriate agarose-BaCl2 reagent (0.02% and 0.5%), was added and let stand for 35 min (at
which time turbidity reaches to its maximum and remains stable for over an hour). Then after
shaking the samples, the optical density was read at 500 nm using a spectrophotometer (14).
2. 4. Fucoidan ointments preparation
After dissolving 750 mg fucoidan powder in 4 ml distilled water, 2% ointments of each fucoidan
sample was prepared using Eucerin® which served as ointment vehicle for the preparation of the
negative control as well.
2. 5. Animals
The study was in accordance with European community guidelines and was approved by the
Animal Ethics Committee of Babol University of Medical Sciences (BUMS). A total of 40 male
Wistar rats (Rattus norvegicus) were used, with body weights range of 200 to 250g, obtained
from the Animal Center of BUMS. The animals’ general health conditions were examined at the
beginning, and received food and water ad libitum. The animals were housed individually in
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transparent polystyrene cages. They were kept at conditioned room temperature (22±2ºC) and a
12-h photoperiod.
2. 5. 1. Burn and excisional wound models
Experimental groups were divided randomly into 4 groups of 10: First group was topically
treated with P. tetrastromatica (right: treatment, left: control) for burn wound, second group was
topically treated with P. boergesenii (right: treatment, left: control) for burn wound, third group
was treated with P. tetrastromatica (right: treatment, left: control) for excision type of wound
and four group was treated with P. boergesenii (right: treatment, left: control) for excisional
wound. To full-thickness burn wound,two equal sized bilateral wounds were created at the back
of rats(n=20) underanesthesia with pentobarbital (50 mg/kg; i.p.). Briefly, using an electric hair
clipper, the rats’ back fur was removed, wiped with warm distilled water (37ºC) and
consequently 70% ethanol to clean the wounds. Using boiling water, a customized iron rod (with
10 mm cut diameter) was heated up to 96ºC, then applied on the shaved rats’ skin for 20s to
cause a 2nd degree burn wound (15).
To produce excisional wound, a standard size (2×2 cm) skin excision was made on rats’ back (n=
20) at each side (right= treatment and left= control) under anesthesia. The fucoidans ointments or
250 mg Eucerin® alone (the ointment base for the control wound) were applied on the lesions
twice a day for 2 weeks (5 days per week) (16).
2. 6. Wound area reduction
All the rats were examined for wound area reduction under anesthesia. The burn or excisional
wounds area was measured manual using a transparent grid at the days 3, 7, 10 and 14
(planimetry method). The wound area reduction was expressed as reduction in percentage of
original wound size (17).
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% wound reduction rate on day-X= [(area on day 0 mm2 − open area on day Xmm2) / area on day
0mm2] × 100
2. 7. Histopathological examination
From each group, five rats on the day 7 and the rest of animals (5 rats) on the day 14 were
euthanized using pentobarbital overdose and the whole wound area skin was removed for
histopathological examination.
The tissue samples (after fixing in formalin and dehydrating by ethanol and xylol) were
embedded in paraffin, sectioned at 5 µm thicknesses, deparaffinized and stained with
hematoxylin and eosin. The stained tissue glass mounted slides were examined using a light
microscope for histopathological alteration in wounds samples.
2. 8. Statistical Analysis
The results were expressed as mean ± standard deviation (SD). All statistical analysis was
performed using the SPSS software package, version 18 (SPSS Inc., Chicago). Student’s paired
and independent t-test was used to test significance of difference between means. p< 0.05 was
interpreted as significant difference between means.
3. Results
3. 1. Fucoidans extraction and compositional analysis
The yields of fucoidan extraction for P. tetrastromatica and P. boergesenii were calculated to be
1 ± 0.5% and 4.5 ± 0.5%, respectively. Fucoidan compositions were mainly made up of fucose
and galactose with significant level of sulfate. As shown in table 1, other monosaccharides
(mainly rhamnose) were minor in both fucoidan samples. Two fucoidan samples showed similar
8
sulfation level and monosaccharide types, however the fucose and glucose content of P.
tetrastromatica was significantly higher than P. boergesenii (Table 1).
3. 2. Effects on wound area reduction
As shown in Figures 1 and 2, the P. boergesenii topical ointment showed significant effects
(P<0.05) on wound area reduction in both burn and excisional wounds. This effect was
observable in excisional wounds from day 3 and reach to its maximum at day 7 (Figure 2). On
the other hand, P. tetrastromatica topical ointment showed significant burn wound area
reduction only on day 7 and the less healing effect was compared to P. boergesenii in excisional
wound model (Figure 2). As shown in Figures 4 and 6, this effect was more obvious with P.
boergesenii specifically in excisional wound model (P<0.05) wound area reduction. With the
burn wounds, the scabs started to come off especially at the boundaries of the wound surface
manifesting a pink scar underneath (Figure 3).
3. 3. Effects on histopathology of the wounds
As shown in Figures 5 and 6, microscopic examinations of wound samples treated by both algal
fucoidans showed marked changes in healing processes. These changes include early fibroblasts
appearance in the wounds' nearby tissues specially on day 7, hair follicles and collagen fibers
formation across the wounded skin, increase in epidermis thickness on day 14 and early
inflammatory cells disappearance in the wound area (Figures 5 and 6).
4. Discussion
The search for new materials with high efficacy of wound healing among natural resources has
been the pillar of many investigations in recent decades. Marine biological products, as a
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potential source of pharmacologically active agents, have attracted the attention of many
researches (18).
In this study, fucoidans compositional analysis showed that the extraction/purification processes
have been quite efficient in isolating the fucose containing sulfated polysaccharide (Table 1).
The yields of fucoidan extraction and purification for both seaweeds were higher than 1% (up to
4.5% for P. boergesenii) which were close to other reported brown seaweeds fucoidan content
values (1.1 – 4.8%) (6, 19). On the other hand, the sulfation extents for both purified fucoidans
are relatively higher than the previously reported fucoidans which is a key factor for their
bioactivities. The position of sulfate group is another important issue that determines the
fucoidan biological properties (20).
Significant wound area reductions, induced by both algal fucoidans, were observable at some
time points (from day 3 to day 14) (Figures 3 and 4). Also, the microscopic findings revealed
improvement in wound healing processes by both algal fucoidans which include shortening the
angiogenesis stage, early fibroblast proliferation, increasing hair follicles and epidermis
formation (Figures 5 and 6).
Fucoidans extrcated from different algae species have shown broad range of pharmacological
activities including antioxidant, antiviral, anticoagulant, and immunomodulating properties (6, 7,
11, 20). The members of Padina genus algae are reach in fucoidans, which have shown several
pharmacological activities (21, 22). For example, as a constituent of a moisten wound dressing,
fucoidans have successfully improved wound healing process, by increasing inflammatory cells
mobilization, in a rat impaired wound healing model (4).
Wound area reduction is an important step in skin wound healing process especially in excisional
wound models (9). In our study the P. boergesenii extractedfucoidans showed significant wound
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area reduction improvement (better than P. tetrastromatica), especially in excisional model
(P<0.05) (Figures 1 and 4). The exact mechanism behind this contraction is not fully understood
but it seems that a complex interaction between contractile fibroblasts called "myofibroblasts"
and the matrix components may be involved (23). An improvement in the matrix formation and
fibroblast migration into wound area, following the primary inflammatory phase, might be
helpful in this step. Due to histopathological differences, contraction changes in excisional
wounds are much more informative than burn wounds (24) and our data with P. boergesenii
support this fact (Figures1 and 2).The sulfate content of P. boergesenii extracted polysaccharides
(32.6 ± 1%) was quite higher than P. tetrastromatica (19 ± 1%). The higher sulfate content
might be attributed to the better wound healing properties (25).
The purified fucoidans may be involved in enhancing the paracrine cellular interactions by
inducing growth factors production or their stabilization, much more like heparin activity (26,
27). Previously, it has been shown that fucoidans bind to some growth factors (e.g. HGF, FGFs,)
and stabilize them (28).
Neovascularization in wound healing process is secondary to secretion of important growth
factors like vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF),
and TGF-β (9). Some investigators believe that angiogenesis is the rate limiting step in wound
healing process (29). An enhanced phase of proliferation might lead to faster angiogenesis and
by consequent improvement in wound remodeling phase, the microscopically observed
vasculatures disappear sooner than in a delayed healing process. It has been shown that basic
fibroblast growth factor (FGF-2), a well known cytokine with mitogenic properties for
fibroblasts and vascular endothelial cells, could induce angiogenesis in vivo (20). In concordance
with this hypothesis, we observed that the extent of vascular beds decreased in histopathologic
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samples obtained from excisional and burn wounded animals treated by both algae fucoidans
(Figures 5 and 6).
Improvement in epithelium formation was observable with both algae fucoidans (Figures 5 and
6). Successful excisional wound re-epithelization depends on the combination of factors
including efficient proliferation and migration of epidermal progenitor cells from the edge of
injured skin and hair follicles (9). Both algae fucoidans might have shown their stimulating
effects on epithelium primarily by improving the fibroblast migration into the wound area and
consequent paracrine effects.
5. Conclusion
We have shown, for the first time that fucoidans purified from two Persian Gulf’s habitats brown
algae (P. boergesenii and P. tetrastromatica) are rich in sulfate and have healing properties on
full-thickness burn and excisional wound models in rat. The topical ointment made of P.
boergesenii’s fucoidan seems to be more effective than P. tetrastromatica especially in
excisional wound healing models, that causes of this might be correlated to its higher sulfate
content.
Acknowledgement
The authors wish to thank the Iran’s Persian Gulf Biotechnology Park (PGBP) staff for their
cooperation in the algae samples collection and the Tehran University of Medical Sciences’
(TUMS) Department of Pharmaceutical Biotechnology for the preparation of the fucoidans and
ointment formulation. This study was funded by a research grant from “Gorgan University of
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Agricultural Sciences and Natural Resources” and “Deputy of Technology and Research - Babol
University of Medical Sciences” (grant No: 9135017).
Conflict of Interest:
There is no conflict of interest.
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Table 1. Monosaccharides and sulfate compositions (mean±SD) (%) of fucoidans derived from P. tetrastromatica
and P. boergesenii
Monosaccharides/Sulfate Compositions (%)(DW)
Samples
Fuc
Rha
Gal
P. tetrastromatica
29.65±3.33
1.09±0.16
17.28±1.01
P. boergesenii
10.72±1.29
3.90±0.58
18.23±2.22
Glc
11.35±0.3
5
4.87±0.51
*Monosaccharide compositions: Fuc=fucose, Rha=rhamnose,
Man=mannose, Glu Acid=glucuronic acid, DW=Dried weight
16
Xyl
Man
Glu Acid
Sulfate
5.63±0.24
4.54±0.36
8.89±0.1
19±1
5.26±0.87
3.99±0.44
7.22±0.89
32.6±1
Gal=galactose,
Glc=glucose,Xyl=xylose,
Figure 1. Burn wound area reduction (%) after application of two algal fucoidans ointments in the rats (CTL:
Control, Pb: P. boergesenii; Pt: P. tetrastromatica). Each column represents Mean ± S.D. * P< 0.05 (compared
to corresponding CTL group)
Figure 2. Excisional wound area reduction (%) after application of two algal fucoidans ointments in the rats
(CTL: Control, Pb: P. boergesenii; Pt: P. tetrastromatica). Each column represents Mean ± S.D. *P<0.05
(compared to corresponding CTL group)
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Figure 3. The photographs of burn wounds area treated by two different algal fucoidans ointment on days 3,
7, 10 and 14. A: Control wounds corresponding to P. tetrastromatica fucoidans ointment, B: Wounds treated
by P.tetrastromatica, C: Control wounds corresponding to P. boergesenii, D: Wounds treated by P.
boergesenii. Both algal fucoidans ointments have significant effects on wound area reduction (wound area
reduction). Specifically P. boergesenii shows significant wound healing effects at the days 7, 10, and 14.
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Figure 4. The photographs of excisional wounds area treated by two different algal fucoidans ointment on
days 3, 7, 10 and 14. A: Control wounds corresponding to P. tetrastromatica fucoidans ointment, B: Wounds
treated by P. tetrastromatica, C: Control wounds corresponding to P. boergesenii, D: Wounds treated by
P.boergesenii. Both algal fucoidans ointments have significant effects on wound area reduction (wound area
reduction). Specifically P. boergesenii shows significant wound healing effects at all days after the treatment
(P<0.05) while P. tetrastromatica show less healing effects in this model at the days: 3, 7, and 14 (P<0.05).
19
20
Figure 5. Light microscopic examination of rats’ burn wounds after 7 days (A, B, C, D) or 14 days (E, F, G,
H) treatment with either P. boergesenii (B, F), or P. tertrastromatica (D, H) or vehicle (A, C, E, G)
A)
Control (the ointment base) treated burn wound after 7 days: Necrotic tissues and scab are still
observable on the wound surface with profound neovascularization (BV=blood vessles), minimal epidermis
and collagen fibers formation.
B)
P. boergesenii treated burn wound after 7 days: Obvious epithelium formation with some growing
hair follicles (Fol) and fibroblast cells (Fib) are observable. Collagen fibers started to reappear in the deeper
tissues.
C)
Control (the ointment base) treated burn wound after 7 days: A thin epithelium layer has been
formed with obvious neovascularization (BV=blood vessels) in under layer tissue and fibroblast (Fib) cells.
Collagen fibers are not formed yet
D)
P. tetrastromatica treated burn wound after 7 days: A thin epithelium layer has covered the wound
surface. Fibroblast cells (Fib) are observable in the wound under layer tissue with scanty hair follicles (Fol)
and minimal collagen fibers formation
21
E)
Control (the ointment base) treated burn wound after 14 days: Epithelium layer has covered the
wound surface along with numerous fibroblast cells (Fib) and some macrophages (Mac), however. Collagen
fibers and other skin structures formation are not completed yet
F)
P. boergesenii treated burn wound after 14 days: The wound surface is completely covered by
epithelium, hair follicles (Fol) and matured collagen (Col) fibers formation are observable
G)
Control (the ointment base) treated burn wound after 14 days: Most of the wound surface is covered
by new epithelium (NE) with remained some scab on the wound surface and extensive deeper tissues
neovascularization (BV=blood vessles)
H) P. tetrastromatica treated burn wound after 14 days: Complete skin formation with normal epithelium,
hair follicles (Fol) and collagen fibers
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Figure 6. Light microscopic examination of rats’ excisional wounds after 7 days (A, B, C, D) or 14 days (E, F,
G, H) treatment with either P. boergesenii (B, F), or P. tertrastromatica (D , H) or vehicle (A, C, E, G)
A)
Control (the ointment base) treated excisional wound after 7 days: Wound surface still covered by
blood clot and the epithelium layer is not complete. Inflammatory cells and Oedema (Od) are still observable
in wound under layer tissue
B)
P. boergesenii treated excisional wound after 7 days: Obvious epithelium formation with some
fibroblast (Fib) along with few inflammatory cells are observable
C)
Control (the ointment base) treated excisional wound after 7 days: A thin epithelium layer has been
formed with obvious neovascularization (BV=blood vessel) and
some inflammatory cells (i.e
macrophage=Mac) in wound area
D)
P. tetrastromatica treated excisional wound after 7 days: Epithelium layer has efficiently covered the
wound surface, though some inflammatory cells (i.e. macrophage=Mac), neovascularization (BV=blood
vessels) and collagen fibers formation are still observable
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E)
Control (the ointment base) treated excisional wound after 14 days: Epithelium layer has completely
covered the wound surface with numerous fibroblast cells (Fib). A reduction in the neovascularization
(BV=blood vessels) and some collagen fibers (Col) formation are observable.
F)
P. boergesenii treated excisional wound after 14 days: Epithelium layer has completely covered the
wound surface with a reduction in the neovascularization. Some hair follicles (Fol) and collagen fibers (Col)
formation are observable.
G)
Control (the ointment base) treated excisional wound after 14 days: The wound surface is covered by
epithelium but some inflammatory cells along with fibroblasts (Fib) and neovascularization (BV=blood
vessels) are observable
H) P. tetrastromatica treated excisional wound after 14 days: A typical normal epithelium with keratin layer
along with well organized collagen fibers (Col) and hair follicles (Fol) are observable
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