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Doctoral Dissertation
Effects of Pycnogenol and Saponin
on Skin Wound Healing
Department of Veterinary Medicine
Graduate School, Chonnam National University
KIM, Youngsoo
August 2012
Effects of Pycnogenol and Saponin
on Skin Wound Healing
Department of Veterinary Medicine
Graduate School, Chonnam National University
KIM, Youngsoo
DirectedbyProfessorBAE,Chunsik
A dissertation submitted in partial fulfillment of the requirements
for the Doctor of Philosophy in Veterinary Medicine.
CommitteeinCharge:
KANG,Seongsoo
JEONG,Moonjin
CHO,Ikhyun
KIM,Sungho
BAE,Chunsik
August 2012
- i -
CONTENTS
List of Figures ...................................................................................... ⅱ
ABSTRACT ............................................................................................ 1
INTRODUCTION .................................................................................... 4
MATERIALS AND METHODS .............................................................. 8
1. Animals ........................................................................................... 8
2. Animal model of incised skin wound .......................................... 8
3. Pycnogenol (PYC) ......................................................................... 9
4. Total ginseng saponin (TGS) ....................................................... 9
5. Histological analysis ..................................................................... 9
6. Statistical analysis ........................................................................ 10
RESULTS ............................................................................................... 11
1. Effects of Pycnogenol on Skin Wound Healing ............................ 11
1.1. Wound healing tendencies after pycnogenol treatment ........... 11
1.2. Histological and morphometric analysis of the skin wound
healing following pycnogenol treatment .................................... 13
1.3. Histological analysis of inflammatory cells and wound
contraction rate during the skin wound healing process ....... 17
1.4. Substrate formation analysis in the wound healing process
........................................................................................................ 21
2. Effects of Saponin on Skin Wound Healing ................................... 24
2.1. Wound healing tendencies after saponin treatment .................. 24
2.2. Histological and morphometric analysis of the skin wound
healing following saponin treatment .......................................... 26
2.3. Histological analysis of inflammatory cells and wound
contraction rate during the skin wound healing process ....... 30
2.4. Substrate formation analysis in the wound healing process
........................................................................................................ 34
DISCUSSION .......................................................................................... 37
CONCLUSION ........................................................................................ 46
REFERENCES ........................................................................................ 47
ABSTRACT (KOREAN) ......................................................................... 54
- ii -
List of Figures
Figure 1. Chemical structures of the main procyanidins present in the
pycnogenol extract and their possible oligomeric assembly ...... 5
Figure 2. Chemical structure of Saponin extracted from Panax
Ginseng ................................................................................... 6
Figure 3. Differences of dermal wound size between control and
pycnogenol-treated groups ................................................... 12
Figure 4. Differences in wound area between control and
pycnogenol-treated groups during skin wound healing ..... 14
Figure 5. Differences in wound area between control and
pycnogenol-treated group ..................................................... 15
Figure 6. Keratinocyte migration in wound tissue .............................. 16
Figure 7. Differences in inflammatory cell recruitment between
control and pycnogenol-treated skin wounds .................... 18
Figure 8. Inflammatory cells count analysis of wound tissues ......... 19
Figure 9. Wound contraction measurements between control and
pycnogenol-treated groups ................................................... 20
Figure 10. Difference of accumulated collagen in healing areas
between control and pycnogenol-treated groups .............. 22
Figure 11. Difference in accumulated collagen in the healing areas
of control and pycnogenol-treated groups ......................... 23
Figure 12. Differences of dermal wound size between control and
saponin-treated groups ......................................................... 25
Figure 13. Differences in wound area between control and saponintreated
groups during skin wound healing ......................... 27
Figure 14. Differences in wound area between control and
saponin-treated group ........................................................... 28
Figure 15. Keratinocyte migration in wound tissue .............................. 29
Figure 16. Difference in inflammatory cell recruitment between
control and saponin-treated skin wounds ........................... 31
Figure 17. Inflammatory cells count analysis of wound tissues ......... 32
Figure 18. Wound contraction measurements between control and
saponin-treated groups ....................................................... 33
Figure 19. Difference of accumulated collagen in healing areas
between control and saponin-treated groups ..................... 35
Figure 20. Difference in accumulated collagen in the healing areas
of control and saponin-treated groups ............................... 36
- 1 -
Effects of Pycnogenol and Saponin
on Skin Wound Healing
KIM, Youngsoo
Department of Veterinary Medicine
Graduate School, Chonnam National University
(Supervised by Professor BAE, Chunsik)
(Abstract)
Cutaneous wound healing involves various cell types for the renewal
of damaged tissues, where strict regulation of soluble and insoluble
factors is required. The medical agents and antibiotics currently used
for wound healing have side effects, such as allergy and potential
infection by super bacteria. Therefore, much effort has been focused
on the investigation of natural plant extracts to replace artificial
chemicals for the application in wound healing. Pycnogenol is a
water-soluble extract of French maritime pine bark that consists of
several types of phenolic compounds and is known to have
antioxidants and anti-inflammatory effects. Also, saponins have been
reported to confer strong anti-inflammatory effects, leading to a
decrease in neutrophilic infiltration.
The purpose of this study was to investigate the effects of
- 2 -
pycnogenol and saponin on the cutaneous wound healing process via
histological analysis. A 48 heads of ICR mice, 6 weeks old were used
for all in vivo experiments. These mice were divided a control,
pycnogenol-, or saponin-treated groups after inducing four equidistant
1 cm full-thickness incisional wounds on the dorsum. The wound site
samples were extracted on days 1, 3, 5, and 7 after induction of
wounding and histomorphometrical analysis was performed, such as
measurement of the wound area, the keratinocyte migration rate,
calculation of infiltrated inflammatory cells, and measurement of wound
contracture.
As a result, the wound areas of the pycnogenol-treated group were
larger than the control group on days 1 to 7, and the keratinocyte
migration rates of the pycnogenol-treated group were lower than the
control group on days 3 to 7. The number of inflammatory cells of the
pycnogenol-treated group was higher than the control group on days 3
to 7. The wound contraction of the pycnogenol-treated group was
lower than the control group on days 1 to 5 but higher on day 7.
Collagen deposition in the pycnogenol-treated group was less than in
the control group but greater than the control group on day 7.
The wound areas of the saponin-treated group were smaller than the
control group on days 3 to 7, and the keratinocyte migration rates of
the saponin-treated group were higher than the control group at day 3
to day 7. The number of inflammatory cells from the saponin-treated
group on day 1 and 3 was lower than the control group. The wound
contraction of the saponin-treated group was higher than the control
group on days 3 to 5. Collagen deposition in the saponin-treated group
- 3 -
was greater in the control group during wound healing.
According to our results, it appears that pycnogenol does not
promote re-epithelialization of the wound site but inhibits the initial
inflammatory response and promotes the substrate synthesis of the
second half of the skin wound recovery process. Also, saponin
promotes re-epithelialization of the wound site and effectively inhibits
the initial inflammatory response and promotes substrate synthesis.
Altogether, we conclude that saponin shows higher efficacy during
the wound recovery process after skin incision.
- 4 -
INTRODUCTION
The recovery process of skin wounds involves complex biological
mechanisms but can be essentially classified into three phases: 1)
inflammatory, 2) granulation formation as the result of cell
proliferation, and 3) tissue remodeling (Stephen and Thomas, 2002).
Many cell types and their interactions are responsible for these
process, including inflammatory cells, fibroblasts, and keratinocytes
which induce microenvironmental changes at the wound site (Wrener et
al., 2007). Tissue remodeling of the dermis involves collagen as well
as antioxidants and a balance of matrix-producing proteins and
protease enzymes essential for the initiation of tissue remodeling
during the recovery process of skin wounds (Blazso et al., 2004).
Pycnogenol (PYC), an aqueous extract of the bark of P. pinaster
(formerly known as P. maritima), is primarily composed of procyanidins
and phenolicacids. Procyanidins are biopolymers of catechin and
epicatechin subunits, which are recognized as important constituents in
human nutrition (Packer et al., 1999) (Fig. 1). PYC contains a wide variety
of procyanidins that range from the monomeric catechin and taxifolin
to oligomers with 7 or more flavonoid subunits. The phenolic acids are
derivatives of benzoic and cinnamic acids (Rohdewald, 2002). PYC
protects against oxidative stress in several cell systems by doubling
the intracellular synthesis of anti-oxidative enzymes and by acting as a
potent scavenger of free radicals (Berryman et al., 2004). Pycnogenol
has multiple biological effects, including antioxidant, anti-inflammatory
and anti-carcinogenic properties (Sime et al., 2004).
- 5 -
Fig. 1. Chemical structures of the main procyanidins present in the
pycnogenol extract (top) and their possible oligomeric assembly (bottom).
Protection against UV-radiation-induced erythema was found in a
clinical study following oral administration of PYC (Blazsóet al., 1997).
PYC can inhibit the expression of the proinflammatory cytokine
interleukin (IL)-1β by regulating redox-sensitive transcription factors
(Cho et al., 2000), leading to significantly curtailing the wound healing
time when applied topically to experimental wounds inflicted by
branding iron to healthy rats (Blazsóet al., 2004).
- 6 -
Fig. 2. Chemical structure of Saponin extracted from Panax Ginseng
Saponins are compounds extensively found in most plants that exist
in a variety of types and are classified based on their internal
structure (Guclu-Ustundag and Mazza, 2007) (Fig. 2). Saponins have
been reported to accelerate numerous biological activities including
hemolytic (Baumann et al., 2000; Oda et al., 2000), anti-bacterial
(Killeen et al., 1998; Konishi et al., 1998), anti-viral (Simoes et al.,
1999; Apers et al., 2001), and anti-oxidative functions (Yogeeswari and
Sriram, 2005). In addition, saponins were also observed to have
anti-inflammatory effects, reducing edema and skin inflammation (Just
et al., 1998; Navarro et al., 2001). A saponin extracted from ginseng,
known as ginsenoside, has been shown to accelerate neovascularization
in burn wounds of the skin in mice and to increase vascular
endothelial growth factor and IL-1β, inflammatory cytokines known to
induce the accumulation of macrophages at skin wound sites and
accelerate wound healing (Kimura et al., 2006).
- 7 -
However, the recovery pattern of skin wounds caused by burns varies
markedly from that caused by physical injury or surgery (Wang et al.,
2008). Pycnogenol and saponin have been shown to be effective in the
wound healing process; however, information on surgical skin wounds
is little to lacking. Thus, this study was aimed at investigating the
effects of pycnogenol and saponin during the healing process at
surgically induced skin wounds using histochemical and
histomorphometric analysis.
- 8 -
MATERIALS AND METHODS
1. Animals
Male ICR mice (n=48) weighing 45 to 55 g were purchased from
Samtako (Osan, Korea). The mice were quarantined and acclimated for
one week prior to use. The animals were housed in polycarbonate
cages in a room maintained at 23±2°C, a relative humidity of 50±5%,
artificial lighting from 08:00 to 20:00 h, and 13 to 18 air changes per
hour. Tap water and commercial rodent chow (Samyang Feed, Daejeon,
Korea) were provided ad libitum. The animals were blindly randomized
into control (n=24), pycnogenol (PYC, n=12) and total ginseng saponin
(TGS, n=12) groups.
2. Animal model of incised skin wound
The animal model with an incised skin wound has been established
and described previously (Guan et al., 2000). Briefly, 6-week-old male
ICR mice were anesthetized by intraperitoneal injection of xylazine
(Rompun®, 2 mg/kg; Bayerkorea, Seoul, Korea) and zolazepam/
tiletamine (Zoletil 50®, 0.1 mg/kg; Virbac, Seoul, Korea). A scalpel was
used to generate four 1-cm-long incisions on the central dorsum skin
layer. Specimens (1.5×2 cm2) were taken from the wound sites while
anesthetized on days 1, 3, 5, and 7 post-wounding (n=3/group).
Experiments conformed to the ‘Principles of Laboratory Animal Care
(National Institutes of Health publication no. 85-23, revised 1985)’ and
sought to minimize both the number of animals used and any suffering
that might be experienced and were performed according to the
- 9 -
Guidelines for the Care and Use of Laboratory Animals of Chonnam
National University.
3. Pycnogenol (PYC)
Pycnogenol was provided by the Horphag Research Ltd., France. The
PYC (1 mg/50 μL/wound) was dissolved in saline and administered once
subcutaneously prior to making the surgical skin wound in the treated
group.
4. Total ginseng saponin (TGS)
TGS was provided by the Korean Ginseng Research Institute, Korea
Ginseng Corporation (KGC, Daejeon, Korea). The provided TGS
contained 11 glycosides known as ginsenosides: Rb1 (15.82%), Rb2
(7.79%), Rc (8.06%), Rd (7.57%), Re (3.21%), Rf (4.72%), Rg1 (1.91%),
Rg2 (22.08%), Rg3 (24.06%), Rh1 (4.63%), and Rh2 (0.15%). The TGS
(1 mg/50 μL/wound) was dissolved in saline and administered once
subcutaneously prior to making the surgical skin wound in the treated
group.
5. Histological analysis
To compare changes between the groups, mice were euthanized on
days 1, 3, 5, and 7 post-wounding. Pictures of the wounds were taken
with a digital camera (DMCFZ5GD; Panasonic, Kadoma, Japan) after
attaching a ruler to the skin wound area. Skin samples from the wound
area were taken and kept in 4% formaldehyde in phosphate-buffered
saline (PBS, pH 7.4) for 24 h, then washed with PBS for 2 h,
- 10 -
dehydrated using graded alcohol, clarified, and finally embedded into
paraffin. The embedded samples were cut into 6-μm-thick sections
with a motorized rotary microtome MT990 (RMC Products, Tucson, AZ,
USA). Hematoxylin and eosin (H&E) staining was performed to
measure the size of the wound area and detect any histomorphometric
changes. To analyze the number of inflammatory cells, Giemsa staining
was performed. To analyze the collagen in the matrix formation,
Masson’s trichrome and picrosirius red staining were used. In the
wound area, measurements were made at the epithelium as well as the
marginal area of the connective tissue. The number of inflammatory
cells was counted at the left, right, and center of the wound area. The
keratinocyte migration rate was determined by measuring the size of
the epithelial tissue rising from the edge of the wound, while the
wound contraction was calculated by measuring the distance between
the edge of the wound and the damaged dermal layer. Stained tissue
samples were observed using a polarizing microscope (Carl Zeiss,
Goettingen, German), and the AxioVisionLE release 4.6 (Carl Zeiss)
image analysis program was used for all analyses.
6. Statistical analysis
Data presented are mean values from three animal ± SD. Statistical
analysis of the data was performed using a Student’ t-test with a
Bonferroni correction for analysis of multiple comparisons. Differences
were considered significant at p<0.05.
- 11 -
RESULTS
1. Effects of Pycnogenol on Skin Wound Healing
1.1. Wound healing tendencies after pycnogenol treatment
To determine the effect of pycnogenol on wound healing, changes in
the wound size were recorded on days 1, 3, 5, and 7 after wound
induction. It was evident that healing was taking place over time
because wounds were being reduced in both the control and the
pycnogenol-treated groups.
In the pycnogenol-treated group, there were no significant changes
in the wound size compared to the control group until day 5. On day
7, the wound size of the control group appeared to reduce more in
size (Fig. 3).
- 12 -
Fig. 3. Differences in the dermal wound size between the control and
the pycnogenol-treated groups. The wound size gradually diminished in
both groups from days 1 to 7. However, there were no differences
between the two groups until day 7.
- 13 -
1.2. Histological and morphometric analysis of the skin wound healing
following pycnogenol treatment
H&E staining was conducted for wound tissues extracted in each
period after inducing skin wounds. Most wound tissues of the control
group appeared to be collapsed and lost on day 1, and scabs caused
by blood clots and fibrous tissues were observed. Removal of scabs
gradually progressed until day 5, and the movement of epithelial cells
was confirmed at this time and most dermal residues disappeared. On
day 7, scabs were completely eliminated, and the wound part was
completely blocked with the outside skin due to re-epithelialization.
Also, the wound site including the derma was markedly reduced
compared to day 1. The pycnogenol-treated group showed a pattern
similar to the control group until day 3 after the wound induction, but
scabs were not removed until day 5 and movement of epithelial cells
was also observed to be lower than that of the control group (Fig. 4).
The wound measurements showed that the wound area of the control
group increased slightly on day 3 compared to day 1, albeit not in a
significant manner, and it decreased on day 5. On day 7, the wound
area decreased significantly. In the pycnogenol-treated group, the
wound size decreased from days 1 to 7, but the reduction in size
decreased on day 7 (Fig. 5).
In regards to the keratinocyte migration rate, both the control and
the pycnogenol-treated groups demonstrated an increasing tendency.
On day 1, however, both the control and the pycnogenol-treated
groups were similar but the pycnogenol-treated group showed lower
values than those of the control group at all other times (Fig. 6).
- 14 -
Fig. 4. Differences in the wound area between the control and the
pycnogenol-treated groups during skin wound healing. There were no
differences between the groups until day 3. However, scar tissues
were observed in the pycnogenol-treated group on day 5. Scale
bar=200 μm
- 15 -
Fig. 5. The wound area of the pycnogenol-treated group was larger
than the control group on days 1 to 7, based upon morphometric
analysis of the wounds tissues from the control and the pycnogenoltreated
groups.
- 16 -
Fig. 6. Keratinocyte migration in the wound tissue. The keratinocyte
migration rate in the pycnogenol-treated group was lower than that of
the control group on days 3 to 7.
- 17 -
1.3. Histological analysis of inflammatory cells and wound contraction
rate during the skin wound healing process
To compare the number of inflammatory cells introduced in the
wound site, tissues that caused wounds were subjected to Giemsa
staining (Fig. 7), which showed the highest number of inflammatory
cells in the control group on day 1. But after day 3, up to day 7, the
number declined sharply.
It could be observed that in the pycnogenol-treated group, there
were less inflammatory cells than in the control group only on day 1,
but it maintained more inflammatory cells than those of the control
group from days 3 to 5, and the number increased on day 7 (Fig. 8).
Also, both the control and the pycnogenol-treated groups showed an
increasing tendency in the analysis of wound contraction. The degree
of contraction in the pycnogenol-treated group was lower than that of
the control group from days 1 to 5 but appeared higher on day 7 (Fig.
9).
- 18 -
Fig. 7. Differences in the recruitment of inflammatory cells between
the control and the pycnogenol-treated skin wounds. Inflammatory cells
in the pycnogenol-treated wounds were increased from days 3 to 7,
compared to the control wound tissue. All tissues were subjected to
Giemsa staining. Scale bar=20 μm
- 19 -
Fig. 8. The inflammatory cell count of the wound tissues. Inflammatory
cells were counted in three distinctive areas (left, right, and middle of
the wound area). The number of inflammatory cells in the
pycnogenol-treated group was lower than the control group on day 1.
However, from days 3 to 5, the number of inflammatory cells of the
pycnogenol-treated group was higher than the control, and on day 7
inflammatory cells of the pycnogenol-treated group were more
increased than the control group.
- 20 -
Fig. 9. Wound contraction in the pycnogenol-treated group was lower
than that of the control group on days 1 to 5. However, wound
contraction in the pycnogenol-treated group was higher than in the
control group on day 7.
- 21 -
1.4. Substrate formation analysis in the wound healing process
To identify how long the reformation of the derma takes during skin
wound recovery, Masson's trichrom staining and picrosirius red staining
were conducted, and then the collagen deposit time was observed. In
the control group, collagen synthesis, stained as light blue, was
identified around the wound from around day 3 after inducing wounds.
In the control and the pycnogenol-treated groups, dark blue staining
was observed in the tissues on days 5 and 7 (Fig. 10).
Quantitative collagen deposit analysis showed clear collagen
synthesis from day 5 and increased on day 7. Compared to the control
group, collagen accumulation in the pycnogenol-treated group was
higher than that of the control group from days 5 to 7 (Fig. 11).
- 22 -
Fig. 10. Difference in collagen accumulation (blue color) in the healing
areas between the control and the pycnogenol-treated groups. Collagen
in the healing area of the control and the pycnogenol-treated groups
gradually increased from days 1 to 7. Collagen deposition in the
pycnogenol-treated group was greater than in the control group on
days 5 and 7. Scale bar=100 μm
- 23 -
Fig. 11. Difference in the accumulated collagen (bright red color) in
the healing areas of the control and the pycnogenol-treated groups.
This result was consistent with the collagen staining. Scale bar=100
μm
- 24 -
2. Effects of Saponin on Skin Wound Healing
2.1. Wound healing tendencies after saponin treatment
To examine the effect of saponin on the wound healing, changes in
the wound size on days 1, 3, 5, and 7 post-wounding were observed
and compared between the control and the saponin-treated groups.
The wound size on day 5 in the saponin-treated group was much
smaller compared to the control group. By day 7, both sides of the
incision were completely joined in the saponin-treated group (Fig. 12).
- 25 -
Fig. 12. Differences in the dermal wound size between the control and
the saponin-treated groups. The wound size gradually reduced in both
groups from days 1 to 7. In the saponin-treated group, the wound size
was more reduced on days 5 and 7 compared to the control group.
- 26 -
2.2. Histological and morphometric analysis of the skin wound healing
following saponin treatment
H&E staining was performed on the wound tissue samples at various
time points post-wounding. Most wound tissues appeared to have
disintegrated in both the control and the saponin-treated groups, and
scabs were observed. However, the amount of scab was smaller,
epithelial cell migration was faster, and less residue in the dermal
layer was present in the saponin-treated group than in the control
group. On day 5 post-wounding, epithelial cell migration was complete
in the saponin-treated group, but not in the control group. The amount
of scab was also less in the saponin-treated group than in the control
group. In addition, the amount of residue in the dermal layer in the
saponin-treated group was much less compared to that of the control
group. By day 7, all scabs had lifted off in both groups. Epithelial cell
migration was fully complete in the saponin-treated group, but the
wound contraction rate appeared to be higher (Fig. 13).
The wound size showed a decreasing trend with time in both groups;
however, by period, the wound sizes in the saponin-treated group
were smaller than those of the control group during the wound healing
process from days 3 to 7 (Fig. 14).
Furthermore, while the keratinocyte migration rate was similar on
day 1 and tended to increase in both groups as time passed, the rate
in the saponin-treated group was higher than that of the control group
(Fig. 15).
- 27 -
Fig. 13. Differences in the wound area between the control and the
saponin-treated groups during skin wound healing. The wound area of
the saponin-treated group was larger than the control group on day 1
but smaller on days 3 to 7. Scale bar=200 μm
- 28 -
Fig. 14. The wound area of the saponin-treated group was larger than
the control group on day 1 but smaller on days 3 to 7. This result
was based upon the morphometric analysis of the wound tissues from
the control and the saponin-treated groups. * p<0.05
- 29 -
Fig. 15. Keratinocyte migration in the wound tissue. The keratinocyte
migration rate in the saponin-treated group was higher than that of the
control group on days 3 to 7. * p<0.05
- 30 -
2.3. Histological analysis of inflammatory cells and wound contraction
rate during the skin wound healing process
Giemsa staining was performed to analyze the number of
inflammatory cells in the wound area at different time periods. The
largest cell numbers were seen on day 1 post-wounding and gradually
decreased over time. The number of inflammatory cells in the
saponin-treated group was noticeably smaller on days 1 and 3 compared
to the control group and tended to dwindle over time (Fig. 16).
However, the number of inflammatory cells in the saponin-treated group
increased starting on day 5 and was maintained until day 7 (Fig. 17).
The control group showed an increase in the rate of wound
contraction on day 7 after being at a constant level from days 1 to 5,
while the saponin-treated group showed a gradual increase in the rate
of wound contraction reaching the maximum level on day 7. Specifically,
the wound contraction rate of the saponin-treated group was lower
than the control group on day 1 but began to increase from day 3
similar to the control group, eventually becoming higher than the
control group on days 5 to 7 (Fig. 18).
- 31 -
Fig. 16. Difference in the recruitment of inflammatory cells between
the control and the saponin-treated skin wounds. Inflammatory cells in
the saponin-treated wounds were reduced from days 1 to 5 compared
to the control wound tissue but increased on day 7. Scale bar =20 μm
- 32 -
Fig. 17. Inflammatory cells were counted in three distinctive areas
(left, right, and middle of wound area). Inflammatory cells of the
saponin-treated group were lower than the control group on days 1
and 3. However, the number of inflammatory cells of the saponintreated
group on day 5 was higher than on day 3. On day 7, the
number of inflammatory cells of the saponin-treated group was higher
than the control group. * p<0.05
- 33 -
Fig. 18. Wound contraction measurements between the control and the
saponin-treated groups. Wound contraction in the saponin-treated
group was higher than that of the control group from days 3 to 7.
- 34 -
2.4. Substrate formation analysis in the wound healing process
To confirm the substrate formation in the dermis, Masson's trichrome
and picrosirius red staining were conducted, and then the collagen
deposit time was observed. In the control group, collagen synthesis,
stained as light blue, was identified around the wound area from day
3, and tended to be active in the tissues on days 5 and 7 (Fig. 19).
To confirm collagen disposition, collagen synthesis was first observed
on day 5 and appeared to be increased on day 7. However, collagen
synthesis was deeper in the saponin-treated group than the control
group for the entire study period (Fig. 20).
- 35 -
Fig. 19. Difference in collagen accumulation (blue color) in the healing
areas between the control and the saponin-treated groups. Collagen in
the healing area of the control and the saponin-treated groups
gradually increased from days 1 to 7. Collagen deposition in the
saponin-treated group was greater than in the control group. Scale
bar=100 μm
- 36 -
Fig. 20. Difference in the accumulated collagen (bright red color) in
the healing areas of the control and the saponin-treated groups. This
result was consistent with the collagen staining. Scale bar=100 μm
- 37 -
DISCUSSION
In mammals, wound healing involves a prompt and efficient process
in which bleeding from the wound is first terminated, followed by
reformation of the damaged tissues. Then, moisture is evaporated
around the wound, and a functional defense membrane is generated to
protect against invasion by microorganisms. Skin wound healing can be
classified into three phases, which include the initial inflammatory
reaction, re-epithelialization and granulation tissue formation, and
finally tissue remodeling (Clark, 1995). These classifications are based
on histological results or functional activities, but in fact, they overlap.
For ideal healing to occur, interactions between cells and tissues that
are deeply involved in these three phases are essential (Toriseva and
Kahari, 2009).
Blood coagulation is initiated by the formation of clots that form in
combination with fibrin fiber bundles activated by blood coagulation
factors and platelets. Fibronectin and vitronectin in the blood plasma
are included in the blood coagulation phase and temporarily form the
substrate for cell migration. Keratinocytes migrate into this substrate
in which scab formation occurs around the wound with the help of
proteinases. The hemotactic stimulus is provided by growth factors and
platelets secreted by the activated blood coagulation factors,
complement components, and damaged cells (Singer and Clark, 1999).
During the blood coagulation process, neutrophils arrive first at the
damaged tissue within a few hours. Major functions of neutrophils
include phagocytosis, which eliminates the infective agents at the site
- 38 -
of damaged tissue, and the stimulation of blood coagulation,
inflammation, and the healing process by secreting various factors
(Eming et al., 2007). Next, monocytes arrive at the wound within two
days and differentiate into macrophage. They also function as
phagocytes and antigen presenting cells, secreting transforming growth
factors- and, basic fibroblast growth factor, and platelet-derived
growth factor (PDGF) to regulate the wound healing process (Eming et
al., 2007).
Re-epithelialization and granulation tissue formation occur
simultaneously within a few hours of the inflammatory reaction.
Keratinocytes that exist around the edges of the wound and in the
residues of skin appendages begin to migrate into the wound and form
a scab (Singer and Clark, 1999). These keratinocytes are
characteristically hyper-proliferative, which enables them to fill the
damaged epithelial layer and reform the basement membrane within
two days after wound initiation, thereby restoring contact between
cells. Through this process, keratinocytes differentiate to form the
epidermal skin layer (Singer and Clark, 1999). Almost simultaneously,
as fibroblasts located around the undamaged dermis begin to
proliferate and migrate upon stimulation by growth factors, granulation
tissue formation is initiated (Singer and Clark, 1999). Fibroblasts that
migrate into the wound and proliferate synthesize proteoglycans,
collagen type III, and collagen type I to reform the extracellular matrix
around the wound (Clark, 1995). Some fibroblasts differentiate into
myofibroblasts and synthesize smooth muscle actin, which provides
mechanical tension to pull the edges of the wound to stimulate
- 39 -
contraction and eventually promote wound closure (Hinz et al., 2001).
New blood vessels appear in the granulation tissue upon migration and
proliferation of the endothelial cells, which is stimulated by
macrophages or keratinocytes and growth factors secreted from
endothelial cells (Singer and Clark, 1999). The dermis remodeling
phase of skin wound healing involves the full growth of the wound
accompanied by collagenous scarring. This phase is characterized by a
reduction in the number of fibroblasts by apoptosis and removal of
damaged blood vessels. The residual fibroblasts rearrange the collagen
fiber to recover the original tension of the skin, repeating collagen
deposition and degradation for several months. However, the original
skin tension before the damage can never be fully recovered (Singer
and Clark, 1999; Hinz, 2007).
There are various medications available to assist in the skin wound
healing, ranging from disinfectants such as ethyl alcohol, iodine, and
ether, to ointments containing various antibiotics and steroid hormones.
This can lead to injury of not only the invading cells but also normal
cells, as well as induce the emergence of resistant bacteria and cause
various hypersensitivity reactions (Kang, 2004). From the ancient
times, various natural substances have been widely used for wound
healing, anti-aging, and the treatment of other diseases (Hsu, 2005).
Among such extracts, polyphenol compounds are known to be strong
antioxidants capable of neutralizing free radicals by combining with
active oxygen (Hsu, 2005). A hydroxyl group attaches to an aromatic
ring of a polyphenol compound that combines with free radicals, which
appear during the metabolic process and form neutralized stable
- 40 -
phenoxyl radicals (Rice-Evans et al., 1996). Free radicals that reacts
with polyphenol compounds include superoxide radical anions, hydroxyl
radicals, lipid peroxyl radicals, nitric oxide radicals, and peroxynitrites.
All of these have been known to play a significant role not only in the
biological environment but also in the treatment of diseases (Packer et
al., 1999).
Radicals produced by wounds are largely superoxide radical anions
produced by neutrophils and macrophages and also play an important
role in the removal of microorganisms and pathogens (Babior, 2000).
Superoxide radical anions are quickly transformed into hydrogen
peroxide (H2O2) which permeates into the cell membranes of pathogens
by superoxide dismutase, promoting the formation of hypochlorous
acid, chloramines, and aldehyde which are more stable than H2O2, and
are characterized by long half-lives. Thus, when H2O2 substances
remain in the wound for an extended period of time, acute
inflammatory reactions may occur that may potentially damage even
normal cells (Sen et al., 2002).
Pycnogenol, which is one of the polyphenol compounds, is a pine
bark extract and has been used as a medicine for wound recovery and
inflammatory diseases since the ancient times (Packer et al., 1999).
Through basic research and clinical studies, pycnogenol was known not
only to be a powerful antioxidant (Chida et al., 1999; Devaraj et al.,
2002) but as an agent to improve vascular integrity and endothelial
function and to stimulate anti-inflammatory responses (Grimm et al.,
2006). Also, pycnogenol regulates nitric oxide production of activated
macrophages and inhibits iNOS activity and inducible macrophage-type
- 41 -
nitric oxide synthase (iNOS) mRNA expression (Virgili et al., 1998).
Based on the abundant evidence, this study aimed to identify the effect
of pycnogenol on wound recovery inducing skin incisions on the dermal
layer by histological analysis.
Observing the speed of wound recovery with the naked eye showed
no difference between the control group and the pycnogenol-treated
groups. Also, the wound measurement was wider than that of the
control group from days 1 to 7 after inducing wounds. The rates of
keratinocyte migration were similar in both groups on day 1 but lower
in the pycnogenol-treated group than the control group thereafter. This
shows that treatment with pycnogenol has no effect on the speed of
wound recovery, which is not consistent with previous reports.
Pycnogenol was demonstrated to promote wound recovery time in mice
with treatment with 5% pycnogenol after induction of burn wounds was
about three days less than the control group, and the measurement of
the scar tissue was also 2.6 times less than the control group (Blazs
et al., 2004). We speculate that the discrepancy may be due to a few
reasons. First, the previous study used a higher concentration of
pycnogenol, 1%, 2% and 5%, compared to our study. Second, there
was difference in the absorption rate of pycnogenol because the
administration of pycnogenol was in conjunction with a gel substance
called Carbomer 934 P in the other studies. In our study, however,
pycnogenol was injected in the form of an aqueous solution. Methods
to induce wounds in wound recovery experiments can be largely
divided into burning, full thickness incision, and excision of some
tissues. In most other studies, the most common form of wound
- 42 -
induction was burning, unlike this current study. Grimm et al. (2006)
reported that pycnogenol promotes inflammatory responses, in that
pycnogenol interferes with matrix metalloproteinase-9 (MMP-9)
secretion by monocytes. Also, pycnogenol has been shown to inhibit the
inflammatory response of UV-induced wounds in a
concentration-dependent manner (Sime et al., 2004). When we
examined the influx of inflammatory cells, there were less
inflammatory cells in the pycnogenol-treated group than in the control
group only on day 1. From days 3 to 5, however, there were more
inflammatory cells in this group than the control group. On day 7, a
higher tendency to increase was observed in the treated group
compared to the control group. Therefore, it appears that pycnogenol
inhibits inflammatory responses in the early period of wound healing,
but as time passes, its effect wanes. However, both the degree of
contraction of wounds and the collagen deposit time were higher in the
pycnogenol-treated group than the control group on day 7. Pycnogenol
also stimulates the formation of a substrate at the second half of the
wound recovery period. Recently, one study said that an Ocimum
sanctum extract, a kind of polyphenol, increases the synthesis of
collagen, which is the main ingredient, by promoting proliferation of
fibroblasts (Shetty et al., 2007). Also, grape seed extracts, another
powerful antioxidant, was demonstrated to stimulate the crosslinking of
collagen produced in the fibroblasts (Han et al., 2003). Altogether,
pycnogenol appears to inhibit the inflammatory response during the
wound recovery process in the early stage and to promote substrate
formation of the derma.
- 43 -
Saponins are plant extracts that have extensive biological activities.
The promotion of anti-oxidants and anti-inflammatory reactions are
included among their functions (Guclu-Ustundag and Mazza, 2007).
Saponins are one of the various types of glycosides that exist in
higher orders of plants (Sparg et al., 2004). Saponin types are
classified and named based on the internal structure. In particular, a
certain saponin referred to as fruticesaponin B is known to have very
high anti-inflammatory activities (Just et al., 1998). Navarro et al.
reported that saponins may have high or low activity depending upon
their internal structure. In fact, only two types of saponins tested were
shown to reduce neutrophil entry to the wound sites, thereby
alleviating chronic skin inflammatory responses. In this study, wound
healing in the saponin-treated group progressed much faster than the
control group on day 5, and both sides of the incisional wounds were
fully joined on day 7. The wound area shrunk more in the
saponin-treated group than the control group at all time points
evaluated with the exception of day 1, and the rate of keratinocyte
migration in the saponin-treated group appeared to be higher than the
control group during all periods except day 1. Other research reported
that the burn wound area in the saponin-treated group gradually
increased up to day 4 then gradually decreased until day 20, while, in
the control group, the burn wound area gradually increased up to day
8 and then tended to diminish in size (Kimura et al., 2006). It is
assumed in this case that the long lead time in healing the burn wound
was the result of an inflammatory reaction around the burn wound,
which persisted longer (Ramzy et al., 1999). In addition, it was
- 44 -
reported that saponin increased the expression of factors relevant to
proliferation, and consequently, promoted the proliferation of epidermal
cells (Choi, 2002). Likewise, in our study, we found the rate of
keratinocyte migration involved in re-epithelialization to be faster in
the saponin-treated group than in the control group. Therefore, it is
assumed that saponin not only enhances epidermal cell proliferation but
also promotes keratinocyte migration. When the influx of inflammatory
cells was measured after wounding, the number of cells in the
saponin-treated group were obviously less and to diminish on days 1
and 3 compared to the control group, but appeared to have a tendency
to increase from day 5. Eventually, the number of inflammatory cells
appeared to be greater in the saponin-treated group than the control
group on day 7. Even in the study by Kimura et al., the number of
leukocytes and macrophages appeared to increase up to day 9 after a
burn wound, reportedly due to the expression of IL-1 by hypoxia
inducible factor-1, which induced the accumulation of macrophages.
Accordingly, we can assume that saponin is involved in the inhibition
of inflammatory responses at the early stage. Furthermore, wound
shrinkage measurements appeared to be sharply increased from day 3
on. Matrix remodeling analysis confirmed that matrix synthesis was
stimulated more in the saponin-treated group compared to the control
group. A recent study revealed that when saponin was used to treat
skin tissue exposed to ultraviolet rays, collagen synthesis of fibroblasts
was increased and the expression of matrix metalloproteinases was
inhibited (Kim et al., 2009). Furthermore, it was also revealed that
saponin increased collagen synthesis in skin fibroblasts through
- 45 -
phosphorylation of the Smad 2 protein (Lee et al., 2007). Thus, it is
assumed that saponin will promote the re-synthesis of matrix at the
site of a skin wound.
During the skin wound recovery process, pycnogenol did not promote
re-epithelialization of the wound site but inhibited the initial
inflammatory response and promoted substrate synthesis of the second
half. Also, saponin promoted re-epithelialization of the wound site and
effectively inhibited the initial inflammatory response and promoted the
substrate synthesis. Taking all results together, it appears that saponin
is more effective for wound recovery in wounds inflicted by incision.
To determine the effect of pycnogenol and saponin treatment on the
expression of inflammation-related cytokines during the wound recovery
process, protein and mRNA expression of proteases such as MMPs
expressed at the wound site can be further investigated in the future.
Also, further investigation can be conducted with different treatment
methods or concentrations of pycnogenol and saponin and other modes
of wound infliction.
- 46 -
Conclusion
To determine the effect of pycnogenol and saponin treatment on the
skin wound healing process, we induced incision wounds of dermal
depth on the back of mice and observed the difference between the
control, pycnogenol-, or saponin-treated groups. We extracted the
wound tissues on days 1, 3, 5, and 7 after inducing the wounds and
performed the analyses for the substrate re-synthesis, wound
contraction, inflammatory cells influx, keratinocyte migration rate,
wound measurements, and histology.
Conclusions based on the experimental results are as follows:
1. Pycnogenol does not promote re-epithelization of the wound site but
inhibits the initial inflammatory response and promotes substrate
synthesis of the second half.
2. Saponin promotes re-epithelization of the wound site and inhibits
the initial inflammatory response and promotes substrate synthesis.
3. Saponin is considered to be more effective than pycnogenol in the
wound recovery process.
- 47 -
REFERENCES
1. Apers S, Baronikova S, Sindambiwe JB, Witvrouw M, De Clercq E,
Vanden Berghe D, Van Marck E, Vlietinck A, Pieters L. Antiviral,
haemolytic and molluscicidal activities of triterpenoid saponins from
Maesa lanceolata: establishment of structure-activity relationships.
Planta Med 67:528-532, 2001.
2. Babior BM. Phagocytes and oxidative stress. Am J Med 109:33-44,
2000.
3. Baumann E, Stoya G, Volkner A, Richter W, Lemke C, Linss W.
Hemolysis of human erythrocytes with saponin affects the
membrane structure. Acta Histochem 102:21-35, 2000.
4. Berryman AM, Maritim AC, Sanders RA, Watkins III JB. Influence
of treatment of diabetic rats with combinations of pycnogenol,
beta-carotene, and alpha-lipoic acid on parameters of oxidative
stress. J Biochem Mol Toxicol 18:345-352, 2004.
5. Blazso G. Gabor M, Rohdewald P. Anti-inflammatory activities of
procyanidin containing extracts from Pinus pinaster Ait. after oral
and cutaneous application. Pharmazie 52(5):380-382, 1997.
6. Blazso G, Gabor M, Schonlau F, Rohdewald P. Pycnogenol
accelerates wound healing and reduces scar formation. Phytother
- 48 -
Res 18:579-581, 2004.
7. Chida M, Suzuki K, Nakanishi-Ueda T, Ueda T, Yasuhara H, Koide
R, Armstrong D. In vitro testing of antioxidants and biochemical
end-point in bovine retinal tissue. Ophthalmic Res 31:407-415,
1999.
8. Cho KJ, Yun CH, Yoon DY, Cho YS, Rimbach G, Packer L, Chung
AS. Effect of bioflavonoids extracted from the bark of Pinus
maritima on proinflammatory cytokine interleukin-1 production in
lipopolysaccharide- stimulated RAW 264.7. Toxicol Appl Pharmacol
168(1):64-71, 2000.
9. Choi S. Epidermis proliferative effect of the Panax ginseng
ginsenoside Rb2. Arch Pharm Res 25:71-76, 2002.
10. Clark RAF. Wound repair: overview and general considerations. In:
Clark RA, Henson PM. The molecular and cellular biology of wound
repair. 2nd ed. New York: Plenum Press, p.513-560, 1996.
11. Devaraj S, Vega-Lóez S, Kaul N, Schölau F, Rohdewald P, Jialal
I. Supplementation with a pine bark extract rich in polyphenols
increases plasma antioxidant capacity and alters the plasma
lipoprotein profile. Lipids 37:931-934, 2002.
12. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair:
- 49 -
molecular and cellular mechanisms. J Invest Dermatol 127:514-525,
2007.
13. Grimm T, ChovanováZ, MuchováJ, SumegováK, LiptáováA,
DurackováZ, Höger P. Inhibition of NF-kappaB activation and
MMP-9 secretion by plasma of human volunteers after ingestion of
maritime pine bark extract (Pycnogenol). J Inflamm (Lond). 27;3:1,
2006.
14. Guan DW, Ohshima T, Kondo T. Immunohistochemical study on Fas
and Fas ligand in skin wound healing. Histochem J 32:85-91, 2000.
15. Guclu-Ustundag O, Mazza G. Saponins: properties, applications and
processing. Crit Rev Food Sci Nutr 47:231-258, 2007.
16. Han B, Jaurequi J, Tang BW, Nimni ME. Proanthocyanidin: a
natural crosslinking reagent for stabilizing collagen matrices. J
Biomed Mater Res A. 65(1):118-124, 2003.
17. Hinz B. Formation and function of the myofi broblast during tissue
repair. J Invest Dermatol 127:526-537, 2007.
18. Hinz B, Mastrangelo D, Iselin CE, Chaponnier C, Gabbiani G.
Mechanical tension controls granulation tissue contractile activity
and myofi broblast differentiation. Am J Pathol 159:1009-1020,
2001.
- 50 -
19. Hsu S. Green tea and the skin. J Am Acad Dermatol
52:1049-1059, 2005.
20. Just MJ, Recio MC, Giner RM, Cuélar MJ, Má5ñz S, Bilia AR, Rís
JL. Anti-infl ammatory activity of unusual lupane saponins from
Bupleurum fruticescens. Planta Med 64:404-407, 1998.
21. Kang JS. Cosmetic surgery. 3rd ed. Seoul: Kunga Press, 2004.
22. Killeen GF, Madigan CA, Connolly CR, Walsh GA, Clark C, Hynes
MJ, Timmins BF, James P, Headon DR, Power RF. Antimicrobial
saponins of Yucca schidigera and the implications of their in vitro
properties for their in vivo impact. J Agric Food Chem
46:3178-3186, 1998.
23. Kim YG, Sumiyoshi M, Sakanaka M, Kimura Y. Effects of ginseng
saponins isolated from red ginseng on ultraviolet B-induced skin
aging in hairless mice. Eur J Pharmacol 602:148-156, 2009.
24. Kimura Y, Sumiyoshi M, Kawahira K, Sakanaka M. Effects of
ginseng saponins isolated from red ginseng roots on burn wound
healing in mice. Br J Pharmacol 148:860-870, 2006.
25. Konishi M, Hano Y, Takayama M, Nomura T, Hamzah AS, Ahmad
RB, Jamani H. Triterpenoid saponins from Hedyotis nudicaulis.
- 51 -
Phytochemistry 48:525-528, 1998.
26. Lee J, Jung E, Lee J, Huh S, Kim J, Park M, So J, Ham Y, Jung K,
Hyun CG et al. Panax ginseng induces human type I collagen
synthesis through activation of Smad signaling. J Ethnopharmacol
109:29-34, 2007.
27. Navarro P, Giner RM, Recio MC, Manez S, Cerda-Nicolas M, Rís
JL. In vivo anti-inflammatory activity of saponins from Bupleurum
rotundifolium. Life Sci 68:1199-1206, 2001.
28. Oda K, Matsuda H, Murakami T, Katayama S, Ohgitani T,
Yoshikawa M. Adjuvant and haemolytic activities of 47 saponins
derived from medicinal and food plants. Biol Chem 38:67-74, 2000.
29. Packer L, Rimbach G, Virgili F. Antioxidant activity and biologic
properties of a procyanidin-rich extract from pine (Pinus maritima)
bark, pycnogenol. Free Radic Biol Med 27:704-724, 1999.
30. Ramzy PI, Barret JP, Herndon DN. Thermal injury. Crit Care Clin
15:333-352, ix, 1999.
31. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant
activity relationships of fl avonoids and phenolic acids. Free Radic
Biol Med 20:933-956, 1996.
- 52 -
32. Rohdewald P. A review of the French maritime pine bark extract
(Pycnogenol), a herbal medication with a diverse clinical
pharmacology. Int J Clin Pharmacol Ther 40(4):158-168, 2002.
33. Sen CK, Khanna S, Gordillo G, Bagchi D, Bagchi M, Roy S.
Oxygen, oxidants, and antioxidants in wound healing: an emerging
paradigm. Ann N Y Acad Sci 957:239-249, 2002.
34. 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 Complement Alternat Med.
5(1):95-101, 2007.
35. Sime S, Reeve VE. Protection from inflammation, immunosuppression
and carcinogenesis induced by UV radiation in mice
by topical Pycnogenol. Photochem Photobiol 79(2):193-198, 2004.
36. Simoes CM, Amoros M, Girre L. Mechanism of antiviral activity of
triterpenoid saponins. Phytother Res 13:323-328, 1999.
37. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med
341:738-746, 1999.
38. Sparg SG, Light ME, van Staden J. Biological activities and
distribution of plant saponins. J Ethnopharmacol 94:219-243, 2004.
- 53 -
39. Stephens P, Thomas DW. The cellular proliferative phase of the
wound repair process. J Wound Care 11:253-261, 2002.
40. Toriseva M, Kahari VM. Proteinases in cutaneous wound healing.
Cell Mol Life Sci 66:203-224, 2009.
41. Virgili F, Kobuchi H, Packer L. Procyanidins extracted from Pinus
maritima (Pycnogenol): scavengers of free radical species and
modulators of nitrogen monoxide metabolism in activated murine
RAW 264.7 macrophages. Free Radic Biol Med. 24(7-8):
1120-1129, 1998.
42. Wang ZY, Zhang J, Lu SL. Objective evaluation of burn and
post-surgical scars and the accuracy of subjective scar type
judgment. Chin Med J (Engl) 121:2517-2520, 2008.
43. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in
wound healing. J Invest Dermatol 127:998-1008, 2007.
44. Yogeeswari P, Sriram D. Betulinic acid and its derivatives: a
review on their biological properties. Curr Med Chem 12:657-666,
2005.
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Pycnogenol과 Saponin이 피부 창상치유에 미치는 효과
김 영 수
전남대학교대학원 수의학과
(지도교수 : 배춘식)
(국문초록)
피부 상처회복은 여러 인자들의 정확한 조절하에 서로 다른 세포들이 손상된
조직을 재생하는 과정을 통해 일어난다. 현재 피부 상처치유를 위해 사용하는 합
성약제들이나 항생제들은 과민반응이나 내성균 출현 같은 여러 부작용을 야기시키
고 있다. 따라서 최근에는 인공약제를 대신하여 식물에서 추출한 천연물들의 상처
치유 효과에 대한 연구가 활발히 진행되고 있다. Pycnogenol은 소나무 껍질에서
추출한 폴리페놀 화합물로서 항산화제 기능뿐만 아니라 염증성 질환에도 효과가
있는 것으로 알려졌다. 또한 saponin은 매우 높은 항염증반응 활성을 가지고 있는
것으로 밝혀졌다. 본 연구에서는 pycnogenol과 saponin의 알려진 기능을 바탕으
로 피부 상처회복과정에서 그 효과를 조직학적으로 밝히고자 하였다.
동일 조건에서 일정기간 사육한 48마리 생쥐의 등쪽 피부에 길이 1cm, 진피
층 깊이까지 외과적 상처를 유발시킨 후 대조군, pycnogenol 또는 saponin을
처리한 실험군으로 나누어 1일, 3일, 5일, 7일 후에 각각 상처 부위 조직을 적출
하였다. 적출한 조직은 조직 표본 제작과정을 거친 후 상처부위의 면적, 각질세
포 이동율, 유입된 염증세포수, 상처 수축정도 그리고 교원질 침작과 같은 조직
계측학적 분석을 실시하였다.
실험결과 pycnogenol 처리군은 대조군에 비해 모든 기간에 걸쳐 상처면적이 더
넓었고 각질세포 이동 정도는 1일째를 제외하고 모두 대조군보다 낮게 나타났다.
또한 상처부위의 염증세포 수를 분석한 결과 pycnogenol 처리군은 대조군에 비해
- 55 -
1일째만 제외하고 모든 기간에 걸쳐 염증세포 수가 더 높았다. 상처 수축도 분석
에서는 pycnogenol 처리군은 1일부터 5일째까지 수축정도가 대조군보다 낮았지만
7일째에는 더 높게 나타났다. 교원질 침작 분석결과 pycnogenol 처리군은 대조군
에 비해서 7일째에 더 높게 나타났다. Saponin 처리군의 상처면적은 1일째를 제
외한 3일에서 7일까지 대조군보다 더 좁게 측정됐고 각질세포 이동율은 대조군에
비해 높게 나타났다. 상처부위의 염증세포수는 saponin 처리군의 대조군에 비해 1
일째와 3일째에 현저히 적게 나타났고 줄어드는 경향을 보였다. 상처 수축도는
saponin 처리군이 대조군보다 더 높은 수치를 나타냈다. 또한 교원질 침작은
saponin 처리군이 대조군에 비해 더 촉진되는 것으로 관찰되었다.
이상의 결과들을 종합해 보면 pycnogenol은 상처부위의 재상피화를 촉진시키
지 않았지만 초기 염증반응을 억제하고 후반부의 기질합성을 증가시키는 것으로
확인되었다. 반면 saponin은 재상피화를 촉진시켰으며 염증반응을 억제하였고
기질 재합성을 위한 교원질 침작을 촉진하였다. 따라서 피부 상처회복과정에서는
pycnogenol 보다 saponin이 전반적으로 효과적인 것으로 생각된다.
