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1031
Introduction
Bellis perennis L. (common daisy) is a herbaceous
perennial herb in the family Asteraceae, which grows in
meadows, lawns and other grassy areas (Panda, 2004).
It is native to western, central, and northern Europe,
but is commonly found as an invasive plant in North
America (Tutin et al., 1976). Common daisy is known as
a traditional wound herb (Leporatti & Ivancheva, 2003;
Al-Douri & Al-Essa, 2010) and it had been used for the
treatment of bruises, broken bones, and wounds by
Crusaders in the Middle Ages (Mitich, 1997). B. perennis
has also been used in the treatment of sore throat (Uysal
et al., 2010), headache (Uzun et al., 2004), common cold,
stomachache, eye diseases, eczema, skin boils, gastritis,
enteritis, diarrhea, bleeding, rheumatism, inammation,
and infections of the upper respiratory tract in tradi-
tional medicine (Cakilcioglu et al., 2010). Furthermore,
astringent, expectorant, diuretic, booster, purgative and
diaphoretic properties have been recorded (Grieve, 1982;
Baytop, 1999; Duke et al., 2002).
RESEARCH ARTICLE
e evaluation of topical administration of Bellis perennis
fraction on circular excision wound healing in Wistar albino rats
Fatma Pehlivan Karakaș1, Alper Karakaș1, Çetin Boran2, Arzu Uçar Türker1, Funda Nuray Yalçin3,
and Erem Bilensoy4
1Department of Biology, Faculty of Arts and Sciences, 2Department of Pathology, Faculty of Medicine, Abant Izzet Baysal
University, Bolu, Turkey, 3Department of Pharmacognosy, Faculty of Pharmacy, and 4Department of Pharmaceutical
Technology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
Abstract
Context: Bellis perennis L. (Asteraceae) has been used traditionally in the treatment of bruises, broken bones, and
wounds by European people.
Objective: To investigate the wound healing activity of B. perennis owers in Wistar albino rats.
Materials and methods: Dried B. perennis owers were extracted with ethanol, then fractioned with n-butanol and
an oinment was prepared. Twelve male adult Wistar rats were used. Six wounds were created for each animal by
using circular excision wound model. The rst two wounds were treated topically with HOTBp (hydrophilic ointment
treatment containing n-butanol fraction). The second two wounds were control group and not treated with anything.
The third two wounds were treated only with HOT (hydrophilic ointment treatment without n-butanol fraction).
Treatments were applied once a day and lasted for 30 days. Wound samples were excised on days 5th, 10th and 30th.
The percentage of wound healing was calculated by Walker’s formula after measurement of the wound area and the
tissue samples were examined histopathologically.
Results: The percentages of wound closure (HOTBp: 100%; HOT: 85% and control: 87%) and histopathological
observations showed that there were statistically signicant dierences between HOTBp, HOT and control groups
(p < 0.05) at 30th day.
Discussion and conclusion: Topically administered ointment prepared from the n-butanol fraction of B. perennis owers
has a wound healing potential without scar formation in circular excision wound model in rats. Thus, traditional
usage of wound healing activity of B. perennis was scientically veried for the rst time.
Keywords: Common daisy, n-butanol fraction, ointment
Address for Correspondence: Fatma Pehlivan Karakaş, Department of Biology, Faculty of Arts and Sciences, Abant Izzet Baysal University,
14280, Bolu, Turkey. Tel: +90 374 254 1000. Fax: +90 374 253 4642. E-mail: fatmapehlivankarakas@gmail.com
(Received 05 October 2011; revised 17 November 2011; accepted 06 January 2012)
Pharmaceutical Biology, 2012; 50(8): 1031–1037
© 2012 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2012.656200
Pharmaceutical Biology
2012
50
8
1031
1037
05 October 2011
17 November 2011
06 January 2012
1388-0209
1744-5116
© 2012 Informa Healthcare USA, Inc.
10.3109/13880209.2012.656200
NPHB
656200
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1032 F. P. Karakaş et al.
Pharmaceutical Biology
e main constituents are triterpenoid saponins
(Hiller et al., 1988; Schopke et al., 1991; Li et al., 2005;
Morikawa et al., 2008; Yoshikawa et al., 2008), essen-
tial oils (Avato et al., 1997; Kavalcioglu et al., 2010)
and avonoids (Gudej & Nazaruk, 2001). Antibacterial
(Avato et al., 1997; Kavalcioglu et al., 2010), antifungal
(Desevedavy et al., 1989; Avato et al., 1997; Kavalcioglu
et al., 2010), antihyperlipidemic (Morikawa et al.,
2010a), antioxidant (Kavalcioglu et al., 2010), postpar-
tum antihemorrhagic (Oberbaum et al., 2005), pan-
creatic lipase inhibitor (Morikawa et al., 2010b) and
cytotoxic activities against HL-60 human promyelo-
cytic leukemia cells (Li et al., 2005) of B. perennis have
also been investigated.
Wound healing involves a series of events namely
chemotaxis, cell division, revascularization, synthe-
sis of new extracellular matrix, and the formation and
remodeling of the scar tissue (Hanna & Giacopelli,
1997). Regeneration involves the restitution of tissue
components identical to those removed or killed. By
contrast, repair is a bro proliferative response that
“patches” rather than restores a tissue. If tissue injury is
severe or chronic, and results in damage of both paren-
chymal cells and the stromal framework of the tissue,
healing cannot be accomplished by regeneration. Under
these conditions, the main healing process is repair by
deposition of collagen and other extracellular matrix
components, causing the formation of a scar. e term
scar is most often used in connection to wound healing
in the skin, but is also used to describe the replacement
of parenchymal cells in any tissue by collagen, as in the
heart after myocardial infarction (Kumar et al., 2010).
When a wound occurs, it may be thoroughly exposed
to the infections and complications. Consequently, the
aim of the wound management is to heal the wound as
quick as possible with minimal scar formation (Clark,
1991). e agents, which shorten the healing process,
are always required in order to contribute for a rapid
and better healing of wounds without hypertrophic
scar. e present study describes, to our knowledge for
the rst time, a wound healing properties of B. perennis
owers.
Materials and methods
Plant material
Common daisy (Bellis perennis L.) owers and pedi-
cels were collected from Abant Izzet Baysal University
Campus, Bolu, Turkey in May of 2009. Identication of
the species was made by Arzu Ucar Turker using “Flora
of Turkey and the East Aegean Islands” (Davis, 1975) and
voucher specimens (collection number AUT-1909) were
deposited at the Abant Izzet Baysal University (AIBU)
Herbarium, Bolu, Turkey.
Preparation of the common daisy n-butanol fraction
Collected common daisy owers and pedicels were dried
in an oven at 40°C and then ground into a powder. Each
25 g plant sample of B. perennis ower was extracted with
250 ml ethanol (96%) at 60°C for 18 h and then ltered.
Ethanol was evaporated under vacuum and residue
was dissolved in water. n-Butanol was added into the
extract in separation funnel and aqueous portion was
discarded. e remaining fraction was ash evaporated
to remove the n-butanol and the residue was suspended
in water. Frozen n-butanol fraction was lyophilized by
using a freeze-dryer at −65°C and residue used for oint-
ment preparation. e yield of fraction (w/w) was 10%
(Yield (%) = weight of extract (g)/25 g of powdered plant
sample × 100).
Preparation of hydrophilic ointment loaded with
B. perennis extract
Ethyl esters wax (12.5 g), white wax (12.5 g), mineral oil
(56 g), sodium borate (0.5 g) and puried water (19 g)
were accurately weighed to obtain approximately 100 g
of hydrophilic cold cream (USP XXI) formulation. Ethyl
esters and white waxes were reduced to small pieces and
melted on a steam bath. Mineral oil was added, and the
mixture was heated until the temperature reached 70°C
to form the oil phase. On the other hand, sodium borate
was dissolved in puried water, warmed to 70°C. e
n-butanol fraction (2.5 g) of B. perennis in powder form
was added to the aqueous phase to dissolve the extract,
mixed and gradually added onto the melted oil phase.
e two phases were stirred rapidly to obtain a homoge-
neous mixture of the phases yielding cold cream contain-
ing 2.5% (w/w) plant fraction.
Animal care
Adult male Wistar albino rats (200–250 g) were obtained
from our laboratory colony maintained at the Abant
Izzet Baysal University (AIBU). ey were exposed from
birth to 12 h of light, 12 h of darkness, lights o at 18:00 h.
Animals were maintained in plastic cages (16 × 31 × 42 cm)
with pine shavings used as bedding. Food pellets and tap
water were accessible ad libitum. e procedures in this
study were carried out in accordance with the Animal
Scientic procedure and approved by the Institutional
Animal care and Use Committee. All lighting was pro-
vided by cool-white uorescent tubes controlled by
automatic programmable timers. Ambient temperatures
in the animal facilities were held constant at 22 ± 2°C in
air-ventilated rooms.
Circular excision wound model
Twelve male Wistar albino rats having same age and
weight were selected. Before surgery, the rats were
anesthetized subcutaneously with ketamine (20 mg/kg,
Sigma Chemical Company, St. Louis, Missouri, USA)
and intraperitoneally with pentobarbital (32.5 mg/kg,
Sigma Chemical Company, St. Louis, Missouri, USA).
e depth of anesthesia was monitored by the frequent
testing of leg reexes and muscle tonus. e back hairs
of the rats were depilated by careful shaving. e cir-
cular wound was created with a 4 mm punch biopsy by
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Wound healing activity of the Bellis perennis 1033
© 2012 Informa Healthcare USA, Inc.
excising only the skin on the dorsal interscapular region
of each animal and wounds were left open (Tramontina
et al., 2002). Six wounds were created for each animal.
e rst two wounds were treated topically with the
HOTBp (hydrophilic ointment treatment containing
the n-butanol fraction of B. perennis). e second two
wounds were control group and not treated with any-
thing. e third two wounds were treated only with the
HOT (hydrophilic ointment treatment without n-butanol
fraction). Treatments were applied once a day and lasted
for 30 days.
Estimation of wound healing (wound closure)
Curative eect on the wound (wound closure) was evalu-
ated by tracing the outer margins of the wound on each
rat. Wound areas were traced manually and calculated in
square millimeters. Wound area was measured at 1st, 5th,
10th and 30th days after wounding and the wound closure
rate was expressed as the percentage of wound area com-
pared with that on post operative day (POD) (100%). e
percentage of wound healing was calculated by Walker’s
formula after measuring the wound area (Walker &
Mason, 1968). e percentage of wound healing was
computed at the beginning of experiments and the next
5th, 10th, and 30th days.
Percentage of wound
areaWound area in
=dday X
Wound area in the first day100
Percentage of wound
×
healing 100 Percentage of wound area =−
Histopathology
e full thickness of skin specimens from each group
were collected for histopathological examination.
Samples were xed in 10% buered formalin, pro-
cessed and embedded in paran, and then serially cut.
e sections were stained with hematoxylin & eosin
(H&E) and Gomori trichrome stains. Histological eval-
uation was done by a pathologist in a blind randomly
numbered fashion. Re-epithelization, the formation
of granulation tissue collogen deposition and connec-
tive tissue remodeling were analyzed. e amount of
collagen was rated on a subjective scale of 0 to 3, with
0, representing no collagen; 1, little collagen; 2 mod-
erate collagen; 3, abundant collagen. It was searched
whether hypertrophic scar formation for the thirtieth
day of the study.
Statistical analysis of the data
All data [3 treatments (control, HOT and HOTBp) × 3 days
(5, 10, 30)] were analyzed by analysis of variance (ANOVA)
with the last factor as a within subject or repeated design
using SPSS version 15 (SPSS Inc., Chicago, IL, USA).
Values were considered statistically signicant at p ≤ 0.05.
Data are presented as mean ± standard error (SE) after
back transforming from ANOVA results. Kruskal-Wallis
test for multiple comparisons and Mann-Whitney U test
for binary comparisons were used for histopathological
data.
Results and discussion
Although there are some studies representing the bio-
logical activities of B. perennis, there is no study about
wound healing eect of this species. We, therefore, aimed
to evaluate the vulnerary activity of B. perennis by an
open wound procedure in rats.
Figure 1A and 1B illustrate the normal histological
skin tissue samples at the beginning of the experiment.
Same tissue properties were observed in all groups of
histological samples at the 5th and the 10th days. Late
phase granulation tissues which were characterized with
the broblastic proliferation and scattered lymphocytes
were existed in the wound at the fth day (Figure 1C). e
collagen bers started to emerge slightly in the granula-
tion tissue (Figure 1D). Collagen brils became more
abundant at the tenth day (Figure 1E). In the thirtieth day,
granulation tissues were converted into hypertrophic
scar formations in all animals of the control and HOT
groups (Figure 1F and 1G). On the other hand, in HOTBp
group, the hypertrophic brous scar tissues disappeared
completely and an absolute amelioration was deter-
mined in all animals (Figure 1H and 1J). e dierences
among groups at the 30th day were statistically signicant
(p = 0.004). Binary comparisons between HOTBp group
and control group, and HOTBp and HOT group were also
statistically signicant (p = 0.008).
Figure 2 represents the percentages of wound healing
after ointment treatments in control, HOT and HOTBp
groups. ere was no statistically signicant dierence
(p > 0.05) in the means of wound healing percentages
between control and HOT animals. Wound healing per-
centage measurement showed a signicant increment
(p < 0.05) in the HOTBp group compared to the controls
and HOT at the 30th day. Hydrophilic ointment loaded
with B. perennis extract accelerated the wound healing
processes at the 30th day of the experiment (Figure 3).
e inammatory phase of the healing begins imme-
diately after wounding and lasts approximately four days
(Albritton, 1991). e proliferating phase begins toward
the end of the inammatory phase and lasts as long as
three weeks. Collagen synthesis starts by the 3th to 5th day
post injury (Cooper, 1990). In the present study, the 5th
and the 10th day samples were similar in all groups when
the inammatory and proliferating phases took place.
In the 5th day histopathology, a granulation tissue was
existed and the collagen started to emerge. At the 10th
day, collagen amount increased. On the other hand, the
dierence among the groups arises at the 30th day of the
experiment when the remodeling phase was going on.
Kanzaki et al. (1998) investigated the accelerator eects
of saponins on the extracellular matrix metabolism, the
activation and synthesis of TGF-B1, and the modica-
tion of TGF-B receptor expressions in broblasts. ey
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1034 F. P. Karakaş et al.
Pharmaceutical Biology
Figure 1. Histological sections of cutaneous wound site obtained from the controls, HOT and HOTBp lesions of the rats (A) normal
histological skin tissue in the rst day. (H&E, ×200) (B) normal histological skin tissue in the rst day samples stained with Gomori trichrome
stain (×200); (C) Late phase granulation tissues which were characterized with broblastic proliferation and scattered lymphocytes in a
fth day subject. Normal dermal tissue is seen at right corner [(rectangular) (Hematoxylin and eosin, ×100); (D) e collagen bers started
to emerge slightly in the granulation tissue in the 5th day samples (Gomori trichrome stain, ×100); (E) a lesion at a 10th day subject. Collagen
brils is seen as more abundant than the 10th day sample (Gomori trichrome stain, ×100), (F) a sample of control lesion in the 30th day.
Healing with brous scar formations is seen (Hematoxylin and eosin, ×100) (G) same tissue of F with Gomori trichrome stain (×100) (H, J)
Near normal skin tissue without bros scar tissue seen in a 30th day sample treated with Bellis perennis (Hematoxylin and eosin, Gomori
trichrome stain, respectively, ×100). (e) epidermis, (d) dermis, (ndt) normal dermal tissue, (cf ) collagen bers.
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Wound healing activity of the Bellis perennis 1035
© 2012 Informa Healthcare USA, Inc.
suggested that saponins stimulate the wound healing
process through the changes of the extracellular matrix
metabolism and is accompanied by modication of TGF
receptor expressions in broblasts (Kanzaki et al., 1998).
One of the main constituents of the B. perennis is triterpe-
noid saponins (Hiller et al., 1988; Schopke et al., 1991; Li
et al., 2005; Morikawa et al., 2008; Yoshikawa et al., 2008).
Wound healing potential of B. perennis may be attributed
to the saponins which might be an additive eect that
accelerates the progress probably during the phases of
wound healing.
Flavonoids are known to promote the rapid wound
healing due to their antimicrobial, antioxidant, anti-
inammatory and astringent properties (Tsuchiya et al.,
1996; Okuda, 2005; Nayak et al., 2009). e avonoid
ingredient of B. perennis (Gudej & Nazaruk, 2001)
may contribute to the vulnerary activity. Inhibition of
lipid per oxidation eect by avonoids, is supposed
to increase the viability of collagen brils, by activat-
ing the DNA synthesis and preventing the cell damage
(Shetty et al., 2008).
Complications in wound healing can arise from
abnormalities in any of the basic components of the
repair process. Immoderate formation of the compo-
nents of the repair process is one of the most important
complications in wound healing. e accumulation of
excessive amounts of collagens may give rise to a raised
scar known as a hypertrophic scar; if the scar tissue
grows beyond the boundaries of the original wound and
does not regress, it is called a keloid. Keloid formation
appears to be an individual predisposition (Kumar et al.,
2010). Hypertrophic scar or keloid is known to be dicult
to treat, and prevention is the best approach (Sanders &
Dickson, 1982).
Scar formation and remodeling to begin in the pro-
liferating phase of repair with the collagen synthesis
by broblasts. Fetal skin has the ability to heal without
a scar. is scar less healing is highly attributed to the
absence of acute inammation and a lack of immune
cell inltration in the rst and the second trimesters of
the fetus (Rowlatt, 1979). Neutrophils, macrophages, T
cells and B cells are all absent in scar less fetal wounds,
and fetal platelets are less reactive (Adolph et al., 1993;
Hopkinson-Woolley et al., 1994). On the other hand, the
brotic healing in the third trimester is attributed to the
Figure 3. Photographs of the dierent rats in dierent days of the experiment. HOT: hydrophilic ointment treatment, HOTBp: hydrophilic
ointment loaded with B. perennis fraction.
Figure 2. Wound closure percentages of the control, HOT and
HOTBp groups at the 5th, 10th and the 30th days of the experiment.
Wound Healing
Days
Percentages (%)
0
20
40
60
80
100
120
Control
HOT
HOTBp
51030
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1036 F. P. Karakaş et al.
Pharmaceutical Biology
presence of inammatory and immune cells (Cass et al.,
1997). In the present study, B. perennis ointment admin-
istered group showed complete wound healing without
the hypertrophic scar. However, HOT and control groups
showed healing with hypertrophic brous scar. Complete
wound healing with B. perennis may be attributed to
its photochemical character which prevents excessive
collagen synthesis and/or improved tissue remodeling
formation. e infections are the most important cause
of disordered healing. It results in persistent inamma-
tion which may result in excessive collagen deposition.
e antimicrobial (Desevedavy et al., 1989; Avato et al.,
1997; Kavalcioglu et al., 2010) eects of the B. perennis
may prevent the wound infection, the inammation and
the excessive collagen synthesis.
e remodeling begins at approximately 21 days post-
injury and continues until 1 to 2 years after the injury
(Cooper, 1990; Albritton, 1991; Canter, 1991). Early in this
phase, broblasts continue to produce collagen. e col-
lagen bundles are synthesized during the proliferating
phase and they are arranged into parallel position. Wound
contraction and the ultimate strength of the healed wound
is determined by the amount of collagen synthesis and the
extent to which cross-linking has occurred between col-
lagen bundles. In the present study, in HOTBp group, the
hypertrophic brous scar tissue disappeared completely,
and an absolute amelioration was recorded in all animals
whereas, the collagenous amount was less and ameliora-
tion was not completed in the scar tissue appearance in
control and HOT groups in the sections of the 13th day.
e results of the present experiment have indicated that
the data coming from the wound closure and the histopa-
thology are consistent with to each other.
Conclusion
Although B. perennis (common daisy) is a vulnerary
herb with a long history of use in folk medicine, there is
no study in terms of the wound healing potential of this
plant in a controlled laboratory experiment. Our ndings
demonstrate that the ointment including the n-butanol
fraction of B. perennis owers have been accelerating
eect on the remodeling of the wound and this ointment
may be applied on the wound in order to obtain a scar
less wound healing. With this study, B. perennis gained a
scientic justication as a vulnerary medicinal herb.
Declaration of interest
e authors report no conicts of interest.
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