Rom J Morphol Embryol 2013, 54(4):10531059 [609279]

Rom J Morphol Embryol 2013, 54(4):1053–1059
ISSN (print) 1220–0522 ISSN (on-line) 2066–8279 OORRIIGGIINNAALL PPAAPPEERR
Evaluation of the wound-healing effect of a
novel Hypericum perforatum ointment
in skin injury
ANCA IRINA PRISĂCARU1), C. V. ANDRIȚOIU2), CORINA ANDRIESCU3),
E. C. HĂVÂRNEANU4), M. POPA1), A. G. M. MOTOC5), ANCA SAVA6)
1)Department of Natural and Synthetic Polymers
2)Department of Chemical Engineering
“Gheorghe Asachi” Technical University, Iassy
3)Department of Pathological Anatomy and Prosecture,
“Sf. Spiridon” Municipal Clinical Emergency Hospital, Iassy
4)Faculty of Psychology and Education Sciences,
“Alexandru Ioan Cuza” University, Iassy
5)Discipline of Anatomy and Embryology,
“Victor Babe ș” University of Medicine and Pharmacy, Timisoara
6)Discipline of Anatomy and Embryology,
“Grigore T. Popa” University of Medicine and Pharmacy, Iassy
Abstract
The present experiment aims to formulate and characterize a new phytotherapy ointment based on a total extract of Hypericum perforatum
included in a novel ointment base. In order to investigate the healing properties of the ointment, in vivo experimental wound models of linear
incision, circular excision and thermal burn were performed on Wistar rats. Topical treatment was achieved daily, for 21 days. Clinical and
macroscopic evaluation, determination of wound contraction rate, period of re-epithelialization, and histopathological examinat ion were
achieved, along with the determination of the particle diameter and particle size distribution of the ointment. The results dem onstrate that
the tested novel ointment has significant wound healing effect in skin injuries and reveals to be safe for use.
Keywords : Hypericum perforatum , incision, excision, thermal burn, wound-healing effect.
 Introduction
Cutaneous wounds are the result of a disruption at the
level of skin integrity. The healing process depends on
local wound factors, systemic mediators, the underlying
disease, and the type of injury, involving a series of
well-organized cellular and molecular events, including
inflammation, angiogenesis, fibroplasia, wound contraction, epithelialization, and matrix remodeling. These factors
combine to determine if physiologic or acute wound
healing occurs, or if there is an abnormal healing process,
also called chronic wound healing. If the process of tissue
repair following an inadequate treatment fails, they become
chronic wounds. Besides the fact that these chronic dermal injuries affect negatively the quality of patients’
life, their management and care need high economical
resources, a rather important problem especially for the
developing countries [1].
The basic principle of optim al wound healing is to
minimize tissue damage and provide adequate tissue
perfusion and oxygenation, proper nutrition and moist wound healing environment to restore the anatomical
continuity and function of the affected part [2]. The last
decades bring natural remedies into the medical forefront,
having as major role the use of plants in the treatment of
different disorders. The concept of phytotherapy treatment is reconsidered by achieving in vivo and in vitro studies
regarding the confirmation of the healing effects of plants, the determination of the activ e principles responsible for
these effects, and the elucid ation of their mechanism of
action [3–5].
The present experiment aims to formulate and prepare
a novel ointment based on the total extract of the aerial parts of St. John’s wort, to determine the particle diameter
and particle size distribution and to evaluate its efficacy
in the re-epithelialization processes on three in vivo
experimental models (incision, excision, and thermal burn).
 Materials and Methods
Chemical and materials
Ethyl alcohol, petrolatum, lanolin, and olive oil ( Olivae
oleum virginale ) were purchased from Sigma-Aldrich.
The aerial parts of Hypericum perforatum (St. John’s
wort) were collected from the Botanical Garden, Iassy,
Romania, and a voucher specimen was identified by the staff of the same institution.
Preparation of the ointment
After collection, the plant material was dried in a dark
room with controlled temperature and relative humidity.
R J M E
Romanian Journal of
Morphology & Embryology
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Anca Irina Pris ăcaru et al.
1054
Samples of the dried plant material with moisture content
of 10% were mechanically ground to obtain a homogenous
drug powder. The oil extract was prepared by weighting
an amount of 50 g powder that afterwards was macerated in 500 mL of virgin olive oil in dark brown jars, at room
temperature, for two weeks. The ethanol extract was
obtained by weighting another quantity of 50 g powder that was macerated in 500 mL of 70% ethanol oil in
dark brown jars, at room temperature, for two weeks.
In the end, the extracts were filtered through gauze and
placed in dark brown jars wi th stoppers. The ointment
base was prepared by mixing petrolatum and lanolin in equal amounts on water bath (40
0C) until a homogenous
base was obtained. Fifteen mL of Hypericum oil extract
and 15 mL of Hypericum ethanol extract were gradually
included into the ointment base, until homogenization was
complete.
Determination of the particles size distribution
Particles size distribution was measured on an aqueous
sample dispersion using a la ser diffraction analyzer
(Shimadzu SALDI-7001), with a refractive index of 1.60–0.10i.
Experimental models of dermal injury
Animals and housing
The experiment was unfold ed on adult male rats,
Wistar strain, having a body weight of 220–250 g. The animals were kept in a light and temperature-controlled room with 12:12 hours light–dark cycles, where the temperature (22±0.5
0C) and relative humidity (65–70%)
were kept constant. Each rat was kept in a separate cage with free access to standard laboratory diet and water.
All the experimental proceedi ngs achieved on laboratory
animals (Wistar rats) in this study were in agreement with the guidelines of animal bioethics from the Act on Animal Experimentation and Animal Health and Welfare Act from Romania and were in compliance with the European Council Directive of 24 November 1986 (86/
609/EEC). The experiment was approved by the Ethics Commission of the “Grigore T. Popa” University of Medicine and Pharmacy, Iassy.
Study design
The experiment included three groups of Wistar rats
(seven animals per group): negative control group (control
group with incision, excision and thermal burn models,
not treated), Hyperici herba ointment group (treated
with the tested Hyperici herba ointment), and ointment
base group (treated with the ointment base). Before performing the experimental models, the animals were
anesthetized with Ketamine i.p. (100 mg/kg) and the
hairs on the dorsal part of the rats were shaved and cleaned with 70% alcohol.
Three experimental wound models (linear incision,
circular excision, thermal burn) were achieved on each animal. The incision wound model applied in this study
was based on a previously described model [6], with
some modifications. Two linear paravertebral incisions (1 cm long) were made with a sterile surgical blade through
the full thickness of the skin at the distance of 1.5 cm
from the midline of each side of the vertebral column. The excision wound model used in this study was based
on a previously described model [6], with some modifi-
cations. The circular wound was created on the dorsal
interscapular region of each animal by excising the skin with an 8 mm biopsy punch; wounds were left open.
The burn model used in the present study was based on a
previously described model [7], with some modifications. The original tip of a 40 W soldering iron was replaced with
a square copper plate measuring 9×8 mm and brought to
100
0C. A laser high performance non-contact thermometer
(Raynger MX4) was used to measure the desired temperature.
The device was placed vertically under its own weight on the back of the rats for nine seconds, in order to induce a
deep-partial thickness burn. I mmediately after each burn
injury, the wound was rinsed with normal saline solution [7].
The Hyperici herba ointment and the ointment base
were applied topically once a day for 21 days, until the
complete healing of the wound.
Evaluated parameters
Clinical and macroscopic evaluation, determination
of wound contraction rate, period of re-epithelialization,
and histopathological examination were performed at
days 1, 2, 3, 6, 9, 12, and 21 of treatment. In the end, a specimen sample of tissue removed from the healed skin
of all rats was taken with a 3 mm biopsy punch in order
to be analyzed by histopathological examination.
Clinical evaluation

Clinical examination evaluated the following parameters:
edema, inflammatory infiltrate, congestion, formation and falling of the scab, the type of scar.
Measurement of wound contraction rate (WCR)

The wound contraction rate (WCR) was calculated as
the percentage of the original wound size (50.27 mm2)
for each animal on days 6 and 9, as shown in formula:
%100
00AA AWCRt
where
A0 – Initial area of wound at day “0” of experiment;
At – Area of wound at day “ t” of experiment.
Digital photography was used to capture all wounds at
days 1, 2, 3, 6, 9, 12, 21, and wound surface areas were measured in mm
2 with SigmaScan software (SigmaScan
Ro, version 5.0).
Measurement of period of re-epithelialization
Falling of scab leaving no raw wound behind was
taken as the end-point of complete epithelialization and
the days required for this were taken as period of epithelialization.
Histopathological examination

In order to collect the samp les of the healed skin for
the ascertainment of the epith elialization process, animals
were anesthetized with Ketamine i.p. (100 mg/kg). The collected samples were fixed in 10% buffered formalin
for at least 24 hours, progressively dehydrated in solutions
containing an increasing percentage of ethanol (60, 80, 90, and 98%, v/v), clarified with amyl alcohol, embedded

Evaluation of the wound-healing effect of a novel Hypericum perforatum ointment in skin injury
1055
in paraffin under vacuum, sectioned at 5 μm thickness,
deparaffinized, and stained with Hematoxylin–Eosin (HE)
and Szekely (Sz).
Statistical analysis
The data obtained from excision wound model were
analyzed by one-way ANO VA followed by Bonferroni
post-test. Statistical analysis was performed using SPSS 15,
where p<0.05 was considered statistically significant.
 Results
Determination of the particles size distribution
The result of the laser diffraction measurement for
the average diameter of the dispersed particles for the Hyperici herba ointment is shown in Figure 1 and Table 1.
The mean values expressed as number distribution and volume distribution show that 75% of the number of particles have a size of 249 nm.
(A)

(B)

Figure 1 – Laser diffraction diagrams of the average
diameter for the Hyperici herba ointment: (A) Number distribution; (B) Volume distribution.
Table 1 – Particles size distribu tion for the Hyperici
herba ointment
Dimension of particle
amount [ μm] Mean 25%D 50%D 75%D
Number distribution (D n) 0.215 0.159 0.193 0.249
Volume distribution (D v) 2.304 1.064 2.375 5.398
Clinical and macroscopic results
The macroscopic evaluation of epidermal lesions for
the Hyperici herba ointment group demonstrated the
efficacy of the treatment with the novel ointmentt containing
the total extract of St. John’s wort. The complete healing occurred after six days of treatment in the case of incision model, and after nine days in the case of excision and thermal burn wound models, the time needed for re-epithelialization of the wounded skin being much shorter than in other studies [8].
Wound contraction rate
The wound areas were measured at days 1, 2, 3, 6, 9,
12, and 21. The results are listed in Table 2 and are expressed as mean ± standard error mean (SEM). Table 2 – The values of the wound contraction rate
Experimental
groups Treatment WCR_day 6
(Mean ±
SEM) WCR_day 9
(Mean ±
SEM)
Hyperici herba
ointment groupHyperici herba
ointment (one
application/day) 80.94±0.31 96.56±0.34*
Ointment base
group Ointment base
(one application/day) 26.74±1.23 33.79±1.28**
Negative
control group No treatment 5.78±1.79 14.85±1.70
*P=0.0001, when compared to the negative control group and the
ointment base; ** P=0.0001, when compared to the negative control
group.
No important changes regarding the contraction of the
wounds took place in the firs t three days of treatment
(as these are the days when inflammatory processes take place). The cellular proliferati on starts after day 3, and
significant reduction in the wound areas ( p<0.001) was
achieved at days 6 and 9. There can be noticed that the treatment with Hyperici herba ointment shows favorable
effects even from the 6
th day of the experiment, when
compared to the group treated with the ointment base (80.94±0.31 versus 26.74±1.23) and especially in compa-
rison with the negative control group (not-treated) (80.94±
0.31 versus 5.78±1.79). In the 9
th day of treatment, the
results are clearly positive, for the Hyperici herba ointment
group the percent of wound closure being of 96.56±0.34
versus 33.79±1.28 (the wound closure for the group treated
with the ointment base) or versus 14.85±1.7 for the
not-treated group. In the 12th day of treatment, the re-
epithelialization process was completed for the excision wound model.
Period of re-epithelialization
Hundred percent wound closure was seen in the 12
th
day of treatment with the Hyperici herba ointment when
compared to the ointment base group (18th day of the
experiment) or to the negative control group which took
21 days for the normal healing.
Histopathological results
The histopathological examination of the skin samples
prelevated from the affected areas that were treated for
21 days with the Hyperici herba ointment revealed the
presence of a mature granulation tissue in almost all the depth of the dermis (for the incision and excision wound
models), while in the case of the thermal burn wound
model, the granulation proce ss took place in one third of
the dermis (Figure 2).
Regarding the group treated with the ointment base,
edema can be observed in the case of the linear incision
wound model, the presence of koilocytes is noticed in
the re-epithelialized areas of excision, and important
stasis is present in the hypo dermis prelevated from the
thermal burn (Figure 2).
For the not-treated group (negative control group),
the epithelialization process is clearly delayed and the results depend on the experi mental wound model: severe
congestion is seen in the hypodermis of the incision
wound model, foreign-body granuloma is present in the reticular dermis of the excision wound model, and stasis with thickening of the blood vessels is seen in the case of the thermal burn wound model (Figure 2).

Anca Irina Pris ăcaru et al.
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Microphotographs and description
Hyperici herba ointment group
(A)
(B)
(C)
Ointment base group
(D)
(E)
(F)
Negative control group
(G)
(H)
(I)
Figure 2 – Histopathological evaluation of the tissue samples removed from the healed skin of the Wistar rats at the
end of the treatment. Hyperici herba oi ntment group: (A) Regenera ted rectilinear epidermis (incision, HE staining,
×200); (B) Regenerated epithelium (excision, HE staining, ×400); (C) Normal aspect of the collagen in the dermis
(thermal burn, Sz staining, ×200). Oi ntment base group: (D) Epidermis an d dermis – collagenization and edema
(incision, HE staining, ×100); (E) Epider mis with koilocytes and der mis (excision, HE staini ng, ×200); (F) Stasis in
the hypodermis – detail (thermal burn, HE staining, ×400) . Negative control group: (G ) Severe conges tion in the
hypodermis (incision, HE staining, ×200) ; (H) Foreign body granuloma in the retic ular dermis (excision, HE staining,
×200); (I) Stasis in the hypodermis, blood vessel with thickened walls (t hermal burn, HE staining, ×200).

 Discussion
The optimal wound healing consists of minimizing
tissue damage and providing adequate tissue perfusion
and oxygenation, proper nutrition and moist wound healing
environment in order to restore the anatomical continuity and function of the affected areas [2]. Wound healing
involves continuous cell–cell and cell–matrix interactions
that allow the process to proceed in three phases: inflammation (0–3 days, consisting of the establishment
of homeostasis and inflammation), cellular proliferation
(3–12 days, consisting of granulation, contraction and epithelialization) and remodeling (that last from three to
six months, which ultimately determines the strength and
appearance of the healed tissue) [9–11]. These phases overlap and ideally a plant-based remedy should affect at
least two different processes before it can be said to have
some scientific support for its traditional use [3, 12].
The total extract of the aerial parts of St. John’s wort
improve the healing of the skin injuries by its anti-
bacterial, antioxidant, and anti-inflammatory effects. Wounds provide an environment for the growth of
microorganisms [13]. An infected wound is less likely to heal, thus removal and prevention of further infection is a key to rapid and effective wound healing [3]. Infected
wounds attract high levels of phagocytic cells, which
release reactive oxygen species in an attempt to fight infection; however, these molecules can damage the host cells and delay the healing process [14]. Hyperforin from the Hyperici herba extracts, with a chemical structure
related to that of the antibacterial keto-enols from the
common hop cones, shows antibacterial properties
against Gram-positive bacteria ( Staphylococcus aureus ,
Corynebacterium diphtheriae ), this effect being sustained
by the presence of tannin, hypericin and essential oil [15].
Although the anti-inflammatory effect of the Hypericum
extracts was attributed mainly to the inhibitory action of quercetin upon the signal tr ansduction pathway, recent
experiments reveal an important role of hyperforin, demonstrating an inhibitory effect upon the lymphocyte reaction at the level of the epidermal cells and upon the

Evaluation of the wound-healing effect of a novel Hypericum perforatum ointment in skin injury
1057
T-lymphocyte proliferation [16]. Hyperforin also interferes
with prostanoid generation in biological systems, particularly with key enzymes participating in prostaglandin (PG) E2 biosynthesis, i.e., cyclooxygenases (COX)-1/2 and
microsomal PGE2 synthase (mPGES)-1 which play key roles in inflammation and tumorigenesis [17]. On the other hand, hyperforin is one of the natural compounds with a strong inhibitory effect upon cyclooxygenase-1 (COX-1) and lipoxygenase-5 (LOX-5) [18]. This dual mechanism offers the rational basis for the traditional use of St. John’s wort in inflammatory dermal disorders. Recent studies demonstrate that hyperforin may interfere with other inflammatory responses of the leukocytes, including the marked inhibition of the reactive oxygen species and release of elastase. These effects seem to be the result of the implications of hyperforin in the G protein-signaling cascade [19].
Burn injuries involve stimulation of intravascular
neutrophils and initiate system ic inflammatory reactions
by producing toxins such as reactive oxygen species
(ROS) almost in every tissue [20, 21]. Lipid peroxide is
thought to be one of the most harmful substances
produced in burn injuries [22, 23]. A strong connection has been demonstrated to exist between the quantity of
lipid peroxidation and the degree of burn complications
such as remote organ damage and shock [24, 25]. A primary effect of lipid peroxidation is decreased
membrane fluidity, which alters membrane properties
and can significantly disrupt membrane-bound proteins [26]. The mechanism of this event is the deformation
of cell membrane phospholipids by oxidizing radicals.
Oxidative damage of DNA causes formation of adducts of base and sugar groups, single- and double-strand
breaks in the backbone, and cross-links to other molecules.
Protein damage includes the oxidation of sulfhydryl
groups, reduction of disulfides, oxidative adduction of
amino acid residues close to metal-binding sites via metal-
catalyzed oxidation, reactions with aldehydes, protein–
protein crosslinking, and peptide fragmentation [27, 28].
Nitric oxide (NO) released by activated macrophages
after burn greatly enhances the oxidative stress [29].
Although many immune cells are able to synthesize NO,
including natural killer cells, mast cells, phagocytes as well as Th1-type cells [30, 31], macrophages in tissues
such as skin represent the main producers of this oxidative
mediator after activation [32]. NO, which represents an important mediator of immunosuppression following burn
injury, could be responsible for the inhibition of the
proliferative response [33]. NO acts directly to inhibit T-cell activity, thus participating in the development of
immunosuppression [34–36].
Burn significantly alters levels of cytokines. The
cytokines produced after burn such as interleukin-1 (IL-1)
and endotoxin activate nuclear factor kappa B (NF-KB),
which induces the synthesis of inducible nitric oxide
synthase (iNOS) which further produces large amounts
of NO and under conditions of substrate or cofactor limitation, may also synthesize superoxide (O
2•¯) [37].
NO becomes a potential pro-inflammatory and cytotoxic
factor by reacting with O 2•¯ to form the toxic product
peroxynitrite (ONOO¯) [38]. ONOO¯ can oxidize/nitrate
other molecules or decay and produce even more damaging species, such as the •OH [39, 40]. Reactive nitrogen
species (RNS), such as NO, react with guanine to yield
the deaminated com pound [41, 42].
The degree of novelty brought by our study consists
in the method of preparation of the ointment, using a new ointment base (made up of equal amounts of petrolatum and lanolin) and the total extract of Hypericum
perforatum (made up of both oil and hydroalcoholic
extracts). The method of preparation aimed to extract
from the aerial parts of Hypericum perforatum all the
active principles (having a hydrophobic or hydrophilic character), mainly hyperforin, considered to be the main
component responsible for the therapeutic effects of the plant.
The extraction step was preferred to the synthesis of
hyperforin, as this compound has a unique molecular architecture [43]. Despite its relatively small size, the structure constitutes a thorny synthetic challenge and remains to this day defiant to chemical synthesis [44, 45]. It contains asymmetric vicinal quaternary centers and a
densely functionalized tetracar bonyl array. Its prenylated
bicyclo[3.3.1]nonanone core is conserved among a number of other acylphloroglucinol derivatives. Synthetic routes to such polycyclic polypre nylated acylphloroglucinols
have been developed [46]. Recently, a novel synthetic sequence to polyfunctional, bridged medium-sized rings
from simple cyclic ketones has been reported [44]. To
date, however, no total synthesis has been described for any of the more than 50 members of this class of
substances.
The wound healing process is evident in all the three
types of dermal affection: incision, excision, and thermal
burn. Regarding the incision, the clinical result consists
in the formation of a mature, slender, pliable scar in comparison with the negative control group, which shows a hypertrophic, rigid scar. It is worth mentioning the positive effect of the Hyperici herba ointment upon the
rate of wound closure, thus suggesting that St. John’s
wort contains active principles that act synergically in the
wound contraction process (one of the most powerful phenomena in the human body).
Maybe the most important observation is regarding
the effect of Hyperici herba ointment upon the thermal
lesion. Besides this effect, the method of treatment of
the thermal burn is a premiere, as it is known the fact
that thermal injuries are treated first of all by the excision of the affected tissue area, and then by the classical local/systemic procedure. In the present experiment, we have started from the premise that the formed scab is a solution of protection of the organism against the
aggressive agent (high temperature in this case). Thus,
we have applied the topical treatment keeping the scab. The ointment containing the total extract of Hypericum
perforatum maintained the moisture degree at the level
of the lesion, thus facilitating the penetration of the active principles through the affected skin. The clinical
evaluation revealed in the firs t three days of treatment a
perilesional inflammatory infiltrate that started to decrease in the 4
th day. Samples of the affected areas were
prelevated along the treatme nt period with a biopsy
punch and studied by electronic microscopy, revealing the benefic result upon the regeneration epithelium.
Following these results, we assume that the healing process

Anca Irina Pris ăcaru et al.
1058
unrolled as follows: after the favorable effect exerted
by the ointment upon the de crease of the inflammatory
infiltrate, the healthy perilesional cells migrated towards the adjacent lesional areas, thus leading to the regeneration
of the epithelium beneath the scab. As the regeneration
process advanced, the healthy tissue pushed the scab away, finally favoring the debridement process. Following the debridement of the scab, the beneath tissue appeared to be newly regenerated, with a specific color, and gradually became mature.
Another advantage of the tested ointment consists
in the reduced diameter of the particles (75% of the particles have a diameter of 249 nm determined through the laser diffraction technique) and a good distribution, thus leading to a superior penetration of the active principles through the skin.
Furthermore, the positive effect of the ointment resulted
into a very good cosmetic appearance of the affected skin areas, thus recommending the Hyperici herba ointment
in the treatment of burns th at affect a small surface of
the skin.
The histopathological results also certify the favorable
implication of the tested ointment in the wound healing
process, with the regeneration of the epithelium and the normal disposal of the collagen fibers. Furthermore, we can sustain that the ointment containing the total extract of Hypericum perforatum reduced the edema at the level
of epidermis and dermis and the congestion degree in
the hypodermis in the case of lesion with loss of
continuity. Moreover, the novel tested ointment had an inhibitory effect upon the stasis in the hypodermis in the case of thermal burn wound.
 Conclusions
The clinical and histopatho logical results, along with
the wound contraction rate and period of epithelialization,
demonstrate the wound-healing effect of the novel
St. John’s wort ointment in linear incisions, circular
excisions and thermal burns . The results are clearly
superior to those cited in other studies.
Acknowledgments
This paper was supported by the project PERFORM-
ERA “Postdoctoral Performance for Integration in the
European Research Area” (ID- 57649), financed by the
European Social Fund and the Romanian Government.
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Corresponding author
Anca Sava, MD, Discipline of Anatomy and Embryology, Facult y of Medicine, “Grigore T. Po pa” University of Medicine
and Pharmacy, 16 Universit ății Street, 700115 Iassy, Romania; Phone +40744–303 678, Fax +40232–301 633,
e-mail: dr_anca_sava@yahoo.com

Received: May 23, 2013
Accepted: December 17, 2013