Surgical Science, 2011, 2, 16-21
doi:10.4236/ss.2011.21005 Published Online January 2011 (http://www.SciRP.org/journal/ss)
The Hemeostatic Efficacy of ANKAFERD after Excision of
Full Thickness Burns: A Comparative Experimental Study
in Rats
Yusuf Kenan Coban1, Elif Ozerol2, Kevser Tanber2, Serkan Erbatur1,
Ahmet Hamdi Aytekin1, Cemal Fırat1
1Inonu Üniversity Medical Faculty, Department of Plastic & Reconstructive Surgery and Burn Unit, Malatya, Turkey.
2Inonu Üniversity Medical Faculty, Department of Biochemistry, Malatya, Turkey.
Received October 14, 2010; revised October 20, 2010; accepted October 24, 2010
Abstract
Objective: In order to evaluate the effects of Ankaferd blood stopper (ABS) on post-excisional burn wound
bleeding via monitoring early blood hemoglobin level changes and to make a comparison with a standart
topical treatment, a controlled experimental study was conducted. Design and Interventions: A contact burn
model of full-thickness injury with 30% TBSA was used. Following the burn injury, excisions of burned ar-
eas were done at postburn 48th hour. 24 male Spraque Dawley rats were divided into control, adrenaline,
ABS-solution and ABS-pad treatment groups. In control group no treatment was done for bleeding, but the
other three groups had topical treatments. Samples were taken at the begining of the study, just before and 1
hour after the burn wound excisions for measurement of haemoglobine (Hb) levels and additionally the ex-
ternal bleeding amounts were measured by weighing the topical pads. Measurements and main results: Base-
line Hb levels of control, adrenaline, ABS-solution and ABS-pad treatment groups were 15.06 ± 0,83, 15,82 ±
0.83, 16.23 ± 1.14 and 15.16 ± 1.46 respectively. At the 48th hour of postburn injury, the Hb levels of these
groups were 13.82 ± 0.58, 13.68 ± 1.26, 13.79 ± 0.90 and 13.57 ± 0.89. Mean blood loss amounts (ml) after
burn wound excisions in groups were 1.44 ± 0.26, 0.65 ± 0.07, 0.53 ± 0.08 and 0.44 ± 0.09. Conclusions:
ABS was found to be as effective as topical adrenaline on reducing excisional bleeding in the experimental
burn injury model.
Keywords: Ankaferd Blood Stopper, Burn Surgery, Bleeding
1. Introduction
Bleeding is a major problem during early excision of
burned skin [1]. Reducing blood loss during burn surgery
is important for decreasing patient morbidity from
hemodynamic derangement and minimizing the risk of
transfusion-associated infections [2]. Different techniques
have been used to reduce the intraoperative blood loss in
burn surgery including warm saline-soaked pads, topical
solutions of thrombin with or without adrenaline, topical
vasoconstructors, compressive dressings or tourniquets,
and vasopressin continuous intravenous infusion [3,4].
Ankaferd blood stopper (ABS) is a herbal extract
which has been used historically as hemostatic agent in
Traditional Turkish medicine. It comprises of standard-
ized mixture of herbs Thymus vulgaris, Glycyrrhiza
glaba, vitis vinifera, alpinia officinarum and urtica dioica.
Exposure to ABS results in a very rapid formation of
network within the plasma and serum without interfering
with any individual clotting factor [5]. ABS seems to be
effective in bleeding control in different tissue injuries.
We tested the hypothesis that ABS could be useful
agent for decreasing blood loss after post-burn wound
excision in a randomized, controlled experimental study.
Change in blood hemoglobin levels was used as a pri-
mary outcome as well as actual blood loss, as it has been
shown that changes in hemoglobin levels is strictly cor-
relates with physiologic parameters of hemorrhage [6].
2. Methods
24 young adult male spraque-dawley rats (mean weight

Y. K. COBAN ET AL.
17
200 ± 20 gram) obtained from Inonu University Animal
Research Center were used in the study. The animals
were kept in a room at a constant temperature of 21°C 12
h dark and 12 h light cycle and fed standard pellet chow
and water, which were avaliable ad libitum. The study
protocol (Figure 1) was approved by Institutional Ethical
Committe (approval no: 2009-25). The experimental
project was furtherly supported by Inonu University
Scientific Research Foundatition (project number,
2010/38). All the rats were allowed free to take water
and ad libitum for post-injury days. Three rats were
housed in each cage during postburn management. The
animals were divided randomly into four groups as con-
trol, adrenaline, ABS solution, and ABS pad.
Prior to burn injury, the animals were anesthesized
with ketamine hydrochloride and xylazine hydrochloride.
Dorsum of each rat was shaved with an electrical clipper
and then the area was burned in order to obtain 30%
TBSA of full-thickness burn injury.
A comb burn model of full-thickness injury with in-
tervening unburned interspaces was used. The method
depended on contact burn injury using a metal brass
thomb heated in boiling water. The dimensions of the
brass comb were 20 × 20 × 60 mm, with four 10 × 20 –
mm rectangular prongs separated by three 5-mm-wide
grooves. The brass comb was preheated in boiling water
(100°C) for 5 minutes and applied without pressure on
one side of the back for a period of 3 minutes [7].
2.1. Resuscitation Fluid Formulation and Its
Estimation
Following to the burning insult, serum physiologic was
subcutaneously given to each rat’s dorsal neck region at
a rate of “4 × kg × burn percentage” for fluid resuscita-
tion. The amount of resuscitation fluid used for each rat
was:
4 × 0, 20 kg × 30 = 24 ± 2 cc
for the first day.
Body surface area is difficult to measure in small
laboratory animals. It is therefore estimated as surface
area (cm2) = 10 x Mass (g 0.66). The prefactor “10” is
empirically adjusted. The prefactor for rats is 9.1.
Therefore to find the surface area of a rat, plug its mass
into the equeation 9.1 × gram 0.66. So “a 300 gram rat”
would have a surface area of 392.6 cm2 [8,9]. In our
model 87 cm2 would corresponds to 30% of TBSA, as
our average rat weight was 200 gr. The preheated brass
comb was applied to the dorsum in order to obtain a
mixed contact burn area of 87 cm2 in repeating sessions.
At the 48 th hour of postburn, the burned areas were
excised with using surgical blade no.10 under ketamine
(Alfamine, Alfasan, Nedherland) and Xylazine Hcl (Al-
fazyne 2%, Alfasan, Nedherland) anesthesia. Ketamine
and xylazine was given to the rats at dose of 50 mg/kg
and 5 mg/kg intraperitoneally in each session. No elec-
trocautery was used during burn wound excision. The
topical treatments were started just after the completition
of surgical debridement in all groups, which were epi-
nephrine or Ankaferd soaked gauze and ankaferd pads or
simply serum physiologic soaked gauzes application to
the wounds following to debridements. In adrenaline
group, the required amount of epinephrine solution
(Adrenalin 0.25 mg/ml, Biofarma, Turkey) was diluted
1:10 with saline (concentration 0.025 mg/ml). Bleeding
or oozing amounts were also macroscopically recorded,
photographized and measured in each group (Figure 2).
Figure 1. Experimental protocol. *Burn injury on rats;
**Burn wound excision time. Black arrows: timing of
obtaining blood samples for hemoglobin measurements,
zero time, just before burn wound excision and 1 hour later
at the second postburn day. 1d; First day, 2d; second day
Figure 2. A-Typical bleeding or oozing from debridement-
sites in rats: from left to right are control, adrenaline,
ABSsoaked gauze, ABS pad.
2.2. Amount of Bleeding Estimation
The amount of bleeding was measured by means of a
soaked gause. The blood was collected on soaked gause,
which was weighed before and after the procedure on a
0.1 g accurate scale. The difference in the weight of the
gause before and after the procedure indicated the amount
of bleeding. The application time over the bleeding area
varied between 45 minutes and 1 hour.

Y. K. COBAN ET AL.
18
2.3. Measurement of Hemoglobin Concentration
in Whole Blood and Blood Sampling
Hemoglobin levels were measured preoperatively, at the
second day of postburn and 1h after completion of the
operation. For measurement of hemoglobin concentration
in whole blood, samples of 0.5 cc. were taken from
hearts of each rat into containers. Lower hemoglobin
concentrations were found in heart blood than in the tail
blood of small rats (less than 100 g body weight). It has
been shown that there was no significant difference in
the haemoglobin values of blood taken from the tail or
from the heart in large rats [10].
The blood samples were send rapidly for hemoglobin
analysis in each session. After obtaining the third and the
last blood samples, all the rats were sacrified.
The levels of hemoglobin in blood were estimated by
using the cyanomethemoglobin method described by
Drabkin in a spectrophotometer [11]. Drabkin’s solution
on mixing with whole blood converts Hemoglobin to
Cyanomethemoglobin. The absorbance of Cyanomethe-
moglobin is proportional to the Hemoglobin concentra-
tion. Drabkin’s solution contains potassium ferricyanide,
potassium cyanide and sodium bicarbonate. This method
is simple, rapid and reliable and measures all types of
hemoglobin except sulfohemoglobin.
2.4. Statistical Analysis
The data were presented as mean ± standart deviance. A
software system of SPSS 11.0 version was used statis-
tical analysis. ANOVA and posthoc Tukey test were
used for multiple comparisions between the groups.
Wilcoxon Signed Rank test was used for comparision of
repeated measures of blood hemoglobin levels. A P value
less than 0,05 was accepted as significant.
3. Findings
Hb levels measured separately in three sessions is shown
in Table 1. Hb levels at the begining of study did not
differ between the groups (p > 0.05). Hb levels obtained
after two days of burn injury did not also show any dif-
ference between the groups, but significantly decreased
in comparision of previous values in all groups (Wil-
coxon signed rank test, p < 0.05). Hb levels obtained 1
hour following to burn excisions from control group was
significantly lower than three other groups (p < 0.05).
There was no difference between the groups of A,
ABS-S, and ABS-P with respect to Hb levels obtained at
third session (p > 0.05).
There were significantly higher amounts of blood loss
in group C than other three groups (p > 0.05). A, ABS-S
Table 1. Hemoglobin levels and blood loss amounts in
groups, and multiple comparisons with ANOVA.
Groups HB1 (g/dl)HB2 (g/dl) HB3 (g/dl) Blood loss
(ml)
C 15.06 ± 0.8313.82 ± 0.58 11.08 ± 0.79 1.44 ± 0.26
A 15.82 ± 1.1313.68 ± 1.26 12.70 ± 0 55 0.65 ± 0.07
ABS-S 16.23 ± 1.1413.79 ± 0.90 12.99 ± 0.83 0.53 ± 0.08
ABS-P 15.16 ± 1.4613.57 ± 0.89 12,57±0,76 0.44 ± 0.09
P
C-A N.S. N.S. 0.007 0.001
C-ABS N.S. N.S. 0.001 0.001
C-ABS padN.S. N.S. 0.012 0.001
A-ABS N.S. N.S. N.S. N.S.
A-ABS padN.S. N.S. N.S. N.S.
ABS-ABS
pad N.S. N.S. N.S. N.S.
N.S. non significant, (According to posthoc Tukey test). C: Control, A:
Adrenaline, ABS-S: Ankaferd blood stopper solution, ABS-P:Ankaferd
blood stopper pad. HB1: first hemoglobin measurements, HB2 second and
HB3 third measurements.
and ABS-P groups showed no difference with respect to
blood loss amounts (p > 0.05).
4. Results and Discussion
Topical hemostatic products containing thrombin are
commonly used in burn surgery to facilitate focal hemo-
stasis and graft adherence. But using these agents some-
times may encounter with unexpected clinical complica-
tions. One example to this type of clinical problem has
been reported by Foster et al. They found coagulopathy
due to factor V deficiency as evidenced by excessive
bleeding and abnormal coagulation parameters following
to repeated applications epineprine solution containing
bovine thrombin in a severe burn patient with 75%
TBSA [12].
A restrictive blood transfusion strategy and the use of
hemostatic agents may decrease morbidity and mortality
in trauma and burn patients [13]. Kahalley was the first
to report diminished intraoperative blood loss with sub-
cutaneous injection of a saline-vasopressor solution un-
der donor sites and debrided areas without severe sys-
temic side reactions [14]. This clinical observation was
further supported with others especially for children with
burn [15,16]. Minimal absorption of adrenaline without
systemic effects using a tumescent technique has been
described. This technique was later modified and found
to be effective in reducing the intraoperative and total
blood transfusion requirements in thermally injured pa-
tients [17]. Anthony et al. studied the effects of adrena-
line on hemodynamics and markers of tissue perfusion in
burned patient with comparing non-burned controls in-
cluded mostly adult age group of patients [18]. They
found increased heart rate, elevated adrenaline serum
levels up to 6h, and hiperlactatemia lasting up to 4 h fol-

Y. K. COBAN ET AL.
19
lowing to topical adrenaline application. Although the
use of topical adrenaline still seems to be a safe way to
be used in burned patients, its potential dangerous side
effects do not make it an ideal haemostatic agent.
Newer products are under investigation for finding the
most ideal haemostatic agent. In vitro studies evaluating
the haemostatic effects of ABS have shown that its addi-
tion to plasma and normal serum has resulted in forma-
tion of an encapsulating protein mesh that acts as a ma-
trix within which erythrocyte aggregation occurs in a
very fast way (less than a second) [19]. Several animal
studies of different experimental models including inci-
sional or excisional traumas have revealed its efficacy in
stopping the bleeding in vivo [20,21].
There are many prospective, retrospective or/and
case-control series studies in which ABS was effective to
stop neoplastic gastrointestinal bleedings, epistaxis, bleed-
ing after knife tonsillectomies, active cutaneous- subcu-
taneous incisional bleedings and bleedings where endo-
scopic variceal ligation failed [22-24]. Clinical reports
are also present of successful ABS usage via endoscope
or oral and rectal routes in gastrointestinal system haem-
orrhagies in where optimal bleeding control could not be
achieved in spite of endoscopic adrenaline injection and
argon plasma coagulation [25,26].
We chose to have 30% TBSA in the model, so as to
have a prominent Hb decline without causing fatal seri-
ous morbidity. Other reasons for chosing this model are
following: First, actual burn injuries seen in clinical
practice are mixed, i.e. some body areas with
full-thickness and some with uninjured. So the model
reflects much actual burn clinical scenario. Second, it is
cheaper and relatively safe for investigators [27]. Fa-
therly, over the course of next few days after injury most
of these ischemic interspace progresses to full-thickness
necrosis. The model has also been shown that gross vis-
ual appearance is good indicator of whether necrosis is
truly present as evidenced histopathologically 7 days
after injury. Resuscitation fluid was given subcutane-
ously instead of giving intraperitoneally, because it has
been shown that subcutaneous fluid administration as
means of rehydration is effective in other species includ-
ing humans [8]. It has also been shown that prophylactic
fluid administered mice via subcutaneous route have
improved survival times after carcinogen treatment [9].
Burn wound excision was done at 48th hour of postburn
injury, so that a closer resembling of early excision in a
clinical picture as early tangential excision of deep burns
is a widely accepted treatment modality. The decrease of
Hb levels seen in control group after burn wound exci-
sion was not observed in adrenaline and ABS treament
groups (p < 0.05).
ABS would be an advantage in traditional theraphies
on several aspects. It has antimicrobial and antifungal
effects which was demonstrated by several in vitro stud-
ies [28,29]. A novel finding of which ABS was found to
be inhibitory for MRSA, Acinetobacter, E. Coli and
Pseudomanas species growth was reported by Saribas et al
recently. This is very important, as most of these organ-
isms are responsible for burn wound sepsis [30].
Regarding to the effects of ABS on wound healing,
literature is scarce. Isler et al. reported ABS decreased
the inflammation and necrosis process and increased the
new bone formation in early bone healing period without
causing any foreign body reaction [31]. ABS application
time was only 1 hour in our study. We did not see any
adverse effects during the observation and could not
make any conclusion on wound healing. In an observa-
tional clinical study, Ozaslan et al. revealed that endo-
scopic application of ABS as a primary therapy in pa-
tients with bleeding due to chronic radiation proctitis was
found to be effective in healing radiation induced ulcers
[32]. The authors had also confirmed their finding with
another case report [33]. For now, the effects of ABS on
wound healing seem to be limited by the observation of a
decreasing effect on microvessel density measurements
in the tissue exposed to ABS, which suggest the presence
of secondary more sustained mechanism of hemostasis
besides the initial protein network [34].
Exposure to ABS in a certain area provides physio-
logical hameostasis process together with tissue oxy-
genation without calling out any individual coagulation
factor. This unique mechanism advantage provides ad-
vantage to ABS compared with others. Bleeding models
in animals pretreated with aspirin, warfarin or heparin
revelaed that ABS had effectively reduced the duration
and amount of bleeding [35,36]. Another most striking
effect of ABS is its in vitro positive effects over mesen-
chymal stem cell proliferation as shown by Kilic et al.
[37]. This would be another potential advantage of this
natural substance over traditional therapies, because a
severe regenerative process is needed during recovery
phase of massive burn injury.
5. Conclusion
Burned patients have a consumption coagulopathy which,
in combination with haemodilution during operation re-
sults in a clinically significant deficiency of coagulation
factors II, VII and X [38]. Any hemostatic agent that
does not interfere with coagulation factors would take
the advantage over other hemostatically active products.
The levels of coagulation factors II, VII, IX, X, XI, and
XIII were not affected by ABS [39]. Furthermore, ABS
as an adjunctive drug has been shown to treat a chal-
lenging upper gastrointestinal bleeding case with a low

Y. K. COBAN ET AL.
20
platelet count [40]. Fibrinogen appears not to be a nec-
essary component for formation of the protein network
of ABS, as it was found to be effective in a patient with
afibrinogenemia [41]. Overt disseminated intravascular
coagulation in which hypofibrinogenemia occurs due to
cosumtion cougulopathy seem to occur in critically ill
burn patients [42,43]. So the use of ABS in severe burn
patients with third degree injury may be useful adjunc-
tive measure to prevent fulminant course.
The experimental study supports the use of ABS in
achieving hemostasis in intraoperative managent of
blood loss at burn wound excision site and but further
studies are needed to clearify the effects of ABS on other
parameters in burn management.
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