The Hemostatic Effect of Ankaferd Blood Stopper in Rat Bleeding Models with Antithrombotic Drug Therapy: An Experimental In Vivo Study/Antitrombotik ilac Tedavisi Uygulanan Sican Kanama Modellerinde Ankaferd Kanama Durdurucusunun Hemostatik Etkisi: In Vivo Deneysel Bir Calisma.
Bleeding is a common challenging problem especially in patients who take an anticoagulant or antiaggregant treatment (i.e., patients with clotting disorders). Severe bleeding can be a life-threatening condition and should be managed effectively in a variety of ways including mechanic sponge pressing on the bleeding area; vessel ligation; applying chemical or electro-cauterization and cryotherapy; using topical haemostatic or vasoconstrictor agents and etc (1-4). Various haemostatic agents have been investigated for their role in haemostasis for decades (2,4).
As a commonly used blood stopper agent in our country, Ankaferd blood stopper [ABS (Ankaferd Health Products Ltd., istanbul, Turkey)] is a traditional folk medicinal plant extract that has been approved in the management of cutaneous, dental and postoperative external bleeding. In addition, it has been reported that ABS had bacteriostatic effects on gram positive and gram negative bacterial flora and induced wound healing (3,5-13). Safety, efficacy, sterility and nontoxicity of the product have been shown (http://www.ankaferd.com) (4).
ABS is a unique mixture of five plant extracts (Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum and Urtica dioica) (2,3,5,6,10,14). One of the noteworthy studies on this topic has revealed that use of ABS caused the rapid formation of protein network within the serum and plasma (5).
The mechanism of the haemostatic action for ABS is the formation of an encapsulated protein network for vital physiological erythrocyte aggregation. This protein network formation with blood cells, particularly erythrocytes, covers the primary and secondary haemostatic system without independently acting on coagulation factors and platelets (5,6). Other than haemostatic effect, Mihmanli et al. (13) has reported a proliferative effect of ABS on human leukocytes.
In the light of the above-mentioned data, this study aimed to investigate the in-vivo haemostatic effect of ABS on warfarin-induced and heparin-induced bleeding model in rats.
Materials and Methods
The study protocol and experimental design were in accordance with the European Community Council Directive of November 24, 1986 (86/609/EEC) and were approved by the institutional review board and Animal Ethics Committee of Gaziosmanpasa University Faculty of Medicine (approval number: 51879863-42).
Ankaferd Blood Stopper[R]
ABS (Ankaferd Health Products Ltd., istanbul, Turkey) products are available as a liquid (solution), spray or in a dressing (tampon) forms that contain a standardized mixture of 5 medicinal plant extracts. The liquid form of ABS was used in the study and it includes active ingredients as follows:
0.16 mg of dried leaf of Vitis vinifera, 0.12 mg of dried root of Urtica dioica, 0.14 mg of dried leaf of Alpinia officinarum, 0.18 mg of dried leaf of Glycyrrhiza glabra, and 0.10 mg of dried leaf of Thymus vulgaris (1).
Forty-eight male Wistar rats, with an average weight of 270-320 g, were used in this study. They were housed in specially designed wire cages and maintained on a 12 h-12 h light-dark cycle with a constant room temperature of 23 [degrees]C. Rats were allowed access to water and standard rodent diet ad libitum. All procedure was performed by the same specialist who authorized to animal experiments.
Because of the limitations of ethics committee on the number of rats, they were not determined by using power analysis. So, 48 male Wistar rats were selected by the directions of Animal Ethics Committee of University and randomly divided into six groups of 8 animals each. The six groups were as follows:
1. Control group (group 1) rats had no pretreatment with any drug.
2. Ankaferd group (group 2) rats had no pretreatment with any drug.
3. Warfarin group (group 3) rats had pretreated with warfarin dissolved in saline (2 mg/kg) orally by a feeding catheter custom-made of silver for 3 consecutive days before experiment.
4. Warfarin-Ankaferd group (group 4) rats had pretreated with warfarin dissolved in saline (2 mg/kg) orally by a feeding catheter custom-made of silver for 3 consecutive days before the experiment.
5. Heparin group (group 5) rats were given an equal volume (0.25 mL) of standard heparin sodium (640 IU/kg) intraperitoneally 3 times a day for 3 consecutive days before the experiment.
6. Heparin-Ankaferd group (group 6) rats were given an equal volume (0.25 mL) of standard heparin sodium (640 IU/kg) intraperitoneally 3 times a day for 3 consecutive days before the experiment.
The maxillary right first molars were extracted under general anaesthesia (Alfamine 10%, Ege-Vet and Rompun[R], Bayer HealthCare AG cocktail) using dental instruments (Figure 1). The extraction comprised fiberotomy, luxation, and tooth removal. The surgical wounds were left for secondary healing. Two ml of saline solution was applied to the each empty tooth sockets in group 1, 3 and 5 immediately after the extraction. Two mL of ABS was applied to the extraction sockets in group 2, 4 and 6 (Figure 1) immediately after the extraction.
The animal number in each group was determined according to literature (11,14) knowledge and local ethic committee advice.
Bleeding Time Assay
The bleeding time was measured with a chronometer and the amount of bleeding was measured with a milligram sensitive scale in all 6 groups just started following the tooth extraction. Bleeding time was detected as the time passed after the start of bleeding just following the tooth extraction to cessation of bleeding. Bleeding times were recorded by a single specialist with using a chronometer.
Amount of Bleeding Assay
The amount of bleeding was measured by means of a cotton wool roll. Each cotton roll was weighed before the procedure on a 0.1-g accurate scale by an investigator blinded to the treatment. Immediately after the extraction, cotton roll was inserted to the socket area and when the roll was filled with blood it is removed and weighed again. This process was carried on until the bleeding stopped. The difference in weight was considered as the amount of bleeding.
The collected data results were analysed and also compared among 6 groups statistically by the ANOVA followed by Tukey test for pair-wise comparisons. P values less than 0.05 were considered as statistically significant.
There were not any complications observed during the study period and surgical procedure.
The mean bleeding time values of all groups were shown in the Table 1. The most prolonged bleeding time was observed in the warfarin group as 47.50 seconds and the shortest bleeding time was observed in Ankaferd Group as 2.87 seconds. Besides, there were not any statistical differences between Ankaferd and Control groups. Regarding the bleeding time, there was no statistically significant difference between warfarin and heparin groups (p>0.05). In the Warfarin and Heparin groups, the bleeding time was higher than those of the control and Ankaferd groups and the difference was statistically significant (p<0.05). The results showed that ABS administration decreased the bleeding time in both Warfarin-Ankaferd and Heparin-Ankaferd groups, but Ankaferd alone achieved no statistically significant difference when compared with control group (Table 1). The bleeding time of Heparin group was higher than those of the Heparin-Ankaferd, and Warfarin-Ankaferd groups (p<0.05).
Although the bleeding time was shortest in the Ankaferd group, there were no statistically significant differences among control, Ankaferd and Heparin-Ankaferd groups (p>0.05).
Amount of Bleeding
The amount of bleeding was highest in the Warfarin group as 0.041 grs, and this result was statistically significant when compared with Control and Ankaferd groups (p<0.05) (Table 1). There were no statistically significant differences among the Control, Ankaferd, Warfarin-Ankaferd, Heparin and Heparin-Ankaferd groups (p>0.05). However, when the results of Warfarin-Ankaferd, Heparin and Heparin-Ankaferd groups were compared with Warfarin group, there were statistically significant differences between them (p<0.05).
Warfarin group came first when both bleeding time and amount of bleeding results of all groups were taken into account.
Purpose of the present study was to evaluate the haemostatic effect of ABS on warfarin and heparin-induced rat bleeding models. Rat bleeding model is a well-known study model for bleeding control studies. Besides, tooth extraction model was used in order to induce bleeding. Tooth extraction causes bleeding, and bleeding normally stops in a few seconds without intervention in rats. In fact, ABS use in not-drugged rats is not essential for bleeding control but it was necessary to compare the amount and time of the bleeding in this study. Tooth extraction was selected for ABS bleeding models due to frequency of its practice in dental surgery area.
Two distinct anticoagulant drugs were utilized to investigate the haemostatic effects of ABS, warfarin and heparin. It is well-known that these drugs, especially warfarin, cause an enormous bleeding in tooth extraction sockets (15). Warfarin is the most commonly prescribed oral anticoagulant in humans especially who require dental extractions (15,16). Considering dental extractions, patients who take warfarin medication for prevention of cardiovascular thrombosis are at an increased risk of perioperative thromboembolism if the medication is interrupted but there is also a possible increased risk of bleeding if the medication is continued (15). Heparin has also similar status of its use. It is usually used by parenteral route (16).
The main indication for using anticoagulant therapy such as warfarin, heparin, low molecular weight heparins and etc. is to prevent or manage for arterial and venous thrombosis (7,16). The use of such anticoagulant agents alters the coagulation cascade in the body (1,16). One of the most important factors in coagulation is the vitamin K. Warfarin is one of the most common used vitamin K antagonist agent and it is quickly adsorbed and well tolerated by oral route. The anticoagulant effect of warfarin is slowly established but has a relatively prolonged effect, through a half-life of at least 48 hours (14,16). Heparin has some different effects on the coagulation but also activates anti-thrombin and other proteases involved in the blood clotting system that causes coagulation problems (1). The results of the present study showed that warfarin and heparin, as anticoagulant drugs, caused an important increase on bleeding time. ABS administration normalized the increase and stopped the bleeding as well. According to results of study, these anticoagulant drugs also increased the amount of the bleeding.
According to previous studies, oral surgical and clinical approach to the patients who take anticoagulant medication made it necessary to stop the anticoagulant treatment for several days or reduce the dose of anticoagulant drug for preventing severe life-threatening hemorrhage (7). But, currently, this radical approach for the treatment of patients undergoing anticoagulant therapy were replaced to an approach that advises to perform the oral surgical procedures without any intervention of anticoagulant treatment but with attention on the local bleeding control methods (7). Therefore, ABS was used as a local haemostatic agent to control warfarin and heparin-induced bleeding in present study. As a result, bleeding control was achieved by using ABS in rats which have been receiving either of these anticoagulant drugs or not.
Ankaferd blood stopper is a unique standardized herbal mixture being used as a haemostatic agent in Turkey for many years. The exact mechanism of ABS is yet unknown but there is a growing body of evidence on the efficacy of ABS on homeostasis (1,3-5,7,8,10-12,14). Additionally, it was found that ABS shortened the bleeding time in the rats receiving and not receiving anti-coagulant therapy both. Meric Teker et al. (17) reported that ABS-induced formation of the protein network affected the physiological haemostatic process without affecting any individual clotting factor. The present study also supports this literature knowledge.
Similar to the present results, Cipil et al. (14) reported that the bleeding time was reduced to 44% with ABS use in animals pretreated with warfarin. Sacco et al. (16) also demonstrated that ABS had a haemostatic effect on the cut tails of rats alone or in the presence of heparin or aspirin medication. Iynen et al. (1) showed that ABS irrigation effectively prevented nasal bleeding in rats pretreated with heparin sodium. Meric Teker et al. (17) reported the ABS as an effective, safe, quick, and easy-to-use alternative to the phenylephrine in patients with anterior epistaxis. But, they also revealed secondary haemorrhage in a few patients with using ABS. Iynen et al. (1) reported that ABS successfully shortened the haemostasis time and decreased the bleeding volume.
Warfarin and heparin have different biochemical anti-haemostatic actions on coagulation cascade mentioned in the literature (1,16). In contrast, the previous results have some contradictions. ABS does not affect the entire blood clotting system including coagulations factors (7,14,18-21). But, heparin and warfarin affect entire clotting system with various different ways (14). ABS has a different level of action on both anti-haemostatic agents.
The present study revealed that ABS more effective on heparin experiment models than warfarin without statistically significance. In addition the previous result of the present study, ABS should be used on warfarin models as a local haemostatic agent due to statistically significance between Warfarin group, Control group, and Warfarin-Ankaferd group in terms of bleeding time and amounts (p<0.05). Similarly, these previous results valid on heparin including groups (p<0.05).
The present study had a limitation as international normalization ratio (INR) test was not used for standardization of anti-coagulation level of blood. According to the literature, Clinicians should attempt to identify contributing factors for prolonged non-therapeutic INR. So, the risk of coagulation can be minimized, as well as costs of hospital stay and laboratory exams can be reduced (22). It is important to achieve the therapeutic levels of INR in the patients treated by anticoagulants (23).
The results of the present study concluded that topically administered ABS to empty tooth sockets immediately after the extraction is effective for homeostasis in warfarin and heparin-induced bleeding in rats. But, efficiency of ABS over warfarin-induced and heparin-induced bleeding patterns and whether if it affects in the same way or not, should be investigated in further studies. Also, the exact mechanism of ABS over haemostasis and the necessity of this agent in clinic use should be investigated in the future studies.
Ethics Committee Approval: Animal Ethics Committee of Gaziosmanpasa University Faculty of Medicine (approval number: 51879863-42).
Informed Consent: It was not taken.
Peer-review: Externally peer-reviewed.
Surgical and Medical Practices: N.A., i.A., M.K.T., Concept: N.A., i.A., Design: N.A., C.A., Data Collection or Processing: N.A., H.E., M.K.T., C.A., Analysis or Interpretation: N.A., H.E., Literature Search: N.A., i.A., H.E., Writing: N.A., H.E.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.
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[iD] Nihat Akbulut (1), [iD] ilker Akar (2), [iD] Hakan Eren (3), [iD] Cemal Asian (2), [iD] Mehmet Kemal Tumer (1)
(1) Gaziosmanpasa University Faculty of Dentistry, Department of Oral and Maxillofacial Surgery, Tokat, Turkey
(2) Gaziosmanpasa University Faculty of Medicine, Department of Cardiovascular Surgery, Tokat, Turkey
(3) Ankara University Faculty of Dentistry, Department of Dentomaxillofacial Radiology, Ankara, Turkey
Ankaferd blood stopper, bleeding, bleeding control, antithrombotic therapy
Ankaferd kanama durdurucu, kanama, kanama kontrolu, antitrombotik tedavi
Received/Gelis Tarihi: 09.11.2017
Accepted/Kabul Tarihi: 04.02.2018
Address for Correspondence/Yazisma Adresi: Hakan Eren MD, Ankara University Faculty of Dentistry, Department of Dentomaxillofacial Radiology, Ankara, Turkey
Phone : +90 312 296 56 26
E-mail : email@example.com
ORCID ID: orcid.org/0000-0001-9006-6836
Table 1. The comparison of the bleeding time and the amount of bleeding in between each group Groups Control Ankaferd Bleeding 14.87[+ or -]5.13 2.87[+ or -]2.10 time (second; sc) Amount of 0.007[+ or -]0.005 0.001[+ or -]0.003 bleeding (gram; gr) Groups Warfarin Warfarin-Ankaferd Bleeding 47.50[+ or -]13.37 (a,b) 18.62[+ or -]12.59 (b,c) time (second; sc) Amount of 0.041[+ or -]0.036 (a,b) 0.009[+ or -]0.018 (c) bleeding (gram; gr) Groups Heparin Heparin-Ankaferd Bleeding 40.50[+ or -]10.14 (a,b,d,e) 9.00[+ or -]4.40 (c) time (second; sc) Amount of 0.015[+ or -]0.007 (c) 00[+ or -]00 (c) bleeding (gram; gr) (a) p<0.05 differences compared to Control, (b) p<0.05 differences compared to Ankaferd, (c) p<0.05 differences compared to Warfarin, (d) p<0.05 differences compared to Warfarin-Ankaferd, (e) p<0.05 differences compared to Heparin
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|Title Annotation:||Original Article|
|Author:||Akbulut, Nihat; Akar, Ilker; Eren, Hakan; Asian, Cemal; Tumer, Mehmet Kemal|
|Publication:||Meandros Medical and Dental Journal|
|Date:||Dec 1, 2018|
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