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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.

Introduction

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).

Animals

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.

Experimental Design

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.

Statistical Analysis

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.

Results

There were not any complications observed during the study period and surgical procedure.

Bleeding Time

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.

Discussion

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).

Study Limitations

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).

Conclusion

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

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.

Authorship Contributions

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.

References

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(2.) I[section]ler SC, Demircan S, Cakarer S, Cebi Z, Keskin C, Soluk M, et al. Effects of folk medicinal plant extract Ankaferd Blood Stopper on early bone healing. J Appl Oral Sci 2010; 18: 409-14.

(3.) Leblebisatan G, Bay A, Karakus SC, Kekilli M, Haznedaroglu IC. Topical Ankaferd hemostat application for the management of oral cavity bleedings in children with hemorrhagic diathesis. Blood Coagul Fibrinolysis 2012; 23: 494-7.

(4.) Tokgoz H, Karakaya K, Hanci V, Abdusoglu M, Erol B, Turksoy O, et al. Protective value of a folkloric medicinal plant extract against mortality and hemorrhage in a life-threatening renal trauma model. Urology 2010; 75: 1515. e9-14.

(5.) Ucar Albayrak C, Caliskan U, Haznedaroglu IC, Goker H. Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper. J Int Med Res 2008; 36: 1447-8.

(6.) Kosar A, Tezel C, Orki A, Kiral H, Arman B. Bronchogenic cysts of the lung: report of 29 cases. Heart Lung Circ 2009; 18: 214-8.

(7.) Cakarer S, Eyupoglu E, Gunes CO, Kuseoglu BG, Berberoglu HK, Keskin C. Evaluation of the hemostatic effects of Ankaferd blood stopper during dental extractions in patients on antithrombotic therapy. Clin Appl Thromb Hemost 2013; 19: 96-9.

(8.) Uz B, Guven GS, Isik A, Kuyumcu ME, Bektas O, Eliacik E, et al. Long-term sustained hemorrhage due to bone marrow biopsy successfully treated with topical ankaferd hemostat in a bleeding-prone patient with secondary amyloidosis. Clin Appl Thromb Hemost 2013; 19: 338-40.

(9.) Huri E1, Haznedaroglu IC, Akgul T, Astarci M, Ustun H, Germiyanoulu C. Biphasic effects of ankaferd blood stopper on renal tubular apoptosis in the rat partial nephrectomy model representing distinct levels of hemorrhage. Saudi Med J 2010; 31: 864-8.

(10.) Kalayci MU, Soylu A, Eroglu HE, Kubilay D, Sancak B, Ugurluoglu C, et al. Effect of ankaferd blood stopper on hemostasis and histopathological score in experimental liver injury. Bratisl Lek Listy 2010; 111: 183-8.

(11.) Kilic O1, Gonen M, Acar K, Yurdakul T, Avunduk MC, Esen HH, et al. Haemostatic role and histopathological effects of a new haemostatic agent in a rat bladder haemorrhage model: an experimental trial. BJU Int 2010; 105: 1722-5.

(12.) Turhan N1, Bilgili H, Captug O, Kurt M, Shorbagi A, Beyazit Y, et al. Evaluation of a haemostatic agent in rabbits. Afr J Tradit Complement Altern Med 2011; 8: 61-5.

(13.) Mihmanli A, Ulker Z, Alpsoy L, Ezirganli S. Evaluation of cytotoxicity of a new hemostatic agent Ankaferd Blood Stopper[R] using different assays. Hum Exp Toxicol 2012; 31: 780-7.

(14.) Cipil HS, Kosar A, Kaya A, Uz B, Haznedaroglu IC, Goker H, et al. In vivo hemostatic effect of the medicinal plant extract Ankaferd Blood Stopper in rats pretreated with warfarin. Clin Appl Thromb Hemost 2009; 15: 270-6.

(15.) Evans IL, Sayers MS, Gibbons AJ, Price G, Snooks H, Sugar AW. Can warfarin be continued during dental extraction? Results of a randomized controlled trial. Br J Oral Maxillofac Surg 2002; 40: 248-52.

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(18.) Kandemir O, Buyukates M, Kandemir NO, Aktunc E, Gul AE, Gul S, et al. Demonstration of the histopathological and immunohistochemical effects of a novel hemostatic agent, Ankaferd Blood Stopper, on vascular tissue in a rat aortic bleeding model. J Cardiothorac Surg 2010; 5: 110.

(19.) Altunhan H, Annagur A, Tokgoz H, Caliskan U, Ors R. Persistent nasal bleeding due to nasal CPAP application in 2 premature newborns successfully treated with topical "Ankaferd blood stopper". Clin Appl Thromb Hemost 2011; 17: E181-2.

(20.) Kosar A, Cipil HS, Kaya A, Uz B, Haznedaroglu IC, Goker H, et al. The efficacy of Ankaferd Blood Stopper in antithrombotic drug-induced primary and secondary hemostatic abnormalities of a rat-bleeding model. Blood Coagul Fibrinolysis 2009; 20: 185-90.

(21.) Bilgili H, Kosar A, Kurt M, Onal IK, Goker H, Captug O, et al. Hemostatic efficacy of Ankaferd Blood Stopper in a swine bleeding model. Med Princ Pract 2009; 18: 165-9.

(22.) Okumura LM, Negretto GW, Carvalho CG. Unusual Warfarin Dose To Achieve Therapeutic INR In a 4-Month Old Child: Non-Genetics Risk Factors Are Still A Challenge. Rev Paul Pediatr 2017; 35: 472-5.

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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

Keywords

Ankaferd blood stopper, bleeding, bleeding control, antithrombotic therapy

Anahtar Kelimeler

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 : dthakaneren@yahoo.com.tr

ORCID ID: orcid.org/0000-0001-9006-6836

doi:10.4274/meandros.73792
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
Article Type:Report
Date:Dec 1, 2018
Words:3546
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