Current Therapeutic Research VOLUME , NUMBER , JUNE
In Vitro Antifungal Activity of Ankaferd Blood Stopper Against Candida albicans Sevgi Ciftci, PhD1; Fahriye Keskin, PhD1; Sema Keceli Ozcan, MD, PhD2; Mehmet Ali Erdem, DDS, PhD3; Burak Cankaya, DDS, PhD3; Recep Bingol, PhD2; and Cetin Kasapoglu DDS, PhD3 1
Department of Microbiology, Faculty of Dentistry, Istanbul University, Istanbul, Turkey; Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey; and 3Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Istanbul University, Istanbul, Turkey 2
ABSTRACT Background: Candida albicans is a memeber of the oral flora that can lead to various complications in immunosupresive patients after oral surgery processes. Ankaferd Blood Stopper® (ABS) is a medical plant extract that is safe to use in patients with dental surgery bleedings in Turkey. Objective: The study evaluated the antifungal activity of ABS medicinal plant extract against C albicans using the agar diffusion and broth microdilution methods. Methods: The plant extract antifungal activity was assessed in vitro either by applying the ABS extract directly and by applying different concentrations of ABS onto Candida culture. For these experiments, an agar diffusion method was used. To determine the minimum inhibitory concentration (MIC), a broth microdilution method was used. Results: Different volumes of the active substance (10, 20, 30, and 40 L) were applied onto Candida (0.5 McFarland solution) cultivated plate; Candida growth was inhibited in accordance with the volumes of ABS. However, when various dilutions of ABS (1:2, 1:20, 1:40, and 1:80) were added as drops containing 20 L, no antifungal effects were found. No MIC values were identified using broth microdilution. When different dilutions of ABS containing 100 L of 0.5 McFarland solution of C albicans were cultured depending on the time (10, 20, 30, and 40 minutes), the effect of the duration was not significant. Conclusion: The various tests were carried out to investigate antifungal effects of ABS on Candida, but none were found. (Curr Ther Res Clin Exp. 2011;72: 120-126) © 2011 Elsevier HS Journals, Inc. All rights reserved. Key words: Ankaferd Blood Stopper, antifungal activity, Candida, in vitro
Accepted for publication April 8, 2011. © 2011 Elsevier HS Journals, Inc. All rights reserved.
120
doi:10.1016/j.curtheres.2011.04.003 0011-393X/$ - see front matter
S. Ciftci et al.
INTRODUCTION
Ankaferd Blood Stopper® (ABS) (Ankaferd Ilaç Kozmetik AS, Istanbul, Turkey) is a medicinal plant extract that has been used in Turkish traditional medicine as a hemostatic agent.1 Ankaferd produces a unique hemostatic effect by promoting very rapid (⬍1 sec) formation of a protein network, which acts as an anchor for vital physiologic erythrocyte aggregation and covers the classic cascade model of the clotting system without acting independently on coagulation factors and platelets.1,2 ABS may be effective in both individuals with normal hemostatic parameters and in patients with deficient primary hemostasis and/or secondary hemostasis.1 Excessive bleeding, particularly in patients with hereditary or acquired hemorrhagic diathesis, is challenging for dental practitioners.3 Antithrombotic, procoagulant, and antifibrinolytic medications are frequently used for the management of bleeding in dentistry.4 ABS as a medicinal product was approved in the management of external hemorrhage and dental surgical bleeding in Turkey based on tolerability and efficacy reports.4,5 ABS includes 5 plants: Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica in a weight ratio of 6, 8, 7, 7, and 5, respectively.1,5 ABS can be used as a spray, solution, or tampon.2 Ankaferd has shown a high inhibitory activity against both gram-negative and -positive bacteria.6 The antimicrobial activity of ABS has been reported in multidrug resistant bacteria such as methicillinresistant Staphylococcus aureus, Enterococcus spp, Enterobacter spp, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii bacteria.7 C albicans is normally located in the oral cavity8 and the oral mucosa. Because it is an opportunistic pathogen, it can cause candidiasis in immunosuppressed patients, such as those with febrile neutropenia or hematologic malignancy and chronic conditions like diabetes mellitus.9,10 Surgery can also cause Candida infection in this group of patients. Because in vitro antifungal activity of ABS is unknown, the activity of ABS against C albicans was evaluated.
MATERIALS AND METHODS Ankaferd Blood Stopper
The standardized vials of ABS (1 vial of 100 mL) used in these experiments were donated by Ankaferd Drug Inc, Istanbul, Turkey. C albicans Isolates
A total of 4 isolates were tested, with the result of 1 standard C albicans (ATCC 90028) and 3 clinical isolates. The clinical isolates were isolated from mouth swabs. The antifungal activity assay was performed by an agar culture method. C albicans stocks were stored at –70°C in their respective broths, supplemented with 3% glycerol. Preparation of Standard Candida Inoculums
Stock inoculum suspensions of standard Candida were prepared in sterile saline (8.5 g/L) sodium chloride from 24- to 48-hour cultures on Sabouraud dextrose agar
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(SDA) plates at 37°C, and the Candida strain concentration was adjusted to 0.5 McFarland standard solution.11 Effect of Ankaferd on the Colony Formatıon of C Albicans
The antifungal activity of the ABS against the Candida strains selected was determined using an agar diffusion method. The standard solution of each tested strain was swabbed over SDA plates. After 10 minutes, various volumes of ABS (10, 20, 30, and 40 L) were placed on 4 different points of the agar surface. All plates were incubated at 37°C for 24 to 48 hours, and the inhibition zone was evaluated. Standard Microdilution Method
The antifungal susceptibility of Candida strains was determined using a broth dilution method as described in Clinical Laboratory Standards Institute standard M27-A2.11 Using this method, the antifungal effect of ABS was investigated. The concentration range of the ABS used was between 0.03 and 350 g/mL. Diluted Active Substance Effect Over Time
Only the standard C albicans strain was used to investigate the effect of ABS. The inoculum strain was adjusted to the turbidity of the 0.5 McFarland standard solution. To observe the effect of ABS on the known colony number of C albicans, 10–3 dilutions of 0.5 McFarland solution were used in these experiments. The dilutions of ABS (1/2, 1/20, 1/40, and 1/80), each containing 100 L of the 10–3dilution of the 0.5 McFarland solution (inoculum), were prepared. The ABS dilutions and inoculums were incubated at 37°C in a water bath for 10, 20, and 40 minutes. At different time points, aliquots of 20 L were dropped onto SDA plates. A tube including only inoculum and no ABS was used as the control, and 20 L of the tube content was also placed onto SDA plates. After the plates dried, they were incubated at 37°C for 24 hours. Additionally, 0.5 McFarland solution and its 3 dilutions (10–1, 10–2, 10–3) were spread on SDA plates. After 10 minutes, 20 L of extract was dropped onto the 4 points. These were incubated for 24 hours at 37°C. Laboratory personnel evaluating the growth of Candida were blind to the concentrations applied.
RESULTS Effect of Ankaferd Blood Stopper on C albicans
Because we investigated only the effect of ABS medicinal plant extract against standard C albicans strain, positive and negative controls were not used. The antifungal activity of ABS was tested in a total of 4 Candida strains. The surface area of the SDA 0.5 McFarland standard solution applied to the 4 different points (10, 20, 30, and 40 L) of ABS was measured so that the inhibition zone diameters were obtained. It was observed that the diameters of zone of inhibition increased with increasing volume of ABS, as shown in Figure 1.
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30 mL 40 mL
20 mL 10 mL
Figure 1. The effect of different volumes of Ankaferd Blood Stopper on Candida albicans colonies.
Standard Microdiluton Method
The test was repeated 3 times, but the MIC values could not be determined. The substances in liquid medium were coagulated by ABS. Diluted Active Substance Effect Over Time
There was no evidence of an effect for duration of (10, 20, and 40 minutes) exposure of ABS on Candida growth (Figure 2). When active substance concentrations (1:2, 12:20, 1:40, and 1:80) were dropped onto a plate containing inoculums, no inhibitory effect was observed (Figure 3).
Figure 2. The numbers of Candida colonies counted on agar plates inoculated with 10–3 dilution of 0.5 McFarland solution of Candida albicans containing Ankaferd Blood Stopper (ABS) and its dilutions after 10-, 20- and 40-minute incubations.
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Figure 3. Inhibitory effect of active substance concentrations.
DISCUSSION The ABS medical plant extract is used in medicine as a hemostatic agent, which has been shown to promote the formation of an encapsulated protein network.1 ABS containing T vulgaris, G glabra, V vinifera, A officinarum, and U dioica plant extracts was investigated separately for antimicrobial and antifungal effects. T vulgaris Candida was used in studies investigating the antifungal effect.12–14 Fatima et al15 found G glabra active against both yeast and filamentous fungi. Furthermore, Pellati et al16 demonstrated the in vitro activity of the compound 18- glycyrrhetinic acid against C albicans strains derived from the root of Glycyrrhiza species. Motsei et al17 reported that G glabra had an antifungal effect on C albicans in clinical specimens, and that it could be used in oral mouthwashes for the treatment of oral candidiasis. Hemorrhagic diathesis during dental surgery can prolong postoperative bleeding, impair wound healing, and increase the risk of infection.3 Bacteria and fungi proliferate in the oral cavity and contamination of the surgical field by this flora can occur very easily. In recent studies, it was shown that ABS inhibited the in vitro growth of gram-negative and -positive bacteria.6,18 In this study, the activity of ABS against C albicans was evaluated. In the first phase of the study, the active substance volume was not diluted by the specific density. When Candida strains were cultivated onto a plate and different concentrations of ABS (40, 30, 20, and 10 L) were dropped onto it, it was found that the greater the concentration of ABS, the greater the inhibition of Candida growth. These results suggested that ABS inhibited the growth of Candida. This effect suggested that there were antifungal properties of T vulgaris and G glabra. However, in other phases of the study with the active substance concentrations (1:2, 1:20, 1:40, and 1:80) and 0.5 McFarland solution Candida strains added as drops of plaque at 20 L, the anti-fungal effects were not determined. It is possible that, because a combination of T vulgaris and G glabra was used, antifungal effects could not be
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obtained. In addition, the different dilutions of ABS against Candida over time (10, 20, and 40 minutes) were evaluated and compared with the control tubes, but the effect of duration was negligible. Therefore, the ABS effects, depending on the duration of the application period, were not considered when it was applied on a wound infected with fungus. The antifungal activity of ABS is not precisely known. Akkoc et al19 assessed the antifungal effect of ABS using an agar well diffusion test. ABS was found to have a high antifungal effect against Zygosaccharomyces bailii, C albicans, Aspergillus flavus, and Aspergillus parasiticus. However, in this study, there was no observed effect of ABS on Candida that was considered to be an antifungal effect. However, when ABS was directly applied onto Candida, it resulted in changes in the growth conditions. The mixture of plant extracts in ABS might have antifungal effects separately; however, when used together, the antifungal effect could not be demonstrated. The limitation of this study was that ABS was only tested against Candida, which is 1 of the most common oral flora. However, to support this conclusion convincingly, additional studies using a large number of clinical specimens need to be performed.
ACKNOWLEDGMENTS Dr. Ciftci contributed to study design, implementation, and interpretation of study analyses, and article preparation. Dr. Keskin contributed to study implementation and interpretation of study analyses. Dr. Keceli-Ozcan contributed to study implementation. Drs. Erdem and Cankaya collected study samples. Dr. Bıngol contributed to study design and implementation and interpretation of study analyses. Dr. Kasapoglu collected study samples and contributed to interpretation of study analysis. The authors have indicated that they have no conflicts of interest regarding the content of this article.
REFERENCES 1. Goker H, Haznedaroglu IC, Ercetin S, et al. Haemostatic actions of the folkloric medical plant extract Ankaferd Blood Stopper. J Int Med Res. 2008;36:163–170. 2. Trakyali G, Oztoprak MO. Plant extract ankaferd blood stopper effect on bond strength. Angle Orthod. 2010;80:570 –574. 3. Israels S, Schwetz N, Boyar R, McNicol A. Bleeding disorders: characterization, dental considerations and management. J Can Dent Assoc. 2006;72:827. 4. Ercetin S, Haznedaroglu IC, Kurt M, et al. Safety and efficacy of Ankaferd Blood Stopper in dental surgery. Int J Hematol Oncol. 2010;20:1–5. 5. Ankaferd Blood Stopper Haemostatic Agent. www.ankaferd.com. Accessed April 30, 2011. 6. Akkoc N, Akcelik M, Haznedaroglu IC, et al. In vitro anti-bacterial activities of Ankaferd medicinal plant extract. Turk J Med Sci. 2009;29:410 – 415. 7. Tasdelen Fisgin N, Tanriverdi Caycı Y, Coban AY, et al. Antimicrobial activity of plant extract Ankaferd Blood Stopper. Fitoterapia. 2009;80:48 –50. 8. Rautemae R, Rusanen P, Richardson M, Meurman JH. Optimal sampling site for mucosal candidosis in oral cancer patients is the labial sulcus. J Med Microbiol. 2006;55:1447–1451. 9. Morace G, Pagano L, Sanguinetti M, et al. PCR-restriction enzyme analysis for detection of Candida DNA in blood from febrile patients with hematological malignancies. J Clin Microbiol. 1999;37:1871–1875.
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10. Musial CE, Cockerill FR, Roberts GD. Fungal infections of the immunocompromised host: Clinical and laboratory aspects. Clin Microbiol Rev. 1998;1:349 –364. 11. Clinical Laboratory Standards Institute (CLSI) (formerly NCCLS). Reference method for broth dilution antifungal susceptibility testing of yeast. Approved Standard, CLSI Document M27 A2, 2nd ed, Wayne, PA: CLSI; 2002. 12. White CM, Fan C, Song J, et al. An evaluation of the hemostatic effect of hydrophilic, alcohol, and lipophilic extracts of notoginseng. Pharmocotherapy. 2001;21:773–777. 13. Bonjor GH. Inhibition of Clotrimazole-resistant Candida albicans by plants used in Iranian folkloric medicine. Fitoterapia. 2004;75:74 –76. 14. Giordani R, Hadef Y, Kaloustian J. Compositions and antifungal activities of essential oils of some Algerian aromatic plants. Fitoterapia. 2008;79:199 –203. 15. Fatima A, Gupta VK, Lugman S, et al. Antifungal activity of Glycyrrhiza glabra extracts and its active constituent glabridin. Phytother Res. 2009;23:1190 –1193. 16. Pellati D, Fiore C, Armanini D, et al. In vitro effect of glycyrrhetinic acid on the growth of clinical isolates of Candida albicans. Phytother Res. 2009;23:572–574. 17. Motsei ML, Lindsey KL, van Staden J, Jäger AK. Screening of traditionally used South African plants for antifungal activity against Candida albicans. J Ethnopharmacol. 2003;86:235–241. 18. Saribas Z, Sener B, Haznedaroglu IC, et al. Antimicrobial activity of Ankaferd Blood Stopper against nosocomial bacterial pathogens. Cent Eur J Med. 2010;5:198 –202. 19. Akkoc N, Akcelik M, Haznedaroglu IC, et al. In-vitro anti-fungal of medicinal plant extracts Ankaferd activity events as [in Turkish]. 34. National Hematology Congress; October 2008; Cesme Izmir, Turkey; S017.
Address correspondence to: Sevgi Ciftci, PhD, Department of Microbiology, Faculty of Dentistry, Istanbul University, 34390 Capa-Istanbul, Turkey. E-mail: [email protected].
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In Vitro Antifungal Activity of Ankaferd Blood Stopper Against Candida albicans Sevgi Ciftci, PhD1; Fahriye Keskin, PhD1; Sema Keceli Ozcan, MD, PhD2; Mehmet Ali Erdem, DDS, PhD3; Burak Cankaya, DDS, PhD3; Recep Bingol, PhD2; and Cetin Kasapoglu DDS, PhD3 1
Department of Microbiology, Faculty of Dentistry, Istanbul University, Istanbul, Turkey; Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey; and 3Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Istanbul University, Istanbul, Turkey 2
ABSTRACT Background: Candida albicans is a memeber of the oral flora that can lead to various complications in immunosupresive patients after oral surgery processes. Ankaferd Blood Stopper® (ABS) is a medical plant extract that is safe to use in patients with dental surgery bleedings in Turkey. Objective: The study evaluated the antifungal activity of ABS medicinal plant extract against C albicans using the agar diffusion and broth microdilution methods. Methods: The plant extract antifungal activity was assessed in vitro either by applying the ABS extract directly and by applying different concentrations of ABS onto Candida culture. For these experiments, an agar diffusion method was used. To determine the minimum inhibitory concentration (MIC), a broth microdilution method was used. Results: Different volumes of the active substance (10, 20, 30, and 40 L) were applied onto Candida (0.5 McFarland solution) cultivated plate; Candida growth was inhibited in accordance with the volumes of ABS. However, when various dilutions of ABS (1:2, 1:20, 1:40, and 1:80) were added as drops containing 20 L, no antifungal effects were found. No MIC values were identified using broth microdilution. When different dilutions of ABS containing 100 L of 0.5 McFarland solution of C albicans were cultured depending on the time (10, 20, 30, and 40 minutes), the effect of the duration was not significant. Conclusion: The various tests were carried out to investigate antifungal effects of ABS on Candida, but none were found. (Curr Ther Res Clin Exp. 2011;72: 120-126) © 2011 Elsevier HS Journals, Inc. All rights reserved. Key words: Ankaferd Blood Stopper, antifungal activity, Candida, in vitro
Accepted for publication April 8, 2011. © 2011 Elsevier HS Journals, Inc. All rights reserved.
120
doi:10.1016/j.curtheres.2011.04.003 0011-393X/$ - see front matter
S. Ciftci et al.
INTRODUCTION
Ankaferd Blood Stopper® (ABS) (Ankaferd Ilaç Kozmetik AS, Istanbul, Turkey) is a medicinal plant extract that has been used in Turkish traditional medicine as a hemostatic agent.1 Ankaferd produces a unique hemostatic effect by promoting very rapid (⬍1 sec) formation of a protein network, which acts as an anchor for vital physiologic erythrocyte aggregation and covers the classic cascade model of the clotting system without acting independently on coagulation factors and platelets.1,2 ABS may be effective in both individuals with normal hemostatic parameters and in patients with deficient primary hemostasis and/or secondary hemostasis.1 Excessive bleeding, particularly in patients with hereditary or acquired hemorrhagic diathesis, is challenging for dental practitioners.3 Antithrombotic, procoagulant, and antifibrinolytic medications are frequently used for the management of bleeding in dentistry.4 ABS as a medicinal product was approved in the management of external hemorrhage and dental surgical bleeding in Turkey based on tolerability and efficacy reports.4,5 ABS includes 5 plants: Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica in a weight ratio of 6, 8, 7, 7, and 5, respectively.1,5 ABS can be used as a spray, solution, or tampon.2 Ankaferd has shown a high inhibitory activity against both gram-negative and -positive bacteria.6 The antimicrobial activity of ABS has been reported in multidrug resistant bacteria such as methicillinresistant Staphylococcus aureus, Enterococcus spp, Enterobacter spp, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii bacteria.7 C albicans is normally located in the oral cavity8 and the oral mucosa. Because it is an opportunistic pathogen, it can cause candidiasis in immunosuppressed patients, such as those with febrile neutropenia or hematologic malignancy and chronic conditions like diabetes mellitus.9,10 Surgery can also cause Candida infection in this group of patients. Because in vitro antifungal activity of ABS is unknown, the activity of ABS against C albicans was evaluated.
MATERIALS AND METHODS Ankaferd Blood Stopper
The standardized vials of ABS (1 vial of 100 mL) used in these experiments were donated by Ankaferd Drug Inc, Istanbul, Turkey. C albicans Isolates
A total of 4 isolates were tested, with the result of 1 standard C albicans (ATCC 90028) and 3 clinical isolates. The clinical isolates were isolated from mouth swabs. The antifungal activity assay was performed by an agar culture method. C albicans stocks were stored at –70°C in their respective broths, supplemented with 3% glycerol. Preparation of Standard Candida Inoculums
Stock inoculum suspensions of standard Candida were prepared in sterile saline (8.5 g/L) sodium chloride from 24- to 48-hour cultures on Sabouraud dextrose agar
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(SDA) plates at 37°C, and the Candida strain concentration was adjusted to 0.5 McFarland standard solution.11 Effect of Ankaferd on the Colony Formatıon of C Albicans
The antifungal activity of the ABS against the Candida strains selected was determined using an agar diffusion method. The standard solution of each tested strain was swabbed over SDA plates. After 10 minutes, various volumes of ABS (10, 20, 30, and 40 L) were placed on 4 different points of the agar surface. All plates were incubated at 37°C for 24 to 48 hours, and the inhibition zone was evaluated. Standard Microdilution Method
The antifungal susceptibility of Candida strains was determined using a broth dilution method as described in Clinical Laboratory Standards Institute standard M27-A2.11 Using this method, the antifungal effect of ABS was investigated. The concentration range of the ABS used was between 0.03 and 350 g/mL. Diluted Active Substance Effect Over Time
Only the standard C albicans strain was used to investigate the effect of ABS. The inoculum strain was adjusted to the turbidity of the 0.5 McFarland standard solution. To observe the effect of ABS on the known colony number of C albicans, 10–3 dilutions of 0.5 McFarland solution were used in these experiments. The dilutions of ABS (1/2, 1/20, 1/40, and 1/80), each containing 100 L of the 10–3dilution of the 0.5 McFarland solution (inoculum), were prepared. The ABS dilutions and inoculums were incubated at 37°C in a water bath for 10, 20, and 40 minutes. At different time points, aliquots of 20 L were dropped onto SDA plates. A tube including only inoculum and no ABS was used as the control, and 20 L of the tube content was also placed onto SDA plates. After the plates dried, they were incubated at 37°C for 24 hours. Additionally, 0.5 McFarland solution and its 3 dilutions (10–1, 10–2, 10–3) were spread on SDA plates. After 10 minutes, 20 L of extract was dropped onto the 4 points. These were incubated for 24 hours at 37°C. Laboratory personnel evaluating the growth of Candida were blind to the concentrations applied.
RESULTS Effect of Ankaferd Blood Stopper on C albicans
Because we investigated only the effect of ABS medicinal plant extract against standard C albicans strain, positive and negative controls were not used. The antifungal activity of ABS was tested in a total of 4 Candida strains. The surface area of the SDA 0.5 McFarland standard solution applied to the 4 different points (10, 20, 30, and 40 L) of ABS was measured so that the inhibition zone diameters were obtained. It was observed that the diameters of zone of inhibition increased with increasing volume of ABS, as shown in Figure 1.
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30 mL 40 mL
20 mL 10 mL
Figure 1. The effect of different volumes of Ankaferd Blood Stopper on Candida albicans colonies.
Standard Microdiluton Method
The test was repeated 3 times, but the MIC values could not be determined. The substances in liquid medium were coagulated by ABS. Diluted Active Substance Effect Over Time
There was no evidence of an effect for duration of (10, 20, and 40 minutes) exposure of ABS on Candida growth (Figure 2). When active substance concentrations (1:2, 12:20, 1:40, and 1:80) were dropped onto a plate containing inoculums, no inhibitory effect was observed (Figure 3).
Figure 2. The numbers of Candida colonies counted on agar plates inoculated with 10–3 dilution of 0.5 McFarland solution of Candida albicans containing Ankaferd Blood Stopper (ABS) and its dilutions after 10-, 20- and 40-minute incubations.
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Figure 3. Inhibitory effect of active substance concentrations.
DISCUSSION The ABS medical plant extract is used in medicine as a hemostatic agent, which has been shown to promote the formation of an encapsulated protein network.1 ABS containing T vulgaris, G glabra, V vinifera, A officinarum, and U dioica plant extracts was investigated separately for antimicrobial and antifungal effects. T vulgaris Candida was used in studies investigating the antifungal effect.12–14 Fatima et al15 found G glabra active against both yeast and filamentous fungi. Furthermore, Pellati et al16 demonstrated the in vitro activity of the compound 18- glycyrrhetinic acid against C albicans strains derived from the root of Glycyrrhiza species. Motsei et al17 reported that G glabra had an antifungal effect on C albicans in clinical specimens, and that it could be used in oral mouthwashes for the treatment of oral candidiasis. Hemorrhagic diathesis during dental surgery can prolong postoperative bleeding, impair wound healing, and increase the risk of infection.3 Bacteria and fungi proliferate in the oral cavity and contamination of the surgical field by this flora can occur very easily. In recent studies, it was shown that ABS inhibited the in vitro growth of gram-negative and -positive bacteria.6,18 In this study, the activity of ABS against C albicans was evaluated. In the first phase of the study, the active substance volume was not diluted by the specific density. When Candida strains were cultivated onto a plate and different concentrations of ABS (40, 30, 20, and 10 L) were dropped onto it, it was found that the greater the concentration of ABS, the greater the inhibition of Candida growth. These results suggested that ABS inhibited the growth of Candida. This effect suggested that there were antifungal properties of T vulgaris and G glabra. However, in other phases of the study with the active substance concentrations (1:2, 1:20, 1:40, and 1:80) and 0.5 McFarland solution Candida strains added as drops of plaque at 20 L, the anti-fungal effects were not determined. It is possible that, because a combination of T vulgaris and G glabra was used, antifungal effects could not be
124
S. Ciftci et al.
obtained. In addition, the different dilutions of ABS against Candida over time (10, 20, and 40 minutes) were evaluated and compared with the control tubes, but the effect of duration was negligible. Therefore, the ABS effects, depending on the duration of the application period, were not considered when it was applied on a wound infected with fungus. The antifungal activity of ABS is not precisely known. Akkoc et al19 assessed the antifungal effect of ABS using an agar well diffusion test. ABS was found to have a high antifungal effect against Zygosaccharomyces bailii, C albicans, Aspergillus flavus, and Aspergillus parasiticus. However, in this study, there was no observed effect of ABS on Candida that was considered to be an antifungal effect. However, when ABS was directly applied onto Candida, it resulted in changes in the growth conditions. The mixture of plant extracts in ABS might have antifungal effects separately; however, when used together, the antifungal effect could not be demonstrated. The limitation of this study was that ABS was only tested against Candida, which is 1 of the most common oral flora. However, to support this conclusion convincingly, additional studies using a large number of clinical specimens need to be performed.
ACKNOWLEDGMENTS Dr. Ciftci contributed to study design, implementation, and interpretation of study analyses, and article preparation. Dr. Keskin contributed to study implementation and interpretation of study analyses. Dr. Keceli-Ozcan contributed to study implementation. Drs. Erdem and Cankaya collected study samples. Dr. Bıngol contributed to study design and implementation and interpretation of study analyses. Dr. Kasapoglu collected study samples and contributed to interpretation of study analysis. The authors have indicated that they have no conflicts of interest regarding the content of this article.
REFERENCES 1. Goker H, Haznedaroglu IC, Ercetin S, et al. Haemostatic actions of the folkloric medical plant extract Ankaferd Blood Stopper. J Int Med Res. 2008;36:163–170. 2. Trakyali G, Oztoprak MO. Plant extract ankaferd blood stopper effect on bond strength. Angle Orthod. 2010;80:570 –574. 3. Israels S, Schwetz N, Boyar R, McNicol A. Bleeding disorders: characterization, dental considerations and management. J Can Dent Assoc. 2006;72:827. 4. Ercetin S, Haznedaroglu IC, Kurt M, et al. Safety and efficacy of Ankaferd Blood Stopper in dental surgery. Int J Hematol Oncol. 2010;20:1–5. 5. Ankaferd Blood Stopper Haemostatic Agent. www.ankaferd.com. Accessed April 30, 2011. 6. Akkoc N, Akcelik M, Haznedaroglu IC, et al. In vitro anti-bacterial activities of Ankaferd medicinal plant extract. Turk J Med Sci. 2009;29:410 – 415. 7. Tasdelen Fisgin N, Tanriverdi Caycı Y, Coban AY, et al. Antimicrobial activity of plant extract Ankaferd Blood Stopper. Fitoterapia. 2009;80:48 –50. 8. Rautemae R, Rusanen P, Richardson M, Meurman JH. Optimal sampling site for mucosal candidosis in oral cancer patients is the labial sulcus. J Med Microbiol. 2006;55:1447–1451. 9. Morace G, Pagano L, Sanguinetti M, et al. PCR-restriction enzyme analysis for detection of Candida DNA in blood from febrile patients with hematological malignancies. J Clin Microbiol. 1999;37:1871–1875.
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10. Musial CE, Cockerill FR, Roberts GD. Fungal infections of the immunocompromised host: Clinical and laboratory aspects. Clin Microbiol Rev. 1998;1:349 –364. 11. Clinical Laboratory Standards Institute (CLSI) (formerly NCCLS). Reference method for broth dilution antifungal susceptibility testing of yeast. Approved Standard, CLSI Document M27 A2, 2nd ed, Wayne, PA: CLSI; 2002. 12. White CM, Fan C, Song J, et al. An evaluation of the hemostatic effect of hydrophilic, alcohol, and lipophilic extracts of notoginseng. Pharmocotherapy. 2001;21:773–777. 13. Bonjor GH. Inhibition of Clotrimazole-resistant Candida albicans by plants used in Iranian folkloric medicine. Fitoterapia. 2004;75:74 –76. 14. Giordani R, Hadef Y, Kaloustian J. Compositions and antifungal activities of essential oils of some Algerian aromatic plants. Fitoterapia. 2008;79:199 –203. 15. Fatima A, Gupta VK, Lugman S, et al. Antifungal activity of Glycyrrhiza glabra extracts and its active constituent glabridin. Phytother Res. 2009;23:1190 –1193. 16. Pellati D, Fiore C, Armanini D, et al. In vitro effect of glycyrrhetinic acid on the growth of clinical isolates of Candida albicans. Phytother Res. 2009;23:572–574. 17. Motsei ML, Lindsey KL, van Staden J, Jäger AK. Screening of traditionally used South African plants for antifungal activity against Candida albicans. J Ethnopharmacol. 2003;86:235–241. 18. Saribas Z, Sener B, Haznedaroglu IC, et al. Antimicrobial activity of Ankaferd Blood Stopper against nosocomial bacterial pathogens. Cent Eur J Med. 2010;5:198 –202. 19. Akkoc N, Akcelik M, Haznedaroglu IC, et al. In-vitro anti-fungal of medicinal plant extracts Ankaferd activity events as [in Turkish]. 34. National Hematology Congress; October 2008; Cesme Izmir, Turkey; S017.
Address correspondence to: Sevgi Ciftci, PhD, Department of Microbiology, Faculty of Dentistry, Istanbul University, 34390 Capa-Istanbul, Turkey. E-mail: [email protected].
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