|Year : 2014 | Volume
| Issue : 4 | Page : 203-207
Evaluation of cytotoxic effects of Anbarnesa on fibroblast L929: Can it be used as a mouthwash?
Hassan Ali Shafiee1, Mohammad Hosein Kalantar Motamedi2, Morteza Mina1, Jamileh Beigom Taheri3, Somayyeh Azimi3, Khojasteh Joharchi4, Zahra Yadegari4, Hamid Reza Rasouli5
1 Department of Orthodontics, Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
2 Department of Oral and Maxillofacial Surgery, Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
3 Department of Oral Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Pharmacology and Biotechnology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Department of Statistics, Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
|Date of Web Publication||19-Dec-2014|
Mohammad Hosein Kalantar Motamedi
Department of Oral and Maxillofacial Surgery, Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran
Source of Support: None, Conflict of Interest: None
Aims: In Iranian traditional medicine Anbarnesa (derived from smoke from burning female donkey's stool) has been used to treat ulcers and inflammatory conditions like stomatitis and ear infections (otitis). We assess the properties of Anbarnesa as an alternative mouthwash.
Materials and Methods: In this experimental study, Anbarnesa smoke was analyzed using aGC-mass device. The smoke collected was dissolved at different densities in propylene glycol and incubated in Dulbecco's modified Eagle's medium in direct contact with fibroblast cells. Assessment of cytotoxicity was done at 1, 24 and 72 h. Cell viability was measured by methyl thiazolyl tetrazolium test, and ELISA Reader machine was used to read the results. Data were analyzed using one-way ANOVA test.
Results: The findings of this study showed Anbarnesa was nontoxic in 1/64, 1/128 and 1/256 dilutions. In 1/32 dilution, toxicity was seen after 72 h. In dilutions, 1/8 and 1/16 toxicity were seen in the 1 st h.
Conclusion: According to the initial results of Anbarnesa may be used as an alternative mouthwash with fewer side-effects for plaque control and prevention of periodontal disease.
Keywords: Cytotoxicity, fibroblast, traditional medicine
|How to cite this article:|
Shafiee HA, Motamedi MK, Mina M, Taheri JB, Azimi S, Joharchi K, Yadegari Z, Rasouli HR. Evaluation of cytotoxic effects of Anbarnesa on fibroblast L929: Can it be used as a mouthwash?. Ancient Sci Life 2014;33:203-7
|How to cite this URL:|
Shafiee HA, Motamedi MK, Mina M, Taheri JB, Azimi S, Joharchi K, Yadegari Z, Rasouli HR. Evaluation of cytotoxic effects of Anbarnesa on fibroblast L929: Can it be used as a mouthwash?. Ancient Sci Life [serial online] 2014 [cited 2019 Oct 18];33:203-7. Available from: http://www.ancientscienceoflife.org/text.asp?2014/33/4/203/147422
| Introduction|| |
In order to prevent periodontal disease, plaque control is important.  Today, mechanical and chemical methods for plaque control are in vogue.  Antibacterial agents such as mouthwash in addition to mechanical methods, are effective in the treatment of periodontal diseases. 
An ideal mouthwash is the one that has no allergic reactions,  no teeth or oral mucosa staining,  possesses anti-plaque and antibacterial properties,  has minimal cytotoxic effects on cells, ,, is sustainable and has a good taste. 
Such a mouth rinse with all the above-mentioned properties is not present yet. Hence, various types of mouthwash have been introduced of which, due to its beneficial effect, chlorhexidine (CHx) has been recommended; , but it has many side-effects, prime among them is cytotoxicity. 
Traditional medicine and herbal remedies have been used in various fields of medicine.  In Iranian traditional medicine Anbarnesa smoke (derived from burning dried dung of the female donkey), is used for the treatment of inflammatory oral lesions such as aphthous ulcer and other inflammatory lesions such as otitis.  Composition of animal manure is organic matter basically organic carbon, water-soluble organic carbon, organic N, carbohydrates, humic acid-like carbon and fulvic acid-like carbon.  Since ancient times, humans have used smoke of medicinal plants to cure illness. In a study shows that in 50 countries, usage of medicine smoke is common, and one type is medicinal smoke of Anbarnesa. 
Fibroblast cells are dominant cells in periodontal ligament (PDL), and are important in maintaining healthy gingiva and surrounding tissues. , The biological effects of the mouth rinses have not been sufficiently studied; the present work was done to determine the cytotoxic effect of Anbarnesa sebum 1 on cultured mice fibroblasts in category L929.
| Materials and methods|| |
In this experimental in vitro study, 198 samples with positive and negative controls of mice fibroblast cells obtained from the Pasteur Institute cell bank (Tehran, Iran) were studied. According to the standard ISO 10993:5, 6 replicates were used. The original concentrations, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256 dilution were prepared and studied. Assessments were done after 1 h, 24 and 72 h after contact with the cells.
First, Anbarnesa smoke was analyzed using aGC-mass device and substances such as hexane, citric acid and dimethylamine were reported. After ensuring the effectiveness of materials, Anbarnesa was burned inside a closed container with its walls coated with propylene glycol. After cooling the environment, substances in the smoke adhered to the walls. This was repeated several times to accumulate enough smoke residue on the walls. Then 10 mL propylene glycol solution was added to the container and mixed well with the material on the walls. Next, the contents were transferred to a falcon tube to separate impurities, and the suspended samples were centrifuged for 30 min. Using a Pasteur pipette, the supernatant fluid was transferred to another tube. This solution was named Anbarnesa sbmu 1. The concentration of the liquid was measured using liquid chromatography and diluted to concentrations of 0.2% and minimum inhibitory concentration values were evaluated against different bacterial species and compared with control samples of propylene glycol. Growth inhibitions on species of Streptococcus mutans, Streptococcus salivarious, Streptococcus sanguinis, and Streptococcus pyogenes after the exposure to this solution were assessed for antibacterial properties.
Complete Dulbecco's modified Eagle's medium (DMEM) with embryonic bovine serum was used for the culture.
After distribution of cells in wells of three plates, the plates were incubated for 24 h. After this, the culture medium was removed from wells and then 200 μl of sample (prepared by dilution) was placed in each of the wells. In each plate, 6 wells of negative controls and 6 wells of positive controls were assigned. The sample plates were incubated at temperatures 37°Cat 98% humidity and 5% CO 2 .
The methyl thiazolyl tetrazolium (MTT) test was used to assess cytotoxicity based on color change and the production of formazan. For the MTT test, tetrazolium bromide salt (Sigma-Aldrich, USA) at a ratio of 5 mg/mL in phosphate buffer solution was mixed with the DMEM at ratio of 1/10. ELISA reader machine (Anthoos, 2020, Australia) was used to read the optical density (OD). Data of OD were divided to OD of negative controls to assess cell viability. The data were statistically analyzed using computer SPSS 17 software (IBM Chicago, IL) and one-way ANOVA test.
| Results|| |
Mean and standard deviation of OD sat different concentrations at 1 h are presented in [Table 1]. For measuring the cell viability, OD at different concentrations are divided to ODs of negative controls shown in Diagram 1.
Cell viability over 70% meant that the concentration was not cytotoxic to fibroblast cells. Cell survival between 50% and 70% meant that the concentration had a cytotoxic effect on half of the fibroblast cells. And cell survival below 50% meant that the concentration is cytotoxic to fibroblast cells.
Means and standard deviations of OD sat different dilutions at 24 and 72 h are presented in [Table 2] and [Table 3], respectively. Furthermore, cell viability at 24 and 72 h is shown in Diagrams 2 and 3 respectively.
There was no toxicity at dilutions of 1/32, 1/64, 1/128 and 1/256. The ANOVA test was used to compare toxicity and significant difference were reported in these four concentrations (P < 0.05).
| Discussion|| |
There are different methods to assess cytotoxicity in vitro: Permeability assay, replication assay, morphologic studies, monolayer agar overlay and functional assay. MTT assay is a subgroup of the functional assay.  MTT assay evaluated levels of cell enzymes or cell mitochondrial function. MTT is a simple colorimetric assay that uses MTT salt to measure changes in mitochondrial enzyme succinyl dehydrogenase. The first salt is pale yellow. Mitochondrial succinyl-dehydrogenase enzymes break the salt and create a new dark blue product called formazan. Thus, only cells that contain vital mitochondria and active metabolism can reduce tetrazolium bromide salt and create formazan deposition. The resulting color change is directly related to cell metabolic activity. ,,
The data in this study showed no toxicity at the 1/64, 1/128 and 1/256 dilutions. At 1/32 dilution, toxicity was seen only after 72 h and in dilutions 1/8 and 1/16 toxicity was not observed in the 1 st h.
Dilutions 1/2 and 1/4 however were cytotoxic. According to the results decreasing dilution, reduces toxicity.
Many studies have been done on a variety of mouthwashes such as CHx; Sanchez et al. showed that all bactericidal concentrations of CHx were lethal to embryonic fibroblasts in vitro.  Damour et al. and Fabreguette et al. suggested that therapeutic concentrations of antiseptics such as CHx are cytotoxic for fibroblasts and keratinocytes. They determined viability using the MTT test. , Boyce et al. demonstrated that CHx gluconate (0.05%) was uniformly toxic to both cultured human fibroblasts cells and microorganisms.  Chang evaluated effect of CHx on cultured human PDL cells in vitro. The result showed that CHx was cytotoxic to human PDL cells. 
In one study, we compared the antibacterial effect of Anbarnesa 0.2% and CHx 0.2% in vitro and result showed that both had similar inhibitory growth zones for different bacterial species which was significantly better than control specimens. CHx 0.2% induced higher minimal inhibitory concentration (MIC) values than Anbarnesa 0.2% for the Streptococcus sanguinis and Enterococcus faecalis species, while no significant differences were found between two agents regarding MIC values against the other bacteria. CHx 0.2% and Anbarnesa 0.2% showed higher growth inhibitory effects than control specimens against all bacteria except for E. faecalis. Hence, we concluded Anbarnesa 0.2% has some antibacterial properties, but it is not as efficacious as CHx 0.2% on some selected species, with no significant effect on the E. faecalis species. 
| Conclusion|| |
Given that satisfactory results of the Anbarnesa sbmu 1 solution that had no cytotoxicity at 1/64 dilution, it may be used as an alternative solution for plaque control with less common side-effects.
| Acknowledgments|| |
This article was based on undergraduate thesis by Dr. Mina which was successfully completed under the supervision of Dr. Shafiee and Dr. Joharchi, with the close cooperation of the oral medicine Department of Dental School of Shahid Beheshti University of Medical Sciences.
| References|| |
Newman MG, Takei HH, Carranza FA. Carranza's Clinical Periodontology. 10 th
ed., Ch. 1, 2, 26, 38, 49. Chicago, IL W.B. Saunders Co.; 2006.
Niederman R, Abdelshehid G, Goodson JM. Periodontal therapy using local delivery of antimicrobial agents. Dent Clin North Am 2002;46:665-77, viii.
Baldo BA, Pham NH, Zhao Z. Chemistry of drug allergenicity. Curr Opin Allergy Clin Immunol 2001;1:327-35.
Moshrefi A. Chlorhexidine. J West Soc Periodontol Periodontal Abstr 2002;50:5-9.
Overholser CD, Meiller TF, DePaola LG, Minah GE, Niehaus C. Comparative effects of 2 chemotherapeutic mouthrinses on the development of supragingival dental plaque and gingivitis. J Clin Periodontol 1990;17:575-9.
Wilken R, Botha SJ, Grobler A, Germishuys PJ. In vitro
cytotoxicity of chlorhexidine gluconate, benzydamine-HCl and povidone iodine mouthrinses on human gingival fibroblasts. SADJ 2001;56:455-60.
Ostad SN, Gard PR. Cytotoxicity and teratogenicity of chlorhexidine diacetate released from hollow nylon fibres. J Pharm Pharmacol 2000;52:779-84.
Hidalgo E, Dominguez C. Mechanisms underlying chlorhexidine-induced cytotoxicity. Toxicol In Vitro
Cury JA, Rocha EP, Koo H, Francisco SB, Del Bel Cury AA. Effect of saccharin on antibacterial activity of chlorhexidine gel. Braz Dent J 2000;11:29-34.
Mandel ID. Antimicrobial mouthrinses: Overview and update. J Am Dent Assoc 1994;125 Suppl 2:2S-10.
Fabricant DS, Farnsworth NR. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 2001;109 Suppl 1:69-75.
Shafiee HA, Mortazavi H, Baharvand M, Eslami G, Bakhtiari S, Taheri S, et al
. Iranian traditional medicine: Comparison of the antibacterial effect of ANNAS 0.2% and chlorhexidine. Am J Ment Retard 2012;6:3600-3.
Moral R, Moreno-Caselles J, Perez-Murcia MD, Perez-Espinosa A, Rufete B, Paredes C. Characterisation of the organic matter pool in manures. Bioresour Technol 2005;96:153-8.
Mohagheghzadeh A, Faridi P, Shams-Ardakani M, Ghasemi Y. Medicinal smokes. J Ethnopharmacol 2006;108:161-84.
Poggi P, Rodrignez Y, Bena R, Rizzo S, Rota MT. Month rinses with alcohol: Cytotioxic effects on human gingival fibroblasts in vitro
. J Periodontal 2007;75:623-9.
Huang FM, Tai KW, Chou MY, Chang YC. Cytotoxicity of resin-, zinc oxide-eugenol-, and calcium hydroxide-based root canal sealers on human periodontal ligament cells and permanent V79 cells. Int Endod J 2002;35:153-8.
Schweikl H, Schmalz G. Toxicity parameters for cytotoxicity testing of dental materials in two different mammalian cell lines. Eur J Oral Sci 1996;104:292-9.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
Keiser K, Johnson CC, Tipton DA. Cytotoxicity of mineral trioxide aggregate using human periodontal ligament fibroblasts. J Endod 2000;26:288-91.
Sanchez IR, Nusbaum KE, Swaim SF, Hale AS, Henderson RA, McGuire JA. Chlorhexidine diacetate and povidone-iodine cytotoxicity to canine embryonic fibroblasts and Staphylococcus aureus
. Vet Surg 1988;17:182-5.
Damour O, Hua SZ, Lasne F, Villain M, Rousselle P, Collombel C. Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes. Burns 1992;18:479-85.
Fabreguette A, Zhi Hua S, Lasne F, Damour O. Evaluation of the cytotoxicity of antiseptics used in current practice on cultures of fibroblasts and keratinocytes. Pathol Biol (Paris) 1994;42:888-92.
Boyce ST, Warden GD, Holder IA. Cytotoxicity testing of topical antimicrobial agents on human keratinocytes and fibroblasts for cultured skin grafts. J Burn Care Rehabil 1995;16:97-103.
Chang YC, Huang FM, Tai KW, Chou MY. The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:446-50.
[Table 2], [Table 3]