Year : 2009 | Volume
: 20 | Issue : 2 | Page : 169--173
Cytotoxicity evaluation of Persica mouthwash on cultured human and mouse cell lines in the presence and absence of fetal calf serum
Saeed Rajabalian1, Mohammad Mohammadi2, Behrooz Mozaffari3,
1 Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran
2 Department of Periodontics, Kerman Dental and Oral Diseases Research Center, Kerman University of Medical Sciences, Kerman, Iran
3 Department of Oral and Maxillofacial Surgery, Dental School, Kerman University of Medical Sciences, Kerman, Iran
Department of Periodontics, Kerman Dental and Oral Diseases Research Center, Kerman University of Medical Sciences, Kerman
Background and Aims: The effectiveness of an ideal antimicrobial agent depends on its ability to kill microbes while causing minimal toxicity to host cells. Several studies have been reported on the antimicrobial effects of chewing sticks (Salvadora persica) on oral bacteria. The purpose of this study was to evaluate the cytotoxic effects of Persica™ and chlorhexidine (CHX) mouthwashes on cultured human and mouse cell lines.
Materials and Methods: This was an experimental study. The toxic effects of four dilutions of Persica™ and CHX mouthwashes on KB, Saos-2, J744 A1, and gingival fibroblast cells were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. The effect of fetal calf serum (FCS) components on the cytotoxicity of these mouthwashes was also investigated.
Statistical Analysis: Analysis of variance and the Kruskal-Wallis test were used to evaluate the results.
Results: The results indicated that Persica™, at concentrations higher than 0.1%, exerted a very significant cytotoxic effect on all the cell lines (P ≤ 0.01). CHX, at a concentration of 0.001%, exerted toxic effects only on gingival fibroblasts; concentrations higher than 0.001% were required to produce significant cell death in the other cell lines. At all the concentrations under study, both Persica™ and CHX exerted significantly greater cytotoxic effects in the absence of FCS than in its presence (i.e., in control culture medium). The toxicities of both mouthwashes were attenuated in the presence of FCS (10%).
Conclusion: Our results indicate that both Persica™ and CHX mouthwashes are toxic to macrophage, epithelial, fibroblast, and osteoblast cells in a concentration-dependent manner.
|How to cite this article:|
Rajabalian S, Mohammadi M, Mozaffari B. Cytotoxicity evaluation of Persica mouthwash on cultured human and mouse cell lines in the presence and absence of fetal calf serum.Indian J Dent Res 2009;20:169-173
|How to cite this URL:|
Rajabalian S, Mohammadi M, Mozaffari B. Cytotoxicity evaluation of Persica mouthwash on cultured human and mouse cell lines in the presence and absence of fetal calf serum. Indian J Dent Res [serial online] 2009 [cited 2015 Mar 31 ];20:169-173
Available from: http://www.ijdr.in/text.asp?2009/20/2/169/52894
The microflora of the oral cavity has great variety. There are almost 500 types of microorganisms in the oral cavity, of which some can cause infectious oral disease.  Preoperative reduction of oral microorganisms is important for decreasing wound and systemic infections.  Mouthwashes are used as antiseptic solutions for decreasing microorganism populations, preventing wound infection, and accelerating the wound healing process. , In recent times, the use of antiseptics has generated a lot of controversy as studies have shown that they are detrimental to wound healing. Many of these studies were carried out on tissue culture specimens and the results differed from that obtained with studies on live animals.  Therefore, when selecting the appropriate mouthwash, in addition to antimicrobial effect the biocompatibility too has to be given due consideration.  Chlorhexidine (CHX) is a mouthwash with a broad spectrum of activity, being active against both gram-positive and gram-negative bacteria. , Long-term maintenance use of CHX can cause local side effects such as calculus formation and staining of teeth, restorations, or tongue. , Furthermore, several investigators have reported delayed wound healing after exposure to CHX. , Because of these undesirable side effects there is now widespread interest in the use of medicinal plants for the maintenance oral hygiene. One candidate plant is Salvadora persica, a traditional and widely used oral hygiene tool in several African and Middle Eastern countries. There are several reports describing the beneficial effects of this plant; it has been shown to be capable of reducing pathogenic microorganisms in the oral cavity and in preventing dental caries. ,,, Some investigators have reported the presence of a considerable amount of chloride, calcium, fluoride, silica, a sulfur compound, vitamin C, tannins, saponins, resins, phytosterols, flavonoids, triterpenes, and alkaloids in this plant. , The high chloride content is believed to reduce calculus formation and prevent staining of the teeth, whereas the isothiocyanate component may inhibit the growth of oral microflora.  In view of these encouraging reports, a mouthwash named Persica™ (Pursina Pharmaceutical Co., Iran) has found a place in the Iranian pharmacopeia. The main components of Persica™ mouthwash are S persica and Achillea millefolium extracts.
Because fibroblasts, osteoblasts, and epithelial and inflammatory cells cooperate in wound healing, we selected these cells to evaluate the cytotoxicity of Persica™ and CHX mouthwashes. In the present study, the cell toxicity of Persica™ mouthwash in the presence and absence of fetal calf serum (FCS) was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and compared with that of CHX.
Materials and Methods
Cell culture and cell lines
We used four cell lines in this study. Three of them-the oral carcinoma cell line (KB), the osteoblastoma cell line (Saos-2), and the mouse macrophage cell line (J774 A)-were purchased from the National Cell Bank of Iran (NCBI). The human gingival fibroblast cell line, designated as MRF, was derived in our lab from the gingival connective tissue of a 30-year-old male in good health. The tissue specimen was dissected into pieces smaller than 2 mm 3 , which were then immersed in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (Serumed, Germany), 25 µg/ml gentamycin, 2.5 µg/ml amphotericin B, and 0.5 mg/ml collagenase (all from Gibco-BRL, UK). After incubation for 18 h at 37°C in atmosphere with 5% CO 2 , the fragments were disaggregated by gentle pipetting, pelleted by centrifugation at 1500 rpm, resuspended in fresh complete growth medium, and then transferred to a 25 cm 2 cell-culture flask (Nunc, Denmark). After expansion of cultures to approximately 75- 80% confluency, the cells were trypsinized and transffered to larger flasks to proliferate more. A number of vials were cryopreserved at the third or fourth cell passage and then, used during this study after defreezing.
The therapeutic classes of Persica™ (Pursina Pharmaceutical Co., Iran) and CHX (0.2%) (Shahre Daru Pharmaceutical Co., Iran) mouthwashes were purchased from a drugstore. They were sterilized by filtration through a 0.22-µm filter (Millipore, USA) and then stored at 4°C. The different concentrations of the drugs were prepared by serial dilution with the FCS-free medium or the medium containing 10% FCS in sterile plastic centrifuge tubes (Nunc, Denmark). These concentrations were as follows: Persica™ - 0.1, 0.5, 1, and 5% (v/v) and CHX - 0.0001, 0.001, 0.01, and 0.03% (w/v). These concentrations were selected on the basis of the results of pilot study performed in our laboratory (data not shown). For each experiment, drug solutions were freshly prepared from the stock solution. For measurement of cytotoxicity, the MTT colorimetric assay was used as per the description given by Mosmann.  Cultures in the exponential growth phase were trypsinized and diluted in culture medium to give a suspension of 1 × 10 6 cells/ml. One hundred microliters of cell suspension was added to each centrifuge tube containing 2 ml of drug solution. One tube containing only cells suspended in complete medium was used as control for cell viability. The tubes were then incubated for 1 h at 37°C in a humidified atmosphere containing 5% CO 2 . Following drug exposure, the cells were washed twice with 10 ml of culture medium to remove any residual drug and then resuspended in 2 ml of fresh complete growth medium. Then 100 µl of suspension was added to the appropriate wells of a sterile 96-well flat-bottomed microtiter plate (Nunc, Denmark). Each drug dilution was assessed in triplicate. Three wells containing only complete medium were used as blank controls for nonspecific dye reduction. The plates were then incubated at 37°C in a humidified atmosphere with 5% CO 2 for 48-72 h. After incubation, 20 µl of a 5 mg/ml solution of MTT (Sigma-Aldrich, USA) was added to each well and the plate was incubated for another 3-4 h. Following incubation, the culture medium was removed from the wells by slow aspiration, and then 100 µl of dimethyl sulfoxide (Sigma-Aldrich, USA) was added to the wells to dissolve the formazan crystals. The plates were then read on a microplate reader (Model Sunrise, Tecan A5082, Austria) at a wavelength of 492 nm and a reference of 630 nm.
The percentage of cells alive for each well was calculated by dividing the absorbance readings by the mean absorbance of eight control wells and multiplying the value by 100. Analysis of variance was performed for each plate, and Kruskal-Wallis analysis was used to evaluate the statistical significance of the difference between the mean percentages of cells alive at each drug concentration in the absence and presence of FCS using the data resulted from three repeated experiments.
[Figure 1] and [Figure 2] illustrate the influence of the drugs on the growth of the cell lines. The results show that at the 0.01% and 0.03% concentrations, CHX induced a very significant inhibition of growth in all the cell lines (P ,, According to a recent study, methanolic extracts of the stems and leaves of S persica showed only weak antifungal activity and no antibacterial activity.  In the same study, the extracts exerted strong activity against Plasmodium falciparum cells and weak activity against mammalian cells.
Giannelli et al. suggest that CHX is highly cytotoxic in vitro and advice a more cautious use of the antiseptic in oral surgical procedures.  The introduction of commercially available concentrations (0.12%) of CHX, or even diluted (to as low as 0.00009% concentration) CHX, to surgical sites for short periods of time prior to wound closure could conceivably have serious toxic effects on gingival fibroblasts and may negatively affect wound healing. 
A recent report on dog skin wound healing showed that CHX has a beneficial effect on wound healing when given in doses that are fatal to fibroblasts in vitro.  In other investigations on gingival wound healing it was concluded that CHX did not interfere with healing. , The results of the present study also suggest that there is some mechanism whereby the organism is protected against CHX. Similar to our results, other studies have also shown that serum protects cells from the cytotoxic effects of CHX. , The binding of CHX to bacteria may decrease the amounts available to bind to the host's cells. The mouth is heavily populated with bacteria and this bacterial challenge may alter the effect of CHX in vivo as compared to the effects seen in vitro. Thus, CHX seems to have a low level of local cytotoxicity.
In the present study, 1-h exposure to as low as 0.1% Persica™ solution induced irreversible cytotoxic effects on the cells involved in the wound healing process. However, the presence of FCS (10%) offered protection from drug toxicity. The decrease in the cytotoxic effect of Persica™ in the presence of FCS is probably due to the binding of potent toxic compounds of the mouthwash to serum proteins. It seems that the toxic compounds of Persica™ solution exert their effects through irreversible binding to cellular proteins, thereby disturbing their function.
S persica has been reported to contain indole alkaloids, flavonoids, the sulphur-containing compound tropaedoin, triterpenes, and phytosterols. , Of these compounds, the alkaloids and flavonoids are probably responsible for the cytotoxic activity observed in this study. As compared to previous reports, our study showed that Persica™ mouthwash exerted a potent cytotoxic effect; we suggest that there may be a synergistic action between the toxic compounds of A millefolium and S persica in this preparation. Further studies are needed to better understand these compounds and their effects on cellular function.
Our results indicate that, at the concentrations available in the market, both Persica™ and CHX mouthwashes are toxic to macrophages and epithelial, fibroblast, and osteoblast cells. On the basis of these results, we suggest that clinical application of both these mouthwashes should be avoided in patients with fresh open oral wounds, which usually heal through secondary intention. We suggest that the mouthwashes should be used only 1 day after surgery because the blood clot that has formed by this time will prevent the direct exposure of the injured tissues to the mouthwashes.
This research was partially supported by The Research Council of Medical Sciences, University of Kerman, Iran.
|1||Theilade E. Advances in oral microbiology. Ann R Australas Coll Dent Surg 1989;10:62-71.|
|2||Debelian GJ, Olsen I, Tronstad L. Systemic diseases caused by oral microorganisms. Endod Dent Troumatol 1994;10:57-65.|
|3||Roldan S, Winkel EG, Herrera D, Sanz M, Van winkelhoff AJ. Clinical effects of a new mouthrinse containing chlorhexidine, cetylpyridinium chloride and zinc-lactate on oral halitosis: A dual-center, double-blind placebo-controlled study. J Clin Periodentol 2003;30:300-6.|
|4||Sharma NC, Charles CH, Qaqish JG, Galustians HJ, Zhao Q, Kumar LD. Comparative effectiveness of an essential oil mouthrinse and dental floss in controlling interpoximol gingivitis and plaque. Am J Dent 2002;15:351-5.|
|5||Drosou A, Falabella A, Kirsner RS. Antiseptics on wounds: An area of controversy. Wounds 2003;15:149-66.|
|6||Tatnall FM, Leigh IM, Gibson JR. Assay of antiseptic agents in cell culture: Conditions affecting cytotoxicity. J Hosp Infect 1991;17:287-96.|
|7||Beyth N, Redlich M, Harari D, Friedman M, Steinberg D. Effect of sustained - release chlorhexidine varnish on streptococcus mutans and Actinomyces visosus in orthodentic patients. Am J Orthod Dentofacial Orthop 2003;123:345-8.|
|8||Budtz - Jorgensen E, Lo?e H. Chlorhexidine as a denture disinfectant in the treatment of denture stomatitis. Scand J Dent Res 1972;80: 457-64.|
|9||Yates R, Jenkins S, Newcombe R, Wade W, Moran J, Addy M. A 6-month home usage trial of a 1% chlorhexidine toothpaste (1): Effects on plaque, gingivitis, calculus and toothstaining. J Clin Periodontal 1993;20:130-8.|
|10||Albandar JM, Gjermo P, Preus HR. Chlorhexidine use after two decades of over-the-counter availability. J Periodontal 1994;65:109-12.|
|11||Saatman RA, Carlton WW, Hubben K, Streett CS, Tuckosh JR, DeBaecke PJ. A wound healing study of chlorhexidine digluconate in guinea pigs. Fundam Appl Toxicol 1986;6:1-6.|
|12||Bassetti C, Kallenberger A. Influence of chlorhexidine rinsing on the healing of oral mucosa and osseous lesions. J Clin Periodontol 1980;7:443-56.|
|13||Darout IA, Albandar JM, Skaug N. Periodontal status of adult Sudanese habitual users of miswak chewing sticks or toothbrushes. Acta Odontol Scand 2000;58:25-30.|
|14||Yarde A, Robinson M. The miswak chewing stick: A traditional oral hygiene aid. NDA J 1996;47:20-1.|
|15||Almas K. The antimicrobial effects of extracts of Azadirachta indica (Neem) and Salvadora persica (Arak) chewing sticks. Indian J Dent Res 1999;10:23-6.|
|16||Almas K. The effect of Salvadora Persica extract (miswak) and chlorhexidine gluconate on human dentin: A SEM study. J Contemp Dent Pract 2002;3:27-35.|
|17||Kamel MS, Ohtani K, Assaf MH, Kasai R, Elshanawani MA, Yamasaki K, et al. Lignan glycosides from stems of Salvadora persica. Phytochemistry 1992;31:2469-71.|
|18||Akhtar MS, Ajmal M. Significant of chewing-sticks (Miswaks) in oral hygiene from a pharmacological view point. J Pak Med Assoc 1981;31:89-95.|
|19||Elvin-Lewis M. The therapeutic potential of plants used in dental folk medicine. Odontostomatol Trop 1982;5:107-17.|
|20||Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55- 63.|
|21||Almas K. The antimicrobial effects of seven different types of Asian chewing sticks. Odontostomatol Trop 2001; 24:17-20.|
|22||al-Bagieh NH, Idowa A, Salako NO. Effect of aquous extract of miswak on the in vitro growth of Candida albicans. Microbios 1994;80:107-13.|
|23||Ali H, Koning GM, Khalid SA, Wright AD, Kaminsky R. Evaluation of selected Sudanese medicinal plants for their in vitro activity against, hemoflagellates, selected bacteria, HIV 1-RT and tyrosine kinase inhibitory and for cytotoxicity. J Ethnopharmacol 2002;83:219-28.|
|24||Mariotti AJ, Rumpf DA. Chlorhexidine-induced changes to human gingival fibroblast collagen and non-collagen protein production. J Periodontol 1999;70:1443-8.|
|25||Giannelli M, Chellini F, Margheri M, Tonelli P, Tani A. Effect of chlorhexidine digluconate on different cell types: A molecular and ultrastructural investigation. Toxicol InVitro 2008;22:308-17.|
|26||Sanchez IR, Swaim SF, Nusbaum KE, Hale AS, Henderson RA, McGuire JA. Effects of chlorhexidine diacetate and povidone-iodine on wound healing in dogs. Vet Surg 1988;17:291-5.|
|27||Hirst RC, Egelberg R, Hornbuckle GC, Oliver RC, Rothbun WE. Microscopic evaluation of topically applied chlorhexidine gluconate on gingival wound healing in dogs. J Southern Calif Dent Assoc 1973;41:311-7.|
|28||Hamp SE, Rosling B, Lindhe J. Effect of chlorhexidine on gingival wound healing in the dog. A histometric study. J Clin Periodontal 1975;2: 43-52.|
|29||Hidalgo E, Dominguez C. Mechanisms underlying chlorhexidine-induced cytotoxicity. Toxical in vitro 2001;15:271-6.|
|30||Babich H, Wurzburger BJ, Rubin YL, Sinensky MC, Blau L. An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol 1995;11:79-88.|