CUMINUM CYMINUM EXTRACT REDUCES DENTAL PULP STEM CELL VIABILITY AT HIGHER CONCENTRATION

Objective: Cuminum cyminum (cumin) extract has potential antibacterial and antifungal activities and is not toxic for mouse fibroblasts. However, to our knowledge, no research exists investigating the toxicity of cumin extract on dental pulp stem cells (DPSCs). Therefore, we compared the viability of DPSCs after treatment with different doses of the cumin extract (0.1, 0.4, 0.7, and 1.0 mg/mL) at 1 h, 24 h, and 4 days. Methods: DPSCs were gently evacuated from exfoliated third molars. Subsequently, cumin seeds were extracted by steam distillation to obtain 0.1, 0.4, 0.7, and 1.0 mg/mL concentrations. Then, the cell viability of DPSCs was analyzed using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay by calculating the absorbance values using a microplate reader, with the optical density (OD) as the final result. Results: There were statistically significant differences in the viability of DPSCs (p<0.05) between 0.1 and 0.4, 0.7, and 1.0 mg/mL cumin extract, but there was no difference (p≥0.05) in the viability of DPSCs at 1 h in each group. Conclusion: The cumin extract at all concentrations did not affect the viability of DPSCs at 1 h. However, the cumin extract at 0.4, 0.7, and 1.0 mg/mL decreased the viability of DPSCs at 24 h and 4 days.


INTRODUCTION
Plants are valuable biological natural resources that can be used as medicines because they contain a wide variety of natural chemical compounds with pharmacological effects [1]. Most materials in dentistry, especially endodontic treatments such as sodium hypochlorite and chlorhexidine, consist of highly irritating synthetic materials that are toxic to stem cells, which are the key to regeneration during treatment. To improve the biologic safety, herbal medicines that chemically have the same compounds that can support the regenerative endodontic treatment are selected [2][3][4][5][6][7].
In general, herbal medicines are considered safer than synthetic drugs. For herbal medicines to be widely used, they must meet the World Health Organization's standardization and quality control criteria to ensure its safety (toxicity) and efficacy locally and systemically [7,8]. The standardization process of herbal medicines consists of various chemical analysis methods based on biocompatibility tests on a medicinal plant extract [1,8]. Cytotoxicity is a sensitive biocompatibility test for assessing toxicity when a substance comes into contact with a particular cell culture (ISO 10993), and it is the best initial step for a biocompatibility test circuit of a substance to a cell or living tissue. A substance is considered nontoxic or biocompatible if the viability test results in a percentage of living cells ≥70% of the control [6][7][8][9][10][11][12].
One herb known to have antibacterial and antifungal effects is cumin [13][14][15][16][17]. A study on cumin extract as a root canal medicament reported it to be effective against Enterococcus faecalis and biocompatible with L929 fibroblasts compared to 2% chlorhexidine. To date, to our knowledge, no one has examined the toxicity of cumin extract to dental pulp stem cells (DPSCs) [11]. Therefore, the effect of cumin extract on the viability of DPSCs was examined.
The procedures were performed in a biohazard cabinet using sterile tools and working procedures. The complete culture medium consisted of Dulbecco's Modified Eagle's Medium (DMEM) containing penicillinstreptomycin-amphotericin (PSA) and 20% fetal bovine serum. The culture medium was filtered using a Sartorius Minisart singleuse syringe with a 50 mL sterile filter with a diameter of 0.2 μm and stored in a refrigerator. This research consisted of several stages: DPSC culture, preparation of the cumin extract, flow cytometry, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay, and statistical analysis.

DPSC culture
The extracted third molars (<24 h) were cleaned and immersed in 20 mL of complete culture medium or phosphate-buffered saline and PSA. Teeth were separated using a sterile mortar and pestle. Pulp tissue was obtained with an extirpation needle, minced into 2.0 mm × 2.0 mm × 1.0 mm fragments, incubated in a 15 mL tube with 2.0 mL of 3.0 mg/mL collagenase type I, and left to stand for 1 h. Subsequently, collagenase was washed by complete DMEM and centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then, the cells were placed on a 10cm Petri dish with complete DMEM. Cells were incubated in 37°C, within 5% CO 2 until 80% confluent.

Preparation of cumin extract
Cumin extract was obtained by steam distillation. Chemical compound analyses were performed with gas chromatography-mass spectrometry. All cumin extracts were placed in a black bottle.

Flow cytometry test
Flow cytometry was performed by placing each cell in a chamber slide, followed by incubating at 37°C and 5% CO 2 and examining for CD73, CD90, and CD105.

MTT assay test
After 24 h of incubation, the cell culture medium was removed and the test material was expressed as 100 μL per well with three replications. Then, the cells were incubated at 37°C and 5% CO 2 for 4-8 h. The specimens were then divided into two groups: The control group (cells without treatment) and the test group (given cumin concentrations of 0.1, 0.4, 0.7, and 1.0 mg/mL).
As much as 100 μL of MTT was added, and the cells were then incubated at 37°C and 5% CO 2 for 4 h. The cell supernatant was removed, and the formed formazan crystals were dissolved with 70% ethanol. Optical density (OD) readings were conducted using a microplate reader with a wavelength of 560 nm. The OD of each test group was then presented against that for the control group to determine cell viability.

Statistical analysis
All data were analyzed using the Kruskal-Wallis and Mann-Whitney U-tests to compare the OD between test groups. p<0.05 was considered to be statistically significant.

RESULTS
The characteristics of DPSCs were seen from the flow cytometry analysis of surface antigen expression. The results were positive for CD90 (98.3%) DPSCs, CD73 (98.5%), and CD105 (71%). Thus, we concluded that the cells are DPSCs.
The test group cells were exposed to cumin extract (concentration: 0.1, 0.4, 0.7, and 1.0 mg/mL) and incubated for 1 h, 24 h, and 4 days. The MTT test and a microplate reader were used to measure the viability. In all groups, there was a significant difference in the viability of DPSCs on the basis of the dose (p≤0.05) but not time (p≥0.05). Furthermore, Mann-Whitney U-test was performed to determine the significance in each group.
In the 1 st h, there was no significant difference (p<0.05) in the viability of DPSCs in all groups. There was a significant difference between Groups 1 and 2, Groups 1 and 3, and Groups 1 and 5 at 24 h and between Groups 1 and 2, Groups 1 and 3, Groups 1 and 4, Groups 1 and 5, and Groups 2 and 3 on day 4; (Table 4).

DISCUSSION
Herein, we studied DPSC cultures. According to the International Society for Cellular Therapy, one criterion for mesenchymal stem cells is that the cells should positively express marker proteins, such as CD73, CD90, and CD105. In our study, flow cytometry was positive for CD90 (98.3%), CD73 (98.5%), and CD105 (71%). In the 1 st h, there was no significant difference (p<0.05) in the viability of DPSCs in all groups ( Table 4), showing that the cumin extract did not cause DPSC death at exposure for up to 1 h.
At 24 h, there was a significant difference between Groups 1 and 2, Groups 1 and 3, and Groups 1 and 5. After 24 h, the number of cells decreased further, especially at concentrations of 0.4 and 1.0 mg/mL. However, at a concentration of 0.1 mg/mL, the number of cells in the test group was higher than that in the controls. The viability of DPSCs at a concentration of 0.7mg/mL did not differ significantly from that of controls.
At 4 days, there were significant differences in Groups 1 and 2, Groups 1 and 3, Groups 1 and 4, Groups 1 and 5, and Groups 2 and 3. After 4 days, the number of cells was very much reduced at the concentrations of 0.4, 0.7, and 1.0 mg/mL compared to controls, with the least number of cells at 1.0 mg/mL, whereas at 0.1 mg/mL, the number of cells was higher than that in the controls.
At 24 h, the average cell viability was reduced, except at 0.1 mg/mL. All cells treated with concentrations above 0.1 mg/mL showed increasingly low viability as time passed. In synthetic drugs, if the concentration had been higher, it would be more effective. Unlike the case with herbs, the graph of the relationship among the concentration, effectiveness, and toxicity is not linear. Therefore, finding the optimum dose of the herbal compound is necessary to determine the maximum effect with minimum toxicity.
Cuminaldehyde (chemical formula: C 6 H 12 O), the main component in cumin (approximately 35-63%), is a volatile compound with a major role in providing a stinging smell to cumin. It has an important antibacterial activity and has an antifungal effect because it inhibits the filament and yeast formation in fungi [17,18].
Aldehydes are electrophilic (electron-deficient species) that forms covalent bonds with nucleophilic (electron-rich) target cells. The resulting formation of these bonds may interfere with the function of the enzymes, DNA, protein structures, and other macromolecules, thus causing the inhibition of cellular processes and ultimately toxicity to cells. Potential of toxic depends on covalent formation which has formed by cell and was decised with a large of aldehyde concentration. Therefore, it can be concluded that the magnitude of the toxicity of the   aldehyde is directly proportional to time and concentration. It will be more toxic to the cells if exposed longer to a higher concentration of aldehyde [19].
Cumin extract at concentrations of 0.1, 0.4, 0.7, and 1.0 mg/mL can be used for 1 h because it does not affect the viability of DPSCs. However, at 24 h and at 4 days, it can reduce the viability of DPSCs, except at the concentration of 0.1 mg/mL. From the result of this study, it is expected that, in the future, cumin extract can be an alternative irrigation material in endodontic treatment.

CONCLUSION
The cumin extract at all concentrations did not affect the viability of DPSCs at 1 h. However, the cumin extract at all concentration decreased the viability of DPSCs at 24 h and 4 days, except for the concentration of 0.1 mg/mL.