|Year : 2020 | Volume
| Issue : 4 | Page : 313-317
Evaluation of the remineralizing potential of Nigella sativa, sodium fluoride and caesin phosphopeptide-amorphous calcium phosphate on enamel: An In vitro study
N Kiran Kumar1, Savitha B Naik1, C H Laxmi Priya1, Seema Merwade1, Biji Brigit1, CN Guruprasad2, Pavithra Prabakaran1
1 Department of Conservative Dentistry and Endodontics, Government Dental College and Research Institute, Bengaluru, Karnataka, India
2 Department of Periodontics, Government Dental College and Research Institute, Bengaluru, Karnataka, India
|Date of Submission||29-Jun-2020|
|Date of Decision||21-Sep-2020|
|Date of Acceptance||14-Oct-2020|
|Date of Web Publication||16-Dec-2020|
C H Laxmi Priya
Government Dental College and Research Institute, Victoria Hospital Campus, City Market, Bengaluru - 560 002, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Dental caries is a multifactorial disease that results from an imbalance between pathological and protective factors. The imbalance produced will favor demineralization of enamel. If detected early, noncavitated lesions can be reverted with the help of remineralizing agents. Aim: To compare remineralizing potential of novel agent-Nigella sativa with conventional remineralizing agents. Materials and Methods: An in vitro study was done to compare remineralizing potential of N. sativa with conventional remineralizing agents. Sixty-four human premolars were covered with nail varnish except for 4 mm × 4 mm exposed window on the facial aspect, which was treated with 37% phosphoric acid for 30 s. Samples are then randomly divided into four groups (n = 16): Group I – N. sativa, Group II – 5% sodium fluoride, Group III – Casein phosphopeptide-amorphous calcium phosphate, and Group IV – Control. The samples in each group were treated with the remineralizing agent, after which they were rinsed with deionised water, dried, and stored in artificial saliva. The same procedure is repeated for the span of 14 days. Depth of demineralization and remineralization were analyzed using the confocal laser scanning microscopy. Data were analyzed using the Statistical Package SPSS 22.0 (SPSS Inc., Chicago, IL) and level of significance was set at P < 0.05. Kruskal–Wallis test was used to analyze the depth of demineralization and remineralization among groups followed by post-hoc Mann–Whitney test to compare demineralization and remineralization between the groups. Results: Group I specimens showed greater depth of remineralization (1558.83) compared to other groups, which is statistically significant (P < 0.001). Conclusion: Herbal product like N. sativa can be considered as a promising remineralizing agent in treating noncavitated demineralized carious lesions.
Keywords: Casein phosphopeptide-amorphous calcium phosphate, Nigella sativa, sodium fluoride, tooth remineralization
|How to cite this article:|
Kumar N K, Naik SB, Priya C H, Merwade S, Brigit B, Guruprasad C N, Prabakaran P. Evaluation of the remineralizing potential of Nigella sativa, sodium fluoride and caesin phosphopeptide-amorphous calcium phosphate on enamel: An In vitro study. J Indian Assoc Public Health Dent 2020;18:313-7
|How to cite this URL:|
Kumar N K, Naik SB, Priya C H, Merwade S, Brigit B, Guruprasad C N, Prabakaran P. Evaluation of the remineralizing potential of Nigella sativa, sodium fluoride and caesin phosphopeptide-amorphous calcium phosphate on enamel: An In vitro study. J Indian Assoc Public Health Dent [serial online] 2020 [cited 2021 Apr 13];18:313-7. Available from: https://www.jiaphd.org/text.asp?2020/18/4/313/303636
| Introduction|| |
Dental caries is a biofilm-mediated, diet modulated, multifactorial, noncommunicable, dynamic disease resulting in the net mineral loss of dental hard tissues. Cariogenic bacteria, fermentable carbohydrates, and salivary dysfunction are scientifically proven as the important pathological factors.
Demineralization and remineralization are balanced processes that normally occur in the oral cavity. Diet variations, oral hygiene, or microbial activity can lead to the predominance of demineralization. Remineralization is favored by the buffering action of saliva, permitting calcium, and phosphate ions to precipitate onto the tooth and form new mineral. Therefore, modulation of the demineralization-remineralization balance is the key to prevention of dental caries.
Various remineralizing agents such as fluorides, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), self-assembling peptides are used to revert initial noncavitated carious lesion. The direction toward herbs and their products is the basis for many unique pharmaceuticals. Nigella sativa is an important drug in traditional systems of medicine such as Unani and Ayurveda. In old Latin, it is called as “Panacea” meaning “cure all.” The use of this miraculous herb in dentistry can be panacea in preventing tooth decay and promoting oral health. The seeds of N. sativa and their oil have been widely used for centuries in the treatment of various ailments throughout the world. N. sativa plant, also known as black seed, black caraway seed, or the blessed Seed, grows in the countries of Mediterranean sea and West of Asia. The chemical composition of black seed is volatile oil, fixed oil, protein, carbohydrates, alkaloids, saponins, amino and fatty acids, trace elements, heavy metals, and minerals. N. sativa contains numerous inorganic elements by range from 1.79% to 3.74% as calcium, phosphorous, potassium, sodium, and iron. Prophet Muhammad, stated “Black seed” can heal every disease except death.
The anticarcinogenic potential and remineralizing properties of CPP-ACP have been exhibited in vitro and in situ studies. CPP-ACP is a technology-based on ACP stabilized by CPPs. The beneficial effect obtained from CPP-ACP is associated with its ability to localize calcium and phosphate in dental plaque in the proximity of the tooth, thus facilitating remineralization.
Fluorides have been conventionally used as remineralizing agents. The topical application of fluoride varnishes provides a noninvasive method for caries control and remineralization. Sodium fluoride has the ability to increase the resistance of enamel to dissolution by the effect of acid which results in the protection of the teeth against decay hence used as a remineralizing agents.
Till date, there is no study comparing this herbal product, N. sativa with conventional remineralizing agents. Hence, this study aimed to compare the remineralizing potential of Novel agent-N. sativa with conventional remineralizing agents like sodium fluoride and CPP-ACP.
| Materials and Methods|| |
This in vitro study was carried out in the department of conservative dentistry and endodontics over a period of 6 months from July 2019 to December 2019. The study was approved by the Institutional Ethical Committee. Sample size estimation was done using G * power, version 3.0.1 (Franz Faul universitat, Kiel, Germany). A sample size of 64 subjects (16 in each group) would yield 90% power to detect the significant differences, with effect size of 0.5 and significance level was set at 0.05.
Sixty-four human premolars extracted for orthodontic purpose and free of caries or cracks were selected for the study. Teeth were cleaned with ultrasonic scaler to remove the tissue remnants, sterilized, and placed in distilled water until use. Teeth were covered with nail varnish except for 4 mm × 4 mm exposed window [Figure 1]. Artificial caries-like lesion was produced by treating the exposed window with 37% phosphoric acid for 30 s. Demineralized samples were randomly divided into four groups consisting of 16 samples in each group. Demineralization depth was analyzed prior remineralization using confocal laser scanning microscopy (CLSM).
Group I: Samples were treated with 4.3% N. sativa (Blessed seed strong black seed oil) for 5 min. This duration of application was chosen to match the time span of other conventional remineralizing agents and to obtain the remineralization in minimum contact time, so that it can be clinically related for future use.
Group II: Samples were treated with 5% sodium fluoride (VOCO varnish) for 4 min according to the manufacturer's instructions.
Group III: Samples were treated with CPP-ACP (GC tooth mousse) for 3 min according to the manufacturer's instructions.
Group IV: Control group. Samples were not treated with any remineralizing agent.
The samples in each group were treated with the remineralizing agent, after which they were rinsed with deionised water, dried, and stored in artificial saliva. The same procedure is repeated for the span of 14 days, as it has been previously reported by Burwell et al. and Vashisht et al. that the average time for remineralization after acid exposure is 14 days., The samples are then subjected to CLSM to analyze for the depth of remineralization.
A power analysis was established by G*power, version 3.0.1 (Franz Faul universitat, Kiel, Germany). A sample size of 64 participants (16 in each group) would yield 90% power to detect significant differences, with effect size of 0.5 and significance level at 0.05.
Data were analyzed using the Statistical Package SPSS 22.0 (SPSS Inc., Chicago, IL, USA), and level of significance was set at P < 0.05. Kruskal–Wallis test was used to analyze the depth of demineralization and remineralization among groups followed by post-hoc Mann–Whitney test to compare demineralization and remineralization between the groups.
| Results|| |
Comparison of the depth of demineralization among the groups is displayed in [Table 1]. Group IV showed higher median values (1698.07) followed by Group II (1681.04), Group I (1660.74), and Group III (1659.67). There was no statistically significant difference among the groups (P = 0.69).
|Table 1: Comparison of the depth of demineralization (microns) among the groups|
Click here to view
Comparison of the depth of remineralization among the groups is displayed in [Table 2]. Group I (1558.83) showed higher median values followed by Group II (1099.16), Group III (989.56), and Group IV (386.90). There was a statistically significant difference among the groups (P < 0.001).
|Table 2: Comparison of the depth of remineralization (microns) among the groups|
Click here to view
Post hoc Mann–Whitney test was applied to compare the demineralization and remineralization between the groups [Table 3]. Statistically significant difference was not seen between the groups for demineralization, whereas there was a statistically significant difference seen between all the groups with respect to remineralization (P < 0.001).
|Table 3: Comparison of demineralization and remineralization among groups|
Click here to view
| Discussion|| |
The dynamic nature of caries makes its management critical and time-specific. With early diagnosis, the disease can be reversed in its initial stages, but it is irreversible in the later stages. Demineralization results in loss of calcium and phosphorous ions from enamel. Reversal can be achieved if the pH is neutralized and by the presence of adequate calcium and phosphate ions, aiding in remineralization.,
Novel treatments aiming to halt or reverse the progression of noncavitated, demineralized lesions involve the topical use of remineralizing agents. These agents provide favorable environment for remineralization of enamel.
This study was done to analyze remineralization potential of novel agent-N. sativa and compare it with remineralizing agents such as sodium fluoride and CPP-ACP. CLSM was used to analyze the remineralization ability of the test agents in the study. CLSM showed significantly greater depth of remineralization in specimens treated with N. sativa (Group I) than other groups [Figure 2].
|Figure 2: Representative confocal laser scanning microscopy images of Groups I, II, III, and IV (a) demineralization and (b) remineralization (white areas denote demineralization; black areas denotes remineralization)|
Click here to view
N. sativa is miracle herb with a rich historical and religious background. It has wide spectrum of pharmacological potential. N. sativa treated specimens (Group I) showed greater remineralization depth (1558.833) compared to other groups which is statistically significant. This could be due to the increased levels of calcium and phosphorus content of this miraculous herb. Furthermore, the active component of N. sativa, thymoquinone is reported to have antimicrobial, anti-inflammatory, analgesic, anticariogenic, and antioxidant properties. These anti-cariogenic minerals might react with outer enamel surface incorporating the ions into the inorganic structure of enamel which might have contributed to remineralization. However, exact mechanism at molecular level is still unclear. N. sativa oil used in the study is a relatively newer agent for remineralisation, so its comparative studies are scarce. However, there is ongoing research related to the use of N. sativa in dentistry.
Omar et al. have proposed the use of N. sativa oil as pulp medicament owing to its favorable histological results due to its potent anti-inflammatory actions. Abd-Awn et al. evaluated the role of N. sativa in inhibiting bacterial adherence to plaque. The results of their study showed that the black seed oil extract has 10% minimum bactericidal concentration against Streptococcus mutans, suggesting its anti-cariogenic property. Thus, N. sativa oil prevents attachment of bacteria to host tissues A study by Jain et al. evaluating N. sativa as an endodontic irrigant has shown that it has a better antimicrobial activity than 2.5% sodium hypochlorite against Enterococcus faecalis. Thus, N. sativa has potential therapeutic actions in oral health.
Specimens treated with 5% sodium fluoride (Group II) showed greater remineralization depth compared to those treated with CPP-ACP (Group III) and control (Group IV), which is statistically significant. McKay and Black in the early 20th century advocated the use of fluoride as a method of reducing enamel susceptibility to decalcification. Fluoride inhibits glycolysis of plaque microorganisms, thereby killing the microorganisms. It also enables the formation of high quality fluorapatite that promotes remineralization. This could be the reason for its greater depth of remineralisation. Similar results are obtained in study done by Chokshi et al. comparing fluoride varnish and CPP-ACP where fluoride varnish proved to be more efficacious than CPP-ACP.
CPP-ACP (Group III) showed least depth of remineralisation when compared to Group I and Group II. But when compared to control specimen (Group IV) which has not been treated with any remineralising agent, it showed significant increase in depth of remineralisation. Similar results are obtained in study done by Girish Babu et al., Indrapriyadharshini et al. and Morales–Vadillo et al. comparing fluoride varnish and CPP-ACP.,, Remineralization mechanism of CPP–ACP involves stabilization of calcium and phosphate as nanoclusters of ions in solution. These ions diffuses into enamel and depress demineralization and promote remineralization., This could have possibly resulted in better remineralisation compared to control group.
Control group showed least depth of remineralisation compared to all groups. Remineralisation seen in this group is minimal and could be due to presence of calcium ions in artificial saliva. Saliva has the ability to exert an important remineralization effect over time.
This is the first study comparing herbal product, N. sativa with conventional remineralising agents. Novel remineralising agent–N. sativa proved to be effective than conventional remineralising agents.
Each of the remineralising agent used in this study promoted enamel remineralization, although the mechanisms of action of the agents were different. Remineralization in vitro may be quite different when compared with dynamic and complex biological system of the oral cavity.
| Conclusion|| |
Novel agent–N. sativa oil used in this study showed better remineralising potential than conventional remineralisation agents. However, further clinical studies with greater sample size and randomized controlled trials have to be performed to invite greater research about use of this novel agent in dentistry.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Machiulskiene V, Campus G, Carvalho JC, Dige I, Ekstrand KR, Jablonski-Momeni A, et al
. Terminology of dental caries and dental caries management: Consensus report of a workshop organized by orca and cariology research group of IADR. Caries Res 2020;54:7-14.
Oliveira GM, Ritter AV, Heymann HO, Swift E Jr., Donovan T, Brock G, et al
. Remineralization effect of CPP-ACP and fluoride for white spot lesions in vitro
. J Dent 2014;42:1592-602.
Soares R, De Ataide IN, Fernandes M, Lambor R. Assessment of enamel remineralisation after treatment with four different remineralising agents: A scanning electron microscopy (SEM) study. J Clin Diagn Res 2017;11:ZC136-41.
Khan SA, Khan AM, Karim S, Kamal MA, Damanhouri GA, Mirza Z. Panacea seed “Nigella”: A review focusing on regenerative effects for gastric ailments. Saudi J Biol Sci 2016;23:542-53.
Hoobi NM, Rzoqi MG. Dissolution of inorganic phosphorous ion from teeth treated with different concentrations of aqueous extract of Nigella sativa
(Black Seed) in comparison with sodium fluoride: An in vitro
study. Int J Sci Res 2017;6:1962-5.
Savas S, KavrÌk F, KucukyÌlmaz E. Evaluation of the remineralization capacity of CPP-ACP containing fluoride varnish by different quantitative methods. J Appl Oral Sci 2016;24:198-203.
Yu OY, Mei ML, Zhao IS, Li QL, Lo EC, Chu CH. Remineralisation of enamel with silver diamine fluoride and sodium fluoride. Dent Mater 2018;34:e344-52.
Burwell AK, Thula-Mata T, Gower LB, Habelitz S, Kurylo M, Ho SP, et al
. Functional remineralization of dentin lesions using polymer-induced liquid-precursor process. PLoS One 2012;7:e38852.
Vashisht R, Kumar A, Indira R, Srinivasan MR, Ramachandran S. Remineralization of early enamel lesions using casein phosphopeptide amorphous calcium phosphate: An ex-vivo
study. Contemp Clin Dent 2010;1:210-3.
] [Full text]
Premnath P, John J, Manchery N, Subbiah GK, Nagappan N, Subramani P. Effectiveness of theobromine on enamel remineralization: A comparative in-vitro
study. Cureus 2019;11:e5686.
Chaudhury T, Ananthakrishna S, Kumari RV, Kour S, Syam A. Comparative study of remineralization potential of three different remineralizing agents on demineralized enamel using light fluorescence and confocal fluorescence microscope: An in vitro
study. Int J Prev Clin Dent Res 2018;5:45-9. [Full text]
Ahmad A, Husain A, Mujeeb M, Khan SA, Najmi AK, Siddique NA, et al
. A review on therapeutic potential of Nigella sativa
: A miracle herb. Asian Pac J Trop Biomed 2013;3:337-52.
Al-Attass SA, Zahran FM, Turkistany SA. Nigella sativa
and its active constituent thymoquinone in oral health. Saudi Med J 2016;37:235-44.
Omar OM, Khattab NM, Khater DS. Nigella sativa
oil as a pulp medicament for pulpotomized teeth: A histopathological evaluation. J Clin Pediatr Dent 2012;36:335-41.
Abd-Awn B, Al-Dhaher Z, Al-Dafaai R. The effect of black seed oil extracts on mutans streptococci in comparison to chlorhexidine gluconate (in vitro
). J Baghdad Coll Dent 2012;24:126-31.
Jain N, Kumar NK, Naik SB, Merwade S, Brigit B, Rashmi K. A comparative study on antimicrobial efficacy of a novel irrigant, Nigella sativa
oil against enterococcus faecalis (ATCC 29212): A preliminary study. Saudi J Oral Dent Res 2019;4:584-8.
Shashikala K, Sheela NV. Qualitative analysis of remineralized carious lesions subjected to fluoride supplement through confocal laser scanning microscope. Open J Stomatol 2011;1:55-60.
Hicks J, Garcia-Godoy F, Flaitz C. Biological factors in dental caries: Role of saliva and dental plaque in the dynamic process of demineralization and remineralization (part 1). J Clin Pediatr Dent 2003;28:47-52.
Chokshi K, Chokshi A, Konde S, Shetty SR, Chandra KN, Jana S, et al
. An in vitro
comparative evaluation of three remineralizing agents using confocal microscopy. J Clin Diagn Res 2016;10:ZC39-42.
Girish Babu KL, Subramaniam P, Teleti S. Remineralization potential of varnish containing casein phosphopeptides-amorphous calcium phosphate with fluoride and varnish containing only fluoride: A comparative study. Saudi J Oral Sci 2018;5:35-40. [Full text]
Indrapriyadharshini K, Madan Kumar PD, Sharma K, Iyer K. Remineralizing potential of CPP-ACP in white spot lesions – A systematic review. Indian J Dent Res 2018;29:487-96.
] [Full text]
MoralesVadillo R, GuevaraCanales JO, GarcÍaRivera HP, BazanAsencios RH, RobelloMalatto JM, Cava-Vergiu CE. In vitro
comparison of the remineralizing effect of casein phosphopeptideamorphous calcium phosphate and fluoride varnish on early carious lesions. J Int Oral Health 2019;11:45-9.
Srinivasan N, Kavitha M, Loganathan SC. Comparison of the remineralization potential of CPP-ACP and CPP-ACP with 900 ppm fluoride on eroded human enamel: An in situ
study. Arch Oral Biol 2010;55:541-4.
Salman NR, ElTekeya M, Bakry N, Omar SS, El Tantawi M. Comparison of remineralization by fluoride varnishes with and without casein phosphopeptide amorphous calcium phosphate in primary teeth. Acta Odontol Scand 2019;77:9-14.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]