Remineralisation Potential Of Grape Seed, Ginger Honey - An In vitro Study

Keywords

Caries; Demineralization; Ginger Honey; Grape Seed; Microhardness; Remineralisation.

Introduction

Minimal invasive dentistry is a conservative approach to preserve the remaining tooth structure as much as possible, in that path of conservative approach comes the remineralisation of caries [1, 2]. Caries starts as a demineralization and then which is followed by organic matter and invasion of microorganisms [3-7]. Despite of world wide improvements in in oral health, dental caries is the major problem in most of the countries which affects about 60-90% of school children. The current concept in cariogenesis is that due to continuous alteration in PH the demineralizationremineralisation cycle takes place.

Remineralisation is possible only when we are able to control the pH in favour of remineralisation , that is the resting PH should be stable [8, 9]. This induce the partialy dissolved crystals to grow by accretion of calcium and phosphate ions from the remineralising agent. It is usually the natural repair process that counteracts the cariogenic progress [9, 10].

Various agents are implemented in past few decades to remineralize, minimize or delay the progression of caries like fluoride, Casein Phosphopeptide - Amorphous Calcium Phosphate(CPPACP), tri calcium phosphate [11-14] etc. Due to various other factors influencing on caries, permanent solution for caries is still not achievable. Caries is one of the main factor which is progressing to other pulp and periapical diseases. If we could able to stop or remineralize the caries , the progression of caries diseases could be kept in control, there by preventing the tooth from undergoing deep caries leading to pulpal involvement and increasing the oral hygine etc.

The fluoride reacts with oral fluids and combine with calcium and phosphate ions to form fluorapatite crystals, the anticaries effect of fluoride depends on the use of concentration and frequency of application.

In the year 1998, Casein Phosphopeptide -Amorphous Calcium Phosphate(CPP-ACP) was introduced as a remineralizing agent. It is a milk protein combined with ACP , which promotes remineralisation by inhibiting the colonization of cariogenic bacteria [14-17].

Natural products are used in folk medicines for thousands of years, in recent years most of the researchers are concentrating on natural products for treating the diseases. As of researches done so far in all fields of treating the oral diseases, it proves to work out with the natural agents with addition of few initiators to enhance the effectiveness. Only a very few studies are reported with natural products on the demineralization and remineralization of dental hard tissues.

Naturally occurring plant metabolite Proanthocyanidins(PA) are widely available in vegetables, fruits, nuts,seeds , flowers and barks. Studies have shown that PA increased the collagen synthesis and accelerates the conversion of soluble collagen to insoluble collagen during development. Thus remineralization with grape seed extract brings changes in the organic matrix by the presence of newly induced collagen cross links [18-21]. It has biocompatibility and many beneficial biological properties, including anticariogenic, antibacterial, anti-inflammatory and immunostimulatory effects [22-25].

Zingiberofficinale (or ginger, Ginger rhizome) is most traditionally used herbs in India, this natural food source possesses antimicrobial and antifungal activities. Few studies have also reported its effect on streptococcus mutans [26-29]. One more traditionally used with herbal medicines in India is honey. It has a PH of about 3.9, which have bacteriostatic effect on pathogens. Studies have shown its efficacy on oral pathogenic bacteria [30, 31].

We have numerous highly cited publications on well designed clinical trials and lab studies [32-45]. The aim of this study is to evaluate the efficiency of remineralisation among chemical and natural remineralising agents.

Materials And Methods

Specimen collection

Extracted 60 single rooted teeth were collected .The teeth were cleaned, and the contaminants were removed with an ultrasonic scaler. The root fragments from below the cementoenamel junction were sealed with an acid-resistant nail varnish except for 3 mm × 4 mm window and 100µm lesion was done using microtome. They were divided into three groups 20 per group, group 1 - CPP-ACP, group 2- ginger powder(1%) with honey(1ml), group 3- grape seed extract (6.5%)

Grape seed extract preparation

Grape seeds were powdered, then 100 gm were extracted with ethanol to water ratio of 70:30 v/v by maceration method. Then the extracts were filtered.

The total phenol content of grape seed extract was estimated by Folin-Ciocalteu method and processed by adding one ml of GSE solution in aceton/water (6/4) in test tube and mixed with0.2ml of Folin-Ciocalteu reagent for 3min. Then 1 ml of 2% (w/v) sodium carbonate was added to it. This mixture is agitated with a vortex mixer and kept in dark for 30mins then they were centrifuged at 1200g for 5 min. the absorbance of this GSE extracts and the prepared blank were measured at 750nm using spectrophotometer. The total phenolic content of GSE was 70 g of gallic acid equivalents (GAE)/100 g.

Demineralization with lactic acid

The baseline microhardness value was measured. Three groups (n=20) in each group was treated with a demineralizing solution containing undersaturated 0.1-M lactic acid buffer solution (pH 4.75) for 30 min and then placed in artificial saliva at 37°C between treatments. These procedures were conducted for 7 days.

Treatment protocol

The specimens in Group1 (n=20) were painted with CPP-ACP paste. The specimens in Group 2 (n=20) were applied with Ginger powder + honey. The specimens in Group 3 (n=20) were applied with grape seed extract for 5 minutes twice a day for 7 days. These specimens were then placed in artificial saliva throughout the duration of study to stimulate oral condition. The solution was kept at room temperature. After that each tooth was rinsed with de-ionized water for two minutes, then teeth were restored in glass container filled with (20 ml) of de-ionized water and stored at a temperature of 37°C in the incubator for the next day. The procedure was repeated daily for seven days.

Surface microhardness measurement

Surface microhardness of specimens was measured at baseline using Vickers microhardness tester. The microhardness tester is equipped with a 400X magnification lens that enables a clear view of the indentation created by the microprobe. The created indentation has 2 diameters of X and Y that are precisely measured by the device and reported as d1 and d2. The microhardness is calculated using the mean d1 and d2 and the formula below:

D = The microhardness number is inversely correlated with the d value.

The greater the d1, d2 and consequently the total d value, the higher the penetration of indenter into the surface and the lower the microhardness number of the object and vice versa.

In order to confirm the accuracy of the obtained microhardness value, each specimen was tested 5 times and the mean of all values was reported as the microhardness number. During the experiment, the solutions had a temperature equal to the room temperature (approximately 30°C). After immersion for 5min, the specimens were rinsed with saline solution, dried and their surface microhardness was measured again. The specimen image was taken after microhardness measurement using fluorescence spectroscopy (Figure 1).

Statistical Analysis

Therefore, surface microhardness was measured and the data were analyzed using one-way ANOVA with Dunnett’s T3 test. p<0.05 was considered statistically significant.

Results And Discussion

The results of this in vitro study are shown in table 1.

---

×

Table 1:

Close

Table 1. This table shows the Micro hardness testing results of treatment groups.

---

The results were expressed as Mean ± SD (n=20). The *p<0.001 statistically significant as compared with after demineralization groups.#p<0.05 statistically significant as compared with CPPACP treatment.ap<0.001 statistically significant as compared with CPP-ACP treatment.

This study results show that all group are statistically significant with their demineralizing groups. Group 2 is statistically significant when compared to CPP-ACP (p<0.05). Grape seed extract (6.5%) is statistically significant to CPP-ACP (p<0.001).

---

×

Figure 1:

Close

Figure 1. Microhardness test fluorescent image, a diamond pyramidal indentation for Vickers hardness measurement was observed on the enamel surface.

---

In this study , grape seed extract of 6.5% has more remineralisation potential than the commonly used CPP-ACP, followed by ginger powder and honey combination.

Mahkameh Mirkarimi et al demonstrated that grape seed has positive effects in remineralization in an in vitro study conducted in human primary teeth. It is a non invasive theraphy of carious lesions [19].

Cheng-fang Tang et al., demonstrated that transient GSE biomodification may promote remineralization (mostly HA crystals) on the superficial surface of acid- etched demineralized dentine. 15% of GSE preconditioner , without PH adjustment gives the best results, that may be ascribed to its higher polyphenolic content [46].

Shiny Benjamin et al., demonstrated the mechanism of grape seed extract with the PH cycling and confocal scanning microscopy. Grape seed extract is better than fluoride and 0.5% CaGP [47]. Grape seed extract contains 98% ofproanthocyanidin (PA). This proanthocyanidin is seen widely in fruits, nuts, seeds, flowers, bark and vegetables. This is an natural plant metabolite , antioxidant and a free radical scavenger. The large molecule structure of PA, bioflavinoid consists of flavin. This PA is found to produce acid production against streptococcus mutans and also increase collagen synthesis by preventing conversion of soluble collagen to insoluble collagen [46, 48].

Ginger rhizome (ginger) has been used as a food spices and medicinal plants for many centuries as tradition in India. Among all the natural food sources, ginger is found to have the more antimicrobial activities. It is a natural material showing no toxicity and are considered ‘generally recognized as safe’ (GRAS) by the US Food and Drug Administration (FDA). The pungent oil component in it consist a series of polyphenolic ketones with many pharmacological activities [49].

Honey has an antibacterial affect on S.mutans, L. acidophillus, A.viscosus, P.aeroginosa, V.alcaligens and S.aureus. The PH of honey is 3.9, hence it is acidic and can inhibit the growth of pathogens because most thrive PH 4.0-4.5. Anyway dilution of honey with saliva will increase the PH and reduce its effect. This increases the enzyme activity and the action of enzyme glucose oxidase and production of hydrogen peroxide, which is an oxidizing agent, will increase [50, 51]. Further studies have to be conducted with incorporation of few agents in the natural remineralizing agents and to provide for clinical usage in future.

Conclusion

Within the limitations of the study , the remineralization of these study groups is greater in grape seed extract , CPP-ACP and ginger honey combination. These study shows that remineralization is achievable even with natural products like grape seed extract and ginger honey.

References

1. Heydecke G, Butz F, Hussein A, Strub JR. Fracture strength after dynamic loading of endodontically treated teeth restored with different post-andcore systems. J Prosthet Dent. 2002 Apr;87(4):438-45.Pubmed PMID: 12011861.
2. Morgano SM, Rodrigues AHC, Sabrosa CE . Restoration of endodontically treated teeth. Dent Clin North Am.2004; 48: vi, 397–416.
3. Siddique R, Nivedhitha MS. Effectiveness of rotary and reciprocating systems on microbial reduction: A systematic review. J Conserv Dent. 2019 Mar-Apr;22(2):114-122.Pubmed PMID: 31142978.
4. Govindaraju L, Neelakantan P, Gutmann JL. Effect of root canal irrigating solutions on the compressive strength of tricalcium silicate cements. Clin Oral Investig. 2017 Mar;21(2):567-571.Pubmed PMID: 27469101.
5. Reeh ES, Messer HH, Douglas WH. Reduction in tooth stiffness as a result of endodontic and restorative procedures. J Endod. 1989 Nov;15(11):512-6. Pubmed PMID: 2639947.
6. Khandelwal A, Palanivelu A. Correlation between dental caries and salivary albumin in adult population in Chennai: An in vivo study. Braz. Dent. Sci. 2019 Apr 30;22(2):228-33.
7. Poorni S, Srinivasan MR, Nivedhitha MS. Probiotic Streptococcus strains in caries prevention: A systematic review. J Conserv Dent. 2019 Mar;22(2):123- 128.
8. Ramarao S, Sathyanarayanan U. CRA Grid - A preliminary development and calibration of a paper-based objectivization of caries risk assessment in undergraduate dental education. J Conserv Dent. 2019 Mar-Apr;22(2):185-190. Pubmed PMID: 31142991.
9. Morgano SM. Restoration of pulpless teeth: application of traditional principles in present and future contexts. J Prosthet Dent. 1996 Apr;75(4):375-80. Pubmed PMID: 8642522.
10. Safavi KE, Dowden WE, Langeland K. Influence of delayed coronal permanent restoration on endodontic prognosis. Endod Dent Traumatol. 1987 Aug;3(4):187-91.Pubmed PMID: 3481569.
11. Alsamadani KH, Abdaziz el-SM, Gad el-S. Influence of different restorative techniques on the strength of endodontically treated weakened roots. Int J Dent. 2012;2012:343712.Pubmed PMID: 22666251.
12. Bergman B, Lundquist P, Sjögren U, Sundquist G. Restorative and endodontic results after treatment with cast posts and cores. J Prosthet Dent. 1989 Jan;61(1):10-5.Pubmed PMID: 2644413.
13. Naumann M, Blankenstein F, Kiessling S, Dietrich T. Risk factors for failure of glass fiber-reinforced composite post restorations: a prospective observational clinical study. Eur J Oral Sci. 2005 Dec;113(6):519-24.Pubmed PMID: 16324143.
14. Azeem RA, Sureshbabu NM. Clinical performance of direct versus indirect composite restorations in posterior teeth: A systematic review. J Conserv Dent. 2018 Jan-Feb;21(1):2-9.Pubmed PMID: 29628639.
15. enarthanan S, Subbarao C. Comparative evaluation of the efficacy of diclofenac sodium administered using different delivery routes in the management of endodontic pain: A randomized controlled clinical trial. J Conserv Dent. 2018 May-Jun;21(3):297-301.Pubmed PMID: 29899633.
16. Nandakumar M, Nasim I. Comparative evaluation of grape seed and cranberry extracts in preventing enamel erosion: An optical emission spectrometric analysis. J Conserv Dent. 2018 Sep-Oct;21(5):516-520.Pubmed PMID: 30294113.
17. Malli Sureshbabu N, Selvarasu K, V JK, Nandakumar M, Selvam D. Concentrated Growth Factors as an Ingenious Biomaterial in Regeneration of Bony Defects after Periapical Surgery: A Report of Two Cases. Case Rep Dent. 2019 Jan 22;2019:7046203.Pubmed PMID: 30805222.
18. Teja KV, Ramesh S, Priya V. Regulation of matrix metalloproteinase-3 gene expression in inflammation: A molecular study. J. Conserv. Dent. 2018 Nov;21(6):592-596.
19. Rajakeerthi R, Nivedhitha MS. Natural Product as the Storage medium for an avulsed tooth–A Systematic Review. Cumhur. Dent. J. 2019;22(2):249- 56.
20. Rajendran R, Kunjusankaran RN, Sandhya R, Anilkumar A, Santhosh R, Patil SR. Comparative evaluation of remineralizing potential of a paste containing bioactive glass and a topical cream containing casein phosphopeptide- amorphous calcium phosphate: An in vitro study. Pesqui. Bras. Odontopediatria Clín. Integr. 2019;19:1-10.
21. Vârlan C, Dimitriu B, Vârlan V, Bodnar D, Suciu I. Current opinions concerning the restoration of endodontically treated teeth: basic principles. J Med Life. 2009 Apr-Jun;2(2):165-72.Pubmed PMID: 20108535.
22. Nagasiri R, Chitmongkolsuk S. Long-term survival of endodontically treated molars without crown coverage: a retrospective cohort study. J Prosthet Dent. 2005 Feb;93(2):164-70.Pubmed PMID: 15674228.
23. Colman HL. Restoration of endodontically treated teeth.Dent Clin North Am. 1979; 23: 647–662.
24. Schwartz RS, Robbins JW. Post placement and restoration of endodontically treated teeth: a literature review. J. Endod. 2004 May 1;30(5):289-301.
25. Ross RS, Nicholls JI, Harrington GW. A comparison of strains generated during placement of five endodontic posts. J Endod. 1991 Sep;17(9):450-6. Pubmed PMID: 1811040.
26. Assif D, Gorfil C. Biomechanical considerations in restoring endodontically treated teeth. J Prosthet Dent. 1994 Jun;71(6):565-7.Pubmed PMID: 8040817.
27. Tikku AP, Chandra A, Bharti R. Are full cast crowns mandatory after endodontic treatment in posterior teeth? J Conserv Dent. 2010 Oct;13(4):246-8. Pubmed PMID: 21217953.
28. Whitworth JM, Walls AW, Wassell RW. Crowns and extra-coronal restorations: endodontic considerations: the pulp, the root-treated tooth and the crown. Br Dent J. 2002 Mar 23;192(6):315-20, 323-7.Pubmed PMID: 15552070.
29. Eckerbom M, Magnusson T. Restoring endodontically treated teeth: a survey of current opinions among board-certified prosthodontists and general dental practitioners in Sweden. Int J Prosthodont. 2001 May-Jun;14(3):245-9. Pubmed PMID: 11484572.
30. Scurria MS, Shugars DA, Hayden WJ, Felton DA. GENERAL DENTISTS'PATTERNS OF RESTORING: ENDODONTICALLY TREATED TEETH. The J Am Dent Assoc. 1995 Jun 1;126(6):775-9.
31. Auswin MK, Ramesh S. Knowledge, attitude, and practice survey on the use of dental operating microscope in endodontics: A cross-sectional survey. Pharm. Educ. Res. 2017;7(3):320-322.
32. Manohar MP, Sharma S. A survey of the knowledge, attitude, and awareness about the principal choice of intracanal medicaments among the general dental practitioners and nonendodontic specialists. Indian J Dent Res. 2018 Nov-Dec;29(6):716-720.Pubmed PMID: 30588997.
33. Janani K, Sandhya R. A survey on skills for cone beam computed tomography interpretation among endodontists for endodontic treatment procedure. Indian J Dent Res. 2019 Nov-Dec;30(6):834-838.Pubmed PMID: 31939356.
34. Naumann M, Kiessling S, Seemann R. Treatment concepts for restoration of endodontically treated teeth: A nationwide survey of dentists in Germany. J Prosthet Dent. 2006 Nov;96(5):332-8.Pubmed PMID: 17098496.
35. Hussey DL, Killough SA. A survey of general dental practitioners' approach to the restoration of root-filled teeth. Int Endod J. 1995 Mar;28(2):91-4. Pubmed PMID: 7665207.
36. Morgano SM, Hashem AF, Fotoohi K, Rose L. A nationwide survey of contemporary philosophies and techniques of restoring endodontically treated teeth. J Prosthet Dent. 1994 Sep;72(3):259-67.Pubmed PMID: 7965899.
37. Stankiewicz N, Wilson PR. The ferrule effect: a literature review. Int. Endod. J. 2002 Jul;35(7):575-81.
38. Sorensen JA, Martinoff JT. Intracoronal reinforcement and coronal coverage: a study of endodontically treated teeth. J Prosthet Dent. 1984 Jun;51(6):780- 4.Pubmed PMID: 6376780.
39. Martin N, Jedynakiewicz N. A radiographic survey of endodontic post lengths [abstract 418]. J Dent Res. 1989;68(Special Issue):919.
40. Deutsch AS, Musikant BL, Cavallari J, Silverstein L, Lepley J, Ohlen K, et al. Root fracture during insertion of prefabricated posts related to root size. J Prosthet Dent. 1985 Jun;53(6):786-9.Pubmed PMID: 3891978.
41. Ruemping DR, Lund MR, Schnell RJ. Retention of dowels subjected to tensile and torsional forces. J Prosthet Dent. 1979 Feb;41(2):159-62.Pubmed PMID: 366115.
42. Mattison GD, von Fraunhofer JA. Angulation loading effects on cast-gold endodontic posts: a photoelastic stress analysis. J Prosthet Dent. 1983 May;49(5):636-8.Pubmed PMID: 6343592.
43. Bitter K, Hambarayan A, Neumann K, Blunck U, Sterzenbach G. Various irrigation protocols for final rinse to improve bond strengths of fiber posts inside the root canal. Eur J Oral Sci. 2013 Aug;121(4):349-54.Pubmed PMID: 23841787.
44. Siddique R, Sureshbabu NM, Somasundaram J, Jacob B, Selvam D. Qualitative and quantitative analysis of precipitate formation following interaction of chlorhexidine with sodium hypochlorite, neem, and tulsi. J Conserv Dent. 2019 Jan-Feb;22(1):40-47.Pubmed PMID: 30820081.
45. Siddique R, Nivedhitha MS, Jacob B. Quantitative analysis for detection of toxic elements in various irrigants, their combination (precipitate), and para-chloroaniline: An inductively coupled plasma mass spectrometry study. J Conserv Dent. 2019 Jul-Aug;22(4):344-350.Pubmed PMID: 31802817.
46. Teja KV, Ramesh S. Shape optimal and clean more. Saudi Endod. J. 2019 Sep 1;9(3):235.
47. Seow LL, Toh CG, Wilson NH. A survey of current practices among general dental practitioners in Manchester in 2002. Prim Dent Care. 2003 Jul;10(3):87-92.Pubmed PMID: 12929337.
48. Saupe WA, Gluskin AH, Radke RA Jr. A comparative study of fracture resistance between morphologic dowel and cores and a resin-reinforced dowel system in the intraradicular restoration of structurally compromised roots. Quintessence Int. 1996 Jul;27(7):483-91.Pubmed PMID: 8941826.
49. Sen D, Poyrazoglu E, Tuncelli B. The retentive effects of pre-fabricated posts by luting cements. J Oral Rehabil. 2004 Jun;31(6):585-9.Pubmed PMID: 15189317.
50. Mendoza DB, Eakle WS, Kahl EA, Ho R. Root reinforcement with a resinbonded preformed post. J Prosthet Dent. 1997 Jul 1;78(1):10-4.
51. Cagidiaco MC, Goracci C, Garcia-Godoy F, Ferrari M. Clinical studies of fiber posts: a literature review. Int J Prosthodont. 2008 Jul 1;21(4):328-336.
52. Ikram OH, Patel S, Sauro S, Mannocci F. Micro-computed tomography of tooth tissue volume changes following endodontic procedures and post space preparation. Int Endod J. 2009 Dec;42(12):1071-6.Pubmed PMID: 19912377.
53. Smidt A, Venezia E. Techniques for immediate core buildup of endodontically treated teeth. Quintessence Int. 2003 Apr;34(4):258-68.Pubmed PMID: 12731611.
54. Faria AC, Rodrigues RC, de Almeida Antunes RP, de Mattos Mda G, Ribeiro RF. Endodontically treated teeth: characteristics and considerations to restore them. J Prosthodont Res. 2011 Apr;55(2):69-74.Pubmed PMID: 20709618.
55. Stavropoulou AF, Koidis PT. A systematic review of single crowns on endodontically treated teeth. J Dent. 2007 Oct 1;35(10):761-7.
56. Fedorowicz Z, Carter B, de Souza RF, Chaves CD, Nasser M, Sequeira-Byron P. Single crowns versus conventional fillings for the restoration of root filled teeth. Cochrane Database Syst Rev. 2012;(5):CD009109.
57. Fokkinga WA, Le Bell AM, Kreulen CM, Lassila LV, Vallittu PK, Creugers NH. Ex vivo fracture resistance of direct resin composite complete crowns with and without posts on maxillary premolars. Int Endod J. 2005 Apr;38(4):230-7.Pubmed PMID: 15810973.
58. Ferrari M, Vichi A, Grandini S, Goracci C. Efficacy of a self-curing adhesiveresin cement system on luting glass-fiber posts into root canals: an SEM investigation. Int J Prosthodont. 2001 Nov-Dec;14(6):543-9.Pubmed PMID: 12066701.