Effect of supplementation of soft drinks with green tea extract on their erosive potential against dentine

Authors


Dr Marília Afonso Rabelo Buzalaf
Al Octávio Pinheiro Brisolla, 9-75
Bauru-SP 17012-901
Brazil
Email: mbuzalaf@fob.usp.br

Abstract

Background:  Matrix metalloproteinase (MMP) inhibitors reduce dentine erosion. This in vitro study evaluated the effect of the supplementation of soft drinks with green tea extract, a natural inhibitor of MMPs, on their erosive potential against dentine.

Methods:  For each drink tested (Coca-Cola™, Kuat™ guarana, Sprite™ and light Coca-Cola™), 40 dentine specimens were divided into two subgroups differing with respect to supplementation with green tea extract at 1.2% (OM24®, 100%Camellia sinensis leaf extract, containing 30 ± 3% of catechin; Omnimedica, Switzerland) or not (control). Specimens were subjected to four pH cycles, alternating de- and remineralization in one day. For each cycle, samples were immersed in pure or supplemented drink (10 minutes, 30 mL per block) and in artificial saliva (60 minutes, 30 mL per block) at 37 °C, under agitation. Dentine alterations were determined by profilometry (μm). Data were analysed by two-way ANOVA and Bonferroni’s test (p < 0.05).

Results:  A significant difference was observed among the drinks tested with Sprite™ leading to the highest surface loss and light Coca-Cola™ to the lowest. Supplementation with green tea extract reduced the surface loss by 15% to 40% but the difference was significant for Coca-Cola™ only.

Conclusions:  Supplementation of soft drinks with green tea extract might be a viable alternative to reduce their erosive potential against dentine.

Abbreviations and acronyms:
CPP-ACP

casein phosphopeptide-amorphous calcium phosphate

EGCG

epigallocatechin-3-gallate

MMP

matrix metalloproteinase

Introduction

A reduction in tooth loss due to caries has been observed in many countries.1 However, the maintenance of teeth in the oral cavity for a longer time, as well as an ageing population favours the occurrence of other dental lesions. Among these, dental erosion has received great attention over the last few years. Dental erosion can be defined as the loss of dental substance due to a chemical process caused by acids or chelating agents, without involvement of micro-organisms, triggered by intrinsic factors (anorexia, bulimia, gastro-oesophageal problems, xerostomia) or extrinsic factors (food, beverages, acid products or acid contamination in the environment).2

Dietary acids play a key role among the aetiological factors for dental erosion.3 Soft drinks and other acidic drinks are particularly important due to recent changes in dietary habits which have seen higher levels of consumption, especially among adolescents.4 The erosive potential of different acidic drinks has been evaluated and depends not only on their pH, but is also influenced by their mineral content, titrable acidity and calcium-chelating properties.5 In addition, the adhesiveness of the drink, which determines its time of contact with the tooth structure, plays a role in erosive potential.6 The temperature, frequency of consumption and duration of contact with the tooth structure is also an important factor on erosivity.7

Many studies have attempted to supplement acidic beverages with compounds to reduce erosivity. These studies involved the addition of ions such as calcium, phosphate, fluoride or iron, alone or in combination,8,9 proteins such as ovalbumin,10 casein11 or its derivative casein phosphopeptide-amorphous calcium phosphate (CPP-ACP),12,13 as well as food-approved polymers such as polyphosphate14 to acids or acidic drinks.

It has recently been reported that matrix metalloproteinase (MMP) inhibitors when added to solutions or gels protect against dentine erosion.15–18 The proposed mechanism of action is that these agents would reduce the degradation of the demineralized organic layer at the time of the erosive challenge. This layer has been attributed to hamper ionic diffusion and act as a buffer to the acids.19 Green tea polyphenols, especially epigallocatechin-3-gallate (EGCG) were found to have distinct inhibitory activity against MMP-2 and MMP-9.20 Taking this into consideration, the present investigation tested the hypothesis that the addition of green tea to marketed soft drinks would reduce their erosive potential against dentine. To accomplish this, an in vitro pH-cycling erosive model was employed using bovine dentine as the substrate and profilometry as the response variable. A secondary aim was to compare the erosive potential of the different soft drinks against dentine.

Materials and Methods

Dentine specimens preparation

Two hundred and fifty dentine specimens (4 × 4 × 3 mm) were prepared initially from extracted sound bovine incisors. One sample was cut from each crown using an ISOMET Low Speed Saw cutting machine (Buehler Ltd., Lake Bluff, IL, USA) and two diamond discs (Extec Corp., Enfield, CT, USA), which were separated by a 4 mm diameter spacer. The enamel surface was removed using a diamond bur (KG Sorensen 4054) and then the dentine surface was ground flat with water-cooled carborundum discs (600 and 1200 grades of Al2O3 papers; Buehler, Lake Bluff, IL, USA) and polished with felt paper wet by diamond suspension (1 μm; Buehler, Lake Buff, IL, USA).

After preparation, the surface hardness of the samples was determined by performing five indentations (Knoop diamond, 25 g, 5 seconds, HMV-2000; Shimadzu Corporation, Tokyo, Japan) for selection and randomized distribution. Dentine samples with a mean microhardness of 38 KHN (±10%) were randomly distributed into four groups (n = 40 per group), which differed according to the type of soft drink tested. The beverages evaluated were: Coca-Cola™, Light Coca-Cola™, Sprite Zero™ and Kuat™ guarana which were tested plain (control) or after supplementation with 1.2% green tea extract (OM24™, Camellia sinensis leaf extract 100%, containing 30% EGCG and 0.39 ppm F, Omnimedica, Zürich, Switzerland). This concentration was chosen based on a previous study by our group15 where it was observed that the use of a rinse containing OM24™ at 0.61% (400 μM of EGCG) was able to reduce bovine dentine erosive loss by approximately 50%in situ/ex vivo. Considering that the rinses with the green tea extract solution in our previous study were conducted for 1 minute, we decided to double the concentration of the active principle in the beverages in order to compensate the reduced time of contact that these drinks would have with the dentine surface. Additionally, 20 other specimens were included and treated with a commercially available soft drink similar to Kuat™ guarana but manufactured with green tea (Kuat ECO™, Coca-Cola Company, Spal, Porto Real, RJ, Brazil).

After allocation to the groups, two layers of nail varnish were applied on two-thirds of the surface of the dentine specimens in order to maintain reference surfaces for lesion depth determination.

Erosive pH-cycling

The blocks were submitted to four pH cycles under agitation in only one day.21 Each cycle consisted of demineralization by immersion in pure or modified beverage for 10 minutes (30 mL per sample) at 25 °C, followed by a rinse in deionized water and remineralization in artificial saliva (30 mL) for 59 minutes at 37 °C. The composition of the artificial saliva was: calcium 1.5 mmol L−1, phosphate 0.9 mmol L−1, KCl 150 mmol L−1 in TRIS buffer 0.1 mol L−1, pH 7.0, containing 0.03 μg/mL fluoride.22

Surface loss determination

After the pH cycles, the specimens were kept moist in a solution of 0.9% NaCl containing 0.02% sodium azide at 4 °C until the surface loss analysis, in order to avoid shrinkage of the dentine organic material. The nail varnish over the reference surfaces was carefully removed using a scalpel blade, taking care to avoid touching the dentine surface. The dentine loss was determined in relation to the reference surfaces (interfaces control-erosion-control) by using a contact profilometer (MarSurf GD 25, Mahr, Göttingen, Germany) and appropriate software (MarSurf XT 20, Mahr, Göttingen, Germany). The diamond stylus moved from one reference to the exposed area and to the other reference area (2.5 mm). The differences in height between reference and exposed areas were quantified in microns. Five profile measurements were performed in the centre of each specimen and averaged.

Statistical analysis

GraphPad Instat version 4.0 for Windows was used (GraphPad Software Inc, San Diego, CA, USA). Data were analysed by two-way ANOVA and Bonferroni’s test for individual comparison. The factors evaluated were type of beverage and supplementation.

ANOVA after logarithmic transformation was used to compare the green tea extract-containing guarana available in the market (Kuat ECO™) with the guarana (Kuat™) pure or supplemented in the laboratory. The significance level was set at 5% in all cases.

Results

Table 1 shows mean (±SD) surface loss values found for all beverages evaluated, supplemented with green tea extract or not. Two-way ANOVA revealed a significant difference among the types of beverages (F = 58.68, p < 0.0001) and between the regular and supplemented ones (F = 17.08, p < 0.0001). The interaction between these factors was not significant (F = 0.50, p = 0.683).

Table 1.   Mean surface loss (μm, ±SD) of dentine blocks submitted to erosive challenges with different soft drinks supplemented or not with 1.2% green tea extract
Supplementation with green tea extractTypes of beverages
Coca-Cola™Kuat™ guaranaSprite Zero™Light Coca-Cola™
  1. Distinct lower case superscripts in the same line indicate significant differences among the types of beverages. Distinct upper case superscripts in the same column indicate significant differences between the regular or supplemented beverages. Two-way ANOVA and Bonferroni’s test (p < 0.05). n = 20.

No3.6 ± 1.1bA2.3 ± 0.8cA5.3 ± 1.7aA2.0 ± 1.1cA
Yes2.5 ± 0.7bB1.9 ± 0.5bcA4.5 ± 1.6aA1.2 ± 0.5cA

The highest surface loss was observed for Sprite™, both in the regular (5.3 ± 1.7 μm) and supplemented (4.5 ± 1.6 μm) forms, which differed significantly from all other beverages. Coca-Cola™ presented the second highest degree of surface loss and was significantly different from Coca-Cola Light™ (regardless of supplementation) and Kuat™ guarana (only for the not supplemented form). The lowest degrees of surface loss were found for Coca-Cola Light™ (2.0 ± 1.1 and 1.2 ± 0.5 μm for supplemented and not supplemented forms, respectively).

The supplementation with EGCG reduced surface loss by 15% to 40% for all types of beverages, but the difference was significant with respect to the not supplemented form for Coca-Cola™ only.

The commercially available soft drink (Kuat ECO™) that contains green tea among its components presented a significantly higher mean surface loss (3.0 ± 1.0 μm) when compared with the supplemented (1.9 ± 0.5 μm) and not supplemented (2.3 ± 0.8 μm) forms of Kuat™.

Table 2 shows the pH of the evaluated beverages, supplemented with green tea extract or not. For all the types of beverages, supplementation with green tea extract (1.2%) reduced the native pH. This reduction was more pronounced for Coca-Cola™. The pH of Kuat ECO™ was 3.2.

Table 2.   pH of the different types of soft drinks supplemented or not with 1.2% green tea extract
Supplementation with green tea extractTypes of beverages
Coca-Cola™Kuat™ guaranaSprite Zero™Coca light™
No2.63.23.03.0
Yes2.03.12.92.9

Three blind subjects tasted all beverages before and after supplementation and could not perceive taste alteration.

Discussion

Several in vitro studies have evaluated the erosive potential of different beverages, using distinct parameters such as pH, buffer capacity, type of acid (pKa), adhesion of the product to the tooth surface, concentrations of calcium, phosphate and fluoride and dental loss as response variables.5 In the present study, among the drinks tested, light Coca-Cola™ caused the lowest surface loss. This is in line with recent in situ data showing that light Coca-Cola™ is less erosive than its regular version.23 However, the mechanism by which it occurs is not completely understood. It has been suggested that the lower erosive potential of light Coca-Cola™ could be due to the presence of the amino acid phenylalanine derived from the hydrolysis of aspartame in the presence of saliva. It was speculated that this amino acid could act as a buffer system, increasing the neutralization and buffering the acids from the drink.23 Another possible reason for this distinctive erosive potential is the difference in pH between regular Coca-Cola™ (2.6) and its light version (3.0) (Table 2). The erosive effects of different acids are pH-dependent and vary greatly between pH 2 and 3, which means that even a very small decline in pH can result in increased loss of enamel24 and dentine.25 Recent unpublished observations suggest that the second hypothesis is more likely. The regular cola was supplemented with aspartame and the light one had its pH lowered to 2.6. It was observed that the impact of lowering the pH of light Coca-Cola™ was greater to decrease the loss of enamel than the protection caused by the admixture of aspartame to the regular cola. Another remarkable difference among the soft drinks tested is the lower erosive potential of Kuat™ guarana when compared with Coca-Cola™ and Sprite Zero™. Kuat™ is made of guarana, a fruit from the Amazon region. The lower erosive potential of soft drinks originated from guarana had already been shown using enamel substrates26 and the present results indicate that the same might be valid for dentine.

Since the central research question posed in the present study was if supplementation of soft drinks with green tea extract would reduce their erosive potential against dentine, no control group with, for example, deionized water instead of soft drinks was included. According to our design, each soft drink not supplemented with green tea extract acted as the control for its supplemented counterpart. This design was successful and allowed the acceptance of our main hypothesis, i.e. the supplementation of soft drinks with green tea extract reduces their erosive potential against dentine. Green tea extract was chosen because it contains 30% of EGCG, a polyphenol that inhibits MMPs.20,27 Inhibition of MMPs in turn has recently been shown to reduce dentine loss.15–18 Additionally, green tea is a natural product, has no side effects as the concentration used and its consumption has been associated with health promotion.28 Also, the flavour of the soft drinks was not altered after supplementation, which is an important characteristic for a potential supplement.

The reduction of the erosive potential by supplementation with green tea extract was more pronounced for the cola drinks. It is noteworthy that supplementation caused a decrease in the pH of the soft drinks which was slight for most of them but more pronounced for Coca-Cola™, the only beverage for which a significant reduction in surface loss could be observed after supplementation. Thus, if attempts were made to avoid the pH reduction due to supplementation, it could be expected that the protective effect of green tea extract would be potentiated, which deserves further investigation. It would also be interesting to test if the addition of higher amounts of green tea extract that are still not associated with potential side effects or taste alteration could ameliorate protection against dentine erosion.

Since a green tea-containing guarana is commercially available in the Brazilian market (Kuat ECO™), the erosive potential of this product against dentine was also evaluated in comparison with the plain guarana (Kuat™) or guarana supplemented with green tea extract. However, the dentine surface loss caused by Kuat ECO™ was significantly higher when compared with Kuat™ guarana, regardless of supplementation. Difference in pH is certainly not responsible for these findings, since Kuat ECO™ had the same pH as regular Kuat™ which is higher than the pH observed for the supplemented Kuat™ guarana. The concentration of EGCG in Kuat ECO™ was not evaluated. It is possible that this concentration was too low to have any impact on MMP inhibition and/or other components of the beverage are responsible for increasing its erosive potential.

In conclusion, our findings indicate that supplementation of soft drinks with green tea extract might be a viable alternative to reduce their erosive potential against dentine, since green tea is a natural product associated with health promotion and the concentrations used cause no side effects or taste alteration of the beverages.

Acknowledgements

The authors thank FAPESP for the concession of a scholarship to the first author.

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