Elsevier

Archives of Oral Biology

Volume 57, Issue 8, August 2012, Pages 1093-1099
Archives of Oral Biology

Effect of pH on Galla chinensis extract's stability and anti-caries properties in vitro

https://doi.org/10.1016/j.archoralbio.2012.04.007Get rights and content

Abstract

Objectives

Considering that Galla chinensis extract (GCE) solution has a low pH, which might dissolve dental enamel, we investigated the effects of elevation of pH on GCE stability, and on its anti-caries properties.

Designs

Stability of GCE solutions, either in H2O (pH less than 4.0) or when buffered at pH 5.5, 7.0 and 10.0, was assessed from UV–VIS spectra. Inhibition of enamel demineralization was determined in a pH-cycling set up, comprising treatments with either GCE solutions or negative control buffers and acid and neutral buffer immersions. Demineralization was assessed by calcium in the acetate buffers. To determine antimicrobial properties, polymicrobial biofilms were formed after saliva inoculation on glass surfaces which were treated after 48 h. Treatment output parameters were lactic acid formation and viability, the latter by colony forming unit (CFU) counts.

Results

At pH 7.0 and higher GCE solutions changed colour and absorption spectra in UV–VIS, indicative of chemical changes. Regarding enamel demineralization, significant inhibitions (P < 0.05) were found for all GCE treatments when compared with corresponding controls. In polymicrobial biofilms, GCE reduced the acid production, compared with the negative controls (P < 0.05). However, this difference was only significant at the lower pH values.

Conclusions

GCE solutions were unstable under neutral and alkaline conditions. pH did not significantly influence the inhibiting effect of GCE on enamel demineralization. However, GCE was not effective on polymicrobial biofilms at alkaline pH (8.5). To avoid enamel damage due to acidic treatment, GCE solutions should be used at about pH 5.5.

Introduction

Galla chinensis, a traditional natural Chinese medicine, is rich in hydrolysable tannins and possesses a wide range of biological activities.1, 2 In recent years, our research group has obtained Galla Chinensis extract (GCE), which contains substantial quantities of polyphenols (e.g. gallotannin, gallic acid).3 Both in in vitro and in vivo experiments GCE was shown to inhibit growth and metabolism of caries pathogens, and also to enhance remineralization and inhibit demineralization of enamel.3, 4, 5, 6, 7, 8, 9, 10, 11 The latter was considered the primary mode of action of GCE as anti-caries agent. Other types of polyphenols, such as those occurring in cocoa, coffee, tea, oat hull, hop bract, mainly contain condensed tannins. Their anti-caries mode of action primarily concerns the physiology of caries pathogens.2, 12 Although GCE is a promising agent for effective caries prevention, in aqueous solution it is generally too acidic; for example, the pH of a 4000 ppm GCE solution is lower than pH 4.0. Normally, the pH of the oral fluids is approximately neutral (pH of individual unstimulated saliva varies between 5.8 and 8.013). A pH below about 5.5 could give rise to demineralization of enamel and a lower pH cause dental erosion.14, 15 Therefore, it is suggested to increase the pH of GCE solutions to make them feasible as anti-caries agent.6

However, increasing the pH of GCE solutions created various problems: (1) When the pH was increased to 7.0, the solution initially turned light-brown but then rapidly became dark-brown, similar to green tea catechins in Krebs–Ringer bicarbonate buffer.16 Such dark-brown solutions would stain enamel, and are therefore not suitable for clinical use. (2) At higher pH some of the polyphenol molecules in GCE could break down and lose their efficacy.16, 17, 18, 19, 20 Based on these observations, the following questions should be addressed: could GCE be applied at neutral pH? Will the colour change, indicative of instability, have a negative impact on GCE's anti-caries properties?

The inhibition of both the demineralization of enamel and the growth and fermentation of bacteria in biofilms on teeth are effective strategies in the prevention of dental caries. In vitro, the former can be tested in pH-cycling experiments with the rate of demineralization generally evaluated by assessment of mineral loss.15 The latter can be evaluated in saliva induced polymicrobial biofilms model.4, 21

The aim of the current study was to analyze the stability of GCE solutions at different pH and to subsequently measure the effects of GCE at different pH on enamel demineralization and on polymicrobial biofilms in vitro. The rationale was to determine the optimal (effective and safe) pH-condition for prospective anti-caries applications of GCE.

Section snippets

G. chinensis sample

GCE was extracted as described in previous studies.3, 4, 5, 6, 7, 8, 9, 10, 11 In brief, G. chinensis was dried at 60 °C for 3 days, powdered, double extracted with distilled water, dissolved in ethanol and subsequently GCE was recovered by evaporation of the ethanol.

Determination of total phenol content

Total phenols (TP) was determined by Folin-Ciocalteu (FC) assay and expressed as gallic acid equivalents (GAE) based on a gallic acid calibration curve. FC assay was performed in accordance with the method of Singleton and Rossi,22

The total phenol and tannin content

The total phenol content of GCE was 763.9 ± 0.2 mg GAE/g, and the tannin content was 164.8 ± 0.4 mg GAE/g (means and standard deviations).

Spectrophotometric analysis

Effect of pH and time (0-time, 1 h, 6 h, 24 h, and 48 h) on GCE (10 μg/mL) in buffer solution was shown by UV–VIS absorption spectra.

The UV–VIS spectra of GCE in H2O (Fig. 1 B), at the intrinsic pH, had absorption maxima at 212 nm and 261 nm, which were similar to the spectra of gallic acid in H2O (λmax1 = 213 nm, λmax2 = 263 nm) (Fig. 1A) and did not show a change in time. At

Discussion

Combining the data from this study we conclude that GCE solutions stored at pH 7.0 and higher, were unstable, although such GCE solutions were still efficacious in inhibiting both enamel demineralization and biofilm virulence. In contrast, GCE at pH 5.5 showed only marginal instability, while retaining a significant efficacy. Increasing the intrinsic pH of the GCE solutions to pH 5.5 would therefore overcome detrimental side effects of GCE solutions at their intrinsic pH (below pH 4.0), which

Funding

National Natural Science Foundation of China (Grant Nos. 30572409 and 30430800), Youth Foundation of Sichuan Province in China (07ZQ026-020)

Competing interests

None declared.

Ethical approval

Not required.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant Nos. 30572409 and 30430800) and Youth Foundation of Sichuan Province in China (07ZQ026-020). The authors are grateful to the West China School of Pharmacy, Sichuan University for providing technical assistance in G. chinensis extraction and fractionation.

References (33)

  • O. Djakpo et al.

    Rhus chinensis and Galla chinensis-folklore to modern evidence: review

    Phytotherapy Research

    (2010)
  • J.G. Jeon et al.

    Natural products in caries research: current (Limited) knowledge, challenges and future perspective

    Caries Research

    (2011)
  • Q. Xie et al.

    Anticaries effect of compounds extracted from Galla chinensis in a multispecies biofilm model

    Oral Microbiology and Immunology

    (2008)
  • L. Cheng et al.

    Effect of Galla chinensis on growth and metabolism of microcosm biofilms

    Caries Research

    (2011)
  • L. Cheng et al.

    Effect of Galla chinensis on the in vitro remineralization of Advanced enamel lesions

    International Journal of Oral Science

    (2010)
  • R.K. Wang et al.

    Inhibitive effect of extracts of Galla Chinesis on caries development in rats

    Journal of Sichuan University (Medical science edition)

    (2008)

    • Cited by (24)

    • Opportunities and challenges of plant extracts in food industry

      2021, Plant Extracts: Applications in the Food Industry

    • Polyphenol uses in biomaterials engineering

      2018, Biomaterials
      Citation Excerpt :

      Quinones are electrophilic molecules, which can react with proteins nucleophilic groups, such as, -SH and -NH2 and result in insoluble metamorphic organic networks [72]. Therefore, GSE PAs are more stable at an acidic pH and exhibited a better effect on dentin remineralization under acidic pH [72,82]. On the other hand, a very low acidic pH (below 4) as that which was used by Tang et al. [72], can damage the enamel, and there should be a balance to preserve the bioactivity of the PA and, at the same time prevent acidic damage to the enamel.

    • Technological aspects and stability of polyphenols

      2018, Polyphenols: Properties, Recovery, and Applications

    • Effect of arginine on the growth and biofilm formation of oral bacteria

      2017, Archives of Oral Biology
      Citation Excerpt :

      The saliva was diluted 2-fold with sterile 60% glycerol and was stored at −80 °C (Exterkate, Crielaard, & ten Cate, 2010; Huang, Exterkate et al., 2012) Polymicrobial dental biofilms were formed in a high-throughput active attachment biofilm model as previously described in detail (Exterkate et al., 2010; Huang, Cheng et al., 2012; Huang, Exterkate et al., 2012). In brief, glass disks were attached to a custom-made lid in such a way that each disk fitted into one well of a polystyrene 24-well flat-bottomed microtiter plate.

    • Characterization of dentin matrix biomodified by Galla chinensis extract

      2013, Journal of Endodontics
      Citation Excerpt :

      Therefore, GCE, instead of its subfractions, was used in this study. The pH of GCE solution was set at 5.5, because GCE was unstable at neutral and alkaline conditions (22). Moreover, this pH value was in the vicinity of the isoelectric point of collagen.

    • The role of grape seed extract in the remineralization of demineralized dentine: Micromorphological and physical analyses

      2013, Archives of Oral Biology
      Citation Excerpt :

      The absorbances of GSE preconditioner dilutions at pH 7.4 and pH 10.0 decreased at the two typical polyphenol bands in the UV–vis spectra, suggesting reduced polyphenolic contents in neutral and alkaline conditions. Owing to the increase in spectra (300–398 nm, similar to that of benzoquinones) under both neutral and alkaline conditions,33,34 we assume that the reductions in polyphenols were possibly due to the formation of quinones. Considering the critical role of polyphenols in the various biological activities of GSE, it is advisable to maintain the PA preconditioner at the original pH for higher polyphenolic contents.

    View all citing articles on Scopus
    View full text
    Copyright © 2012 Elsevier Ltd. All rights reserved.