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Effect of compounds of Galla chinensis on remineralisation of initial enamel carious lesions in vitro
journal of dentistry 35 (2007) 383–387
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Effect of compounds of Galla chinensis on remineralisation of initial enamel carious lesions in vitro J.P. Chu a, J.Y. Li b, Y.Q. Hao a, X.D. Zhou b,* a b
Key Laboratory of Oral Biomedical Engineering of Ministry of Education, Sichuan University, Chengdu, China West China College of Stomatology, Sichuan University, Chengdu, China
article info
abstract
Article history:
Objective: To evaluate the effect of compounds of Galla chinensis on the remineralisation of
Received 11 August 2006
initial enamel carious lesions in vitro.
Received in revised form
Methods: Sixty bovine enamel blocks with early lesions were prepared and randomly
5 November 2006
divided into six treatment groups. The lesions were subjected to a pH-cycling regime for
Accepted 11 November 2006
12 days. Each daily cycle included 4 1 min applications with one of six treatments; 1000 ppm F aq. (as NaF, positive control); deionized water (negative control); or 4000 ppm aqueous solutions of four G. chinensis extracts (GCEs); GCE, GCE-B, GCE-B1, or GCE-B2.
Keywords:
Surface enamel microhardness was measured on the enamel blocks before and after
Galla chinensis
demineralisation, and after pH-cycling, and percentage surface microhardness recovery
Chemical compounds
(%SMHR) was calculated. The enamel specimens were then sectioned (thickness ca. 80 mm)
Early enamel lesions
and examined by polarized light microscopy.
Remineralisation
Results: All samples rehardened significantly compared to baseline. Fluoride had a significantly greater effect than all other treatments. In the GCEs groups, %SMHR was significantly greater than DDW for the GCE, GCE-B and GCE-B1 groups. There was no significant difference between the GCE-B2 group and DDW. Polarized light microscopy showed that the thickness of the surface layer increased obviously in all specimens including NaF group, GCE group, GCE-B group and GCE-B1 group. Negative birefringent band appeared in the lesions body and the depth of the lesions was obviously reduced. Conclusion: The present study has demonstrated the potential of three GCEs (GCE, GCE-B and GCE-B1) to effect net rehardening of artificial carious lesions under dynamic pH-cyclic conditions. # 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental caries in enamel is unique amongst diseases in that enamel is acellular and avascular. In contrast to other tissues, enamel cannot heal itself by a cellular repair mechanism and must rely on repair by a physicochemical process involving inorganic constituents from saliva or solutions.1 Therefore the concept that remineralisation, especially with fluoride present, can repair damage caused by demineralisation (before
cavitation occurs) is of crucial importance for the management of preventive care. For enamel the prime mode of action of fluoride in caries prevention is the inhibition of demineralisation and the enhancement of remineralisation.2 Over the last 25 years, the decline in dental caries experienced in most industrialized countries can be attributed largely to the widespread use of fluoride.3 However, there is still a need to seek alternative, effective anti-caries agents, for although fluoride has had a profound effect on the level of caries
* Corresponding author. Tel.: +86 28 85501439; fax: +86 28 85582167. E-mail addresses: [email protected], [email protected] (X.D. Zhou). 0300-5712/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2006.11.007
384
journal of dentistry 35 (2007) 383–387
prevalence, it is far from a complete cure. Further, fluoride has the potential to cause fluorosis through overexposure,4 and although fluoride presents no problems to the normal individual, when used properly, among certain groups there has been the suggestion that fluoride exposure should be limited. In recent years, studies on the properties of Chinese herbal medicines have included their evaluation as anti-caries agents.5–9 Galla chinensis, a natural non-toxic traditional Chinese medicine, formed when the Chinese sumac aphid Baker (Melaphis chinensis Bell) parasitizes the leaves of Rhus chinensis Mill,10 is a potentially interesting agent. It is widely distributed in China, mainly produced in Sichuan province. It is usually harvested in the autumn seasons and manufactured into a traditional Chinese medicine by cooking and drying after removing the larvae. G. chinensis has the potential to accelerate blood coagulation, act as an anti-bacterial/antiviral agent, and act as a detoxifying agent by combining with various ions of metals, alkaloids or glycosides to form insoluble compounds. It has long been considered in China to have natural medicinal properties, with numerous beneficial effects. During previous studies it was proposed that G. chinensis had anti-bacterial properties to inhibit the grow and acid production of certain cariogenic bacteria including Streptococcus mutans 3a3 (serotype c, clinical isolate) and Lactobacillus rhamnosus AC 413,11,12 and in particular it had an ability to inhibit enamel demineralisation in vitro.13 In addition, crude aqueous extract of G. chinensis has been proven effective in inhibiting the development and enhancing the remineralition of dentinal caries.14 The purpose of this study was to examine the effect of chemical compounds of G. chinensis on in vitro remineralisation of artificial early enamel caries under a pHcycling regimen and to determine which of its constituent chemical species confer a potential anti-caries benefit.
2.
Materials and methods
2.1.
G. chinensis sample
G. chinensis (1 kg) produced in the Sichuan province of the People’s Republic of China was dried in an oven at 60 8C for 3 day, finely powdered, added to 600 ml of distilled water. The mixture was stirred for 10 h at 65 8C and then filtered. The extract was re-extracted with distilled water under the same conditions. Then the extract was dissolved in 500 ml of ethanol (100%). After filtration and evaporation of the ethanol, the remaining extract was lyophilized to give a powder (G. chinensis extract, GCE) (yield, 160 g). The GCE preparation (160 g) was further fractionated by adsorption chromatography by using a Diaion HP-21 column (8 cm 20 cm; Mitsubishi Chemical Industries, Tokyo, Japan). The column was eluted with deionized water (10 l), then with 30% ethanol (5 l), and finally with 100% acetone (5 l), and fraction, GCE-B (2.7 g), were obtained. A portion of GCE-B was further purified by successive column chromatography with a Diaion HP-20 column (8 cm 20 cm) and a Sephadex LH-20 column (3 cm 120 cm; cm; Pharmacia-LKB Biotechnology, Uppsala, Sweden). The
column was eluted by acetone–water (2:8, 3:7, 4:6, v/v) and two active compounds were obtained, characterized as gallic acid (1) and methyl gallate (2) by spectroscopic methods including MS and NMR (Fig. 1).
2.2.
Specimen preparation
In this study bovine incisors were employed. Immediately after extraction the teeth were rinsed under tap water and stored at 4 8C in water containing 0.05% thymol until required. The crowns were separated from the roots using a diamondcoated band saw under continuous water cooling (Struers Minitom; Struers, Copenhagen, Denmark). From each tooth crown two halves were prepared in a mesiodistal direction. One rectangular slab (approximately 4 mm 4 mm 3 mm) was prepared from each half. Subsequently the enamel surfaces were ground flat and hand-polished using aqueous slurries of progressively finer grades of silicon carbide, up to 4000 grit (Struers, Copenhagen, Denmark), thereby removing about 150 mm of the outer enamel layer. In this way flat surfaces without surface contaminations were obtained. (Lesions produced in this type of subsurface enamel are much more reproducible than lesions created in enamel at the anatomical surface.15)
2.3.
Baseline microhardness
Mineral changes in superficial enamel layers are directly related to microhardness alterations, i.e. remineralisation of enamel carious lesions is associated with an increase of enamel surface microhardness.16,17 Therefore, after polishing, the baseline surface microhardness (SMH) of 120 blocks was measured using a Knoop diamond indenter under a 50 g load. Five indentations were averaged on each surface of the individual specimens for SMH determination. After measuring the baseline surface microhardness, 35 blocks were discarded because of their excessively high or low Knoop hardness numbers (KHNs), leaving a total of 85 blocks with KHNs of between 298.30 and 313.80 for preparation of early artificial carious lesions.
2.4.
Preparation of early artificial caries lesions
All tooth surfaces of the blocks, except the polished enamel surface, were protected with acrylic resin. Early artificial caries lesions were produced in the enamel, basically according to ten Cate and Duijsters.18 A solution containing 2.2 mM Ca(NO3)2, 2.2 mM phosphate as KH2PO4, 0.1 ppm NaF
Fig. 1 – Structure of two active principles from GCE-B, gallic acid (1) and methyl gallate (2).
385
journal of dentistry 35 (2007) 383–387
and 50 mM acetic acid, pH adjusted to 4.5 using 2 M KOH, was used for demineralizing the enamel. During demineralisation, the solution was stirred at about 100 rpm and specimens were demineralized for 72 h, 5 samples per 100 ml solution. After artificial caries preparation, 60 blocks with baseline KHN values (SMH1) between 166.80 and 176.50 were selected for pH-cycling. Then half of each specimen was covered with an acid-resistant varnish to maintain the baseline lesion.
2.7.
2.5.
2.8.
pH-cycling
The specimens were then randomly divided (10 specimens/ group) into six treatment groups: 1000 ppm NaF aq. (positive control), distilled and deionized water (DDW, negative control), and four 4000 ppm aqueous solutions of GCE, GCEB, GCE-B1, or GCE-B2. They were then pH-cycled using the regime reported by White.19 Artificial saliva was used as a remineralizing solution (pH 7.0) according to ten Cate and Duijsters,15 which included 1.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 1 mM NaN3, 20 mmol/l HEPES. The cycling daily de/remineralisation regime consisted of a 2 h/day acid challenge in the demineralization solution (2.2 mM Ca(NO3)2, 2.2 mM phosphate as KH2PO4 and 50 mM acetic acid, pH 4.5) at 11:00 am–1:00 pm, with four 1-min test solution treatment periods per day at 08:00 am, 09:00 am, 3:00 pm and 4:00 pm. During the remaining time (approx. 22 h/day) the specimens were placed in remineralizing solution. After each treatment, the blocks were washed with deionized water. The regimen was repeated for 12 days and temperature maintained at 37 8C.
2.6.
Surface microhardness analysis
After pH-cycling, SMH was measured on all enamel blocks (SMH2). Five indentations spaced by 100 mm, from the baseline and from those made after the artificial caries development, were made. A microhardness tester (Duramin-1/-2; Struers, Copenhagen, Denmark) with a Knoop diamond indenter was used with a 50 g load for 15 s. The mean values of all five measurements at the three different times (baseline, after demineralisation and after pH-cycling) were then compared and the percentage surface microhardness recovery (SMHR) was calculated as 20: %SMHR ¼
100ðSMH2 SMH1 Þ SMH1 SMH
Polarized microscopy examination
After SMH analysis, thin planoparallel sections about 80 mm thick were prepared from the central part of each specimen. Representative specimens were mounted on a glass microscope slide, then examined under a polarized light microscope at 10 magnification (ECLIPSE ME600L, Nikon, Tokyo, Japan) after imbibition in deionized water. Digital images were taken with Nikon ACT-1 for L-1 software (Nikon, Japan).
Statistical analysis
Data were computerized and analysed using SPSS 11.0 software. Student’s paired t-test was used to compare Knoop surface microhardness before and after the treatments. Surface microhardness recovery (%SMHR) among treatments were analyzed by ANOVA, followed by the Newman–Keuls test. The significance limit was set at 5%.
3.
Results
The analysis of enamel blocks with regard to SMH is shown in Table 1. Comparing surface microhardness before and after remineralisation, it was found that lesions in all treatment groups had re-hardened significantly (Table 1). Further, all treatments except the deionized water group and the GCE-B2 group were statistically different from each other. In the GCE groups, the highest percentage SMHR was found for the treatment with GCE and the lowest with GCE-B2 group (Fig. 2). Representative lesions developed in specimens of treatment groups before and after the pH-cycling are shown in Fig. 3. When the sections were examined using polarized light microscope after imbibition in water (RI = 1.33), all increasingly demineralized areas (with enhanced tissue porosity) appeared positively birefringent. It was apparent that the lesions had intact surface layers (seen as an area of negative birefringence), above positively birefringent lesion bodies. After remineralisation, the thickness and density of the surface layer increased obviously in all specimens in the NaF group, GCE group, GCE-B group and GCE-B1 groups. Negative birefringent bands similar to that of the surface layer appeared in the lesions body and the depth of the lesions was obviously reduced. However, for the GCE-B2 group and deionized water group, little change had taken place except that the density of the surface layer increased slightly.
Table 1 – Surface microhardness analysis of enamel blocks according to the treatments Treatments NaF GCE GCE-B GCE-B1 GCE-B2 DDW *
Baseline SMH 306.0 5.0 306.9 4.7 306.9 5.2 308.1 4.8 308.5 5.4 306.3 6.2
Before pH-cycling 171.9 2.8 171.6 3.1 171.2 2.8 171.8 2.7 170.9 3.3 172.3 2.9
After pH-cycling *
256.3 3.7 229.9 5.1* 211.1 3.2* 191.9 3.9* 180.3 2.4* 180.0 3.0*
%SMHR 63.0 3.6 a 43.1 3.9 b 29.4 2.7 c 14.8 2.0 d 6.8 2.0 e 5.7 1.6 e
Show difference between before and after pH-cycling for surface microhardness (SMH) for each treatment ( p < 0.05). Treatments whose means are followed by distinct letters (a–e) differ statistically ( p < 0.05).
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journal of dentistry 35 (2007) 383–387
Fig. 2 – The percentage surface microhardness recovery (%SMHR) of all treatment groups. Treatments with different letters differ statistically ( p < 0.05).
4.
Discussion
In recent years, G. chinensis has been widely discussed as a kind of anticaries Chinese herbal medicine. In our previous work, we have shown that the aqueous extract of G. chinensis was able to influence the demineralisation and remineralisation of dental hard tissues.13,14 However, no research has analyzed the effect of chemical compounds of G. chinensis on remineralisation of early enamel caries lesion. In the present study, an in vitro pHcycling model was used to evaluate the ability of compounds of G. chinensis to remineralize early carious lesions. The results confirm the ability of the artificial saliva to aid in remineralizing enamel because even specimens from the DDW group rehardened significantly.
Our preliminary chemical analyses of compounds extracted from G. chinensis have revealed that GCE contained significant quantities of monomeric and polymeric polyphenols (e.g., gallotannin, gallic acid) and some other components (carbohydrates, proteins and other constituents). GCE-B purified from GCE contained only polymeric polyphenols, and GCE-B1 further purified from GCE-B has been characterized as gallic acid.12 The results of the present study showed GCE was the most efficient, recovering 43.1% of the enamel hardness (%SMHR) that had been reduced by the artificially induced caries, with GCE-B next, then GCE-B1. Hence, it was suggested that both GCE and GCE-B contained some additional components possessing effectiveness of the enhancement of remineralisation. Polarized light evaluations of enamel sections have been useful in describing the early caries lesion and alterations in structure upon further demineralisation or remineralisation.21 In the current study, artificial caries lesions were examined using polarized light microscopy. Before pH-cycling, it could be easily observed that the lesion has an intact surface layer (seen as an area of negative birefringence) on top of the positively birefringent body of the lesion. This confirmed that the artificially induced lesions used in this study were similar to natural caries lesions, based on the most important pathological feature of natural lesion that mineral loss occur underneath the enamel surface. After the pH-cycling, a hypomineralized band (negative birefringent band similar to the surface layer) was observed in the lesions body for NaF group, GCE group, GCE-B group and GCE-B1 group, and the depth of the caries lesions was obviously reduced. As for GCEB2 group and deionized water group, little change had taken place after remineralisation except that the density of the
Fig. 3 – Polarized light microscopic pictures of enamel sections from the groups before and after the pH-cycling: (A) NaF group, (B) GCE group, (C) GCE-B group, (D) GCE-B1 group, (E) GCE-B2 group, (F) DDW group (scale bar = 100 mm). The white arrows on the top picture indicate the approximate boundaries of early artificial caries lesions and 12-day remineralisation regime in all pictures.
journal of dentistry 35 (2007) 383–387
surface layer increased slightly. This also supported the result of the above surface microhardness analysis. In the present study fluoride was employed as a positive control and was clearly effective. Fluoride’s anti-caries efficacy is well-proven. In preventing and arresting caries, fluoride acts via two principal mechanisms: inhibiting demineralisation when fluoride is present at the crystal surfaces during an acid challenge and enhancing remineralisation and thereby forming a low solubility veneer similar to the acid-resistant mineral fluorapatite or FAP, on the remineralized crystals.22 So far, G. chinensis’s mechanism of action is still unknown. However, it has been established that the essential compounds of G. chinensis are polyphenol compounds (e.g., tannin and gallic acid), whilst interactions can occur between tannic acid and calcium ions, forming indissolvable complex.23 From the combined data from this study we proposed that although its mechanism of action is unknown, G. chinensis is believed to combine with calcium in aqueous solution. Furthermore, G. chinensis actually prevents decalcification by inhibiting the translocation of dissolved Ca2+ and PO43 ions from lesions.13 Thus, chemical compounds of G. chinensis, which are GCE, GCE-B and GCE-B1, might act as a Ca2+ ion carrier supplying the middle layers of caries lesion with Ca2+ ions from the remineralizing solution, thereby enhancing remineralisation by providing the Ca2+ required for crystal repair. In conclusion, the present has demonstrated the potential of three GCEs; GCE, GCE-B and GCE-B1; to effect net rehardening of artificial carious lesions under dynamic pH-cyclic conditions. Although not as effective as fluoride, G. chinensis might still be a promising adjunct or alternative to fluoride.
Acknowledgments The authors are grateful to Prof. F. Wang and Dr. J. Wang (The Institute of Naturally Occurring Drugs of Sichuan University, Chengdu, China) for kindly providing chemical compounds extracted from Galla chinensis. This investigation was supported by the National Natural Science Foundation of China (Grant No. 30430800).
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references 19. 1. Zero DT. Dental caries process. Dental Clinics of North America 1999;43:635–64. 2. ten Cate JM, Featherstone JDB. Mechanistic aspects of the interactions between fluoride and dental enamel. Critical Reviews in Oral Biology and Medicine 1991;2:283–96. 3. Brambilla E. Fluoride—is it capable of fighting old and new dental diseases? An overview of existing fluoride compounds and their clinical applications. Caries Research 2001;35:6–9. 4. Liang L. The biochemistry and physiology of metallic fluoride:action, mechanism, and implications. Critical Reviews in Oral Biology and Medicine 2003;14:100–14. 5. Ooshima T, Osaka Y, Sasaki H, Osawa K, Yasuda H, Matsumura M, et al. Caries inhibitory activity of cacao bean
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husk extract in in-vitro and animal experiments. Archives of Oral Biology 2000;45:639–45. Leitao DP, Filho AA, Polizello AC, Bastos JK, Spadaro AC. Comparative evaluation of in-vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutans. Biological & Pharmaceutical Bulletin 2004;27:1834–9. Yatsuda R, Rosalen PL, Cury JA, Murata RM, Rehder VL, Melo LV, et al. Effects of Mikania genus plants on growth and cell adherence of mutans streptococci. Journal of Ethnopharmacology 2005;97:183–9. Nostro A, Cannatelli MA, Crisafi G, Musolino AD, Procopio F, Alonzo V. Modifications of hydrophobicity, in vitro adherence and cellular aggregation of Streptococcus mutans by Helichrysum italicum extract. Letters in Applied Microbiology 2004;38:423–7. Duarte S, Koo H, Bowen WH, Hayacibara MF, Cury JA, Ikegaki M, et al. Effect of a novel type of propolis and its chemical chemical fractions on glucosyltransferases and on growth and adherence of mutans streptococci. Biological & Pharmaceutical Bulletin 2003;6:527–31. Ou M. Chinese–English manual of common-used in traditional Chinese medicine. Guangzhou: Guangdong Science & Technology Press; 1992. p. 1–72. Huang ZW, Zhou XD, Li JY, Liu TJ, Li H, Zhu B. The effects of traditional Chinese medicines on the adherence of Streptococcus mutans to salivary acquired pellicle in vitro. Journal of Sichuan University (Medical Sciences Edition) 2003;34:135–7. Xie Q, Li JY, Zuo YL, Zhou XD. The effect of Galla chinensis on the growth of cariogenic bacteria in vitro. West China Journal of Stomatology 2005;23:82–4. Liu ZH, Liu TJ, Li JY, Zhou XD, Zhang J. The effect of Galla chinensis on the demineralization of enamel. Journal of Sichuan University (Medical Sciences Edition) 2003;34:507–9. Li YL, Tang RY, Lu X. Effects of Chinese nutgall extractum on degradation of collagen in dentine caries. Chinese Journal of Conservative Dentistry 2002;5:252–4. ten Cate JM, Duijsters PP. Alternating demineralisation and remineralisation of artificial enamel lesions. Caries Research 1982;16:201–10. Feagin F, Koulourides T, Pigman W. The characterization of enamel surface demineralisation, remineralisation, and associated hardness changes in human and bovine material. Archives of Oral Biology 1969;14:1407–17. White DJ, Chen WC, Nancollas GH. Kinetic and physical aspects of enamel remineralisation—a constant composition study. Caries Research 1988;22:11–9. ten Cate JM, Duijsters PP. Influence of fluoride in solution on tooth demineralisation. I. Chemical data. Caries Research 1983;17:193–9. White DJ. Reactivity of fluoride dentifrices with artificial caries I. Effect on early lesions. F uptake hardening and remineralisation. Caries Research 1987;21:126–40. Cury JA, Simo˜es GS, Del Bel Cury AA. Effect of a calcium carbonate-based dentifrice on in situ enamel remineralisation. Caries Research 2005;39:255–7. Wefel JS, Harless JD. Comparison of artificial white spots by microradiography and polarized light microscopy. Journal of Dental Research 1984;63:1271–5. Featherstone JD. The science and practice of caries prevention. The Journal of the American Dental Association 2000;131:887–99. Calixto J, Nicolau M, Rae G. Pharmacological actions of tannic acid. I. Effects on isolated smooth and cardiac muscles and on blood pressure. Planta Medica 1986;32:32–5.
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Effect of compounds of Galla chinensis on remineralisation of initial enamel carious lesions in vitro J.P. Chu a, J.Y. Li b, Y.Q. Hao a, X.D. Zhou b,* a b
Key Laboratory of Oral Biomedical Engineering of Ministry of Education, Sichuan University, Chengdu, China West China College of Stomatology, Sichuan University, Chengdu, China
article info
abstract
Article history:
Objective: To evaluate the effect of compounds of Galla chinensis on the remineralisation of
Received 11 August 2006
initial enamel carious lesions in vitro.
Received in revised form
Methods: Sixty bovine enamel blocks with early lesions were prepared and randomly
5 November 2006
divided into six treatment groups. The lesions were subjected to a pH-cycling regime for
Accepted 11 November 2006
12 days. Each daily cycle included 4 1 min applications with one of six treatments; 1000 ppm F aq. (as NaF, positive control); deionized water (negative control); or 4000 ppm aqueous solutions of four G. chinensis extracts (GCEs); GCE, GCE-B, GCE-B1, or GCE-B2.
Keywords:
Surface enamel microhardness was measured on the enamel blocks before and after
Galla chinensis
demineralisation, and after pH-cycling, and percentage surface microhardness recovery
Chemical compounds
(%SMHR) was calculated. The enamel specimens were then sectioned (thickness ca. 80 mm)
Early enamel lesions
and examined by polarized light microscopy.
Remineralisation
Results: All samples rehardened significantly compared to baseline. Fluoride had a significantly greater effect than all other treatments. In the GCEs groups, %SMHR was significantly greater than DDW for the GCE, GCE-B and GCE-B1 groups. There was no significant difference between the GCE-B2 group and DDW. Polarized light microscopy showed that the thickness of the surface layer increased obviously in all specimens including NaF group, GCE group, GCE-B group and GCE-B1 group. Negative birefringent band appeared in the lesions body and the depth of the lesions was obviously reduced. Conclusion: The present study has demonstrated the potential of three GCEs (GCE, GCE-B and GCE-B1) to effect net rehardening of artificial carious lesions under dynamic pH-cyclic conditions. # 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental caries in enamel is unique amongst diseases in that enamel is acellular and avascular. In contrast to other tissues, enamel cannot heal itself by a cellular repair mechanism and must rely on repair by a physicochemical process involving inorganic constituents from saliva or solutions.1 Therefore the concept that remineralisation, especially with fluoride present, can repair damage caused by demineralisation (before
cavitation occurs) is of crucial importance for the management of preventive care. For enamel the prime mode of action of fluoride in caries prevention is the inhibition of demineralisation and the enhancement of remineralisation.2 Over the last 25 years, the decline in dental caries experienced in most industrialized countries can be attributed largely to the widespread use of fluoride.3 However, there is still a need to seek alternative, effective anti-caries agents, for although fluoride has had a profound effect on the level of caries
* Corresponding author. Tel.: +86 28 85501439; fax: +86 28 85582167. E-mail addresses: [email protected], [email protected] (X.D. Zhou). 0300-5712/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2006.11.007
384
journal of dentistry 35 (2007) 383–387
prevalence, it is far from a complete cure. Further, fluoride has the potential to cause fluorosis through overexposure,4 and although fluoride presents no problems to the normal individual, when used properly, among certain groups there has been the suggestion that fluoride exposure should be limited. In recent years, studies on the properties of Chinese herbal medicines have included their evaluation as anti-caries agents.5–9 Galla chinensis, a natural non-toxic traditional Chinese medicine, formed when the Chinese sumac aphid Baker (Melaphis chinensis Bell) parasitizes the leaves of Rhus chinensis Mill,10 is a potentially interesting agent. It is widely distributed in China, mainly produced in Sichuan province. It is usually harvested in the autumn seasons and manufactured into a traditional Chinese medicine by cooking and drying after removing the larvae. G. chinensis has the potential to accelerate blood coagulation, act as an anti-bacterial/antiviral agent, and act as a detoxifying agent by combining with various ions of metals, alkaloids or glycosides to form insoluble compounds. It has long been considered in China to have natural medicinal properties, with numerous beneficial effects. During previous studies it was proposed that G. chinensis had anti-bacterial properties to inhibit the grow and acid production of certain cariogenic bacteria including Streptococcus mutans 3a3 (serotype c, clinical isolate) and Lactobacillus rhamnosus AC 413,11,12 and in particular it had an ability to inhibit enamel demineralisation in vitro.13 In addition, crude aqueous extract of G. chinensis has been proven effective in inhibiting the development and enhancing the remineralition of dentinal caries.14 The purpose of this study was to examine the effect of chemical compounds of G. chinensis on in vitro remineralisation of artificial early enamel caries under a pHcycling regimen and to determine which of its constituent chemical species confer a potential anti-caries benefit.
2.
Materials and methods
2.1.
G. chinensis sample
G. chinensis (1 kg) produced in the Sichuan province of the People’s Republic of China was dried in an oven at 60 8C for 3 day, finely powdered, added to 600 ml of distilled water. The mixture was stirred for 10 h at 65 8C and then filtered. The extract was re-extracted with distilled water under the same conditions. Then the extract was dissolved in 500 ml of ethanol (100%). After filtration and evaporation of the ethanol, the remaining extract was lyophilized to give a powder (G. chinensis extract, GCE) (yield, 160 g). The GCE preparation (160 g) was further fractionated by adsorption chromatography by using a Diaion HP-21 column (8 cm 20 cm; Mitsubishi Chemical Industries, Tokyo, Japan). The column was eluted with deionized water (10 l), then with 30% ethanol (5 l), and finally with 100% acetone (5 l), and fraction, GCE-B (2.7 g), were obtained. A portion of GCE-B was further purified by successive column chromatography with a Diaion HP-20 column (8 cm 20 cm) and a Sephadex LH-20 column (3 cm 120 cm; cm; Pharmacia-LKB Biotechnology, Uppsala, Sweden). The
column was eluted by acetone–water (2:8, 3:7, 4:6, v/v) and two active compounds were obtained, characterized as gallic acid (1) and methyl gallate (2) by spectroscopic methods including MS and NMR (Fig. 1).
2.2.
Specimen preparation
In this study bovine incisors were employed. Immediately after extraction the teeth were rinsed under tap water and stored at 4 8C in water containing 0.05% thymol until required. The crowns were separated from the roots using a diamondcoated band saw under continuous water cooling (Struers Minitom; Struers, Copenhagen, Denmark). From each tooth crown two halves were prepared in a mesiodistal direction. One rectangular slab (approximately 4 mm 4 mm 3 mm) was prepared from each half. Subsequently the enamel surfaces were ground flat and hand-polished using aqueous slurries of progressively finer grades of silicon carbide, up to 4000 grit (Struers, Copenhagen, Denmark), thereby removing about 150 mm of the outer enamel layer. In this way flat surfaces without surface contaminations were obtained. (Lesions produced in this type of subsurface enamel are much more reproducible than lesions created in enamel at the anatomical surface.15)
2.3.
Baseline microhardness
Mineral changes in superficial enamel layers are directly related to microhardness alterations, i.e. remineralisation of enamel carious lesions is associated with an increase of enamel surface microhardness.16,17 Therefore, after polishing, the baseline surface microhardness (SMH) of 120 blocks was measured using a Knoop diamond indenter under a 50 g load. Five indentations were averaged on each surface of the individual specimens for SMH determination. After measuring the baseline surface microhardness, 35 blocks were discarded because of their excessively high or low Knoop hardness numbers (KHNs), leaving a total of 85 blocks with KHNs of between 298.30 and 313.80 for preparation of early artificial carious lesions.
2.4.
Preparation of early artificial caries lesions
All tooth surfaces of the blocks, except the polished enamel surface, were protected with acrylic resin. Early artificial caries lesions were produced in the enamel, basically according to ten Cate and Duijsters.18 A solution containing 2.2 mM Ca(NO3)2, 2.2 mM phosphate as KH2PO4, 0.1 ppm NaF
Fig. 1 – Structure of two active principles from GCE-B, gallic acid (1) and methyl gallate (2).
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journal of dentistry 35 (2007) 383–387
and 50 mM acetic acid, pH adjusted to 4.5 using 2 M KOH, was used for demineralizing the enamel. During demineralisation, the solution was stirred at about 100 rpm and specimens were demineralized for 72 h, 5 samples per 100 ml solution. After artificial caries preparation, 60 blocks with baseline KHN values (SMH1) between 166.80 and 176.50 were selected for pH-cycling. Then half of each specimen was covered with an acid-resistant varnish to maintain the baseline lesion.
2.7.
2.5.
2.8.
pH-cycling
The specimens were then randomly divided (10 specimens/ group) into six treatment groups: 1000 ppm NaF aq. (positive control), distilled and deionized water (DDW, negative control), and four 4000 ppm aqueous solutions of GCE, GCEB, GCE-B1, or GCE-B2. They were then pH-cycled using the regime reported by White.19 Artificial saliva was used as a remineralizing solution (pH 7.0) according to ten Cate and Duijsters,15 which included 1.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 1 mM NaN3, 20 mmol/l HEPES. The cycling daily de/remineralisation regime consisted of a 2 h/day acid challenge in the demineralization solution (2.2 mM Ca(NO3)2, 2.2 mM phosphate as KH2PO4 and 50 mM acetic acid, pH 4.5) at 11:00 am–1:00 pm, with four 1-min test solution treatment periods per day at 08:00 am, 09:00 am, 3:00 pm and 4:00 pm. During the remaining time (approx. 22 h/day) the specimens were placed in remineralizing solution. After each treatment, the blocks were washed with deionized water. The regimen was repeated for 12 days and temperature maintained at 37 8C.
2.6.
Surface microhardness analysis
After pH-cycling, SMH was measured on all enamel blocks (SMH2). Five indentations spaced by 100 mm, from the baseline and from those made after the artificial caries development, were made. A microhardness tester (Duramin-1/-2; Struers, Copenhagen, Denmark) with a Knoop diamond indenter was used with a 50 g load for 15 s. The mean values of all five measurements at the three different times (baseline, after demineralisation and after pH-cycling) were then compared and the percentage surface microhardness recovery (SMHR) was calculated as 20: %SMHR ¼
100ðSMH2 SMH1 Þ SMH1 SMH
Polarized microscopy examination
After SMH analysis, thin planoparallel sections about 80 mm thick were prepared from the central part of each specimen. Representative specimens were mounted on a glass microscope slide, then examined under a polarized light microscope at 10 magnification (ECLIPSE ME600L, Nikon, Tokyo, Japan) after imbibition in deionized water. Digital images were taken with Nikon ACT-1 for L-1 software (Nikon, Japan).
Statistical analysis
Data were computerized and analysed using SPSS 11.0 software. Student’s paired t-test was used to compare Knoop surface microhardness before and after the treatments. Surface microhardness recovery (%SMHR) among treatments were analyzed by ANOVA, followed by the Newman–Keuls test. The significance limit was set at 5%.
3.
Results
The analysis of enamel blocks with regard to SMH is shown in Table 1. Comparing surface microhardness before and after remineralisation, it was found that lesions in all treatment groups had re-hardened significantly (Table 1). Further, all treatments except the deionized water group and the GCE-B2 group were statistically different from each other. In the GCE groups, the highest percentage SMHR was found for the treatment with GCE and the lowest with GCE-B2 group (Fig. 2). Representative lesions developed in specimens of treatment groups before and after the pH-cycling are shown in Fig. 3. When the sections were examined using polarized light microscope after imbibition in water (RI = 1.33), all increasingly demineralized areas (with enhanced tissue porosity) appeared positively birefringent. It was apparent that the lesions had intact surface layers (seen as an area of negative birefringence), above positively birefringent lesion bodies. After remineralisation, the thickness and density of the surface layer increased obviously in all specimens in the NaF group, GCE group, GCE-B group and GCE-B1 groups. Negative birefringent bands similar to that of the surface layer appeared in the lesions body and the depth of the lesions was obviously reduced. However, for the GCE-B2 group and deionized water group, little change had taken place except that the density of the surface layer increased slightly.
Table 1 – Surface microhardness analysis of enamel blocks according to the treatments Treatments NaF GCE GCE-B GCE-B1 GCE-B2 DDW *
Baseline SMH 306.0 5.0 306.9 4.7 306.9 5.2 308.1 4.8 308.5 5.4 306.3 6.2
Before pH-cycling 171.9 2.8 171.6 3.1 171.2 2.8 171.8 2.7 170.9 3.3 172.3 2.9
After pH-cycling *
256.3 3.7 229.9 5.1* 211.1 3.2* 191.9 3.9* 180.3 2.4* 180.0 3.0*
%SMHR 63.0 3.6 a 43.1 3.9 b 29.4 2.7 c 14.8 2.0 d 6.8 2.0 e 5.7 1.6 e
Show difference between before and after pH-cycling for surface microhardness (SMH) for each treatment ( p < 0.05). Treatments whose means are followed by distinct letters (a–e) differ statistically ( p < 0.05).
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Fig. 2 – The percentage surface microhardness recovery (%SMHR) of all treatment groups. Treatments with different letters differ statistically ( p < 0.05).
4.
Discussion
In recent years, G. chinensis has been widely discussed as a kind of anticaries Chinese herbal medicine. In our previous work, we have shown that the aqueous extract of G. chinensis was able to influence the demineralisation and remineralisation of dental hard tissues.13,14 However, no research has analyzed the effect of chemical compounds of G. chinensis on remineralisation of early enamel caries lesion. In the present study, an in vitro pHcycling model was used to evaluate the ability of compounds of G. chinensis to remineralize early carious lesions. The results confirm the ability of the artificial saliva to aid in remineralizing enamel because even specimens from the DDW group rehardened significantly.
Our preliminary chemical analyses of compounds extracted from G. chinensis have revealed that GCE contained significant quantities of monomeric and polymeric polyphenols (e.g., gallotannin, gallic acid) and some other components (carbohydrates, proteins and other constituents). GCE-B purified from GCE contained only polymeric polyphenols, and GCE-B1 further purified from GCE-B has been characterized as gallic acid.12 The results of the present study showed GCE was the most efficient, recovering 43.1% of the enamel hardness (%SMHR) that had been reduced by the artificially induced caries, with GCE-B next, then GCE-B1. Hence, it was suggested that both GCE and GCE-B contained some additional components possessing effectiveness of the enhancement of remineralisation. Polarized light evaluations of enamel sections have been useful in describing the early caries lesion and alterations in structure upon further demineralisation or remineralisation.21 In the current study, artificial caries lesions were examined using polarized light microscopy. Before pH-cycling, it could be easily observed that the lesion has an intact surface layer (seen as an area of negative birefringence) on top of the positively birefringent body of the lesion. This confirmed that the artificially induced lesions used in this study were similar to natural caries lesions, based on the most important pathological feature of natural lesion that mineral loss occur underneath the enamel surface. After the pH-cycling, a hypomineralized band (negative birefringent band similar to the surface layer) was observed in the lesions body for NaF group, GCE group, GCE-B group and GCE-B1 group, and the depth of the caries lesions was obviously reduced. As for GCEB2 group and deionized water group, little change had taken place after remineralisation except that the density of the
Fig. 3 – Polarized light microscopic pictures of enamel sections from the groups before and after the pH-cycling: (A) NaF group, (B) GCE group, (C) GCE-B group, (D) GCE-B1 group, (E) GCE-B2 group, (F) DDW group (scale bar = 100 mm). The white arrows on the top picture indicate the approximate boundaries of early artificial caries lesions and 12-day remineralisation regime in all pictures.
journal of dentistry 35 (2007) 383–387
surface layer increased slightly. This also supported the result of the above surface microhardness analysis. In the present study fluoride was employed as a positive control and was clearly effective. Fluoride’s anti-caries efficacy is well-proven. In preventing and arresting caries, fluoride acts via two principal mechanisms: inhibiting demineralisation when fluoride is present at the crystal surfaces during an acid challenge and enhancing remineralisation and thereby forming a low solubility veneer similar to the acid-resistant mineral fluorapatite or FAP, on the remineralized crystals.22 So far, G. chinensis’s mechanism of action is still unknown. However, it has been established that the essential compounds of G. chinensis are polyphenol compounds (e.g., tannin and gallic acid), whilst interactions can occur between tannic acid and calcium ions, forming indissolvable complex.23 From the combined data from this study we proposed that although its mechanism of action is unknown, G. chinensis is believed to combine with calcium in aqueous solution. Furthermore, G. chinensis actually prevents decalcification by inhibiting the translocation of dissolved Ca2+ and PO43 ions from lesions.13 Thus, chemical compounds of G. chinensis, which are GCE, GCE-B and GCE-B1, might act as a Ca2+ ion carrier supplying the middle layers of caries lesion with Ca2+ ions from the remineralizing solution, thereby enhancing remineralisation by providing the Ca2+ required for crystal repair. In conclusion, the present has demonstrated the potential of three GCEs; GCE, GCE-B and GCE-B1; to effect net rehardening of artificial carious lesions under dynamic pH-cyclic conditions. Although not as effective as fluoride, G. chinensis might still be a promising adjunct or alternative to fluoride.
Acknowledgments The authors are grateful to Prof. F. Wang and Dr. J. Wang (The Institute of Naturally Occurring Drugs of Sichuan University, Chengdu, China) for kindly providing chemical compounds extracted from Galla chinensis. This investigation was supported by the National Natural Science Foundation of China (Grant No. 30430800).
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