846 Asian Pac J Trop Biomed 2016; 6(10): 846850

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Asian Pacic Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Original article http://dx.doi.org/10.1016/j.apjtb.2016.08.005

Effectiveness of some herbals on initial enamel caries lesion

Gulcin Bilgin Gocmen1, Funda Yanikoglu2, Dilek Tagtekin2*, George K. Stookey3, Bruce R. Schemehorn3,
Osman Hayran4
1
Kartal Kosuyolu Education and Research Hospital, Istanbul, Turkey
2
Department of Restorative Dentistry, Marmara University, Istanbul, Turkey
3
Therametric Technologies, Inc., Noblesville, Indiana, USA
4
Department of Health Science, Istanbul Medipol University, Istanbul, Turkey

A R TI C L E I N F O ABSTRACT

Article history: Objective: To evaluate the effectiveness of herbal medicaments such as ginger, rosemary
Received 2 Feb 2016 and honey on remineralization of initial enamel lesion.
Received in revised form 23 Feb, 2nd Methods: Demineralized human enamel specimens were measured for baseline surface
revised form 24 Mar 2016 microhardness and uorescence methods. Ten specimens in each of four groups were
Accepted 10 Jun 2016 used in this in vitro recycling study with the following treatments which applied three
Available online 26 Aug 2016 times a day: 1) sodium uoride toothpaste (Ipana, Procter & Gamble, Turkey), 2) ginger-
honey (Arifoglu Herbals, Anzer Honey, Turkey), 3) ginger-honey-chocolate (Bind
Chocolate, Turkey), 4) rosemary oil (Arifoglu Herbals, Turkey). Treatment regimens of
Keywords:
demineralization and remineralization cycle were applied for 21 days. The post-treatment
Remineralization
data were obtained by measurements of surface microhardness and uorescence methods.
Ginger
Data were statistically analyzed by ANOVA test with Tukey's honest signicant differ-
Rosemary
ence test.
Chocolate
Results: Enhanced remineralization was observed with several of the treatment systems
Honey
including ginger + honey and rosemary. Signicant differences between treatments were
FluoreCam
observed by microhardness and FluoreCam uorescence assesment, compared to the
QLF
positive control group (NaF dentifrice). Signicantly, greater remineralization was
Initial enamel lesion
observed with the honey + ginger treatment regimen. No signicant differences between
groups were observed using the uorescence assessment method, quantitative light-
induced uorescence.
Conclusions: Herbals (ginger, honey and rosemary) have enhanced remineralization of
initial enamel lesion.

1. Introduction should be treated with non-invasive remineralization strategies.

On this purpose, topical gels, varnishes, mouthwashes and
The rst sign of tooth caries, opaque lesion dened as dentifrices contain uoride being used by dentists for the treat-
subsurface enamel porosity from carious demineralization is ment of white spot lesions [1].
manifested clinically by a milky white opacity. This subsurface Fluoride is proved agent for caries prophylaxis, however,
porosity is being caused by an imbalance between the dynamic excess use of uoride causes uorosis, and hardening of carti-
biological processes of de- and remineralization. In minimally lage. Moreover, the usage of bactericides or antibacterial agents
invasive dentistry paradigm, incipient enamel carious lesions has several negative effects on gastrointestinal system with
increased resistance to these chemicals. Due to nancial situa-
*Corresponding author: Prof. Dr. Dilek Tagtekin, Department of Restorative tion, developing countries need biocompatible and cost effective
Dentistry, Marmara University, Istanbul, Turkey. preventive methods. Therefore, instead of using articial anti-
Tel: +90 5423124294 biotics and bactericides, it has been proposed to use medicinal
E-mail: [email protected]
Peer review under responsibility of Hainan Medical University. The journal plant extracts which have an effect on causative bacteria of tooth
implements double-blind peer review practiced by specially invited international decay [2].
editorial board members.

2221-1691/Copyright 2016 Hainan Medical University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
 Gulcin Bilgin Gocmen et al./Asian Pac J Trop Biomed 2016; 6(10): 846850 847

Among natural food sources, ginger rhizome (Zingiber of- amount and Carbopol 907 as 0.2%, 50% hydroxyapatite-
cinale Roscoe, Zingiberaceae) and rosemary (Rosmarinus of- saturated in volume and adjusted to pH 5.0 using NaOH [17].
cinalis L., Lamiaceae) are natural herbals with their This procedure resulted in lesions approximately 3550 mm in
antimicrobial activities. Additionally, they do not show any depth.
toxicity approved by generally recognized as safe in the Food
and Drug Administration of the United States. Pungent oil 2.2. Study design
contents of these herbals harbor some polyphenolic ketones with
many pharmacological activities. Their antifungal and antimi- The specimens were divided randomly to six groups (10
crobial effects on oral cavity pathogens have been reported in specimens/group) with the treatment materials. Treatment
many studies [37]. However, there is no study in current regimen was designed with approximate pH oral environment and
literature about the effect of these herbal medicaments on modied by Dunipace et al. [18]. The demineralization and
remineralization of initial enamel caries. remineralization cycles showed episodes as observed in Table 1.
Another regimen used in most ancient cultures for nutritional Each cycle contained 3 h of demineralization to simulate the daily
and/or medicinal aim is honey. It is believed that honey is a acid challenges in oral cavity. The samples were kept in laboratory
nutrient and can be used as a drug for a long time. Honey is a produced saliva which consisted of 2.00 g/L methyl p-hydrox-
supersaturated sugar solution with low water activity that does ybenzoate, 10.0 g/L sodium carboxymethyl cellulose, 8.38 mmol/
not support the growth of bacteria [8]. The average pH value of L KCl, 0.29 mmol/L MgCl2$6H2O, 1.13 mmol/L CaCl2$2H2O,
honey is 3.9, and can show bacteriostatic effect on pathogens as 4.62 mmol/L KH2PO4, 2.40 mmol/L K2HPO4; and adjusted pH
most thrive at pH between 4.0 and 4.5 [9]. However, dilution of was 7.0 using KOH and there was not any precipitation observed
honey, for example by saliva will increase the pH and reduce during the experiment [19]. Repeated treatment regimen lasted
this effect. On the other hand, dilution results in 2 50050 000 during 21 days. This saliva was changed each day and these
times increase in enzyme activity and this glucose oxidase treatment materials were freshly prepared in every application.
enzyme is the production of hydrogen peroxide, an oxidizing All the time except applications, the samples were kept in
agent. Hydrogen peroxide is present in honey in small articial saliva that was mixed by a magnetic stirring machine
amounts, yet is still very effective antibacterial agent (Multipoint HP15P, Variomag, USA).
compatible with cellular preservation [10]. There are a few
studies on efcacy of honey on oral pathogenic bacteria [1113] 2.3. Treatment materials
and none is about effect of honey on remineralization of initial
enamel caries. Ipana, NaF toothpaste consisted of 1 450 mg/kg uoride and
Methylxanthines are plant-produced natural products. Most was used as a positive control group. Based on the previous
plants used for preparation of beverages on human consumption studies that have found the minimum inhibition concentration
are enriched in methylxanthines [14]. The antioxidants of (MIC) of ginger (58 mg/mL) [4,20], we used ginger in powder
Theobroma cacao beans have psychoactive effects at high form (Arifoglu Herbals, Turkey) and applied 8 mg into 1 mg
amounts because of methylxanthines [15]. Besides, theobromine honey (Anzer honey, Turkey). Chocolate (Sokella, Turkey)
could show protective effect on enamel surface of human molars was added as 1 mg into the mixture of ginger + honey. All
as shown by a pilot study [16]. This protection was attributed to materials were mixed homogeneously and applied on the
carbohydrate content of cacao which can be metabolized and a surfaces by smearing. Rosemary (Arifoglu Herbals, Turkey)
trap for bacteria to protect dental enamel from caries. oil was applied with an applicator. All pastes were prepared
The purpose of this study is to evaluate remineralization freshly at each application of remineralization materials.
potential of herbals (ginger, rosemary and also honey).
In addition to our main purpose, we investigated the efciency 2.4. Assessment of mineral content FluoreCam & QLF
of new detection device (FluoreCam), for demineralization and
remineralization of human dental enamel by microhardness, also Before and after each test period, assessments of the mineral
quantitative light-induced uorescence (QLF) systems. content of demineralized area of each specimen were obtained
using both the FluoreCam (Daraza, Corporate Headquarters,
2. Materials and methods
Indiana, USA) and QLF (Inspektor Pro, Inspektor Research
Systems, Amsterdam, Holland) systems. Using the FluoreCam
2.1. Enamel specimens and preparation of subsurface instrument, the images were collected with and without
lesions

A total of 40 human enamel specimens were used in this

Table 1
study. Extracted teeth obtained from oral surgeons were used; the
The pH-cycling model in the experiment.
teeth were stored in 0.10% thymol solution immediately after
extraction and maintained in this solution prior to use. The sound Time Application
enamel specimens required for this study were 3 mm in diameter 08:0009:00 Lactic acid
and 1.62.0 mm thick from surface of enamel. These enamel 09:0009:01 Treatment materials
cores were mounted on acrylic rods. Surfaces of specimens were 09:0113:00 Articial saliva
polished by a 600-grit grinding disk and with a slurry of 0.05 mm 13:0014:00 Lactic acid
14:0014:01 Treatment materials
gamma alumina polishing gel. Articial subsurface carious le- 14:0119:00 Articial saliva
sions were formed on each enamel specimen by placing the 19:0020:00 Lactic acid
specimens individually for 72 h at 37  C in 7.0 mL of a 20:0020:01 Treatment materials
demineralizing solution containing lactic acid as 0.1 molar 20:0108:00 Articial saliva
848 Gulcin Bilgin Gocmen et al./Asian Pac J Trop Biomed 2016; 6(10): 846850

dehydration for 5 s and analyzed using the specially designed rosemary oil group and NaF toothpaste but the results showed that
software. The parameters to be assessed included uorescence rosemary oil group was almost equal on effect to positive control
loss (%), area (mm2) and lesion volume (mm2 %). Any sig- group, NaF toothpaste (P > 0.05). Ginger + honey + chocolate
nicant change in uorescence indicated that remineralization group showed still some demineralization but there was no sta-
(or demineralization) had taken place. tistical difference with NaF toothpaste (P > 0.05).

2.5. Surface microhardness measurements 4. Discussion

Following the lesion formation procedure, the surface Main process occurs at the outer layer of enamel which is
microhardness was measured (LECO LM247AT microhardness principally in contact with oral environment [21]. The
tester). The parameters were 200 g force for 15 s with a Vickers remineralizing agents applied on the surface of enamel
indenter. Four indentations were made on each specimen (one in specimens were evaluated by QLF, FluoreCam and surface
each quadrant) and averaged for an average specimen value. microhardness test methods. They are quick, easy and simple,
This value had provided a baseline surface hardness value. By moreover nondestructive methods, giving the mineral changes
performing the indentations prior to the polycrystalline diamond following the treatments. In addition to these, they provide
inserts assessments, the indentation marks were in both the pre- repeated measurements of same sample at any other time by
test and the post-test determinations. eliminating any variation in the experiment.
Following the removal of the specimens, they were individ- QLF has shown great promise as an early caries detection
ually mounted on plexiglas rods as at as possible so post-test method. The study concluded that it is suitable for monitoring
hardness and mineral content determinations could be made. mineral changes in vivo except the cost of the system [22]. Also
After the post-test polycrystalline diamond inserts measure- there are some factors conning its success such as dehydration
ments, the post-test surface microhardness indentations were [23] and angulation [24]. It seems there is conicting results with
made in the same manner as described above. Any signicant ginger + honey group which was found highly remineralized in
increase in hardness over the test period was indicative for microhardness and FluoreCam test methods (P < 0.001) while
remineralization and any signicant softening was indicative for QLF has shown no signicance between the treatment
demineralization. materials (P > 0.05). The FluoreCam system is an innovative
approach for quantication of enamel called uorescence
2.6. Statistical analysis enamel imaging. Surface of a tooth is induced with a high
intensity light and the instrument has sent the uorescent
For comparison of any remineralization effects of the treat- image and measurements to a computer. Determining the
ment materials in each group, ANOVA repeated measures tests suspected de- or remineralization area is automatically done by
were conducted, with Tukey's multiple tests. The statistical an- FluoreCam software.
alyses were processed using SPSS 15.0 for Windows (SPSS Nowadays, more phytochemicals, especially antibacterial
Inc., Chicago, IL) with signicant level of 0.001. agents, have been derived from edible plants. Many reports
revealed some antibacterial activities of ones, against Strepto-
3. Results coccus mutans (S. mutans), main pathogen of dental caries.
Ohara et al. searched 81 edible plants' antibacterial activities
Over time almost all treatment values in the groups were against S. mutans in polarity-differing solvents (hexane and
signicantly higher than in the baseline following three weeks. ethyl acetate) and ginger is found to be effective (MIC 23 mg/g
The mean DF (lesion depth) and DQ (lesion volume) values and 8 mg/g) [4]. Moreover, after boiling 10 min at 100  C or
( SEM) for QLF were Ipana (1.01 1.22)% and (15.25 after storage for 1 week at 4  C, ginger protects its
8.75)% mm2, ginger + ;honey (1.89 0.57)% and (21.30 antibacterial activity [1]. White found that in glycolic or
6.67)% mm2, ginger + honey + chocolate (0.38 1.03)% and hydroalcoholic solvents, 5 mg/mL MIC of ginger is effective
(10.45 6.00)% mm2, and rosemary oil (0.23 0.80)% on S. mutans [17]. Honey is potentially antibacterial agent and
and (4.55 4.56)% mm2. Although all samples resulted studies demonstrated that manuka honey is likely to be non-
in remineralization, there was not any signicant difference cariogenic. Patel et al. reported ginger and honey are more
between the groups with QLF (P > 0.05). Mean DF and DQ effective than gentamycin on S. mutans. They found the MIC is
values ( SEM) for FluoreCam were Ipana (3.53 2.05)% and ginger 31.25 mg/mL, while honey 1:2 (%, v/v), and
(7.75 5.03)% mm2, ginger + honey (12.12 1.72)% and ginger + honey 15.63 mg/mL [13]. Our study was consistent with
(38.49 4.65)% mm2, ginger + honey + chocolate these reports demonstrating that ginger + honey (8 mg/mL) was
(9.00 1.39)% and (27.22 2.84)% mm2, and rosemary oil a strong remineralizing agent. The obtained high
(7.86 1.39)% and (22.17 4.58)% mm2. In contradiction for remineralization is probably due to antimicrobial properties of
FluoreCam assessment, we observed signicantly greater remi- ginger which might be the result of high amount of uoride
neralization in ginger + honey group (P < 0.001). content (79 mg/kg uoride in 8 mg). By addition of honey,
The surface microhardness results showed signicant differ- the content of uoride has decreased to 23.7 mg/kg.
ences between all treatment materials. The mean Vickers hardness Additionally, pH of ginger and honey content was quite high
number values were Ipana (6.76 1.96), ginger + honey with 6.35 (Therametric Technologies, Inc., Indiana, USA).
(11.69 1.19), ginger + honey + chocolate (10.72 2.34), and Even though NaF toothpaste had much more uoride
rosemary oil (2.72 3.71). Among all treatment materials, (1 450 mg/kg), it has provided less remineralization than
ginger + honey group showed the greatest remineralization ginger and honey mixture. These results were consistent with
(P < 0.001). There was not signicant difference between the in situ study done by Bilgin et al. [25].
 Gulcin Bilgin Gocmen et al./Asian Pac J Trop Biomed 2016; 6(10): 846850 849

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