European Journal of General Dentistry

Manuscript: EJGD-0-0-0 - (360)

Title: Antibacterial Properties of Herbal Toothpaste on the Market
Keywords: antibacterial, herbal, Streptococcus mutans, toothpaste
Type: Original/Research Article
 Antibacterial Properties of Herbal Toothpaste on the Market

Abstract

Objectives: Effective plaque control is one approach to prevent caries, which can be achieved

through mechanical plaque removal such as tooth brushing. As advancements in science and

technology continue, toothpaste manufacturers are actively exploring innovations by

incorporating additional beneficial ingredients, including herbs. Numerous studies have

reported the positive impact of herbal toothpaste on reducing dental plaque attributed to its

antimicrobial properties. The objective of this study was to investigate the antibacterial

properties of several herbal toothpastes that are readily accessible in the market.

Materials and Methods: This study was a laboratory experiment that utilized four different

herbal toothpastes available in the market. The herbal toothpaste samples included toothpaste

A, which contained clove oil as its main ingredient; toothpaste B, consisting of betel leaf, lime,

and salt; toothpaste C, containing miswak as the primary ingredient; and toothpaste D, which

contained betel leaf extract. The control toothpaste was a non-herbal toothpaste containing

sodium fluoride as its active ingredient.

Statistical analysis: The ANOVA test analysis was used to evaluate different inhibitory zones

among the toothpaste groups with significant levels of p<0.05.

Results: The herbal toothpaste A exhibited the highest average inhibition of Streptococcus

mutans with a measurement of 25.65. This was followed by the control toothpaste with an

average inhibition of 23.19, the herbal toothpaste C with 22.43, the herbal toothpaste D with

20.15, and the herbal toothpaste B with the smallest average inhibition of 14.60.

Conclusions: All of the tested toothpaste demonstrated inhibitory effects on Streptococcus

mutans. Among them, the herbal toothpaste containing clove exhibited the highest inhibition,

while the herbal toothpaste containing betel leaf, lime, and salt showed the lowest inhibition.

Keywords: toothpaste – herbal – antibacterial - Streptococcus mutans

Introduction
Caries or cavities are tooth decay which is a global public health problem.1 Caries are

caused by an imbalance in the oral flora (biofilm) due to fermentable dietary carbohydrates

that deposit on the tooth surface over time. Streptococcus mutans, a gram-positive bacterium,

is widely recognized as one of the primary causes of dental caries. It is a facultative anaerobe

and serves as the predominant pathogenic agent responsible for dental caries.2 This bacterium

grows rapidly at 37°C, and some strains grow at 45°C. Streptococcus mutans produces lactic

acid as a byproduct of fermenting the carbohydrates in the host food in order to maintain a pH

of 4.5 to 5.5, which results in demineralization of tooth enamel and increased caries

formation.3

Caries can be prevented by knowing the cause. One way to prevent caries is to control

plaque, a collection of microbes that colonize the tooth surface in a structured and organized

biofilm, which is the leading cause of caries. Plaque control can be achieved by mechanical

plaque removal.4,5 Tooth brushing is the most common oral hygiene practice for mechanical

plaque control. Tooth brushing habit is one of the factors associated with caries. People who

brush their teeth less than twice a day have a higher risk of experiencing caries than those who

brush their teeth more than twice a day.6

Toothpaste used when brushing teeth removes dental biofilm (plaque) and increases

tooth remineralization.7 Toothpaste is a complex mixture of abrasives and surfactants;

antimicrobial agents such as triclosan and chlorhexidine, anti-tartar agents; pH buffers;

humectants (to prevent dryness and improve mouthfeel); binder, and fluoride. In a study

conducted by Tomasz (2013), fluorine can assist in the process of preventing the growth of

cariogenic bacteria that are resistant to acidic conditions, such as Streptococcus mutans which

is cariogenic by inhibiting acid production and glucosyltransferase.8

 In light of the progress in science and technology, toothpaste manufacturers are actively

pursuing innovation by incorporating additional beneficial ingredients to promote dental

health. One of the substances commonly added to toothpaste is herbal ingredients. Herbal

toothpaste has received much public attention, and many studies have reported the beneficial

effects of herbal toothpaste on dental plaque.9 This is due to the ability of several types of

herbs to inhibit microbial growth. In addition, the components of herbal toothpaste are derived

from natural plants.10

Many kinds of toothpaste available in the market today contain herbal ingredients such

as betel leaf, lime, miswak, etc. Betel leaves contain lots of essential oils, catechins, and

tannins. Tannins are polyphenolic compounds that act as preservatives and inhibit the

bioactivity of cariogenic bacteria such as Streptococcus mutans. In addition, the content of

lime also causes a decrease in the plaque index, namely flavonoids. A study showed that

flavonoid compounds' properties are more acidic than alkaline, so they can kill all types of

cells, including plaque-forming bacterial cells.11,12 Miswak is very good to use to clean the

mouth because Miswak contains chemical substances such as sulfates, silicon, bile substances,

fluoride substances, calcium, phosphates, trimethylamine, alkaline acids, glycolytic, vitamin

C, tannins, and waxes. Miswak wood consists of antibacterial acids, such as astringents and

abrasives, which destroy bacteria and prevent infection, according to research done by

Apriansi M. (2017). In addition, the antibacterial compound Nitran (NO3) found in miswak

wood can interfere with the bacterium Streptococcus mutans' active transport of oxidative

phosphorylation and oxygen uptake.13,14 There is also toothpaste containing cloves. Clove leaf

essential oil has antibacterial and anti-biofilm activity. The eugenol compound is the active

ingredient that acts as an antibacterial in clove essential oil. According to research by Susi et

al. (2015), clove oil's inhibitory ability against gram-positive bacteria was better than gram-

negative bacteria.14
Materials and Methods

This research has received ethical approval with number 0132/ PL.09/ KEPK FKG-

RSGM UNHAS/ 2022. The type of research used is laboratory experimental. The research

samples used were four herbal toothpastes available on the market and one non-herbal

toothpaste as a control. The toothpaste sample consists of 1) herbal toothpaste A with the main

ingredient of clove oil; 2) herbal B toothpaste with main ingredients of betel leaf, lime, and

salt; 3) herbal C toothpaste with the main ingredient miswak; 4) herbal D toothpaste with the

primary ingredient betel leaf extract; and 5) control toothpaste, namely non-herbal toothpaste

containing active ingredients sodium fluoride.

Research flow: a) the tools and materials needed are sterilized using an autoclave at a

temperature of 121 ºC with generally 30 minutes of sterilization time; b) preparation of Nutrient

Agar (NA) bacterial media. NA media was prepared by dissolving 20 grams of powdered NA

in 1-liter aquades. The media is homogenized with a stirrer and simultaneously heated using a

hot plate, then sterilized by autoclaving at 121 ºC for 15 minutes to obtain a sterile NA medium;

c) Isolate rejuvenation Streptococcus mutans by taking NA and putting it in a test tube.

Streptococcus mutans originating from the stock is taken with a sterile loop and then streaked

into a test tube containing NA with a slanted surface then, incubated for 24 hours, then observed

whether the bacteria Streptococcus mutans pure has grown; d) preparation of toothpaste

preparations by taking 1 gram of each toothpaste, then diluting it with 1 ml aquades; e) to know

the sensitivity Streptococcus mutans for toothpaste, do the following: prepare three Petri dishes

and 15 paper dish with a diameter of 5 mm. Paper dish soaked for 5 minutes in diluted

toothpaste (each toothpaste five paper dish), then in each petri dish containing NA media and

isolates of Streptococcus mutans placed five paper dishes of 5 kinds of pasta. Then put in an

incubator at 37 ºC for 24 hours.

Results
 Observations on the inhibitory ability (width of the zone of inhibition) of several herbal

toothpastes indicated that all of these had an inhibitory effect on Streptococcus mutans with

varying inhibition zones (Table 1). Calculation results with the ANOVA test, a p-value <

𝛼=0.05 was obtained, indicating a significant difference in the inhibition of Streptococcus

mutans of the toothpastes tested. Because there were differences, a further difference test was

carried out with the Least Significant Difference (LSD), the results of which are shown in Table

2.

Table 2 shows that the average inhibition of S. mutans bacteria in herbal toothpaste

containing clove was the highest, namely 25.65; then followed by control toothpaste, namely

23.19; herbal toothpaste containing miswak, namely 22.43; herbal toothpaste containing betel

leaf, namely 20.15 and the smallest, namely herbal toothpaste containing betel leaf, lime, and

salt, namely 14.60.

The calculations using one-way ANOVA showed significant differences in the

inhibition of bacteria Streptococcus mutans among the five toothpastes tested (p=0.000;

p<0.05). Because there were significant differences, it was continued with the LSD test to

determine whether one group had a significant difference from the others (Table 3).

Table 3 shows that herbal toothpaste A and herbal toothpaste B obtained results of p =

0.000 and α = 0.05, where p <α means a significant difference. Herbal toothpaste A and herbal

toothpaste C obtained results of p=0.015 and α=0.05 where p<α means there is a significant

difference. Herbal toothpaste A and herbal toothpaste D obtained p=0.001 and α=0.05 where

p<α means a significant difference. Finally, herbal toothpaste A and control toothpaste obtained

p=0.049 and α=0.05, where p<α means a significant difference.

Herbal toothpaste B and herbal toothpaste C obtained results of p=0.000 and α=0.05

where p<α means there is a significant difference. Herbal toothpaste B and herbal toothpaste

D obtained p=0.000 and α=0.05 where p<α means a significant difference. Herbal B toothpaste
with control toothpaste obtained results of p=0.000 and α=0.05 where p<α means there is a

significant difference.

Herbal toothpaste C and herbal toothpaste D obtained p=0.064 and α=0.05 where p>α

means no significant difference. Herbal C toothpaste with control toothpaste obtained results

of p=0.053 and α=0.05 where p>α means no significant difference. Herbal toothpaste D and

control toothpaste obtained results of 0.020 α = 0.05, where p <α means there is a significant

difference.

Discussion

In this study, the inhibitory power against Streptococcus mutans bacteria showed that

the highest inhibitory power was toothpaste containing clove, followed by non-herbal

toothpaste, miswak toothpaste, betel leaf toothpaste, and the lowest in this study was herbal

toothpaste containing betel leaf, lime, and salt.

The four toothpaste samples showed a clear zone (zone of inhibition). This indicates

that toothpaste samples containing herbs can inhibit the growth of Streptococcus mutans

bacteria. In measuring the clear zone (zone of inhibition), herbal toothpaste samples containing

clove (clove) had a higher inhibition of Streptococcus mutans growth than other herbal

toothpaste samples.

Cloves contain clove oil, an antimicrobial agent that can fight oral bacteria, usually the

source of dental caries and periodontitis. Clove oil can inhibit gram-positive and gram-negative

bacteria. Clove oil has phytochemicals in the form of essential oil with the highest eugenol

content. The high eugenol content in clove oil can inhibit the growth of bacteria. Eugenol has

hydrophobic properties that can penetrate the lipopolysaccharide of the bacterial cell membrane

and damage the cell structure.15

In addition, cloves have leaves that contain antibacterial properties. Clove leaves

contain chemical compounds like saponins, phenols, and tannins. According to the research,
saponins activity as an antibacterial with a working mechanism, causing leakage of proteins

and enzymes from inside the cell. This is because saponins' active components, which are

identical to those found on the surfaces of detergents, can affect membrane permeability by

reducing the surface tension of bacterial cell walls. The cell membrane is disrupted and

becomes less stable as the saponins permeate through the outer and cell walls. This results in

the death of both gram-negative and gram-positive bacterial cells by causing cytoplasm to leak

out of the cell.16,17

Phenol has many biological roles, one of which is as an antibacterial. Phenol can

damage the bacterial cell wall into lysis by denaturing the proteins in bacteria so that the

bacterial cells will be damaged due to a decrease in the permeability of the bacterial cell wall,

which causes inhibited cell growth, and bacterial cells will experience death.18 Tannins have

activity as an antibacterial with a working mechanism, interfering with cell permeability. This

results in cells being unable to carry out living activities, so their growth is stunted, and they

die. Tannin compounds can induce the formation of tannin bond complexes to metal ions,

increasing tannins' toxicity.17 Haji and Rahim (2006) found that clove extract had antimicrobial

activity against Streptococcus mutans. While this may indicate the specificity of clove extract

for Streptococcus mutans, more research is required to determine how clove extracts affect

other factors that contribute to cariogenicity, such as adherence..19

The following herbal toothpaste that has inhibitory power against Streptococcus mutans

is herbal toothpaste containing miswak. Siwak is very good for cleaning the mouth because it

contains chemicals such as trimethylamine, fluoride, silica, sulfur, and small amounts of

tannins, saponins, flavonoids, and sterols. Silica, as an abrasive in miswak, can remove the film

attached to the surface of the teeth, and the presence of sulfur gives it a warm feeling and a

distinctive aroma. Tannins reduce the adhesion of bacteria to the tooth surface by inhibiting the

glucosyltransferase enzyme produced by Streptococcus mutans.20

 Trimethylamine and thiocyanate contained in miswak also have a bactericidal effect

which can prevent the formation of acids produced by Streptococcus mutans, which can

prevent bacterial growth. Trimethylamine reduces bacterial adhesion to the tooth surface and

plaque accumulation.20 Fluoride in miswak can prevent bacterial growth and convert

hydroxyapatite to fluorapatite, which is more resistant to acid dissolution, thereby preventing

the formation of dental caries.19

In third place in this study was toothpaste containing betel leaves. Betel leaf (Piper

betel) is a natural ingredient with an antimicrobial effect that can kill bacteria and prevent their

growth. Toothpaste with betel leaf extract is effective in reducing plaque and gingivitis. The

antibacterial power of betel leaf essential oil is due to the presence of phenolic compounds and

their derivatives, such as eugenol and chavicol, which can denature bacterial cell proteins. Due

to the antibacterial properties of phenol, the number of Streptococcus mutans bacteria on the

tooth surface is reduced.21 The eugenol compound has a bactericidal effect by increasing the

permeability of the bacterial membrane. In addition to the chavicol compound, which gives the

betel leaf its distinctive aroma, it also has antibacterial properties five times stronger than other

phenolic compounds.20,21

The herbal toothpaste with the slightest inhibition in this study was betel leaf, lime, and

salt. Toothpaste containing betel leaf extract is effective in reducing plaque and gingivitis. Betel

leaves contain essential oils whose main components are phenol and its derivatives, such as

chavicol and eugenol. Aside from essential oils, betel leaves contain carotene, thiamin,

riboflavin, nicotinic acid, vitamin C, and tannins. Based on research conducted by Jahovah T

et al. (2021) shows that betel leaf essential oil is known to have an antibacterial effect due to

the presence of phenolic compounds and their derivatives which can change the protein

properties of bacterial cells.22

 Another ingredient in this herbal toothpaste is lime. Lime is a herbal substance that is

added to toothpaste because of its ability to inhibit microbial growth. The chemical components

of lime that reduce the plaque index are flavonoids. Flavonoids are the most extensive

polyphenolic compounds that can act as antioxidants and antibacterial substances by denaturing

bacterial cell proteins and damaging bacterial cells.22,23 In a study conducted by Anggina DN,

et al. (2018), it was shown that the flavonoid compounds in lime are naturally more acidic than

alkaline so that they can kill all types of cells, including plaque-shaped bacterial cells.23

Apart from betel leaf and lime, this herbal toothpaste also contains salt. Salt is formed

as sodium chloride (NaCl) by reactions between acids and bases but undergoes no chemical

process. The content of salt that is useful for improving dental health is calcium and

phosphorus, which play a role in the formation of bones and teeth, as well as fluoride, which

plays a role in the formation of tooth enamel which protects against all kinds of dental diseases.

Salt can be used not only as a food additive or flavor enhancer but also as an additive to

toothpaste as a substitute for fluoride.23

The research results concluded that all the toothpastes tested had an inhibitory effect on

Streptococcus mutans and differed significantly (p<0.05). Clove herbal toothpaste produced

the highest and lowest inhibition on betel leaf, lime, and salt herbal toothpaste. In addition, the

herbal toothpaste had higher inhibition than the non-herbal toothpaste used in the control

toothpaste containing sodium fluoride. It was, therefore, more effective than non-herbal

toothpaste in inhibiting Streptococcus mutans.

Conflicts of interest

There are no conflicts of interes

References

1. Cui T, et al. Progress of antimicrobial discovery against the major cariogenic pathogen
streptococcus mutans. Curr Issues Mol Biol, 2019; 32.
2. Ritter AV, Boushell LW, Walter R. Sturdevant’s art and science of operative dentistry. 7th
ed. St.Loui: Elseiver; 2019. p. 40-1.
3. Naka S, Wato K, Misaki T. Streptococcus mutans induces IgA nephropathy-like
glomerulonephritis in rats with severe dental caries. Sci Rep, 2021; 11(1).
4. Zhou X, Li Y. Atlas of oral microbiology: from healthy microflora to disease. 2nd ed.
Singapore: Springer; 2020. p.121.
5. Li Z, Zhang C, Li C. S-glutathionylation proteome profiling reveals a crucial role of a
thioredoxin-like protein in interspecies competition and cariogenecity of Streptococcus
mutans. PLoS Pathog, 2020; 16(7).
6. Gutiérrez-Venegas G, et al. Effect of flavonoids on antimicrobial activity of
microorganisms present in dental plaque. Heliyon, 2019; 5(12).
7. Janakiram C, Taha F, Joe J. The efficacy of plaque control by various toothbrushing
techniques-a systematic review and meta-analysis. Journal of Clinical and Diagnostic
Research, 2018; 11(12).
8. Yu OY, et al. Nonrestorative management of dental caries. Dent J (Basel), 2021; 9(10).
9. Epple M, Enax J, Meyer F. Prevention of caries and dental erosion by fluorides-a critical
discussion based on physico-chemical data and principles. Dent J (Basel), 2022; 10(1).
10. Sultana A, et al. A review on potassium nitrate, chlorhexidine gluconate and hydrogen
peroxide toothpaste formulation to treatsensitivity, bleeding gums & whitening of teeth. J
Global Trends Pharm Sci, 2021; 12(3).
11. Epple M, Enax J, Meyer F. Prevention of caries and dental erosion by fluorides-a
critical discussion based on physico-chemical data and principles. Dent J (Basel). 2022;
10(1).
12. Kiruthika S, et al. A review on herbal toothpaste for plaque and gingivitis. International
Journal of Research in Engineering, Science and Management, 2022; 3(2).
13. Korsowannawong S, et al. Comparison of enamel surface roughness after brushing with
herbal and no-herbal toothpaste. World Journal of Dentistry, 2020; 11(3).
14. Apriansi M. Effect of siwak wood powder extract (salvadora persica) on the growth of
streprococcus mutans bacteria. Jurnal Agroqua, 2017; 15(2).
15. Akbar AF, Cahyaningrum SE. Characterization and anti-bacterial activity testing of
nano hydroxyapatite-clove (eugenia caryophyllus) against streptococcus mutans bacteria.
Indonesian Journal of Chemical Science, 2022; 11(1).
16. Rashad JM. Effect of water clove extract on streptococci and mutans streptococci, in
comparison to chlorhexidine gluconate: a comparative in vitro and in vivo study. MDJ,
2008; 5(4).
17. Mirpour M, Siahmazgi ZG, Kiasaraie MS. Antibacterial activity of clove, gall nut
methanolic and ethanolic extracts on streptococcus mutans ptcc 1683 and streptococcus
salivarius ptcc 1448. Journal of Oral Biology and Craniofacial Research 5, 2015; 7(10).
18. Elgamily H, Safy R, Makharita R. Influence of medicinal plant extracts on the growth
of oral pathogens streptococcus mutans and lactobacillus acidophilus: an in-vitro study.
Maced J Med Sci, 2019; 7(14).
19. Sukkarwalla A, et al. Efficacy of miswak on oral pathogens. Dental Research Journal,
2013; 10(3).
20. Florenly, et al. Nano-green betel leaf extracts (piper betle l.) Inhibits the growth of
streptococcus mutans and staphylococcus aureus. e-GiGi, 2022; 10(2).
21. Subri LM, Dewi W, Satari MH. The antimicrobial effect of piper betel leaves extract
against streptococcus mutans. Padjadjaran Journal of Dentistry, 2012; 24(3).
22. Elsa L, Supriyana S, Sunarjo L. The potential of lime peel extract mouthwash as a non-
pharmacological preparation inhibits caries bacteria. Journal Health Sains, 2023; 4(4).
23. Alexa VT, et al. Synergistic/antagonistic potential of natural preparations based on
essential oils against streptococcus mutans from the oral cavity. Molecules 2019; 24, 4043.
 Table 1. Toothpaste sample composition

Toothpaste Composition

Herbal Toothpaste Water, sorbitol, hydrated silica, PEG-32, zinc citrate,

A (clove) sodium lauryl sulfate, flavor, clove oil, cellulose gum,
sodium fluoride, mica, sodium chloride, sodium
saccharin, ci 16255, ci 19140, ci 77891. Active
ingredient: 0,32% sodium fluoride

Herbal Toothpaste Calcium carbonate, water, sorbitol, hydrated silica,

B (betel leaf, lime, sodium lauryl sulfate, flavor, sodium
and salt) monofluorophosphate, cocamidopropyl betaine,
cellulose gum, potassium citrate, sodium silicate,
sodium chloride, sodium saccharin, DMDM hydantoin,
citrus aurantifolia fruit extract, glycerin, piper betle leaf
oil, CI 47005, CI 74260

Herbal Toothpaste Calcium carbonate, water, sorbitol, hydrated silica,

C (miswak) sodium lauryl sulfate, sodium monofluorophosphate,
flavor, cellulose gum, sodium silicate, benzyl alcohol,
potassium citrate, sodium saccharin, salvadora persica
bark/root extract, CI 16255, CI 19140, CI 73360, CI
77268:1, CI 77492, CI 77891, contains fluoride. Active
ingredient: 1.12% sodium monofluorophosphate

Herbal toothpaste Sorbitol, glycerin, aqua, hydrated silica, piper betel leaf
D (betel leaf) extract, sodium lauryl sulfate, PEG-400, carrageenan,
saccharin, melaleuca, alternifolia (tea tree) leaf water,
potassium sorbate, menthol, mentha piperita
(peppermint) oil, C.I. 19140, C.I. 42090.

Herbal Toothpaste Sorbitol, water, hydrated silica, PEG-32, sodium lauryl

E (control, non- sulfate, flavor, cellulose gum, sodium fluoride, sodium
herbal) saccharin, CI 19140, CI 42090.

Table 2. The difference in inhibition (width of the zone of inhibition in mm) vs

Streptococcus mutans from toothpaste

Toothpaste N Mean Standard Deviation p

Herbal A (Clove) 3 25,65 1,390 0,000*

 Herbal B (Betel leaf, 3 14,60 0,856
lime & salt)

Herbal C (Miswak) 3 22,43 1,549

Herbal D (Betel leaf) 3 20,15 0,146

Control (Non Herbal) 3 23,19 1,986

Oneway anova. *Significant (p<0.05)

Table 3. Differences in inhibition of Streptococcus mutans between herbal-containing
toothpastes and control toothpastes

Toothpaste Herbal A Herbal B Herbal C Herbal D Control

Herbal A
- 0,000* 0,015* 0,001* 0,049*
(clove)

Herbal B
(Betel leaf, 0,000* - 0,000* 0,000* 0,000*
lime & salt)

Herbal C
0,015* 0,000* - 0,064 0,503
(Miswak)

Herbal D
0,001* 0,000* 0,064 - 0,020*
(Betel Leaf)

Control (Non
0,049* 0,000* 0,053 0,020* -
herbal)
Least Significant Difference (LSD). *Significant (p<0.05)

Figure 1. Results of the inhibition test of herbal toothpaste against Streptococcus mutans in a
petri dish
Graph 1. Differences in inhibition (width of the zone of inhibition in mm) against S. mutans
from toothpaste after 24 hours
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Manuscript Title: Antibacterial Properties of Herbal Toothpaste on the Market

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relationship or indicate none (add rows as needed) made to you or to your institution)

11 Stock or stock ☒ None

options

12 Receipt of ☒ None
equipment,
materials, drugs,
medical writing,
gifts or other
services
13 Other financial or ☒ None
non-financial
interests

Please place an “X” next to the following statement to indicate your agreement:

☒ I certify that I have answered every question and have not altered the wording of any of the questions on this form.

3 12/13/2021 ICMJE Disclosure Form

 ICMJE DISCLOSURE FORM
Date: 6/22/2023

Your Name: Asmidar Anas

Manuscript Title: Antibacterial Properties of Herbal Toothpaste on the Market

Manuscript Number (if known): Click or tap here to enter text.

In the interest of transparency, we ask you to disclose all relationships/activities/interests listed below that are related to the
content of your manuscript. “Related” means any relation with for-profit or not-for-profit third parties whose interests may be
affected by the content of the manuscript. Disclosure represents a commitment to transparency and does not necessarily
indicate a bias. If you are in doubt about whether to list a relationship/activity/interest, it is preferable that you do so.

The author’s relationships/activities/interests should be defined broadly. For example, if your manuscript pertains to the
epidemiology of hypertension, you should declare all relationships with manufacturers of antihypertensive medication, even if
that medication is not mentioned in the manuscript.

In item #1 below, report all support for the work reported in this manuscript without time limit. For all other items, the time
frame for disclosure is the past 36 months.

Name all entities with whom you have this Specifications/Comments (e.g., if payments were
relationship or indicate none (add rows as needed) made to you or to your institution)

Time frame: Since the initial planning of the work

1 All support for the ☒ None

present
manuscript (e.g.,
funding, provision
of study materials, Click the tab key to add additional rows.
medical writing,
article processing
charges, etc.)
No time limit for
this item.
Time frame: past 36 months

2 Grants or ☒ None
contracts from
any entity (if not
indicated in item
#1 above).

3 Royalties or ☒ None
licenses

1 12/13/2021 ICMJE Disclosure Form

 Name all entities with whom you have this Specifications/Comments (e.g., if payments were
relationship or indicate none (add rows as needed) made to you or to your institution)

4 Consulting fees ☒ None

5 Payment or ☒ None
honoraria for
lectures,
presentations,
speakers
bureaus,
manuscript
writing or
educational
events

6 Payment for ☒ None

expert testimony

7 Support for ☒ None

attending
meetings and/or
travel

8 Patents planned, ☒ None

issued or
pending

9 Participation on ☒ None
a Data Safety
Monitoring
Board or
Advisory Board

10 Leadership or ☒ None
fiduciary role in
other board,
society,
committee or
advocacy group,
paid or unpaid

2 12/13/2021 ICMJE Disclosure Form

 Name all entities with whom you have this Specifications/Comments (e.g., if payments were
relationship or indicate none (add rows as needed) made to you or to your institution)

11 Stock or stock ☒ None

options

12 Receipt of ☒ None
equipment,
materials, drugs,
medical writing,
gifts or other
services
13 Other financial or ☒ None
non-financial
interests

Please place an “X” next to the following statement to indicate your agreement:

☒ I certify that I have answered every question and have not altered the wording of any of the questions on this form.

3 12/13/2021 ICMJE Disclosure Form

 ICMJE DISCLOSURE FORM
Date: 6/22/2023

Your Name: Asdar Gani

Manuscript Title: Antibacterial Properties of Herbal Toothpaste on the Market

Manuscript Number (if known): Click or tap here to enter text.

In the interest of transparency, we ask you to disclose all relationships/activities/interests listed below that are related to the
content of your manuscript. “Related” means any relation with for-profit or not-for-profit third parties whose interests may be
affected by the content of the manuscript. Disclosure represents a commitment to transparency and does not necessarily
indicate a bias. If you are in doubt about whether to list a relationship/activity/interest, it is preferable that you do so.

The author’s relationships/activities/interests should be defined broadly. For example, if your manuscript pertains to the
epidemiology of hypertension, you should declare all relationships with manufacturers of antihypertensive medication, even if
that medication is not mentioned in the manuscript.

In item #1 below, report all support for the work reported in this manuscript without time limit. For all other items, the time
frame for disclosure is the past 36 months.

Name all entities with whom you have this Specifications/Comments (e.g., if payments were
relationship or indicate none (add rows as needed) made to you or to your institution)

Time frame: Since the initial planning of the work

1 All support for the ☒ None

present
manuscript (e.g.,
funding, provision
of study materials, Click the tab key to add additional rows.
medical writing,
article processing
charges, etc.)
No time limit for
this item.
Time frame: past 36 months

2 Grants or ☒ None
contracts from
any entity (if not
indicated in item
#1 above).

3 Royalties or ☒ None
licenses

1 12/13/2021 ICMJE Disclosure Form

 Name all entities with whom you have this Specifications/Comments (e.g., if payments were
relationship or indicate none (add rows as needed) made to you or to your institution)

4 Consulting fees ☒ None

5 Payment or ☒ None
honoraria for
lectures,
presentations,
speakers
bureaus,
manuscript
writing or
educational
events

6 Payment for ☒ None

expert testimony

7 Support for ☒ None

attending
meetings and/or
travel

8 Patents planned, ☒ None

issued or
pending

9 Participation on ☒ None
a Data Safety
Monitoring
Board or
Advisory Board

10 Leadership or ☒ None
fiduciary role in
other board,
society,
committee or
advocacy group,
paid or unpaid

2 12/13/2021 ICMJE Disclosure Form

 Name all entities with whom you have this Specifications/Comments (e.g., if payments were
relationship or indicate none (add rows as needed) made to you or to your institution)

11 Stock or stock ☒ None

options

12 Receipt of ☒ None
equipment,
materials, drugs,
medical writing,
gifts or other
services
13 Other financial or ☒ None
non-financial
interests

Please place an “X” next to the following statement to indicate your agreement:

☒ I certify that I have answered every question and have not altered the wording of any of the questions on this form.

3 12/13/2021 ICMJE Disclosure Form

 Author Contribution Form
European Journal of General Dentistry (EJGD)
Please fill this form and upload it along with your submission on https://www.manuscriptmanager.net/ejgd

Dr. Nejdet Adanir, DDS, PhD

(Editor in Chief - European Journal of General Dentistry)
Associate Professor, Department of Restorative Dentistry
School of Dentistry, King Faisal University
Al Ahsa, KSA, 31982
necdethan@gmail.com

Manuscript ID #:EJGD
Authors:
Nurlindah Hamrun, Anggun Dwitia Ramadhani, Rafika Hasyim, Asmidar Anas, Asdar Gani, Filzah Azalia

Manuscript Title:
Antibacterial Properties of Herbal Toothpaste on the Market

This form must be completed and signed by ALL authors contributing to the manuscript

Individual Conflict of Interest: Each author must confirm that there is no financial conflict of interest with any
commercial enterprise whose products are in any way described in the manuscript, except for possible funding in
support of the study as disclosed under “Acknowledgements”. It must be disclosed if (1) an author is full or part-
time employee of a company, (2) an author has an ongoing advisory relationship with a company, (3) an author
has received any financial remuneration in connection with this manuscript, (4) an author or immediate family
member has an equity interest in a company or owns patents licensed to a company. If a conflict of interest is
indicated, a box on the title page of the article will read “Dr. X discloses that he/she has a financial relationship
with company Y.”
Acknowledgements: Any funding by grants or otherwise, as well as any financial, technical, statistical or other
assistance is acknowledged at the end of the text.
Freedom of Investigation: This statement confirms freedom from any outside interests in study design, data
acquisition and analysis, as well as in interpretation of the data and full disclosure of all results, especially if
“negative”.
Scientific Responsibility Statement: Each author must sign the form specifying his/her contribution to the study
as indicated.
Exclusive Publication Statement: Each author must sign this form thereby certifying that no part of the
manuscript has been previously published in either print or electronic form, and that there has been no
coexisting submission or consideration for publication elsewhere.
Warranties: The authors warrant that the article is their original work and has not been published before. The
authors also warrant that the article does not infringe on the rights of others. If copyrighted material is included
(e.g. figures), the authors have obtained written permission from the copyright owners and have credited the
sources in the article.
I agree with the preceding conditions and provide the appropriate information below.

Corresponding Author’s Name: Nurlindah Hamrun

Signature: Date: June 22nd, 2023
Conflict of Interest: Yes_ No ✔ If yes, specify:
I had unlimited freedom of investigation: Yes _ No ✔
Undisclosed authors have participated in this work: Yes No ✔
My contributions to this study included (check all that apply): ✔ study design; ✔ collection
of data; ✔ data analysis/interpretation; ✔ writing of the manuscript; other, please
specify:

Author’s Name: Anggun Dwitia Ramadhani

Signature: Date: June 22nd, 2023
Conflict of Interest: Yes_ No ✔ If yes, specify:
I had unlimited freedom of investigation: Yes _ No ✔
My contributions to this study included (check all that apply): study design; ✔ collection
of data; data analysis/interpretation; ✔ writing of the manuscript; other, please
specify:

Author’s Name: Rafika Hasyim

Signature: Date: June 22nd, 2023
Conflict of Interest: Yes_ No ✔ If yes, specify:
I had unlimited freedom of investigation: Yes _ No ✔
My contributions to this study included (check all that apply): study design; collection
of data; ✔ data analysis/interpretation; ✔ writing of the manuscript; other, please
specify:

Author’s Name: Asmidar Anas

Signature: Date: June 22nd, 2023
Conflict of Interest: Yes_ No ✔ If yes, specify:
I had unlimited freedom of investigation: Yes _ No ✔
My contributions to this study included (check all that apply): ✔ study design; collection
of data; ✔ data analysis/interpretation; writing of the manuscript; other, please
specify:
Author’s Name: Asdar Gani
Signature: Date: June 22nd, 2023
Conflict of Interest: Yes_ No ✔ If yes, specify:
I had unlimited freedom of investigation: Yes _ No ✔
My contributions to this study included (check all that apply): ✔ study design; collection
of data; ✔ data analysis/interpretation; writing of the manuscript; other, please
specify:

Author’s Name: Filzah Azalia

Signature: Date: June 22nd, 2023
Conflict of Interest: Yes_ No ✔ If yes, specify:
I had unlimited freedom of investigation: Yes _ No ✔
My contributions to this study included (check all that apply): ✔ study design; collection
of data; data analysis/interpretation; ✔ writing of the manuscript; ✔ other, please
specify: Publication

Author’s Name:
Signature: Date:
Conflict of Interest: Yes_ No If yes, specify:
I had unlimited freedom of investigation: Yes _ No
My contributions to this study included (check all that apply): study design; collection
of data; data analysis/interpretation; writing of the manuscript; other, please
specify:

Author’s Name:
Signature: Date:
Conflict of Interest: Yes_ No If yes, specify:
I had unlimited freedom of investigation: Yes _ No
My contributions to this study included (check all that apply): study design; collection
of data; data analysis/interpretation; writing of the manuscript; other, please
specify:

Author’s Name:
Signature: Date:
Conflict of Interest: Yes_ No If yes, specify:
I had unlimited freedom of investigation: Yes _ No
My contributions to this study included (check all that apply): study design; collection
of data; data analysis/interpretation; writing of the manuscript; other, please
specify:
Author’s Name:
Signature: Date:
Conflict of Interest: Yes_ No If yes, specify:
I had unlimited freedom of investigation: Yes _ No
My contributions to this study included (check all that apply): study design; collection
of data; data analysis/interpretation; writing of the manuscript; other, please
specify:

Author’s Name:
Signature: Date:
Conflict of Interest: Yes_ No If yes, specify:
I had unlimited freedom of investigation: Yes _ No
My contributions to this study included (check all that apply): study design; collection
of data; data analysis/interpretation; writing of the manuscript; other, please
specify:

Author’s Name:
Signature: Date:
Conflict of Interest: Yes_ No If yes, specify:
I had unlimited freedom of investigation: Yes _ No
My contributions to this study included (check all that apply): study design; collection
of data; data analysis/interpretation; writing of the manuscript; other, please
specify:

Author’s Name:
Signature: Date:
Conflict of Interest: Yes_ No If yes, specify:
I had unlimited freedom of investigation: Yes _ No
My contributions to this study included (check all that apply): study design; collection
of data; data analysis/interpretation; writing of the manuscript; other, please
specify:

In case of additional authors, please photocopy this form and attach the completed sheets as needed.