BOHR International Journal of Current research in Dentistry
2023, Vol. 2, No. 1, pp. 22–28
DOI: 10.54646/bijcrid.2023.14
www.bohrpub.com
ORIGINAL RESEARCH
Investigation on the antioxidant, antibacterial, and antibiofilm
properties of three distinct herbal tooth powders
A. S. Smiline Girija 1 , Pachamuthu Balakrishnan 1 , Shanmugam Saravanan 1 , R. Aravindhan 2 , P. Selvam 2 and
Rajesh Kanna Gopal 1*
1 Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences,
Chennai, Tamil Nadu, India
2 R&D Center, Aravindh Herbals Pvt. Ltd., Rajapalayam, India
*Correspondence:
Rajesh Kanna Gopal,
[email protected]
Received: 07 September 2023; Accepted: 29 September 2023; Published: 05 December 2023
Introduction: Oral health is entirely dependent on removing dental plaque on the tooth surface formed by various
unique micro-organisms. Herbal toothpaste is equivalent to non-herbal fluoride toothpaste in removing dental
plaque. Additionally, herbal toothpaste is advantageous in the strengthening of gums.
Methods: In this study, three herbal tooth powders, Brindha tooth powder (BTP), Red tooth powder (RTP),
and Kosali tooth powder (KTP), were tested for antioxidant, antibacterial, and antibiofilm activities against
clinical isolates.
Results: Brindha tooth powder (BTP) has the greatest free-radical scavenging percentage of 52.78%, followed
by 40.9% for RTP, and 39.2% for KTP. Comparatively, RTP and BTP showed significant inhibition of Enterococcus
faecalis, Streptococcus mutans, and Pseudomonas aeruginosa at three different concentrations (10, 5, and 2.5 mg).
Among them, RTP showed a higher antibacterial effect with no inhibitory effect by KTP. However, antibiofilm activity
was not observed with the toothpaste tested.
Conclusion: The study findings revealed promising and varying degrees of antioxidant and antimicrobial properties
of the three different tooth powders.
Keywords: herbal tooth powder, antioxidant activity, antimicrobial activity, antibiofilm activity
1. Introduction Some of the oral pathogens are Candida albicans,
which forms biofilm along with Streptococcus mutans
It been estimated that 3,500 million people are affected causing dental plaque (4), Porphyromonas gingivalis
by untreated oro-dental diseases due to poor oral (Gingivitis), Lactobacillus (Tooth-caries) (5, 6), and
hygiene, but it was neglected in the global health Staphylococcus aureus (7). Other anaerobic pathogens
agenda (1). Thus, the World Health Organization has include Atopobium, Leptotrichia, Lactobacillus salivarius,
recently adopted a resolution on the promotion of and Prevotella (8).
global oral health (2). Approximately 700 different Oral pathogens were also reported to mediate other
kinds of microbes have been reported in the human systemic diseases such as endocarditis (9), rheumatoid
oral microbiota, including pathogenic and probiotic arthritis (10), and some other autoimmune diseases.
species; the most common organisms found in the Oral pathogens are now understood to play a vital role
oral cavity are Firmicutes, Fusobacteria, Gracilibacteria, in the pathogenesis of several oral disorders, including
Saccharibacteria, Chlamydiae, Bacillus, Actinomycetes, oral cancer, endodontic infections, dental caries, and
Proteobacteria, Spirochaetes, and Synergestitis (3). periodontal diseases. In some cases, the virulence
22
10.54646/bijcrid.2023.14 23
of pathogenic oral microbes is enhanced in patients done in triplets, and the average mean was interpreted and
affected with diabetes (11). Through the esophagus, resulted.
the oral microbiota directly enters the digestive system,
disrupting the intestinal micro-ecology and altering the
digestive system. Tooth caries is a general condition 2.2. Determination of in vitro antimicrobial
that infects people of all age group and has a higher
activity
rate of incidence. Children are more likely than adults
to develop dental caries, which is intimately tied to the About 1 g samples of BTP, RTP, and KTP were extracted with
oral microbiome. According to a prior study, youngsters 100 mL of 100% methanol for 2 days in a glass conical flask.
who frequently eat sweets before bed are more likely to After filtering the crude solvent with Whatman No. 1 filter
develop dental caries. paper, the filtrate was left to sit in an incubator overnight
Antibiotic treatment is effective but antimicrobial at 40◦ C to allow the solvent to evaporate and produce the
resistance (AMR) is triggered among such pathogens in extract. 200 mg samples of BTP, RTP, and KTP extracts were
hospitals and is an alarming threat to global health (12). diluted and vortexed in 1 mL of sterile DMSO for analysis.
Therefore, effective removal of dental plaque is required to Three different concentrations of the extracts were prepared
maintain proper oral hygiene (13). for the study, viz., 10, 5, and 2.5 mg.
Toothpaste plays an important role in removing For the antimicrobial bio-assay, fresh broth suspensions
pathogenic microbes in our oral cavity in our daily life were prepared for the test microorganisms up to 0.5
and improving our quality of life (14). Herbal tooth McFarland standards. The test microorganisms chosen for
powder or plant extracts are used in rural villages the study were clinical strains of Pseudomonas aeruginosa
of South Asian countries and are reported to possess (Gram-negative) (PA) and Enterococcus faecalis (EF). Briefly,
antipyretic, analgesic, antibacterial, antiviral, antioxidant, the test microorganisms were made as lawn cultures onto
anti-carcinogenic, and anti-inflammatory properties sterile Mueller Hinton (MH) agar plates, and wells were cut
(15, 16). In addition to this, herbal toothpaste is using a sterile agar cutter. Next, 50 µL extracts at three
effectively equivalent to non-herbal fluoride toothpaste different concentrations were added to the corresponding
in removing dental plaque and strengthening the gums wells. After incubation at 37◦ C for 24 h, the ZOI (clear
(17–19). zone) in and around the wells was measured and recorded.
Therefore, in the present investigation, three different The tests were done in triplets, and the average mean was
herbal tooth powders, Red tooth powder (RTP), Brindha interpreted and recorded.
tooth powder (BTP), and Kosali tooth powder (KTP), were
tested for their antioxidant and antimicrobial activity on
three different clinical isolates of Enterococcus faecalis
(EF), Streptococcus mutans (SM), and Pseudomonas 2.3. Determination of antibiofilm activity
aeruginosa (PE).
Before the commencement of the assay, fresh MH broth
suspension of test microorganisms was prepared up to 0.5
McFarland Standards. Then the 96-well plate was loaded with
2. Materials and methods MH broth, sample, and inoculum to reach a total volume of
200 µL per well (80 µL broth + 100 µL test sample + 20 µL
2.1. Evaluation of antioxidant activity inoculum) (Rows A to F at different concentrations from 5 to
(DPPH free-radical scavenging assay) 0.1 5 mg). Row G is control, and H is sterile control, where
the former had 180 µL of broth and 20 µL of inoculum,
The antioxidant potential of the samples Brindha tooth and the latter contained 200 µL of the broth alone. The
powder (BTP), Red tooth powder (RTP), and Kosali tooth test microorganisms involved in this study were Enterococcus
powder (KTP) (Aravindh Labs Pvt. Ltd.) was determined faecalis (EF), Streptococcus mutans (SM), and Pseudomonas
based on the DPPH assay (20). About 100 µL of the test aeruginosa (PE). The complete design of the antibiofilm
samples each was taken in separate wells in a microtitre assay for this study is clearly illustrated in Figure 1. After
plate, added with the same volume of DPPH (in 0.1% incubation for 48 h in an incubator at 37◦ C, the wells
methanol), and incubated for 30 min under dark conditions. were washed with dis. H2 O, and each well received 50 µL
Ascorbic acid (Vitamin C) was used as a control. After of 0.1 percent crystal violet staining solution, which was
incubation, the change in color of the solution was observed then incubated for 10 min. The wells were washed with
at 520 nm in an ELISA plate reader and the absorbance dis. H2 O and about 100 µL of 70% ethanol was added
(ABS) values were recorded. The percent DPPH radical to each well and again incubated for 5 min. Finally, an
scavenging activity was calculated as [(ABS of ctrl–ABS ELISA plate reader was used to measure the absorbance
of the test sample)/(ABS of ctrl)] × 100. The tests were values at 595 nm.
24 Girija et al.
FIGURE 1 | A template of a 96-well plate in which the antibiofilm assay was designed, where, BTP: Brindha tooth powder; RTP: Red tooth
powder; KTP: Kosali tooth powder; EF: Enterococcus faecalis; SM: Streptococcus mutans; PE: Pseudomonas aeruginosa; C: control; SC: sterile
control; NA: not applicable.
TABLE 1 | DPPH free-radical scavenging activity assay results. (Brindha tooth powder) had shown a moderate inhibition of
all three strains with a ZOI of 18, 15, and 16 mm for 10 mg
S. no. Samples Percentage DPPH free-radical scavenging
concentration for EF, SM, and PE, respectively. It was 14, 12,
1 BTP 52.78% and 13 mm for 5 mg, and 9, 11, and 10 mm for 2.5 mg for EF,
2 RTP 39.2% SM, and PE, respectively (Figure 3). A low rate of inhibition
3 KTP 40.9% was recorded from Kosali tooth powder (KTP) with 11, 12,
4 Ascorbic acid 78.7% and 8 mm for EF, SM, and PE strains, respectively, at 10 mg,
whereas, no zone of inhibition was found at 5 and 2.5 mg
concentrations (Figure 3).
3. Results
3.1. Evaluation of antioxidant activity 3.3. Determination of antibiofilm activity
(DPPH free-radical scavenging assay)
Although the samples BTP, RTP, and KTP had shown
Greater results for DPPH free-radical scavenging were antimicrobial activity against EF, SM, and PE strains, all three
obtained in BTP with 52.78%, followed by KTP (40.9%), and samples did not exhibit an antibiofilm effect against the three
RTP (39.2%) (Table 1). The control ascorbic acid showed bacterial strains under study (Figure 4).
scavenging activity at 78.7%.
4. Discussion
3.2. Determination of in vitro antibacterial
activity Oro-dental disorders arise as a result of a conglomeration of
polymicrobial nature leading to various oral infections like
Based on the results obtained from the in vitro antibacterial dental caries, periodontal diseases, oro-mucosal diseases, and
assay, RTP (Red tooth powder) showed the maximum periapical periodontitis (21). The human oral microbiome
rate of inhibition of all three bacterial strains when consists of both pathogenic and probiotic microbes including
compared to the other two tooth powders. RTP showed archaea, protozoa, fungi, viruses, and bacteria (including
promising antibacterial activity with an average zone of anaerobic). Concurrently, the imbalance between pathogenic
inhibition (ZOI) of 20 mm at 10 mg concentration, and probiotic microbes causes severe oral pathogenic
16 mm at 5 mg concentration, and 12, 11, and 10 mm diseases. Unenviably, oral pathogens are the culprits in
for 2.5 mg concentration for Enterococcus faecalis (EF), causing infective endocarditis leading to atherosclerosis
Streptococcus mutans (SM), and Pseudomonas aeruginosa and coronary heart disease (22). This affects especially the
(PA), respectively (Figures 2, 3). However, the sample BTP population of low economic status in taking care of their oral
10.54646/bijcrid.2023.14 25
FIGURE 2 | Antibacterial activity assay—zone of inhibition, where BTP: Brindha tooth powder; RTP: Red tooth powder; KTP: Kosali tooth
powder; ZOI: zone of inhibition; EF: Enterococcus faecalis; SM: Streptococcus mutans; PE: Pseudomonas aeruginosa.
FIGURE 3 | Antimicrobial activity of (A) Brindha tooth powder (BTP), (B) Red tooth powder (RTP) and (C) Kosali tooth powder (KTP) against the
test micro-organisms Streptococcus mutans, Pseudomonas aeruginosa, and Enterococcus faecalis.
hygiene and medical expenses. In addition, the prolonged use day to prevent dental plaque because “prevention is better
of antibiotics to treat oral pathogens inversely results in the than cure.” (23).
generation of antibiotic-resistant pathogens (12). As a result, Recently, natural products have been given increased
the Centers for Disease Control and Prevention (CDC) urges attention as potential preventative measures for oral
people to practice brushing and flossing their teeth twice a disorders. Due to the adverse consequences of the usage of
26 Girija et al.
FIGURE 4 | Antibiofilm assay showing the (A) microtitre plate after 48 h of incubation and (B) crystal violet assay with no biofilm activity for the
three toothpastes at varying concentrations.
some dangerous chemicals in the majority of toothpastes (25). However, herbal toothpaste has innate antioxidant
and powders on the market, mainly plaque-related disorders activity based on the secondary metabolites of medicinal
such as dental caries have produced an alarming scenario, plants (26, 27). In the present study, the average percentage
especially among young children. Herbal toothpaste is of DPPH free-radical scavenging activity was 52.78, 40.9,
prepared from natural products and effectively controls and 39.2% for BTP, RTP, and KTP, respectively. In a similar
dental plaque and gingivitis which is equivalent to non- study on the herbal formulation (Dabur Red toothpaste), the
herbal and fluoride toothpaste (17, 24). In this study, three activity of scavenging free radicals was 78%, and no activity
different tooth powder formulations, Brindha tooth powder was found in non-herbal fluorinated toothpaste.
(BTP), Red tooth powder (RTP), and Kosali tooth powder In the current investigation, the results obtained from
(KTP), were tested for their efficacy against three common in vitro antimicrobial activity assay (ZOI) indicate that RTP’s
pathogenic bacteria Enterococcus faecalis (EF), Streptococcus inhibition activity is the greatest of all three bacterial strains
mutans (SM), Pseudomonas aeruginosa (PE) isolated from [Enterococcus faecalis (EF), Streptococcus mutans (SM), and
the oral cavity of dental patients at Saveetha Dental College Pseudomonas aeruginosa (PE)]. Similarly, both RTP and
and Hospitals (SDC), SIMATS, Chennai, India. BTP inhibited the proliferation of all three bacterial strains
It is a known fact that the progression of leukocytes and at high and low concentrations, but KTP failed to inhibit
free-radical generation has resulted in periodontal diseases. proliferation at lower concentrations.
Hence, antioxidants in toothpaste must be able to encounter In this line, the Unani polyherbal toothpaste “Sunoon
the plaque-causing pathogenic bacteria and scavenge free Zard” inhibited Staphylococcus aureus (15.33 mm),
radicals. Interestingly, in a recent study, no antioxidant Streptococcus mutans (17.66 mm), Streptococcus pyogenes
activity was recorded in non-herbal fluorinated toothpaste (14.66 mm), Streptococcus viridans (17.33 mm), Streptococcus
10.54646/bijcrid.2023.14 27
epidermidis (14.33 mm), Corynebacterium xerosis Ethical waiver issued from
(18.33 mm), Bacillus cereus (14.66 mm), E. coli (14.33 mm),
Klebsiella pneumoniae (20.33 mm), Pseudomonas aeruginosa
IRB/ethical committee
(22.33 mm), and Proteus vulgaris (13.33 mm) (28). In
Ref. No. IHEC/SDC/WAIVER CERT-FACULTY/22/01.
co-relation with these studies, the present investigation
Reg. No. ECR/1698/Inst/TN/2022 (Registered with
has shown that RTP and BTP inhibit Enterococcus faecalis, Govt. of India).
Streptococcus mutans, and Pseudomonas aeruginosa at
20 mm/10 mg for RTP, and 18, 15, and 16 mm/10 mg
for BTP, respectively, whereas, less activity was recorded
for KTP at 11, 12, and 8 mm/10 mg. In another study, Authors contributions
among nine different herbal toothpaste formulations, a
formulation constituting of extract from pomegranate peel RG: executed the work and drafted the manuscript.
and clove oil demonstrated a high inhibition rate of 26 mm, AG: conceived, design, executed, drafting, evaluated, and
27 mm, and 25 mm against Candida albicans, Streptococcus approved the manuscript in its final form. PB and SS:
reviewed and approved the manuscript in its entirety.
mutans, and Staphylococcus aureus, respectively (29). In
RA and PS: provided the test compounds for the study
another study, some commercial toothpaste had shown
and granted ultimate approval of the article after review.
antimicrobial activity with the zone of inhibition 25.4, 23,
All authors contributed to the article and approved the
and 22.6 mm for Neem, Colgate, and Meswak toothpaste,
submitted version.
respectively, with inhibitory activity against Streptococcus
mutans and Actinobacillus actinomycetemcomitans (30).
An interesting study was carried out in Brazil on the
antimicrobial activity of toothpaste with natural extracts, Acknowledgments
chlorhexidine, and triclosan. Among three different
kinds of toothpaste, triclosan toothpaste hampers the The authors are thankful to the Aravindh Herbal
proliferation of gram-positive bacteria and yeast at a greater Labs for providing the tooth powder samples for the
rate, but no such activity was seen against Pseudomonas present investigation.
aeruginosa. Natural extract toothpaste inhibited all the
bacteria including P. aeruginosa, but no activity was
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