Journal of Dr. NTR University of Health Sciences

: 2019  |  Volume : 8  |  Issue : 4  |  Page : 244--249

Antimicrobial efficacy of herbal extracts combined with zinc oxide eugenol as an obturating material in primary teeth – An in vitro study

Senapathi Navaneet1, Sridhar Muktineni2, Sai Sankar J. Avula2, Pranitha Kakarla2, Harish C Kommineni2, K Amruthavalli3,  
1 Department of Pedodontics and Preventive Dentistry, Sree Sai Dental College, Srikakulam, Andhra Pradesh, India
2 Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India
3 Department of Botany and Microbiology, Archarya Nagarjuna Unviversity, Guntur, Andhra Pradesh, India

Correspondence Address:
Dr. Sai Sankar J. Avula
Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur - 522 509, Andhra Pradesh


Background and Aims: The aim of this study is to evaluate the antimicrobial efficacy of synthetic obturating material (Zinc oxide eugenol) individually and combined with medicinal plant extracts (Triphala, Aloevera, Tulsi) against four bacterial strains (Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli). Methods: The herbal extracts were independently mixed in different ratios with zinc oxide powder, followed by mixing with eugenol to know the effective concentration using antibacterial assay. Initially different concentrations of each test material were placed into Agar diffusion wells and zones of inhibition were measured against the selected bacteria. The concentration with maximum zone of inhibition for each test material was assessed and grouped as Group I- Triphala and zinc oxide eugenol (80:20), Group II- Aloevera and zinc oxide eugenol (80:20), Group III- Tulsi and zinc oxide eugenol (50:50) and Group IV- Plain zinc oxide eugenol. Results: All the groups exhibited varied antimicrobial activity against test microorganisms which were statistically significant. Highest antimicrobial efficacy was noticed in the group I followed by group II, III and the least with group IV samples. Conclusion: The formulations obtained by incorporating Triphala, Aloevera, Tulsi in Zinc oxide exhibited substantial antimicrobial activity when compared to Zinc oxide eugenol alone. Even though, the tested materials showed good antibacterial activity in this in vitro study, clinical trials need to be conducted before recommending them as obturating materials in primary teeth.

How to cite this article:
Navaneet S, Muktineni S, Avula SS, Kakarla P, Kommineni HC, Amruthavalli K. Antimicrobial efficacy of herbal extracts combined with zinc oxide eugenol as an obturating material in primary teeth – An in vitro study.J NTR Univ Health Sci 2019;8:244-249

How to cite this URL:
Navaneet S, Muktineni S, Avula SS, Kakarla P, Kommineni HC, Amruthavalli K. Antimicrobial efficacy of herbal extracts combined with zinc oxide eugenol as an obturating material in primary teeth – An in vitro study. J NTR Univ Health Sci [serial online] 2019 [cited 2020 Feb 27 ];8:244-249
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Full Text


The usage of plants and plant products as medicines could be outlined way back to the beginning of human civilization. Secondary metabolites and essential oils which are of therapeutic importance are abundantly found in the medicinal plants. Indispensable advantages with the use of medicinal plants in various ailments because they are safe, economical, less toxic, easily available, good shelf life and less chances of developing microbial resistance.[1],[2]

Inspite of all the claimed advantages of natural products, the enthusiasm for synthetic and semi synthetic products to combat infectious diseases has drastically increased after the invention of antibiotics. This is because of their quick and promising results in eliminating infections. In the present era, development of drug resistant microbes, the evolution of superbugs, attainment of nosocomial infections, projected toxicity and side effects of these medicaments spurred the world to look at alternative health remedies.[3]

The usage of herbal extracts in dentistry as irrigants and intracanal medicaments has gained popularity in the recent decades because of their antimicrobial activity, biocompatibility, anti-inflammatory and anti-oxidant properties. In the literature, it has been shown that herbal extracts have an exceptional role as root canal medicaments to overcome disadvantages of allopathic conventional drug formulations.[4]

The reason behind the failure of endodontic treatment might be the persistent infection in the root canals even after chemo mechanical preparation and placement of intracanal medicament. To combat this, it is advisable to use obturating material with good antimicrobial activity. Thus, theIn Vitro study was planned to evaluate the antimicrobial activity of herbal formulations i.e. Triphala, Aloe Vera and Tulsi by mixing with zinc oxide and eugenol that can be used as an obturating material in the primary dentition.


The presentIn Vitro microbiological study was designed and the institutional ethical clearance was obtained. The herbal extracts, Triphala (Baidyanath Tiphala churna, Mfg. Lic. No.ND/AYU/60, Nagpur), Aloevera (Aloevera powder-Anjaneya herbals, Mfg. Lic. No.A-1511/Ayur, Vijayawada) and Tulasi (Tulasi Choornam, Mfg. Lic. No.T-1635/Ayur, Grovel Herbal and Ayurvedic products, Medak) were procured in powdered form. Freeze dried forms of microorganisms Streptococcus mutans (MTCCNo. 497), Staphylococcus aureus (MTCC No. 1144), Escherichia coli (MTCC No. 452) and Enterococcus faecalis (MTCC NO.439) were obtained from microbial type culture collection, Chandigarh, India. These lyophilized forms of four different bacterial species were activated by growing them on their respective selective media.

Antimicrobial assay

Powder and liquid ratios were standardized according to the formula given by Tchaou et al. (1995)[5] i. e., 0.2 gm of zinc oxide powder mixed with 0.07 ml of eugenol. Based on this formula herbal powders were measured by weight and mixed in different ratios [Table 1] with zinc oxide powder for effective concentration. Digital weighing machine and a micropipette were used to measure the exact amount of powder and liquid to be dispensed. Each test material thus prepared was mixed with eugenol to obtain a creamy mix and used for antibacterial assay.{Table 1}

Initially, different concentrations of each test material were placed into agar diffusion wells [Figure 1] and zones of inhibition were measured against all the test microorganisms. The concentration with maximum zone of inhibition for each test material was assessed. The maximum inhibition zones were observed at 80:20 for Triphala: ZnOE (Group I), 80:20 for Aloevera: ZnOE (Group II), 50:50 for Tulsi: ZnOE (Group III). The plain zinc oxide mixed with eugenol was used as control (Group IV).{Figure 1}

Later, agar well diffusion method prescribed by National committee for clinical Laboratory Standards (NCCLS 2000)[6] was employed in antimicrobial susceptibility testing for the aqueous extract concentrations of each test material. Agar media (100 ml) was sterilized in separate conical flasks, cooled and inoculated with 0.1 ml of the respective test bacterial suspension. After thorough mixing, the inoculated medium was transferred into sterilized petri dishes and on solidification of agar medium, four wells of about 6 mm diameter were punched with a sterilized cork borer.

Just before agar diffusion assay each of the test materials with pre-designated concentration was spatulated on a dry pre sterilized glass slab using a cement spatula at room temperature. 200 mg of each test material was back loaded into a sterile 2 ml syringe and placed into the wells. The inoculated bacterial plates were incubated at 37o C, triplicates were prepared for each test material against each bacterial strain to attain the consistency. The diameter of thus obtained inhibition zones were measured after 24 hours using vernier calipers of accuracy 0.5 mm [Figure 2]. The results obtained were tabulated and subjected to statistical analysis.{Figure 2}


Statistical analysis was performed using ANOVA test and Posthoc Tukey test at 0.05 significance level. Group I showed maximum zone of inhibition against all the test microorganisms followed by group II, III and IV, with statistical significant difference [Table 2]. However, significant difference in antimicrobial efficacy was observed between groups I and III; I and IV; II and IV; II and III (except S. aureus). Intragroup comparison betweenI and II; III and IVexhibited significant difference in S. aureus and E. faecalis [Table 3].{Table 2}{Table 3}


In the prehistoric period, medicines have profoundly propped upon herbal products to treat common ailments which later shifted to allopathic medicines. However, in the recent past, there is revival of interest in traditional medicine because of its safety and efficacy.[7]

The problem under focus nowadays is the emergence of multi drug resistant bacterial and fungal strains with the use of allopathic conventional medication. Antibiotic resistant inhibitors produced from medicinal plants can be used to reduce the resistance of microorganisms to antibiotics. Prabhat et al. (2010)[8] stated that plant extracts have target sites which are active against drug resistant pathogens. During the last three decades the activities of plant extracts against bacteria have been studied extensively. Plants exhibit antimicrobial effects by virtue of phytoconstituents which are non-nutritive plant chemicals having protective or disease preventive properties.[9]

Triphala [tri- three; phala- fruits] has potent antibacterial and anti-inflammatory properties. Tannic acid present in triphala has bacteriostatic and bactericidal properties. It was found to have significant antimicrobial activity against E. faecalis biofilm formed on tooth substrate.[10] Garg P et al. (2014)[11] stated that triphala is as effective as 5.25% sodium hypochlorite against E. faecalis biofilm which might be due to its free radical scavenging action.

Aloevera (Aloe barbadensis) belongs to Liliaceae family. It contains a clear gel and green part of the leaf which surrounds the gel is used to produce dried powder. The main chemical constituents of aloevera are alloins and barbadoins. It was found to have inhibitory effects on S. pyogens and E. faecalis.[12] Gupta et al. (2010)[13] stated that aloevera has anti-inflammatory, antibacterial, antifungal, antiviral, moisturizing, wound healing and pain relief properties. Kurian et al. (2016)[14] showed that minimal inhibitory concentration (MIC) of aloevera was superior to calcium hydroxide in eliminating E. faecalis and its anti-bacterial activity increased with time period.

Jain and Ranjan (2014)[15] revealed that chloroform extract of aloevera had significant antimicrobial effect when compared to alcohol and water against the resistant microorganisms commonly found in the root canal like E. faecalis. Ethanolic extracts of aloevera also has significant antibacterial property against S. mutans and L. acidophilus.[16]

Tulsi (Ocimum sanctum) is used as a medicinal plant in day-to-day practice in Indian homes for various disorders. The principal constituent present in Ocimum sanctum leaves is Eugenol (l-hydroxy-2-methoxy-4-allylbenzene), which is mainly responsible for its therapeutic potential. It also acts as a strong COX-2 inhibitor and the analgesic property of tulsi made it utilized for treatment of dental and mucosal pain.[17] Methanolic extract of tulsi has significant antimicrobial activity against S. mutans, E. faecalis and S. aureus.[18]

With due consideration to available evidence pertaining to medicinal values of the three plants, the present study was carried out to evaluate the antibacterial potential of these plant extracts mixed with zinc oxide and eugenol against four bacterial strains.

Complete disinfection of the root canal space is an important prerequisite for achieving long term success of nonsurgical endodontics. But, the root canal anatomy of the primary teeth is complex and it is challenging to make it an infection free state. This can be done by proper chemomechanical preparation and obturating the root canals with nontoxic materials having good antimicrobial action.

Zinc oxide eugenol (ZOE) is the widely used root canal filling material in primary teeth as it has significant antimicrobial activity against E. faecalis, S. mutans, E. coli and S. aureus.[19],[20] Hashieh et al. (1999)[21] studied the beneficial effects of eugenol and stated that the amount of eugenol released in the periapical area immediately after placement was 104 and falls to 10-6 after 24 hrs, reaching zero after one month. Within these concentrations eugenol is said to have anti-inflammatory and analgesic properties. Considering these valid opinions, the present study was designed to evaluate whether there is any improvement in the antibacterial activity by combining the herbal extracts with ZOE.

According to Schleifer (1984),[22] Ryan (2004)[23] anaerobes (69%) and gram positive bacteria (67.8%) are more prevalent in primary root canal infections. Streptococci (20%), Staphylococci (17.3%), E. coli (11.43%) have been isolated in a large proportion of primary endodontic infections, whereas 29-77% of secondary endodontic infections are comprised of E. feacalis.[22],[23] Thus, these organisms were selected for this study.

Antimicrobial efficacy of any natural or synthetic agent can be evaluated using Broth dilution method, Agar dilution method, Disc diffusion method, Cup plate method or Agar well diffusion method and Ditch plate method. However, agar well diffusion method was used in the present study as it depends upon the diffusion of the tested material to such an extent that growth of the added microorganism is prevented entirely in a zone around the well containing the test material. Similarly Kriplani et al. (2013)[6] also employed agar well diffusion method to evaluate the antimicrobial efficacy of different groups as it is more reliable, acceptable and easy to perform.

In the present study group I showed significant antimicrobial activity against all the test organisms but maximum antibacterial activity was found against S. aureus followed by S. mutans, E. coli and E. faecalis. Compared to plain ZOE, more antibacterial activity was noticed when ZOE was mixed with herbal extracts [Table 2].

Group II samples exhibited maximum antibacterial activity against S. mutans followed by E. faecalis, S. aureus and E. coli whereas group III showed maximum antibacterial activity against S. aureus followed by E. faecalis, S. mutans and E. coli. In group IV maximum antibacterial activity was observed against S. aureus followed by S. mutans, E. coli and E. faecalis [Table 3]. The mean score for zones of inhibition observed with group II and IV in the present study were similar to the study results by Kriplani et al. (2013).[6]

The Zone of inhibition values obtained in the present study with Triphala formulation against E. faecalis were superior to the values obtained in the study by Bhargava et al. (2015).[24] These variations could be attributed to the difference in the formulations. The mean Zone of inhibition against S. aureus with aloevera formulation observed in this study almost coincided with the study results of Thiruppathi et al. (2010)[25] However, the zone of inhibition against S. mutans was less than that obtained by Sharma et al. (2015).[26]

Tulsi formulation in the present study exhibited a superior zone of inhibition against E. faecalis when compared to the study conducted by Subbiya et al. (2013).[27] Nevertheless the zone of inhibition obtained against S. mutans, E. faecalis and S. aureus were inferior than those obtained in the previous study by Mistry et al. (2014).[28] These variations in the results can be attributed to the formulation used and to some extent the difference in the tulsi species selected.

The antimicrobial efficacy of any test agent depends upon its diffusion capacity through the solid agar medium which will elicit the zone of inhibition. The variability in the inhibition zones noticed in the present study can be attributed to the formulations used (powdered form) that provided a compatible mix with zinc oxide eugenol. In contrast, ethanolic or methanolic extract forms of herbs were used in previous studies. These alterations can enhance or reduce the actual antimicrobial activity of these formulations. Another reason could be the type of organism selected. In the present study, lyophilized forms were used whereas in the earlier studies the organisms have been isolated directly from the root canals.

Apart from this, the clinical applicability of these antimicrobial agents of plant origin mixed with zinc oxide eugenol needs to be considered as it has certain limitations like obtaining a homogenous mix, use of preservatives for better shelf life and biological issues like tissue irritation and resorption pattern. Further studies are required to evaluate their biocompatibility and safety concerns before recommending them as an obturation material in primary teeth.


The formulations obtained by incorporating Triphala showed superior antimicrobial activity against all the tested microorganisms followed by Aloevera, Tulsi and plain Zinc oxide eugenol. However, the synergetic effect needs to be further evaluated for clinical application as obturating materials in primary teeth.

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Conflicts of interest

There are no conflicts of interest.


1Bhateja S, Arora G. Therapeutic benefits of holy basil (tulsi) in general and oral medicine: A Review. Int J Res Ayur Pharm 2012;3:761-4.
2Williams MG, Simon OR, West ME. The past and present use of plants for medicines. West Indian Med J 2006;55:217-8.
3Fabricant DS, Fransworth NR. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 2001;109:69-75.
4Pujar M, Patil C, Kadam A. Comparison of antimicrobial efficacy of Triphala, Green tea polyphenols and 3% of sodium hypochlorite on Enterococcus faecalis biofilms formed on tooth substrate: In vitro study. J Int Oral Health 2011;3:23-30.
5Tchaou WS, Turng BF, Minah GE, Coll JA. Inhibition of pure cultures of oral bacteria by root canal filling materials. Pediatr Dent 1996;18:444-9.
6Kriplani R, Thosar N, Baliga MS, Kulkarni P, Shah N, Yeluri R. Comparative evaluation of antimicrobial efficacy of various root canal filling materials along with aloevera used in primary teeth: A microbiological study. J Clinc Pediatr Dent 2013;37:257-62.
7Puja C, Shyam S, Nagesh L. Evaluation of antibacterial potential of Aloe vera extracts against streptococcus mutans and lactobacillus acidophilus: An in vitro study. J Indian Ass Public Health Dent 2012;19:8-12.
8Prabhat, Ajaybhan, Navneet, Chauhan A. Evaluation of antimicrobial activity of six medicinal plants against dentalpathogens. Rep Opin 2010;2:37-42.
9Megalaa N, Kayalvizhi G, Silas AJ, Sajeev R, Saravana Kumar MS. Role of herbal leaf extracts in caries prevention. Int J Contemp Med Res 2014;1:71-8.
10Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K, Sehgal PK, Sukumaran VG. Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and Green tea polyphenols), MTAD, and 5% Sodium hypochlorite against Enterococcus faecalis biofilm formed on tooth substrate: An In Vitro study. JOE 2010;36:83-6.
11Garg P, Tyagi SP, Sinha DJ, Singh UP, Malik V, Maccune ER. Comparison of antimicrobial efficacy of propolis, morinda citrifolia, azadirachta indica, triphala, green tea polyphenols and 5.25% sodium hypochlorite against enterococcus fecalis biofilm. Saudi Endod J 2014;4:122-7.
12Wynn RL. Aloe Vera gel: Update for dentistry. Generaldentistry 2005:53:6-9.
13Gupta N, Bhat M, Devi P, Girish M. Aloe vera: A natures gift to children. Int J Clinc Pediatr Dent 2010;3:87-92.
14Kurian B, Swapna DV, Nadig RR, Ranjini MA, Rashmi K, Bolar SR. Efficacy of calcium hydroxide, mushroom, and Aloe vera as an intracanal medicament against Enterococcus faecalis: An in vitro study. Endodontology 2016;28:137-42.
15Jain P, Ranjan M. Role of herbs in root canal irrigation-A review. J Pharm Bio Sci 2014;9:6-10.
16Mahabala KY, Shrikrishna SB, Natarajan S, Nayak AP. Ethanolic extracts of Aloe vera and propolis as cavity disinfectants: An in vitro study. Dent Hypotheses 2016;7:61-6.
17Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum linn (tulsi) with a note on eugenol and its pharmacological actions: A short review. Indian J Physiol Pharmacol 2005;49:125-31.
18Shetty RM, Anita G, Bhavana G, Tamrakar A. An antimicrobial efficacy of guava and tulsi against Streptococcus mutans and Enterococcus faecalis: In Vitro study. Int Res J Nat and App Sci 2015;2:89-97.
19Praveen P, Anantharaj A, Venkataraghavan K, Prathibha Rani S, Sudhir R, Jaya AR. A review of obturating materials for primary teeth. Streamdent 2011. Available from:
20De Queiroz AM, Filho PN, Da Silva LAB, Assed S, Da Silva RAB, Izabel ITO. Antibacterial activity of root canal filling materials for primary teeth: Zinc oxide and eugenol cement, Calen paste thickened with Zinc oxide, Sealapex and EndoREZ. Braz Dent J 2009;20:290-6.
21Hashieh IA, Pommel L, Camps J. Concentration of eugenol apically released from zinc oxide-eugenol-based sealers. J Endod 1999;25:713-5.
22Schleifer KH, Kilpperbalz R. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int J Syst Bacteriol 1984;34:31-4.
23Ryan K, Ray CG. Sherris Medical Microbiology: An Introduction to Infectious Diseases. 3rd ed. New York: McGraw-Hill Medical; 2004.
24Bhargava K, Kumar T, Aggarwal S, Zinzarde S, Sanap A, Patil P. Comparative evaluation of the antimicrobial efficacy of neem, green tea, triphala and sodium hypochlorite: An in vitro study. J Dent Res Rev 2015;2:13-6.
25Thiruppathi S, Ramasubramanian V, Sivakumar T, Arasu T. Antimicrobial activity of Aloevera (L.) Burm. f. against pathogenic microorganisms. J Bio sci Res 2010;1:251-8.
26Sharma M, Dorwal R, Bhat KG, Kashyap N, Chandrasekhar P, Bagri S. Comparative evaluation of the antibacterial efficacy of the Aloevera and Tulsi: An in vitro Study. J Res Adv Dent 2015;4:170-5.
27Subbiya A, Mahalakshmi K, Pushpangadan S, Padmavathy K, Vivekanandan P, Sukumaran VG. Antibacterial efficacy of Mangifera indica L. kernel and Ocimum sanctum L. leaves against Enterococcus faecalis dentinal biofilm. J Conserv Dent 2013;16:454-7.
28Mistry KS, Sanghvi Z, Paramar G, Shah S. The antimicrobial activity of Azadirachta indica, Mimusops elengi, Tinospora cardifolia, Ocimum sanctum and 2% Chlorhexidine gluconate on common endodontic pathogens: An In Vitro study. Eur J Dent 2014;8:172-7.