ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10005-2631
International Journal of Clinical Pediatric Dentistry
Volume 16 | Issue S-2 | Year 2023

Effect of Passion Fruit Juice in Removal of Smear Layer in Root Canal of Ex Vivo Human Teeth: A Scanning Electron Microscopic Study

Vinothini Venkatachalamoorthi¹, Prathima G Shivashankarappa², Sanguida Adimoulame³, Kayalvizhi Gurusamy⁴, Kavitha Muthukrishnan⁵, Ezhumalai Govindan⁶

¹Vinu Dental Clinic, Gobichettipalayam, Tamil Nadu, India

^2,3Department of Pediatric and Preventive Dentistry, Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, India

⁴Department of Pediatric & Preventive Dentistry, Mathrusri Ramabai Ambedkar Dental College and Hospital, Bengaluru, Karnataka, India

⁵Department of Pediatric & Preventive Dentistry, Mahatma Gandhi Postgraduate Institute of Dental Sciences, Govt of Puducherry Institution, Puducherry, India

⁶Department of Statistics, Mahatma Gandhi Medical College and Research Institute, Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, India

Corresponding Author: Prathima G Shivashankarappa, Department of Pediatric and Preventive Dentistry, Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, India, Phone: 04256250387, e-mail: prathimag@igids.ac.in

ABSTRACT

Context: The smear layer produced during the instrumentation of the root canal contains both organic and inorganic components, and it is recommended to be removed as it has a mixture of bacteria and their byproducts. Irrigants like sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), endodontic irrigant, citric acid, etc., possess the ability to remove the smear layer. Considering the antimicrobial effect, antiinflammatory, cavity disinfectant, nontoxicity, better taste, and aroma of herbal products, a novel approach has been introduced in the field of endodontics.

Aims: To assess the efficacy of passion fruit juice in removing the smear layer and its erosive potential in root canals after instrumentation by scanning electron microscopy.

Materials and methods: About 35 single-rooted premolars were divided into five groups—30% passion fruit extract (PFE), 17% EDTA, 17% EDTA + 5.25% NaOCl, 30% PFE + 17% EDTA, and saline. Following irrigation with the above irrigants, each tooth was split into two halves and examined and scored for smear layer removal (modified Torabinejad’s criteria) using a scanning electron microscope.

Statistical analysis: Overall comparison of the irrigant’s action on removing the smear layer and their erosive potential was done using the Kruskal–Wallis test, and intergroup comparison of the irrigant action was done using the Mann–Whitney U test. All the statistical analyses were set with a significance level of p < 0.05.

Results: A total of 30% PFE produced less erosion and statistically significant smear layer removal in the coronal and middle third of the root. When combined with 17% EDTA, it effectively removed the smear layer in all three-thirds of the root. Around 30% PFE + 17% EDTA is less erosive when compared to 17% EDTA + 5.25% NaOCl.

Conclusion: This study revealed that 30% PFE and 30% PFE + 17% EDTA are promising irrigants as root canal disinfectants in endodontics.

How to cite this article: Venkatachalamoorthi V, Shivashankarappa PG, Adimoulame S, et al. Effect of Passion Fruit Juice in Removal of Smear Layer in Root Canal of Ex Vivo Human Teeth: A Scanning Electron Microscopic Study. Int J Clin Pediatr Dent 2023;16(S-2):S190–S194.

Source of support: Nil

Conflict of interest: None

Conflict of interest: Dr Prathima G Shivashankarappa is associated as the National Editorial Board member of this journal and this manuscript

Keywords: Erosion, Ethylenediaminetetraacetic acid, Passion fruit extract, Smear layer, Sodium hypochlorite

INTRODUCTION

The smear layer in the root canal prevents the penetration of irrigants and intracanal medicaments into dentinal tubules and affects the adherence of the sealer to canal walls.¹^,² Irrigants like sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), endodontic irrigant (MTAD), and citric acid possess the ability to remove the smear layer; nevertheless, no single irrigant dissolves organic tissues and at the same time demineralize the smear layer.³^-⁵ Reports indicate that EDTA and NaOCl are associated with dentinal erosion of canal wall.⁶^,⁷ NaOCl causes complications like chemical burns and tissue necrosis when extruded beyond the root apex, has a disagreeable smell, taste, high toxicity, allergic potential,⁶ and causes corrosion of instruments.⁸ This led to the search for alternative solutions, especially those that are naturally occurring, such as extracts of Morinda citrifolia, German chamomile, tea tree oil, oregano, etc.⁹^,¹⁰ Passion fruit (Passiflora edulis) is an exotic creeper that possesses antibacterial, antifungal, anticarcinogenic, antianxiety, antispasmodic, antihypertensive, cholesterol, lipid-lowering, antiinflammatory, and antioxidant properties.¹¹ Its antimicrobial activity is attributed to the “Passicol” content. It has some properties to be used as an endodontic irrigant, such as antimicrobial activity, ability to remove the smear layer,¹²^-¹⁴ and nontoxic nature.¹⁵ It has an acceptable taste and aroma, which offer added advantages over conventional irrigants.¹²^,¹³ This study aimed to assess the efficacy of passion fruit extract (PFE) in removing the smear layer and also evaluate its erosive potential using scanning electron microscopy.

MATERIALS AND METHODS

This study was approved by the Institutional Ethical Committee. A total of 35 mature, caries-free, single-rooted premolars with a single canal of curvature <10° extracted for orthodontic purposes were collected after obtaining written consent from patients. About 30% alcoholic extract of passion fruit was prepared (Fig. 1).¹³ To check if 30% PFE possessed smear layer-removing properties, and if present, to rule out the possible contribution of alcohol to smear layer removal, a pilot study was done using ethyl alcohol alone and 30% PFE alone as irrigants. Scanning electron microscopic evaluation showed smear layer removal in 30% PFE irrigated samples and no smear layer removal in alcohol-irrigated samples. It was also verified that no chemical reaction occurred between PFE and EDTA. Root lengths of all the teeth were standardized to 15 mm (decoronation). The apices were sealed (sticky wax), and instrumentation was done up to #40 K-file using the step-back technique (Fig. 2). Saline irrigation (3 mL) was done using 30-gauge side vent needles that were inserted into the canal until 2 mm short of the apex (Fig. 3). The teeth were then divided into five test irrigant groups and the investigator was blinded to the allotment (envelop technique). Group I—30% PFE, group II—17% EDTA, group III—17% EDTA + 5.25% NaOCl as a positive control, group IV—30% PFE + 17% EDTA, and group V—saline (negative control), final irrigation of the samples was done with 3 mL of the test irrigant for 1 minute followed by irrigation with 10 mL of distilled water.

Fig. 1: Preparation of alcoholic extract of passion fruit, passion whole fruit and pulp

Fig. 2: Decoronation of the samples and root apex covered with sticky wax

Fig. 3: Working length estimation and irrigation with saline

Canals were dried, the entrance closed with a cotton pellet, and roots were split into halves with chisel and mallet after making deep grooves on buccal and lingual surfaces with care taken not to perforate the root canals. The 70 specimens were then dehydrated using ethyl alcohol (30% for 10 minutes, 50% for 20 minutes, 70% for 20 minutes, 90% for 30 minutes, 100% for 30 minutes, and 100% for 30 minutes) dried and each half was divided into three parts using graphite; apical, middle, and coronal at distances 5, 10, and 15 mm from the apex, respectively. After assembly on coded stubs, the specimens were placed in a vacuum chamber and sputter-coated with a 300 A gold layer (Fig. 4). Both halves of all samples were examined using scanning electron microscope (MAKE - JEOL INDIA Pvt. Ltd/MODEL- JSM–6610LV) and scored using modified Torabinejad’s criteria.

Fig. 4: Samples after sputtering on stubs

Smear Layer

No smear layer: No smear layer on the surface of the root canal; all tubules were clean and open.
Moderate smear layer: No smear layer on the surface of the root canal and/or >75% of dentinal tubules contain debris.
Heavy smear layer: The smear layer covered the root canal surface and the tubules.

Erosion

No erosion: All tubules looked normal in appearance and size.
Moderate erosion: The peritubular dentin was eroded.
Severe erosion: The intertubular dentin was destroyed and tubules were connected to each other.

The scoring was done by the blinded investigator. The images were observed under ×2000 magnification. In each of the 70 samples, scores were recorded at four places for each of the coronal, middle, and apical thirds in order to avoid examiner bias. In the present study, an additional scoring criteria was used to assess the effectiveness of the individual test irrigant.

Smear Layer

Modified Torabinejad scores 1 and 2: Effective.
Modified Torabinejad score 3: Not effective.

Erosion

Modified Torabinejad score 1: Effective (not causing dentin erosion).
Modified Torabinejad scores 2 and 3: Not effective (causing dentin erosion).

Kruskal–Wallis test was used to compare smear layer removal and erosive potential of the five irrigants and pairwise intergroup comparison of the irrigants was done using the Mann–Whitney U test at p < 0.05.

RESULTS

Table 1 shows the mean smear layer removal and erosion scores for each group at each region. Overall comparison for both smear layer removal and erosive potentials showed statistically significant differences among the five irrigants for all the parts of the root canal (coronal, middle, and apical) (p = 0.001). Intergroup comparison for smear layer removal at the coronal, middle, and apical third of the root canal showed that the removal of smear layer in coronal and middle thirds of the root canal was high in 17% EDTA + 5.25% NaOCl group when compared to 30% PFE (p = 0.001), 17% EDTA (p = 0.001), 30% PFE + 17% EDTA (p = 0.002), and saline (p = 0.001). However, smear layer removal in the apical third was high in 30% PFE + 17% EDTA group when compared to 30% PFE (p = 0.015), 17% EDTA (p = 0.038), 17% EDTA + 5.25% NaOCl (p = 0.004), and saline (p = 0.002). Around 30% PFE was better than saline in all the areas (Fig. 5).

**Table 1:** Mean smear layer removal and erosion scores for each group at each region
Groups	Smear layer			Erosion
Groups	Coronal	Middle	Apical	Coronal	Middle	Apical
30% PFE	1.1429	1.6429	1.6429	1.0	1.0	0.4286
17% EDTA	1.0	1.2143	2.6429	2.1071	1.9286	0.5714
17% EDTA + 5.25% NaOCl	1.0	1.0	1.1786	2.7857	2.1429	1.8929
30% PFE + 17% EDTA	1.0	1.1786	1.9286	1.8929	1.0	0.7500
Saline	2.1429	2.7500	2.8929	0.8929	0.2857	0

Fig. 5: Representative SEM images for smear layer removal and erosion of dentinal tubules at the coronal, middle, and apical third of the sample

Intergroup comparisons of erosion at the coronal, middle, and apical third of the root canal demonstrated that 17% EDTA showed less erosion in the coronal, middle, and apical third of the root canal when compared to 17% EDTA + 5.25% NaOCl (p = 0.002) (Fig. 6). Around 30% PFE + 17% EDTA showed less erosion in the coronal, middle, and apical third of the root canal when compared to 17% EDTA + 5.25% NaOCl (p = 0.002) (Fig. 6). Around 30% PFE produced less erosion (coronal and middle thirds) compared to 17% EDTA and 17% EDTA + 5.25% NaOCl.

Fig. 6: Mean erosion scores for each group at each region

DISCUSSION

The persistent increase in antibiotic-resistant strains and side effects of chemicals has impelled researchers to look for herbal substitutes for cleaning root canals. Ress et al. in 2006¹² showed that peach and passion fruit tea possess smear layer removing capacity and demonstrated the antibacterial effect of Passiflora group against Enterococcus faecalis (E. faecalis).¹³^-¹⁵ To the best of our knowledge, this is the first study to assess the efficacy of PFE in terms of smear layer removal and its erosive capacity on canal walls. Jayahari et al.¹³ showed that there were no differences in the antibacterial efficacy of alcoholic and aqueous extracts of passion fruit and that colony-forming units were slightly less in the alcoholic extract groups. Another study used different percentages of alcoholic extracts of passion fruit to assess their antimicrobial action on E. faecalis and found 30% extract to be effective.¹⁴

In the present study 30% PFE was effective in removing the smear layer from coronal to middle thirds but not the apical third of the root canals. The smear layer removing capacity of 30% PFE could be attributed to the acidic nature of the extract. Phytochemical analysis of the alcoholic extract of PEE should be done to understand the mechanism of the smear layer removing property. Studies have proved that 17% EDTA does not remove the smear layer in the apical third of the root canals owing to the high surface tension of EDTA.¹⁶ However when 30% PFE was combined with 17% EDTA the smear layer removing efficacy was better than 30% PFE alone. This could be attributed to the combined chelating action of the EDTA. In the apical third 30% PFE + 17% EDTA was more efficient than 17% EDTA and 17% EDTA + 5.25% NaOCl. This is an important observation as studies have generally shown poor penetration of irrigants into the narrow apical portion of the root canal.¹⁷^,¹⁸ Results could vary with variations in the instrumentation technique (manual, rotary, or activation of irrigant), volume of irrigant used, time of irrigation, and needle used for irrigation. Similar to a few studies¹⁹^,²⁰ the present study also found the EDTA and NaOCl combination to be effective in the apical third.

It was observed that 30% PFE produced less erosion when compared to 17% EDTA and 17% EDTA + 5.25 NaOCl in coronal and middle thirds. When 30% PFE was combined with 17% EDTA the erosion was less compared to 17% EDTA, 17% EDTA + 5.25% NaOCl in both coronal and middle thirds, and less compared to 17% EDTA + 5.25% NaOCl in the apical third. Calt and Serper have suggested that using EDTA for a shorter time or in a smaller volume does not cause erosion.⁴ Around 30% PFE + 17% EDTA produced more erosion as compared to 30% PFE. This could be due to the better removal of the smear layer by the PFE and EDTA combination which could have facilitated the demineralizing effects of EDTA.

Around 30% PEE when used alone could be a promising irrigant in terms of causing less damage to root dentin. Increasing the concentration of the extract, volume of irrigation, and contact time might increase the smear layer removing the efficiency of PEE while maintaining dentin integrity. However according to Smita et al.,¹⁵ as the concentration of passion fruit juice was increased, the zone of inhibition decreased.

The sequence of irrigation of EDTA and NaOCl also has an important role in damaging root dentin.²¹ Studies have demonstrated marked erosion of root canal dentin when NaOCl was used after EDTA.⁴^,⁷ In the present study irrigation with 30% PFE followed by 17% EDTA did not cause much erosion compared to the irrigation with EDTA followed by NaOCl. This study suggests that the PFE along with EDTA could be a safe alternative to NaOCl for smear layer removal.

On comparing the effectiveness of test irrigants in terms of erosive potential in the coronal, middle, and apical thirds, 30% PFE was considered more effective as it did not cause erosion in all the samples examined. Except for four samples in the coronal third and two samples in the apical third, all the remaining samples irrigated with 30% PFE + 17% EDTA did not show erosion.

One of the limitations of this study is that it was done in vitro. Blood, tissue remnants, and a multitude of other variables may affect the actions of agents in the root canal system. Canal curvatures can pose significant challenges and posterior teeth which have narrow canals may give different results. Though passion fruit has several advantages, its tissue dissolving capacity is questionable. More in vitro studies and clinical studies are thus warranted. Moreover, this study was conducted with a small sample size of seven per group. Examination of smear layer removal was done using scanning electron microscopy which has its own limitations being subjective, less comparative, and low reproducibility. Future research can also aim at using passion fruit pulp as an intracanal medicament.¹⁵

CONCLUSION

In terms of smear layer removal, 30% PFE + 17% EDTA was effective than 30% PFE. In terms of erosive potential 30% PFE was least erosive followed by 30% PFE + 17% EDTA and 30% PFE + 17% EDTA was less erosive compared to the gold standard irrigant combination, and 17% EDTA + 5.25% NaOCl.
Around 30% PFE and 30% PFE + 17% EDTA are promising irrigants for root canal disinfection. A larger sample size is required to come to a more definitive conclusion and drawbacks of the scanning electron microscope (SEM) could be overcome by using digital imaging analysis.

Key message: The smear layer during instrumentation of root canal prevents the penetration of irrigants and intracanal medicaments into dentinal tubules and compromises the fluid-tight seal in root canal obturation. The combination of irrigants used currently removes the smear layer but is not without disadvantages, such as eroding dentin, causing chemical burns, and tissue necrosis. PFE irrigant, a herbal alternative, has been found to possess antimicrobial properties.

REFERENCES

1. Teixeira CS, Felippe MC, Felippe WT. The effect of application time of EDTA and NaOCl on intracanal smears layer removal: an SEM analysis. Int Endod J 2005;38(5):285–290. DOI: 10.1111/j.1365-2591.2005.00930.x

2. Yamada RS, Armas A, Goldman M, et al. A scanning electron microscopic comparison of a high volume final flush with several irrigating solutions: part 3. J Endod 1983;9(4):137–142. DOI: 10.1016/S0099-2399(83)80032-6

3. Mello I, Kammerer BA, Yoshimoto D, et al. Influence of final rinse technique on ability of ethylenediaminetetraacetic acid of removing smear layer. J Endod 2010;36(3):512–514. DOI: 10.1016/j.joen.2009.11.004

4. Calt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod 2002;28(1):17–19. DOI: 10.1097/00004770-200201000-00004

5. Kandaswamy D, Venkateshbabu N. Root canal irrigants. J Conserv Dent 2010;13(4):256–264. DOI: 10.4103/0972-0707.73378

6. Spencer HR, Ike V, Brennan PA. Review: the use of sodium hypochlorite in endodontics potential complications and their management. Br Dent J 2007;202(9):555–559. DOI: 10.1038/bdj.2007.374

7. Hariharan VS, Nandlal B, Srilatha KT. Efficacy of various root canal irrigants on removal of smear layer in the primary root canals after hand instrumentation: a scanning electron microscopy study. J Indian Soc Pedod Prev Dent 2010;28(4):271–277. DOI: 10.4103/0970-4388.76157

8. Pujar M, Patil C, Kadam A. Comparison of antimicrobial efficacy of Triphala, (GTP) Green tea polyphenols and 3% of sodium hypochlorite on Enterococcus faecalis biofilms formed on tooth substrate: in vitro. J Int Oral Health 2011:3(2):23–29.

9. Gupta A, Duhan J, Sangwan P, et al. The effectiveness of three different plant extracts used as irrigant in removal of smear layer: a scanning electron microscope study. J Oral Health Dent 2015;9(1):16–22.

10. Ok E, Adanir N, Ozturk T. Antibacterial and smear layer removal capability of oregano extract solution. Eur J Dent 2015;9(1):20–24. DOI: 10.4103/1305-7456.149633

11. Patel SS. Morphology and pharmacology of Passiflora edulis – a review. J Herb Med Toxicol 2009;3(1):1–6.

12. Rees JS, Loyn T, Rowe W, et al. The ability of fruit teas to remove smear layer: an in vitro study of tubule patency. J Dent 2006;34(1):67–76. DOI: 10.1016/j.jdent.2005.03.006

13. Jayahari NK, Niranjan NT, Kanaparthy A. The efficacy of passion fruit juice as an endodontic irrigant compared with sodium hypochlorite solution: an in vitro study. J Investig Clin Dent 2014;5(2):154–160. DOI: 10.1111/jicd.12023

14. Gayathri K, Prathima GS, Sajeev R. Antimicrobial efficacy of passion fruit extract against Enterococcus faecalis- an in vitro study. Int J Sci Res 2020;76–78.

15. Singh S, Das D. Passion fruit: a fetched passion for dentists. Int J Pharm Sci Res 2013;4(2);754–757.

16. Kuruvilla A, Jaganath BM, Krishnegowda SC, et al. A comparative evaluation of smear layer removal by using EDTA, etidronic acid, and maleic acid as root canal irrigants: An in vitro scanning electron microscopic study. J Conserv Dent 2015;18(3):247–251. DOI: 10.4103/0972-0707.157266

17. Jindal V, Palekar A, Jindal D. An in vitro comparative evaluation of smear layer removal using NaOCl, EDTA and MTAD solution as a final rinse- a scanning electron microscopic study. J Oral Health Res 2011;2(2):61–65.

18. Abou-Rass M, Patonai FJ Jr. The effects of decreasing surface tension on the flow of irrigating solutions in narrow root canals. Oral Surg Oral Med Oral Pathol 1982;53(5):524–526. DOI: 10.1016/0030-4220(82)90470-4

19. Lui JN, Kuah HG, Chen NN. Effect of EDTA with and without surfactants or ultrasonics on removal of smear layer. J Endod 2007;33(4):472–475. DOI: 10.1016/j.joen.2006.12.007

20. O’Connell MS, Morgan LA, Beeler WJ, et al. A comparative study of smear layer removal using different salts of EDTA. J Endod 2000;26(12):739–743. DOI: 10.1097/00004770-200012000-00019

21. Bohora A, Hegde V, Kokate S. Comparison of the antibacterial efficiency of neem leaf extract and 2% sodium hypochlorite against E. faecalis, C. albicans and mixed culture - an in vitro study. Endodontology 2010;22(1):8–12. DOI: 10.4103/0970-7212.351972

________________________
© The Author(s). 2023 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.