Grapefruit Seed Extract as an Alternative to Eliminate E. Faecalis in No Instrumented Endodontic Technique Volume 8 - Issue 3

Sandoval Palazuelos CY¹, Rayos Verdugo JY¹, Rivera Chaparro F², Ramírez Álvarez M¹, Loyola Rodríguez JP³, Soto Sainz JE³ and Silva Benítez EL³*

• ¹Facultad de Odontología, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, México
• ²Facultad de Ciencias Químico-biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, México
• ³Maestría en Rehabilitación Oral Avanzada, Facultad de Odontología, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, México

Received: February 14, 2022;   Published: February 24, 2023

*Corresponding author: Silva Benítez Erika de Lourdes, PhD, Ciudad Universitaria, Josefa Ortiz de Domínguez S/N y Avenida de las Américas, CP. 80010. Culiacán, Sinaloa, México

DOI: 10.32474/IPDOAJ.2023.08.000287

Abstract PDF

No instrumentation endodontic treatment (NIET) is a new therapy to treat primary dental organs using a mix of different antibiotics to eliminate microorganisms present in necrotic pulp tissue. Until now, there are several publications that show promising clinical results with this technique but is still controversy the use of this drugs may cause bacterial resistance; for this reason, we consider that the use of substances of natural origin could be a new option.

Objective: To evaluate the E. faecalis inhibition of grapefruit seed extract and their capacity to potentiate the antimicrobial effect of CTZ paste.

Materials and methods: Different natural vehicles were evaluated alone and mixed with CTZ (propylene glycol, grapefruit seed extract, eugenol and as negative control 0.9% saline solution) A clinical isolated of E. faecalis was cultivated and distributed throughout plates with Müller Hilton agar to place a triplicate of absorbent paper disks embedded with the different vehicles and the mixes and incubated for 24 h at 37 ºC. Data were analyzed and One-way ANOVA and Tukey test were used with a significance level of p<0.05.

Results: For only vehicles, grapefruit seed extract presented the greatest inhibition with a halo of 30.9 mm showing a statistically significant difference with the rest of the vehicles. When vehicles were mixed with the CTZ paste, propylene glycol showed a greater inhibition with a statistically significant difference with the rest of the vehicles.

Conclusion: To the inhibition of E. faecalis could be possible the introduction of natural products in the application of NIET.

Keywords: Non instrumented endodontic technique; CTZ; grapefruit seed extract; E. faecalis

Dental caries is the most prevalent oral disease in children, if this bacterial infection is not treated rapidly could involve dental pulp causing necrosis. To eradicate this polymicrobial infection, a has been proposed the noninstrumentation endodontic treatment (NIET) using a mixture of antibacterial drugs placed over the pul par floor [1] with the objective to prevent over instrumentation of root canals and irritation of periapical tissue decreasing chair time to only one visit [2]. In this context, CTZ (chloramphenicol, tetracycline, zinc oxide and eugenol) paste has been suggested for treatment of deciduous teeth with pulp necrosis [3]. Although this and other mixes of antibiotics have shown promising results [4,5] the risk of hypersensitivity and increment of bacterial resistance lead us to evaluate natural origin alternatives with the capacity to eliminate microorganisms as Enterococcus faecalis (E. faecalis), a gram positive bacteria resistant to endodontic treatment [6] for their capacity to invade dentinal tubules, resist nutritional deprivation [7], as well as, delayed penetration of antimicrobial agents by the presence of its enterococcal surface protein (Esp) [8]. Grapefruit seed extract is a natural product obtained of Citrus paradisi, grinding their seeds, pulp and white membranes mixing them with glycerin [9]. This extract has shown a powerful antimicrobial activity [10] mainly attributed to the presence of polyphenolic compounds, flavonoids, citric and ascorbic acid, tocopherol and limonoid [11]. Propylene glycol (1,2-propanediol), a dihydric alcohol [12], has bactericidal activity [13], as a vehicle has demonstrated to improve the diffusing property of medicaments [14]. Therefore, the aim of this study is to evaluate if these natural products inhibit E. faecalis by themselves and have the capacity to potentiate the antimicrobial effect of CTZ paste.

A clinical isolate was cultivated and characterized through PCR assay, confirming the presence of E. faecalis; this was maintained at standard conditions. The CTZ (Farmacia Galenico, Mexico) was commercially obtained to avoid the variation in the formulation and the presence of excipients. The evaluated natural products were 46% grapefruit seed extract (Nutribiotic, Mexico), 99.5% propylene glycol (Harleco, Mexico), as positive control 37.5% eugenol (Viarden, Mexico) and as negative control 0.9% saline solution (PISA, Mexico). E. faecalis was adjusted to 0.5 McFarland standard, a bacterial suspension of 100 μl was distributed throughout Müller Hilton agar (Becton Dickinson, USA) to place a triplicate of absorbent paper disks of 5 mm in diameter impregnated with 5 μl of the different natural products and incubated 24 h at 37 ºC. To evaluate if these natural products potentiate the antimicrobial effect of CTZ paste, 500 mg of CTZ were mixed with 400 μl of different natural products to obtain a paste with a firm and adhesive consistency as clinically applied. Inoculated plates with a triplicate of absorbent paper disk impregnated in each mixture were incubated 24 h at 37 ºC. After incubation period inhibition zone was measured in millimeter with a digital vernier (CALDI-6MP, Truper). For the data analysis t-test and normality Shapiro-Wilk test were used with a significance level of p<0.05 using Sigma Plot Software, version 11 (Systat, Software Inc.).

Eugenol was used as a positive control to evaluate the bacterial inhibition increment against E. faecalis. In this study grapefruit seed extract showed the highest inhibition activity (2.17 times of increment), which had a statistically significant difference (p< 0.05). On the other hand, propylene glycol showed a negative value (-0.03) for bactericidal activity increment compared with eugenol solution (Table 1). Results of combined preparations showed a slight increase in bactericidal activity with CTZ + grapefruit seed extract compared with CTZ + eugenol, which es the vehicle normally used for the application of this paste (Table 1). CTZ + propylene glycol has a statistically significant difference (p< 0.05).

Table 1: Comparison of bacterial inhibition between the different products tested against E. faecalis.

Our findings suggests that grapefruit seed extract could be used directly without combining with other materials, could even be used in the cleanising phase in endodontic procedures in primary and permanent teeth that involve periodontal tissues without side effects since is biocompatible. This green technology extracts are already being evaluated as future option as intracanal irrigants, fruit extracts such blueberry and wild strawberry [15] and the obtention of enzymes from the peel of pineapple, orange and papaya [16] has been proposed as an alternative to the use of sodium hypochlorite, avoiding damage and toxic effects caused by the use of this substance. In relation to combined preparations results, propylene glycol showed better results, this vehicle is broadly used in combination with antibiotics such as tri-antibiotic paste (TAP: ciprofloxacin, metronidazole and minonocycline) to eliminate complex root canal infections [17–19]. Our results coincide with previous reports of clinical studies where the combination of this vehicle with antibiotic pastes shown radiographical and clinical success [20], mainly for the bacterial elimination in the area. We suggest that propylene glycol could be an excellent vehicle for CTZ as it could increase the clinical success of this paste. Although NIET proposes the use of topical antibiotic mixtures to treat deciduous teeth with chronic infection, these pastes may develop antibiotic resistance or cannot been used in resistant patients, therefore other medicament options should be explored. Grapefruit seed extract could be a promissory option, their natural origin makes it biocompatible and in the same time this single solution has antimicrobial capacity against E. faecalis, a very resistant bacteria present in the oral microbiome associated with active dental infections, highly associated with failure in endodontic treatment of primary teeth [21].

The present study shows that grapefruit seed extract has antibacterial activity against an endodontic clinical isolate of E. faecalis; we conclude that this extract could be a future excellent natural option for NIET.

We sincerely thank Dist. de Prod. y Mat. Prim. Alim. Hidalgo S. A. de C. V. for the donation of the grapefruit seed extract used in this work.

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