Evaluation of Antimicrobial Activity of Different Essential Oil Gutta–Percha Solvents Against Enterococcus faecalis and Candida albicans

Abstract

Essential oils are a common alternative to chloroform for dissolving gutta–percha. This study evaluated the antimicrobial effects of chloroform and six essential oil gutta–percha solvents: eucalyptus oil, orange oil, clove oil, rosemary oil, grapefruit oil, and castor oil, against Enterococcus faecalis and Candida albicans by using disk diffusion techniques. The impregnated sterile disk with 10 μL of pure, tested solvents was inoculated on agar plates at three time contacts: 3 min, 10 min, and 24 h. The mean diameter of the zone of inhibition (ZOI) of each solvent was measured after 24 h of incubation. Against Enterococcus faecalis, in both 3 min and 10 min contact, rosemary oil had the largest ZOI (11.40 ± 0.90 and 11.55 ± 0.68 mm), and orange oil showed the smallest ZOI (7.90 ± 0.31 and 9.05 ± 0.68 mm), respectively. Eucalyptus oil exhibited ZOI with persistence, while grapefruit oil and castor oil showed no ZOI. After 24 h of contact, the largest ZOI was recorded for orange oil. Against Candida albicans, at all three time points, clove oil produced the largest ZOI (20.25 ± 0.82, 23.10 ± 0.93, 30.59 ± 0.74 mm) and chloroform the smallest (10.4 ± 0.77, 9.85 ± 0.62, 11.6 ± 0.65 mm), for 3 min, 10 min, and 24 h, respectively. Conclusively, clove oil, orange oil, and rosemary oil exhibit significant antimicrobial activity like chloroform.

1. Introduction

Root canal treatment (RCT) is a commonly performed dental procedure aimed at saving a tooth that has become infected or is irreversibly damaged [1,2]. However, despite the advancements in techniques and materials, RCT failure remains a significant challenge in clinical practice [3,4]. Failure can occur due to incomplete cleaning of the root canal system, microbial reinfection, or poor sealing of the canal [5,6]. Among the reasons for RCT failure, the persistence of microbial pathogens such as predominant Enterococcus faecalis and Candida albicans within the root canal is a prominent factor [7]. These microorganisms can form biofilms and resist conventional treatments, complicating the healing process [8,9].

E. faecalis, a facultative anaerobe Gram-positive coccus, is frequently identified in secondary endodontic infections (77%) [10]. It is also present, though in smaller quantities (40%), in the bacterial microbiota of primary endodontic infections [3,10,11,12]. This bacterium is found in large quantities both before and after mechanical preparation, indicating its resistance to endodontic procedures [12]. C. albicans, a type of fungus that can switch between yeast and hyphal forms, may become pathogenic under certain circumstances. This transformation can lead to endodontic treatment failure. It is often identified in about 20% of the cases of secondary endodontic infections, especially in situations associated with periradicular lesions and persistent symptoms [13,14].

Gutta–percha (GP) is the most widely used material for root canal obturation due to its biocompatibility, ease of handling, and long-term stability [15]. However, in some cases, retreatment is necessary. The endodontic retreatment procedure includes the removal of the existing obturation GP material by using organic solvents and retreatment rotary instruments and disinfecting the root canal to eliminate any remaining contaminated tissue, bacteria, or fungi responsible for the endodontic failure before restoring the canals once more [16,17].

Various solvents are used to dissolve GP during root canal retreatment [18,19]. Chloroform has traditionally been one of the most effective solvents for GP due to its ability to quickly dissolve the material [20,21]. Chloroform is a volatile organic compound, and prolonged exposure may lead to significant health risks, including liver damage and carcinogenicity [22,23]. Due to these health risks, many dental professionals have searched for safer alternatives, such as essential oil-based solvents. Essential oils are composed of volatile compounds and are biosynthesized by plants. In recent years, essential oils have been widely used in the field of medicine and dentistry due to their effectiveness, non-toxicity, biocompatibility, antimicrobial properties, and non-carcinogenic nature. Several of these oils have been used as effective gutta–percha solvents and alternatives to chloroform in root canal treatments [3,20,24,25].

According to the previous literature, the following essential oils were reported as effective GP solvents: eucalyptus oil [26], orange oil [27], clove oil [28], rosemary oil [29], grapefruit oil [30], and castor oil [31]. However, the antibacterial properties of most of these essential oils, GP solvents, which help to disinfect surfaces while dissolving GP, were not evaluated. This study aims to evaluate the antimicrobial properties of the aforementioned six essential oils and chloroform, specifically against the persistent endodontic pathogens E. faecalis and C. albicans, and identify those that are most effective in reducing the microbial count while facilitating gutta–percha removal.

2. Materials and Methods

2.1. Microorganisms

The microorganisms used in this study, E. faecalis (ATCC 29212) and C. albicans (ATCC-10231), were provided by the pathology laboratory, Medya Diagnostic Center.

2.2. Tested Gutta–Percha Solvents

Seven GP solvents were chosen to assess their antibacterial activity against E. faecalis and antifungal activity against C. albicans. They were chloroform (99%) (Scharlau, Sentmenat, Spain), eucalyptus oil (99%) (Clinix, Tremblay-en-France, France), orange oil (100%) (MIAROMA, Nuneaton, England, UK), clove oil (100%) (MIAROMA, Nuneaton, England, UK), rosemary oil (100%) (MIAROMA, Nuneaton, England, UK), grapefruit oil (100%) (MIAROMA, Nuneaton, England, UK), and castor oil (100%) (MIAROMA, Nuneaton, England, UK).

2.3. Antimicrobial Effectiveness of Solvents Through Disk Diffusion Method

Each E. faecalis and C. albicans strain was reactivated by transferring it into Thioglycolate broth and incubating at 37 °C for 24 h. After 18 h of culturing at 37 °C, the inoculum was adjusted to the 0.5 McFarland standard (1.5 × 10⁸ CFU/mL). The suspension was then evenly spread over Mueller–Hinton agar plates with 500 μL [32]. Sterile, empty disks (6 mm in diameter) were soaked with 10 μL of pure GP solvents. The antimicrobial activities of selected solvents were evaluated at three time intervals: 3 min, 10 min, and 24 h. For the 3 min and 10 min intervals, the impregnated disks were placed on the agar for 3 min and 10 min, respectively, before being removed. At 24 h intervals, the impregnated disks with gutta–percha solvents were left on the inoculated agar. Then, all plates were incubated at 37 °C for 24 h. After overnight incubation, the diameters of the inhibition zones were measured using a millimeter ruler with an accuracy of 0.5 mm [14].

2.4. Statistical Analysis

The data were assessed for normality using both the Shapiro–Wilk and Kolmogorov–Smirnov tests. The inhibition zones were calculated as the mean and standard deviation for all studied GP solvents at three time intervals. The data were analyzed using one-way ANOVA and the Kruskal–Wallis test to compare all groups. Tukey post hoc test and Mann–Whitney U-test were used for pairwise comparisons, using SPSS version 27 (IBM Corp., Armonk, NY, USA). The significance level was set at p < 0.05.

3. Results

3.1. Antibacterial Properties of GP Solvents Against E. faecalis

The ZOI was measured for all seven GP solvents against E. faecalis. At both 3 min and 10 min intervals, there were statistically significant differences in the ZOI among the study solvents, as determined by the Kruskal–Wallis test (p < 0.001) and ANOVA (p < 0.001), respectively. In both time frames, rosemary oil had the largest ZOI, with means of (11.40 ± 0.90 mm) and (11.55 ± 0.68 mm), respectively. However, at the 3 min mark, the ZOI was observed with persistence. On the other hand, the smallest ZOI was recorded for orange oil, with means of (7.90 ± 0.31 mm) at 3 min and (9.05 ± 0.68 mm) at 10 min. At the 24 h mark, the results showed that orange oil produced the largest ZOI with a mean of (15 ± 0.47 mm), and grapefruit oil (7.6 ± 0.31 mm) produced the smallest ZOI. Castor oil recorded no inhibition zone and was excluded from statistical analysis. The differences in ZOI between the solvents were statistically significant, as confirmed by the Kruskal–Wallis test (p < 0.001) (

Table 1. Descriptive statistics ZOI of GP solvents against E. faecalis. * ZOI with persistence (p).

GP Solvents	Time	N	Mean (mm)	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
						Lower	Upper
Chloroform	3 min	10	10.45	±0.76	0.24	9.90	10.99	9.50	12.00
Eucalyptus oil		10	8.90 * p	±0.69	0.22	8.39	9.40	8.00	10.00
Orange oil		10	7.90	±0.31	0.10	7.67	8.12	7.50	8.50
Clove oil		10	9.45	±0.43	0.13	9.13	9.76	9.00	10.00
Rosemary oil		10	11.40 * p	±0.90	0.28	10.75	12.04	10.00	12.50
Grapefruit oil		10	0	0	0	0	0	0	0
Castor oil		10	0	0	0	0	0	0	0
Chloroform	10 min	10	10.1	±0.65	0.20	9.62	10.57	9.00	11.00
Eucalyptus oil		10	10.5 * p	±1.08	0.34	9.72	11.27	9.00	12.50
Orange oil		10	9.05	±0.68	0.21	8.55	9.54	8.00	10.00
Clove oil		10	10.2	±0.75	0.23	9.66	10.73	9.00	11.50
Rosemary oil		10	11.55	±0.68	0.21	11.05	12.04	10.50	12.50
Grapefruit oil		10	0	0	0	0	0	0	0
Castor oil		10	0	0	0	0	0	0	0
Chloroform	24 h	10	10.85	±0.41	0.13	10.55	11.14	10.5	11.5
Eucalyptus oil		10	8.05	±0.55	0.17	7.65	8.44	7.5	9.5
Orange oil		10	15	±0.47	0.14	14.66	15.33	14	15
Clove oil		10	14.55	±0.55	0.17	14.15	14.94	13.5	15.5
Rosemary oil		10	12.95	±0.43	0.13	12.63	13.26	12.5	13.5
Grapefruit oil		10	7.6	±0.31	0.1	7.37	7.82	7	8
Castor oil		10	0	0	0	0	0	0	0

, Figure 1 and Figure 2).

At the 3 min mark, the Mann–Whitney U-test showed a significant difference between all solvent groups (p < 0.05), except for the comparisons between chloroform and both clove oil and rosemary oil, as well as between eucalyptus oil and both orange oil and clove oil. However, at the 10 min mark, post hoc intergroup analysis showed no significant differences between chloroform, eucalyptus oil, and clove oil (p > 0.05). At the 24 h mark, Mann–Whitney comparisons also revealed significant differences between all groups (p < 0.05), except for the comparison between chloroform and both eucalyptus oil and rosemary oil, as well as between clove oil and both orange oil and rosemary oil), and the comparison between eucalyptus oil and grapefruit (

Table 2. Intergroup comparisons of disk diffusion test of GP solvents against E. faecalis. The mean difference is significant at the p < 0.05 level.

GP Solvents	Time	Chloroform	Eucalyptus Oil	Orange Oil	Clove Oil	Rosemary Oil	Grapefruit Oil
Chloroform	3 min	_	0.006	0.000	0.071	0.259	_
Eucalyptus oil		0.006	_	0.077	0.334	0.000	_
Orange oil		0.000	0.077	_	0.006	0.000	_
Clove oil		0.071	0.334	0.006	_	0.003	_
Rosemary oil		0.259	0.000	0.000	0.003	_	_
Chloroform	10 min	_	0.331	0.003	0.775	0.000	_
Eucalyptus oil		0.331	_	0.002	0.480	0.018	_
Orange oil		0.003	0.002	_	0.002	0.000	_
Clove oil		0.775	0.480	0.002	_	0.000	_
Rosemary oil		0.000	0.018	0.000	0.000	_	_
Chloroform	24 h	_	0.115	0.000	0.004	0.191	0.022
Eucalyptus oil		0.115	_	0.000	0.000	0.004	0.479
Orange oil		0.000	0.000	_	0.516	0.026	0.000
Clove oil		0.004	0.000	0.516	_	0.115	0.000
Rosemary oil		0.191	0.004	0.026	0.115	_	0.000
Grapefruit oil		0.022	0.479	0.000	0.000	0.000	_

).

Mann–Whitney U-test and post hoc multiple comparisons were also used to compare the study solvents across different time intervals: 3 min, 10 min, and 24 h. Significant differences were observed between all three time points for all solvents (p < 0.05), except for a non-significant difference found between 3 min and 10 min intervals for chloroform, clove oil, and rosemary oil (p = 0.437, p = 0.124, and p = 0.883, respectively), as well as between the 3 min and 24 h intervals for chloroform (p = 0.343) (

Table 3. Intergroup comparisons of disk diffusion test of GP solvents against E. faecalis at three time intervals (3 min, 10 min, and 24 h). The mean difference is significant at the p < 0.05 level.

Groups	Chloroform	Eucalyptus Oil	Orange Oil	Clove Oil	Rosemary Oil
3 min–10 min	0.437	0.013	0.022	0.124	0.883
3 min–24 h	0.343	0.044	0.000	0.000	0.000
10 min–24 h	0.033	0.000	0.007	0.002	0.000

).

3.2. Antifungal Properties of GP Solvents Against C. albicans

Kruskal–Wallis statistical analysis revealed a significant difference in the effectiveness of GP solvents tested against C. albicans at three tested time intervals (p < 0.001). At all three time points, clove oil produced the largest average ZOI diameters (20.25 ± 0.82, 23.10 ± 0.93, 30.59 ± 0.74 mm), followed by orange oil (14.55 ± 0.36, 14 ± 1.24, 24 ± 0.81 mm) for 3 min, 10 min, and 24 h, respectively. The smallest ZOI averages were observed with chloroform (10.4 ± 0.77, 9.85 ± 0.62, 11.6 ± 0.65 mm) at the same intervals. Persistence was noted in both grapefruit oil at 3 min and 10 min and in eucalyptus oil at only 3 min. Castor oil showed no inhibition zone and was excluded from the statistical analysis (

Table 4. Descriptive statistics zone of inhibition of GP solvents against C. albicans. * ZOI with persistence (p).

GP Solvents	Time	Mean (mm)	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
					Lower	Upper
Chloroform	3 min	10.40	±0.77	0.24	9.84	10.95	9.50	11.50
Eucalyptus oil		12.50 * p	±0.70	0.22	11.99	13.00	11.00	13.00
Orange oil		14.55	±0.36	0.11	14.28	14.81	14.00	15.00
Clove oil		20.25	±0.82	0.26	19.65	20.84	19.00	21.50
Rosemary oil		12.95	±0.43	0.13	12.63	13.26	12.50	13.50
Grapefruit oil		12.10 * p	±0.45	0.14	11.77	12.42	11.50	12.50
Castor oil		0	0	0	0	0	0	0
Chloroform	10 min	9.85	±0.62	0.19	9.40	10.29	9.00	11.00
Eucalyptus oil		12.25	±0.88	0.28	11.61	12.88	10.50	13.50
Orange oil		14.00	±1.24	0.39	13.10	14.89	11.00	15.00
Clove oil		23.10	±0.93	0.29	22.42	23.77	22.00	24.50
Rosemary oil		12.85	±1.13	0.35	12.04	13.65	10.50	14.00
Grapefruit oil		10.90 * p	±0.45	0.14	10.57	11.22	10.00	11.50
Castor oil		0	0	0	0	0	0	0
Chloroform	24 h	11.60	±0.65	0.20	11.12	12.07	10.50	12.50
Eucalyptus oil		15.50	±0.57	0.18	15.08	15.91	14.50	16.50
Orange oil		24.00	±0.81	0.25	23.41	24.58	22.50	25.50
Clove oil		30.59	±0.74	0.23	30.05	31.12	29.50	31.50
Rosemary oil		15.30	±0.42	0.13	14.99	15.60	14.50	16.00
Grapefruit oil		14.25	±0.42	0.13	13.94	14.55	13.50	14.50
Castor oil		0	0	0	0	0	0	0

, Figure 3).

Mann–Whitney U-test intergroup comparisons revealed significant differences between all solvent groups (p < 0.05), with a few exceptions. No significant differences were found between grapefruit oil and both chloroform and eucalyptus oil at all three points (p > 0.05). Additionally, no significant differences were observed between rosemary oil and eucalyptus oil and between clove oil and orange oil at all three points. A non-significant difference was also found between rosemary oil with both orange oil at 3 min and 10 min, grapefruit oil at 3 min and 24 h, and eucalyptus oil with orange oil at 10 min and 24 h (

Table 5. Mann–Whitney intergroup comparisons of disk diffusion test of GP solvents against C. albicans.

GP Solvents	Time	Chloroform	Eucalyptus Oil	Orange Oil	Clove Oil	Rosemary Oil	Grapefruit Oil
Chloroform	3 min	_	0.011	0.000	0.000	0.001	0.104
Eucalyptus oil		0.011	_	0.011	0.000	0.495	0.364
Orange oil		0.000	0.011	_	0.198	0.064	0.001
Clove oil		0.000	0.000	0.198	_	0.002	0.000
Rosemary oil		0.001	0.495	0.064	0.002	_	0.112
Grapefruit oil		0.104	0.364	0.001	0.000	0.112	_
Chloroform	10 min	-	0.006	0.000	0.000	0.001	0.195
Eucalyptus oil		0.006	-	0.082	0.000	0.504	0.142
Orange oil		0.000	0.082	-	0.081	0.284	0.001
Clove oil		0.000	0.000	0.081	-	0.005	0.000
Rosemary oil		0.001	0.504	0.284	0.005	-	0.033
Grapefruit oil		0.195	0.142	0.001	0.000	0.033	-
Chloroform	24 h	-	0.001	0.000	0.000	0.002	0.169
Eucalyptus oil		0.001	-	0.066	0.002	0.787	0.054
Orange oil		0.000	0.066	-	0.199	0.035	0.000
Clove oil		0.000	0.002	0.199	-	0.001	0.000
Rosemary oil		0.002	0.787	0.035	0.001	-	0.097
Grapefruit oil		0.169	0.054	0.000	0.000	0.097	-

).

A statistical analysis of the three time intervals showed significant differences for all solvents when comparing 24 h with both 10 min and 3 min (p < 0.05). However, when comparing 3 min to 10 min, significant differences were observed only for clove oil (p = 0.011) and grapefruit oil (p = 0.016) (

Table 6. Intergroup comparisons of disk diffusion test of GP solvents against C. albicans at three time intervals (3 min, 10 min, and 24 h).

Groups	Chloroform	Eucalyptus Oil	Orange Oil	Clove Oil	Rosemary Oil	Grapefruit Oil
3 min–10 min	0.194	0.625	0.606	0.011	0.951	0.016
3 min–24 h	0.002	0.000	0.000	0.000	0.000	0.008
10 min–24 h	0.000	0.000	0.000	0.011	0.000	0.000

).

4. Discussion

Solvents are solutions used in endodontic retreatment to soften the GP root-filling material. Various types of GP solvents have been used, including chloroform, xylene, halothane, trichloroethane, and tetrachloroethylene [28,31,33]. However, none of these solvents fulfills all the criteria of an ideal solvent, which should be nontoxic and non-carcinogenic to surrounding tissues, the patient, and clinicians; should effectively soften GP; and should exhibit antibacterial properties [3,18,34,35].

As a promising alternative to synthetic GP solvents, several essential oils have been reported as effective GP solvents, including eucalyptus oil, orange oil, clove oil, rosemary oil, grapefruit oil, and castor oil [3,28,33]. A few studies [3,36] have explored the antibacterial properties of GP solvents against E. faecalis, while only one study [14] compared their antifungal activity against C. albicans. However, none of these studies assessed the antimicrobial activities of various GP solvents or examined their effectiveness over different time intervals.

This study compared the antimicrobial activities of seven GP solvents: chloroform, eucalyptus oil, orange oil, clove oil, rosemary oil, grapefruit oil, and castor oil. For the first time, the antimicrobial properties of four of these essential oils—GP solvents, clove oil, rosemary oil, grapefruit oil, and castor oil—were compared with the more efficient and commonly used solvent chloroform [28,29,36]. Unlike previous studies [3,14,36,37], which measured the activities after 24 h, this study took into account the actual clinical scenario by assessing their antimicrobial effects at time intervals of 3 min, 10 min, and 24 h based on the data collected, which indicated that the time needed for retreatment ranged from 1.5 to 10.8 min [38]. GP solvents typically remain in the canals for 3 to 10 min to soften the obturating GP before removal [18,31,39]. In this study, the effectiveness of a 24 h interval of GP solvents was also assessed for comparison to previous studies, particularly in terms of its impact when contact is sustained over a long period.

The results obtained were classified into four groups. Group 1 (ZOI with no persistence) included chloroform, orange oil, and clove oil for E. faecalis and chloroform, orange oil, clove oil, and rosemary oil for C. albicans. The results align with the findings of Maria et al. (2021), who observed that orange oil had a significantly greater ZOI than chloroform at the 24 h mark [3]. These results suggest that each solvent produced the largest ZOI at a specific time interval. This is consistent with Chouhan et al. (2017), who emphasized that the antibacterial effectiveness of essential oils is influenced by several factors, such as their chemical composition, concentration, and interaction with bacterial cell membranes over time [40].

In contrast, when evaluating the GP solvents against C. albicans, the results showed greater consistency. The ZOI ranked from highest to lowest as follows: clove oil, orange oil, rosemary oil, and chloroform at all three time intervals. Clove oil was significantly more effective against C. albicans. This could be attributed to eugenol, the most abundant ingredient in clove oil, which is recognized as one of the key active ingredients in essential oils effective against C. albicans, while chloroform was considerably less effective among the other three GP solvents at all three time intervals. These results are somewhat consistent with Dutta et al. (2023), who demonstrated that chloroform exhibited lower antifungal activity compared to eucalyptus oil, xylene, and turpentine oil and showed similar antifungal activity to orange oil [14] while increasing the ZOI for all GP solvents against C. albicans over time, most probably related to stability and sustained release of active components, and their antifungal mechanisms of action include different cellular targets. For example, the chemical stability of essential oil components, such as eugenol in clove oil and terpene derivatives in rosemary oil, enables them to continue releasing their antifungal effects even after initial exposure and inhibits the hyphae formation of Candida species. This sustained release contributes to prolonged antifungal activity against C. albicans [41,42].

Group 2 (ZOI with persistence) included oil and rosemary oil for E. faecalis and eucalyptus oil and grapefruit oil for C. albicans. Rosemary oil demonstrated persistence at the 3 min time point only against E. faecalis, while eucalyptus oil exhibited ZOI with persistence at both 3 min and 10 min time intervals against E. faecalis and only at 3 min against C. albicans. The persistence of E. faecalis and C. albicans to some essential oils at short contact times (such as 3 min or 10 min) can be attributed to several factors related to the chemical composition and mode of action of essential oils, as well as the inherent properties of the microorganisms [43]. For example, the major constituent of eucalyptus oil is oxygenated monoterpenes (1,8-cineole) (87.32%) and monoterpenes hydrocarbons (12.45%). Most of its strong antimicrobial activity is due to the presence of 1,8-cineole [3,44,45]. Rosemary essential oil is primarily composed of monoterpenes and their derivatives, making up 95–98% of its content, with the remaining 2–5% consisting of sesquiterpenes [46,47,48]. Essential oils with higher levels of monoterpenes may act more slowly, whereas those with phenolic compounds may have faster action [40]. Although eucalyptus oil and rosemary oil possess potent antimicrobial properties [3,45,47,48], their antimicrobial activities may take more time to become highly effective. The efficacy of essential oils can be dependent on concentration and exposure time. At shorter contact times, the concentration of these active compounds may not reach levels high enough to induce cell membrane disruption, inhibition of enzyme activity, or other cellular damage, allowing the bacteria to adapt and resist the effects. It has been reported that compounds with slow action require 30–60 min to exhibit effective antimicrobial activity [49]. In addition, both E. faecalis and C. albicans have relatively robust cell walls and membranes that can limit the uptake of essential oils. E. faecalis, a Gram-positive bacterium, has a thick peptidoglycan layer that can act as a barrier to many antimicrobial agents [50,51]. In addition, E. faecalis is known to harbor various resistance mechanisms, such as efflux pumps and changes in membrane permeability [52,53], which can decrease susceptibility to essential oils. These resistance mechanisms may be activated or more pronounced at shorter contact times. Similarly, C. albicans has a complex cell wall that includes polysaccharides, like glucan [54], which can reduce the effectiveness of antimicrobial agents during brief exposure. Moreover, brief contact may allow these organisms to repair any damage or activate resistance mechanisms [55].

Group 3 (ZOI only at specific time intervals) included grapefruit oil at the 24 h mark for E. faecalis. Grapefruit oil did not produce any ZOI against E. faecalis at the 3 min and 10 min intervals, while ZOI of 7.6 ± 0.31 was observed against E. faecalis at the 24 h interval, which was significantly lower than all the other tested GP solvents, except for castor oil, which showed no ZOI. Grapefruit oil primarily consists of limonene (88.6–91.5%), β-pinene (0.8–1.2%), linalool (1.1–0.7%), and α-terpinene (0.7–1.0%) [56]. This means that over 96% of grapefruit oil is made up of monoterpenes [57], which tend to act more slowly and require longer contact times to exhibit noticeable effects, such as a ZOI [40].

Group 4 (no ZOI) included castor oil for both E. faecalis and C. albicans. Castor oil did not produce ZOI at any time intervals tested for both E. faecalis and C. albicans. Castor oil is a type of vegetable oil extracted from the seeds of the castor plant (Ricinus communis Linn). The primary fatty acid components in castor oil include ricinoleic acid (92%), oleic acid (3.53%), linoleic acid (2.90%), stearic acid (1.02%), and myristic acid (0.55%). The castor oil derivatives, K-soap, free fatty acids, and methyl esters are responsible for their antibacterial activity, especially against Escherichia coli and Staphylococcus aureus bacteria [58]. Some studies [59,60,61] have shown that castor oil exhibits antimicrobial properties against various bacterial and fungal species. However, its activity against E. faecalis and C. albicans specifically is not always significant. Salles et al. (2015) concluded that castor oil had a non-significant action on E. faecalis but could reduce colony counts for other microorganisms like C. albicans and Staphylococcus aureus [60]. Momoh et al. (2012) showed that fungi were less susceptible to the castor oil extract than bacteria [59]. Alezzi et al. (2022) indicated in their review that castor oil could reduce but not eliminate the colony count of E. faecalis and C. albicans in comparison to the significant antimicrobial activity of NaOCl and chlorhexidine [61]. These might indicate that although castor oil may have a mild inhibitory effect on certain microorganisms, it may fail to produce ZOI in some laboratory studies. The antimicrobial properties of castor oil can vary based on factors such as concentration, the study design, the methodology used, and specific strains of microorganisms tested.

The current study also compared the impact of the duration of application of the tested GP solvents on E. faecalis and C. albicans. For orange oil, clove oil, and rosemary oil, the ZOI increased as the duration of application lengthened, with the highest ZOI observed at 24 h, followed by 10 min and 3 min, respectively. In contrast, the order was different for chloroform and eucalyptus oil. Chloroform exhibited the highest ZOI at 24 h (10.85), followed by 3 min (10.45) and 10 min (10.1). For eucalyptus oil, ZOI was the highest at 10 min (10.5), followed by 3 min (8.95) and 24 h (8.05). However, both the 10 min and 3 min applications showed a higher ZOI than the 24 h application, which was associated with persistence. These results, in agreement with Chouhan et al. (2017), indicated that the antibacterial activity of essential oils can change according to the duration of their application [40]. The time factor is crucial because the longer the essential oil is in contact with the bacteria, the more likely it is that its bioactive compounds (such as terpenes, aldehydes, limonene, phenols, and eugenol) will penetrate the bacterial cell membranes and exert their antimicrobial effects. With short contact times, there may not be enough time for these compounds to have a significant effect [40,43,62,63] and be associated with persistence. The duration of application can influence the bacteria’s growth phase. Essential oils may be more effective during specific phases of bacterial growth (such as the exponential growth phase) and less effective if the bacteria are in a dormant state [64].

5. Conclusions

The findings suggest that essential oils such as clove oil, orange oil, and rosemary oil exhibit significant antimicrobial activity similar to chloroform against both E. faecalis and C. albicans, even with a brief 3 min application, but without the adverse effects of chloroform. As the duration of application increases, the ZOI for most of the tested essential oils significantly increases, while eucalyptus oil shows ZOI with persistence against both E. faecalis and C. albicans at short application of 3 min, whereas grapefruit oil demonstrates antibacterial effects against E. faecalis only after a prolonged 24 h exposure. In contrast, castor oil does not exhibit any antimicrobial activity against E. faecalis or C. albicans at any tested duration.