Antimicrobial Effects of Aloe Vera Mouthwash in Adults with Dental Caries: A Preliminary Clinical Trial

ABSTRACT

Aim:

To investigate the antimicrobial efficacy of an aloe vera (ALOE)-based mouthwash against key cariogenic pathogens in adults with dental caries, comparing its effects to a standard chlorhexidine (CHX) mouthwash.

Materials and Methods:

In this single-blind, randomized clinical trial, 54 adults with active dental caries (decayed, missing, and filled teeth [DMFT] ≥3) were assigned to either an ALOE (n = 27) or a 0.12% CHX (n = 27) mouthwash group for a 7-day regimen. Unstimulated saliva was collected at baseline and post-intervention. Salivary levels of Streptococcus mutans, Candida, and Lactobacilli were semi-quantitatively assessed using a modified dip-slide test. Statistical analyses, including the Wilcoxon signed-rank and Mann–Whitney U-tests, were performed with significance set at P < 0.05.

Results:

Post-intervention, the ALOE group demonstrated a significant reduction in salivary Candida levels (P = 0.008). While a decrease in S. mutans was observed, it was not statistically significant (P = 0.096). The CHX group showed significant reductions in both S. mutans (P = 0.046) and Candida (P = 0.002). Neither group exhibited a significant change in Lactobacilli levels, nor significant differences in microbial counts were found between the two groups after the 7-day period.

Conclusion:

ALOE mouthwash demonstrates significant antifungal activity against salivary Candida in adults with dental caries, positioning it as a potential natural alternative for managing oral fungal loads. While its effect on S. mutans was not as pronounced as CHX in this short-term trial, its efficacy against Candida warrants further investigation into its long-term benefits for oral health maintenance.

INTRODUCTION

Dental caries is one of the leading causes of the oral disease burden globally.[1] It is considered a multifactorial disease involving dysbiosis of the oral microbiome and changes in the oral microenvironment that favor an acidic condition.[2,3] Major caries-associated microorganisms include acid-tolerant species, such as the streptococcal species Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, and Streptococcus mitis; Lactobacillus spp.; and Candida spp.[4] Mouthwashes containing antimicrobial agents have been used as adjuncts to conventional oral hygiene regimens. However, chlorhexidine (CHX) gluconate, the gold standard mouthwash in dental care, has side effects when used over the long term, such as taste alteration and tooth staining.[5] In recent years, naturally derived compounds with antimicrobial properties against cariogenic microorganisms have attracted considerable interest owing to their potential for preventing or controlling dental caries and other oral infections.[6,7]

Aloe vera (ALOE) is a medicinal plant with various therapeutic benefits, including the promotion of wound healing and the exhibition of anti-inflammatory, antibacterial, antifungal, and antiviral properties.[8] Most studies on the antimicrobial effects of ALOE against oral microorganisms have been conducted in vitro, targeting both cariogenic and periodontal pathogens such as S. mutans, Aggregatibacter actinomycetemcomitans, Porphylomonas gingivalis, and Bacteroides fragilis.[9] Additionally, the antifungal activity of ALOE against three species of Candida has been demonstrated in vitro,[10] including Candida albicans, which contributes to the progression of severe early childhood caries and root caries.[11,12] The association between oral Candida carriage and the occurrence of dental caries has been revealed in people of a wide range of ages, including children, adolescents, adults, and elderly persons.[13,14] Furthermore, the synergistic effect of ALOE and fluoride in toothpaste on the remineralization process has been proposed.[15] Nevertheless, clinical studies evaluating the antimicrobial effects of ALOE mouthwash on oral microorganisms remain limited and have predominantly focused on pediatric populations.[16,17]

Caries risk assessment based on microbiological analysis can be performed using salivary microbial tests, including the conventional agar plate technique, dip-slide test, and molecular methods.[4] The modified dip-slide test developed by Thaweboon et al.[18] has been shown to be a reliable and simple method for determining salivary levels of mutans streptococci, Candida spp., and Lactobacilli spp. Collectively, this preliminary study aimed to evaluate the antimicrobial effects of an ALOE mouthwash containing 0.11% sodium fluoride and to compare them with those of a 0.12% CHX mouthwash in a 7-day regimen in adults with dental caries. Salivary levels of S. mutans, Candida, and Lactobacilli were assessed using the modified dip-slide test. The null hypothesis stated that there would be no significant difference between the effects of the ALOE and CHX mouthwashes on salivary levels of S. mutans, Candida, and Lactobacilli.

MATERIALS AND METHODS

ETHICAL APPROVAL

This interventional clinical study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. The study protocol was approved by the Human Research Ethics Committee at Srinakharinwirot University (SWUEC/E-365/2022) and was registered with the Thai Clinical Trials Registry (TCTR20250301001). All participants were fully informed about the study procedures and provided written informed consent before participation.

SAMPLE SIZE CALCULATION

The sample size was calculated based on the study by Mohamed et al.[17] The primary outcome variable used to estimate the effect size was the mean total bacterial count. A statistical power of 80% and an alpha error of 5% were set, with an effect size of 0.4, using statistical power analysis software (G*Power 3.1, Bonn, Germany). The required total sample size was 54 (27 per group).

MOUTHWASH PREPARATIONS

Two mouthwash formulations were developed for this study. The ALOE mouthwash contained 89% w/w ALOE extract, 0.11% w/w sodium fluoride, 1% w/w poloxamer, 9 mL glycerin, 0.1% w/w sodium benzoate, sodium saccharin, peppermint spirit, a coloring agent, and distilled water. The CHX mouthwash contained 0.12% w/w CHX and was visually matched to the ALOE mouthwash. The excipients used in the CHX formulation were similar to those in the ALOE mouthwash, except that sodium fluoride was excluded to replicate a commercially available CHX mouthwash.

PARTICIPANT RECRUITMENT

The inclusion criteria for selecting volunteers to participate in the research project were as follows: individuals aged 18 years or older, with at least 10 teeth present in the oral cavity, and a minimum of two active carious lesions involving dentin (International Caries Detection and Assessment System [ICDAS] score ≥4), according to the ICDAS.[19] Participants were also required to have a decayed, missing, and filled teeth (DMFT) index of ≥3.[20] Individuals with orthodontic appliances or removable dentures were excluded. Exclusion criteria included the presence of underlying medical conditions such as diabetes mellitus or other significant systemic diseases; known allergies to CHX, ALOE, garlic, onions, or tulips; and the use of antibiotics within the past 3 months. Pregnant or lactating individuals, as well as those with a history of alcoholism, were also excluded.

All participants were randomly assigned to either the CHX mouthwash group (n = 27) or the ALOE mouthwash group (n = 27), using a single-blind design. Demographic data, including age, sex, and body mass index (BMI), were collected. The number of remaining teeth and the DMFT index were recorded by a calibrated investigator (ST), in accordance with World Health Organization criteria.[20]

SALIVA COLLECTION

Unstimulated whole saliva samples were collected at baseline and after the 7-day mouthwash regimen between 8:00 AM and 11:00 AM using the spit method.[21] All participants were instructed to refrain from drinking, eating, or performing oral hygiene for at least two hours before saliva collection. They rinsed their mouths with distilled water for one minute and then expectorated. Subsequently, participants were asked to spit into a 15 mL centrifuge tube (Corning Life Sciences, Tewksbury, MA, USA) until a volume of 2 mL was reached. The time required to collect this volume was recorded and used to calculate the salivary flow rate (mL/min).

Following baseline sample collection, participants were instructed to use the assigned mouthwash (15 mL), measured using a graduated plastic cup provided by the researcher. The mouthwash was used twice daily—once in the morning and once before bedtime—for seven consecutive days. A fluoride-based toothpaste (Fluocaril Bi-fluoride Original, Unilever, Thailand) was provided for use throughout the study period.

Saliva samples were collected again after completion of the 7-day mouthwash regimen. The study workflow is illustrated in Figure 1.

Figure 1.

Study workflow

MICROBIOLOGICAL CULTURE

Microbial counts were assessed using the modified dip-slide method to evaluate salivary levels of S. mutans, Candida, and Lactobacilli.[18] A total of 2 mL of each undiluted saliva sample was applied to the three compartments of the modified dip-slide test kit, which contained selective culture media: Mitis-Salivarius Bacitracin agar for S. mutans, Sabouraud dextrose agar for Candida spp., and Rogosa SL for Lactobacilli spp. The saliva was evenly spread across the surface of the culture media to form a film, and any excess was removed by gently blotting the edge of the dip-slide on absorbent paper. The slides were incubated at 37°C for 72 h. Microbial levels were scored semi-quantitatively on a scale from 0 to 4 by comparing colony densities with a reference chart provided with the test kit. All test results were independently verified by three investigators.

STATISTICAL ANALYSIS

Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test was used to assess the normality of continuous data. Categorical variables were analyzed using the chi-square test. Between-group comparisons of continuous variables were conducted using the Mann–Whitney U test, depending on data distribution. Within-group comparisons (pre- and post-intervention) were analyzed using the Wilcoxon signed-rank test, as appropriate. Two-sided P-values less than 0.05 were considered statistically significant.

RESULTS

PARTICIPANT CHARACTERISTICS

This study recruited 54 participants who were randomly allocated to the CHX group and the ALOE group, with 27 participants in each group. There were no dropouts among the participants during the 7-day study period.

At baseline, there were no significant differences in age, sex, BMI, or the DMFT index between the CHX group and the ALOE group (P > 0.05). However, the unstimulated salivary flow rate in the ALOE group was significantly higher than that in the CHX group (P = 0.019) [Table 1].

Table 1.

Characteristics of participants in this study

Variables	Total (N = 54)	Group		P value
		CHX (n = 27)	ALOE (n = 27)
Age (years)	34 (24.75, 49.75)	35.0 (26.0, 48.0)	33.0 (24.0, 52.0)	0.945
Sex				0.564
Female	36 (66.7%)	19 (35.2%)	17 (31.5%)
Male	18 (33.3%)	8 (14.8%)	10 (18.5%)
BMI (kg/m2)	23.38 (20.55, 26.56)	24.03 (21.48, 26.67)	22.66 (20.50, 26.22)	0.452
Unstimulated salivary flow rate (mL/min)	0.52 (0.35, 0.78)	0.40 (0.31, 0.61)	0.65 (0.40,1.00)	0.019*
D (decay)	6 (3.75, 8.0)	5 (4,7)	6 (3,9)	0.433
M (missing)	1 (0, 3.25)	1 (0,2)	1 (0,4)	0.593
F (filling)	0 (0, 3)	0 (0,2)	1 (0,5)	0.274
T (total tooth number)	28.5 (27.0, 31.25)	29.0 (27.0, 31.0)	28.0 (27.0, 32.0)	0.979
DMFT	9 (5.75, 14.25)	9.0 (5, 13)	11.0 (7,16)	0.134

ALOE = Aloe vera, CHX = chlorhexidine, DMFT = decayed, missing, filled teeth

Data are presented as medians (first quartile, third quartile) or as counts with percentages. Intergroup comparison (Mann–Whitney U test, chi-square, independent t test)

^*P < 0.05 = significant

MICROBIOLOGICAL LEVELS AT BASELINE

The levels of salivary S. mutans, Candida, and Lactobacilli, as determined by the modified dip-slide test, were categorized into counts ranging from 0 to 4.[18] At baseline (T0), there was no significant difference in the median levels of any microorganisms between the CHX group and the ALOE group (P > 0.05) [Table 2].

Table 2.

Levels of salivary Streptococcus mutans, Candida, and Lactobacilli according to the modified dip-slide test in the chlorhexidine and aloe vera mouthwash groups at baseline (T0) and after seven days of mouthwash use (T1)

Microbes	Group				P value
	CHX (n = 27)		ALOE (n = 27)
	Median (Q1, Q3)	Mean	Median (Q1, Q3)	Mean
S. mutans
T0	1 (1, 2)	1.48	1 (1, 2)	1.33	0.641
T1	1 (1, 1)	1.07	1 (1, 1)	1.11	1.000
P value	0.046*		0.096
Candida
T0	1 (1, 3)	1.74	1 (1, 2)	1.63	0.629
T1	1 (1, 2)	1.19	1 (1, 2)	1.26	0.937
P value	0.002*		0.008*
Lactobacilli
T0	1 (1, 2)	1.67	1 (1, 2)	1.67	0.899
T1	1 (1, 2)	1.44	1 (1, 2)	1.41	0.931
P value	0.177		0.088

CHX = chlorhexidine mouthwash group, ALOE = aloe vera mouthwash group

Data are presented as median (first quartile, third quartile) and mean. Intragroup comparison: Wilcoxon signed-rank test; intergroup comparison: Mann‒Whitney U test

^*P < 0.05 = significant

INTRAGROUP COMPARISON OF MICROBIOLOGICAL LEVELS AT BASELINE AND FOLLOWING THE SEVEN-DAY MOUTHWASH REGIMEN

There was a significant decrease in S. mutans levels in the CHX group after the 7-day mouthwash regimen compared with the baseline levels (P = 0.046). No significant change in S. mutans levels was observed in the ALOE group after the 7-day study period (P = 0.096) [Table 2].

Significant decreases in Candida levels were observed after the 7-day study period in both the CHX group (P = 0.002) and the ALOE group (P = 0.008) [Table 2].

Nevertheless, no significant change in the levels of Lactobacilli was observed in either group (P > 0.05) [Table 2].

INTERGROUP COMPARISON OF MICROBIOLOGICAL LEVELS AT BASELINE AND FOLLOWING THE SEVEN-DAY MOUTHWASH REGIMEN

No significant differences in the levels of any of the assessed microorganisms were observed between the two groups following the 7-day mouthwash regimen (T1) (P > 0.05) [Table 2]. Both groups exhibited comparable salivary levels of S. mutans, Candida, and Lactobacilli following the intervention.

Representative microbiological specimens from each group, collected at baseline and after the 7-day mouthwash intervention, are depicted in Figure 2.

Figure 2.

Representative specimens showing colonies of Streptococcus mutans (A), Candida (B), and Lactobacilli (C) with their corresponding scores according to the modified dip-slide test in the chlorhexidine and aloe vera groups at baseline (T0) and following the intervention (T1)

DISCUSSION

One of the key preventive strategies for dental caries involves the reduction of salivary pathogenic microorganisms to shift the balance toward protective factors. In this context, ALOE mouthwash demonstrated antimicrobial properties that can aid in lowering salivary levels of pathogenic microorganisms. This clinical trial investigated the effects of the 7-day ALOE mouthwash regimen on salivary levels of oral microorganisms, including S. mutans, Candida, and Lactobacilli, in individuals with dental caries. The findings indicated that ALOE mouthwash significantly reduced salivary Candida levels in adults with dental caries. Although a statistically significant reduction in S. mutans and Lactobacilli was not observed, both microorganisms exhibited a decreasing trend in the ALOE group. In contrast, CHX mouthwash demonstrated efficacy in reducing both S. mutans and Candida levels, although no significant reduction in Lactobacilli was observed. Therefore, the null hypothesis of this study was rejected.

At baseline, participants in both the ALOE and CHX groups were comparable in terms of age, sex, BMI, and the DMFT index. However, the ALOE group had a significantly higher unstimulated salivary flow rate compared with the CHX group. Despite this, baseline levels of S. mutans, Candida, and Lactobacilli were similar in the two groups. This similarity may be attributed to the fact that mean salivary flow rates in both groups remained within the normal range, exceeding 0.01 mL/min.[22] Furthermore, no significant correlations were observed between salivary flow rate and changes in the levels (T0–T1) of any of the microbes. Collectively, this study ensured that the baseline characteristics were well balanced to minimize potential confounding factors.

The results of this study demonstrated the efficacy of ALOE mouthwash in reducing salivary concentrations of Candida in individuals with dental caries. This finding was consistent with that of a previous clinical study, which reported that a 7-day regimen of ALOE mouthwash effectively reduced C. albicans colonies, comparable to reductions achieved with 0.06% CHX mouthwash.[23] The association between Candida carriage and decayed, missing, and filled surfaces had been reported in adults, supporting the role of Candida in the pathogenesis of dental caries in this age group.[13] Moreover, the synergistic interactions between C. albicans and S. mutans have been found to contribute to the pathogenesis of dental caries.[24] In addition, other oral fungal infections may benefit from ALOE mouthwash due to its antifungal and immunomodulatory properties.[25] Therefore, the reduction in salivary Candida observed in this study highlighted the potential of ALOE mouthwash for promoting oral health. Further long-term studies involving participants with oral candidiasis are required to confirm its antifungal efficacy.

Although the ALOE group did not show a statistically significant reduction in S. mutans and Lactobacilli levels, decreasing trends for both microorganisms were observed following the intervention. In contrast, the CHX group demonstrated a significant reduction in S. mutans levels. This finding aligned with that of a previous study[26] reporting that 0.2% CHX mouthwash significantly reduced salivary S. mutans levels after a single use, whereas ALOE mouthwash only showed a decreasing trend. Nevertheless, some clinical studies have suggested that ALOE mouthwash can reduce dental plaque and salivary S. mutans levels to a degree comparable to that of CHX mouthwash.[16,27,28] These contradictory findings may be attributed to variations in the intervention duration, the type of biological samples collected (such as saliva or dental plaque), and differences in study population characteristics. The oral microbiomes can be altered throughout life by genetics and environmental factors, including diet, stress, and oral hygiene practices.[29] Therefore, further studies that control for these factors are recommended to improve the validity of research on the effectiveness of antimicrobial agents in mouthwashes.

This study had several limitations. Firstly, this study applied a semi-quantitative score (0 to 4) rather than colony-forming-unit counts, which may have hindered the evaluation of the mouthwash’s efficacy in reducing microbial levels. Secondly, participants were recruited based on their dental caries status rather than their salivary microbial levels. As a result, the baseline microbial levels of most participants were relatively low, classified as levels 1 to 2 [Table 2].[18] This may have contributed to the lack of a significant reduction in S. mutans and Lactobacilli levels, as well as the lack of significant differences in all microbial levels between the two groups at the end of the study. Further longitudinal studies in which participants are recruited who have high salivary levels of microorganisms, or those who have scores of 3 to 4 on the modified dip-slide test, and the application of the conventional agar plate method to investigate the antimicrobial effect of ALOE mouthwash should be encouraged to confirm these results. In addition, the sustained effects of ALOE mouthwash and its potential long-term impacts on the oral microbiome and oral tissues should be further determined.

CONCLUSIONS

ALOE mouthwash significantly reduced salivary Candida levels in adults with dental caries, suggesting its potential as an alternative antifungal agent. Although a trend in reductions in S. mutans and Lactobacilli was observed, it was not statistically significant. Further studies using a conventional culture method and participants with higher baseline microbial levels are needed to confirm the antimicrobial effects of ALOE mouthwash.

ACKNOWLEDGEMENTS

The authors would like to thank Miss Thunpicha Buengpaisansomboon, Miss Patcharaporn Junlaphan, and Miss Jidapa Burenoi for their assistance in data collection. We also extend our gratitude to all volunteers for their valuable support.

CONFLICTS OF INTEREST

There are no conflicts of interest.

AUTHOR CONTRIBUTIONS

AK, BT, and ST conceived the research idea. PS developed the mouthwash formulations. AK and ST collected and analyzed the data. ST created the figures and tables. AK and ST wrote the first draft. AK, PS, and ST edited the manuscript. All authors have approved the final version of the article.

ETHICAL POLICY AND INSTITUTIONAL REVIEW BOARD STATEMENT

The study protocol was approved by the Ethics Committee for research involving human participants at Srinakharinwirot University, Thailand (SWUEC/E-365/2022) on May 30, 2023. This study was registered with the Thai Clinical Trials Registry (register number: TCTR20250301001). All procedures in this study were performed in accordance with the international guidelines for human research, along with the laws and regulations of Thailand.

PATIENT DECLARATION OF CONSENT

All participants were informed about the research procedures and provided their written consent to participate.

DATA AVAILABILITY STATEMENT

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

LIST OF ABBREVIATIONS

• ALOE Aloe vera

• BMI Body mass index

• CHX Chlorhexidine

• DMFT Decayed, missing, filled teeth

• ICDAS The International Caries Detection and Assessment System

Funding Statement

This study was supported by a grant to S. Techatanawat from the Faculty of Dentistry, Srinakharinwirot University. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.