In Vitro Comparative Study of Antimicrobial Activity of Chia Seeds (Salvia hispanica) with Fennel Seeds (Foeniculum vulgare) and 0.2% Chlorhexidine Against Streptococcus mutans

Abstract

Background

Caries is a dental disease caused by pathogenic bacteria, such as Streptococcus mutans, which produces the enzyme glucosyltransferase (GTF), essential for the formation of glucan. Chia (Salvia hispanica) and fennel (Foeniculum vulgare) seeds are well-known seeds due to their antibacterial activity, but there is limited research linking the antibacterial activity of these seeds to their effectiveness against Streptococcus mutans.

Aim

In this investigation, the aim is to assess the comparative antimicrobial potency of S. hispanica (chia) and F. vulgare (fennel) seed extracts versus 0.2% chlorhexidine in inhibiting S. mutans.

Materials and methods

Aqueous extracts of chia seeds and fennel seeds were prepared through cold maceration, and the resulting crude residue was collected. A 0.2% chlorhexidine solution was used as the negative control. S. mutans was cultured on Brain Heart Infusion (BHI) agar, and wells were created in the agar plates into which 100 µL of each extract was introduced. Antimicrobial activity was assessed by measuring the zone of inhibition and minimum inhibitory concentration (MIC) for group I: chia seeds (100%), group II: fennel seeds (100%), group III: chia seeds (50%), group IV: fennel seeds (50%), group V: chia seeds (25%), group VI: fennel seeds (25%), and group VII: chlorhexidine (0.2%). Statistical analysis was performed using the Kruskal–Wallis test, followed by pairwise comparisons utilizing the Mann–Whitney U test.

Results

Chia seed and fennel seed extracts both exhibited antimicrobial properties; however, fennel seed extract showed significantly higher activity against S. mutans compared to chia seed extract (p = 0.050), as determined by statistical analysis.

Conclusion

This study's observations point to the viability of herbal extracts as a practical alternative to conventional antimicrobial products.

Clinical significance

The use of synthetic antimicrobial agents has led to side effects and the development of antimicrobial resistance due to improper usage. In contrast, herbal extracts may offer safer and more effective alternatives, provided they are backed by scientifically validated evidence.

Introduction

Dental caries, a condition with multifactorial origins, involves the action of microorganisms such as Streptococcus mutans, which is central to the pathogenesis and caries development.¹ Diminishing S. mutans levels may represent an effective measure for caries prevention.¹ Dental practitioners are constantly seeking innovative, practical strategies for managing this disease, and one potential avenue is exploring the antimicrobial properties of medicinal plants.² The use of plant-based treatments has ancient origins, with numerous cultures employing native plant extracts in medicinal applications.³ Today, many pharmaceuticals, especially those in the United States, are derived from plants, although their use as antimicrobial agents remains limited.²

Salvia hispanica, commonly called chia, is a biennial member of the Lamiaceae family native to Central and South America.⁴ Its seeds are densely packed with essential nutrients—especially omega-3 and omega-6 polyunsaturated fatty acids, high-quality proteins, dietary fiber, and a spectrum of minerals.^4,5 Although both animal and human investigations have highlighted chia's myriad health-promoting effects,^4,5 its antimicrobial efficacy—particularly against oral pathogens such as S. mutans—remains largely uncharted.⁴

Foeniculum vulgare, commonly called fennel, is a member of the Apiaceae (formerly Umbelliferae) family and has enjoyed centuries of use in traditional herbal medicine.⁶ Fennel seeds are known for their digestive benefits and are commonly used as breath fresheners.⁶ Their key chemical constituents include transanethole, fenchone, estragole, and d-limonene.^7,8 Despite their traditional uses, little research has explored the antimicrobial potential of fennel seeds, especially against oral bacteria such as S. mutans.^9,10

Chlorhexidine, a widely studied antimicrobial agent, is considered the gold standard in combating S. mutans and dental caries.^11–13 Its established potency makes it a reliable positive control in studies evaluating the antimicrobial properties of other agents.^14,15

This study seeks to explore the antimicrobial potential of chia and fennel seed extracts, comparing their effectiveness with chlorhexidine. The null hypothesis suggests that these extracts will show no significant antibacterial activity against S. mutans.

Materials and Methods

Study Design

The study was conducted in the Department of Microbiology at SMBT Institute of Dental Sciences and Research. All experimental procedures were completed over a period of three months.

Methodology

Extraction process was adapted from Agarwal et al.¹⁶ The study was repeated thrice following the checklist for reporting in vitro studies (CRIS) guidelines.¹⁷

Collection and Drying of Plant Materials

Salvia hispanica and Foeniculum vulgare specimens were authenticated by a botanist via morphological examination. The seeds were then cleaned, rinsed with sterile water, and left to air-dry in the shade. Once completely desiccated, they were pulverized into a coarse powder using a sterilized blender and stored in airtight containers until further use.

Preparation of Extract

To prepare the aqueous extract, 100 grams of chia seed powder were accurately measured and soaked in 600 mL of distilled water in a sterilized glass vessel. This cold maceration process was carried out over 48 hours with periodic agitation. Following the extraction, the mixture was passed through Whatman filter paper to obtain a clear solution, which was subsequently concentrated using a water bath maintained at 60°C, yielding the crude extract of S. hispanica.¹⁸

For F. vulgare, the seeds were initially pulverized using an electric grinder. Then, 100 grams of the fennel powder were immersed in 400 mL of distilled water for 24 hours under cold maceration. The resultant mixture was filtered, concentrated, and subsequently dried using vacuum evaporation. The final extract was stored at a temperature between 2°C and 8°C for preservation. From this procedure, a yield of 3.2 grams of dried F. vulgare extract was obtained per 100 grams of raw fennel seeds.¹⁹

Stock Solution

Stock solutions of aqueous extracts from S. hispanica (chia) and F. vulgare (fennel) seeds were formulated and stored in sterile vessels for microbiological evaluation.

Cultivation of Microorganisms

Streptococcus mutans (ATCC 890) pure isolates were procured from the microbial type culture collection (MTCC) in Chandigarh. The strains were activated and cultured on Brain Heart Infusion (BHI) agar, maintained at an incubation temperature of 37°C. The bacterial suspension was then adjusted to a concentration of 10⁵ CFU/mL, in accordance with the 0.5 McFarland turbidity standard.

Sterile agar plates were uniformly inoculated with the standardized S. mutans suspension using a swabbing technique. Circular wells, 10 mm in diameter, were created in the solidified agar using a sterile borer. Each well received a 100 µL portion of the respective extract. The plates were subsequently incubated for 24 hours at 37°C to assess antimicrobial activity.

Evaluation of Antibacterial Properties

To assess the antimicrobial potential of the extracts, the disk diffusion technique was employed. The minimum inhibitory concentration (MIC) for each test solution was established using the tube dilution assay. These MIC values were subsequently compared to those of 0.2% chlorhexidine, which served as the positive control, against S. mutans. The diameter of the inhibition zones (ZOI) around each sample was recorded with the aid of an antibiotic zone measurement scale (Fig. 1).

Fig. 1.

Measurement of zone of inhibition using the antibiotic zone scale

Statistical Analysis

All data were coded and entered into a computerized database, followed by thorough proofreading to ensure accuracy. The compiled data were organized using Microsoft Excel (version 2019; Microsoft Corporation, Redmond, WA, USA) and subjected to statistical analysis using IBM SPSS Statistics (version 26.0; IBM Corp., Armonk, NY, USA). Descriptive statistics, including mean and standard deviation (SD), were computed for continuous variables. Owing to the small sample size, nonparametric statistical methods were applied. Intergroup comparisons involving more than two groups were performed using the Kruskal–Wallis ANOVA, followed by pairwise comparisons with the Mann–Whitney U test. A p-value of <0.05 was considered statistically significant. The level of significance was set at α = 0.05 and β = 0.20, providing the study with a statistical power of 80%. Statistical significance was denoted as follows: *p < 0.05 (significant), **p < 0.01 (highly significant), and ^#p > 0.05 (not significant). There was a statistically highly significant difference seen for the values between the groups (p <0.01), with higher values in group II (Tables 1 and 2). Pairwise comparisons revealed no statistically significant differences among most group pairs (p > 0.05), except for comparisons between groups I vs VII, II vs VII, III vs VII, and IV vs VII, which demonstrated statistically significant to highly significant differences (p < 0.05 and p < 0.01, respectively) (Figs 2 and 3).

Table 1.

Intergroup comparison of the zone of inhibition of all the groups

Groups	N	Mean	Std deviation	Std error	95% CI lower bound	95% CI upper bound	Mean rank	Chi-square value	p-value of the Kruskal–Wallis test
I	3	3.866667	0.2516611	0.1452966	3.241506	4.491828	16.00	18.055	0.006**
II	3	4.533333	0.2516611	0.1452966	3.908172	5.158494	20.00
III	3	3.000000	0.2000000	0.1154701	2.503172	3.496828	10.50
IV	3	3.633333	0.4041452	0.2333333	2.629381	4.637286	14.83
V	3	2.500000	0.1000000	0.0577350	2.251586	2.748414	3.00
VI	3	2.766667	0.2516611	0.1452966	2.141506	3.391828	7.33
VII	3	2.633333	0.1154701	0.0666667	2.346490	2.920177	5.33

**p < 0.01 (highly significant)

Table 2.

Intergroup pairwise comparison using Mann–Whitney U test

Group	vs group	Mann–Whitney U value	Z value	p-value of Mann–Whitney U test
I	II	0.000	−1.964	0.050#
I	III	0.000	−1.964	0.050#
I	IV	0.000	−0.655	0.513#
I	V	0.000	−1.964	0.050#
I	VI	0.000	−1.964	0.050#
I	VII	0.000	−1.995	0.046*
II	III	0.000	−1.964	0.050#
II	IV	0.000	−1.964	0.050#
II	V	0.000	−1.964	0.050#
II	VI	0.000	−1.964	0.050#
II	VII	0.000	−1.995	0.046*
III	IV	0.500	−1.771	0.077#
III	V	0.000	−1.964	0.050#
III	VI	2.000	−1.124	0.261#
III	VII	0.000	−1.995	0.046*
IV	V	0.000	−1.964	0.050#
IV	VI	0.000	−1.964	0.050#
IV	VII	0.000	−1.995	0.046*
V	VI	1.500	−1.328	0.184#
V	VII	1.500	−1.328	0.178#

*p < 0.05 (significant); ^#p > 0.05 (not significant)

Fig. 2.

Agar plates showing zone of inhibition of the chia seeds and fennel seeds extract at different concentrations

Fig. 3.

Showing intergroup comparison of zone of inhibition

Results

The findings from this analysis highlight the superior antimicrobial potential of natural plant-based agents, particularly fennel seeds in their unprocessed form. All test groups showed varying degrees of antimicrobial activity against S. mutans, as indicated by the ZOI. The Kruskal–Wallis test revealed a statistically highly significant difference among the groups (χ² = 18.055, p = 0.006). Fennel seed extract (group II) exhibited the highest mean ZOI (4.53 ± 0.25 mm), followed by chia seed extract (group I) with a mean ZOI of 3.87 ± 0.25 mm. Pairwise comparison using the Mann–Whitney U test showed a significant difference between fennel and chia seed extracts (p = 0.050), confirming greater antimicrobial efficacy of fennel seeds. Additionally, statistically significant or highly significant differences (p < 0.05 or p < 0.01) were observed between several other groups when compared to the control (chlorhexidine) and diluted seed extracts.

Discussion

This study explored the antimicrobial potential of aqueous extracts from S. hispanica (chia) and F. vulgare (fennel) seeds against S. mutans, a primary pathogen responsible for dental caries. These findings suggest that fennel may serve as a more effective natural agent against cariogenic bacteria.

The enhanced antibacterial activity observed in fennel may be attributed to its phytochemicals, including anethole, estragole, and fenchone, which have been documented for their antimicrobial efficacy. Chia seeds, on the other hand, contain polyphenols, omega-3 fatty acids, and flavonoids that contribute to their antimicrobial action, albeit to a lesser extent than fennel. The differential activity can be explained by variations in bioactive compound concentrations and extraction efficiencies.

This investigation supports the potential role of widely accepted herbal extracts—particularly fennel and chia seeds—as promising anticaries agents for pediatric populations. Emphasis is placed not only on their antibacterial efficacy but also on critical formulation factors such as concentration optimization, taste acceptability, and biocompatibility. Fennel extract, with its favorable sensory properties and minimal toxicity, stands out as a strong candidate for incorporation into child-friendly oral hygiene products such as mouthwashes.

Globally, dental caries remains the most prevalent oral disease, largely driven by the cariogenic action of S. mutans. Conventional management relies heavily on synthetic antimicrobial agents such as chlorhexidine. However, prolonged or improper use of such agents is associated with several drawbacks, including tooth staining, altered taste sensation, mucosal irritation, and the emergence of antimicrobial-resistant strains. This has created an imperative for the development of safer alternatives. Herbal extracts, if scientifically validated, can offer effective and sustainable solutions with fewer side effects.

These findings align with previous research highlighting the potential of plant-derived substances in combating oral pathogens. Kripalani et al. demonstrated the inhibitory effects of plant extracts such as E. officinalis and P. guajava against S. mutans, supporting their potential as alternative therapeutics.²⁰ Similarly, Kajjari et al. found that herbal mouthwashes made from Mangifera indica and Mentha arvensis significantly reduced S. mutans counts in children, though chlorhexidine remained more effective.²¹

A randomized crossover study by Shah et al. compared chlorhexidine and a herbal mouth rinse in children with mixed dentition and concluded that the herbal rinse significantly reduced S. mutans levels, highlighting its clinical relevance.²² Palit et al. also demonstrated the efficacy of Terminalia chebula-based rinses in lowering salivary S. mutans counts among schoolchildren.²³

Additional support comes from studies on other botanicals, such as green tea. Salama and Alsughier found that a green tea extract mouthwash significantly reduced S. mutans in preschool children.²⁴ Likewise, Mon et al. evaluated herbal water, ozonated water, and chlorhexidine, concluding that herbal formulations could positively impact oral hygiene in children.²⁵

Though the current study's findings are promising, it is important to note that it has an in vitro design. Laboratory conditions cannot fully replicate the dynamic oral environment, which is influenced by salivary flow, pH fluctuations, microbial diversity, and host immunity. Future investigations should include in vivo clinical trials to validate efficacy, assess long-term safety, and explore the potential for integrating these extracts into practical oral care products.

Further research into the antimicrobial properties of these herbal extracts could be advanced by isolating their bioactive compounds and establishing optimal dosages for clinical use.

Conclusion

The antibacterial activity of the aqueous extracts from chia seeds (S. hispanica) and fennel seeds (F. vulgare) was observed against S. mutans. This presents an opportunity for pediatric dentists to offer treatments with minimal side effects, making them a viable option for children. To fully understand the potential of fennel and chia seed extracts in preventing oral infections, further in vitro and in vivo research is required. If these extracts prove effective, they could serve as valuable natural antimicrobial agents in oral care products such as toothpaste, gels, or mouth rinses, helping prevent and treat dental caries. This highlights the need for further research to enhance their effectiveness and substantivity for use in pediatric dentistry, especially as we move toward a more holistic, integrated approach to medicine, with herbal solutions becoming increasingly popular.

Clinical Significance

While newer technologies for dental caries restoration continue to emerge, prevention remains a priority, as “prevention is always better than cure.” Maintaining good oral hygiene is key to preventing oral diseases, and this can be achieved by using mouthwashes with antibacterial properties. The materials used in this study are cost-effective, easily accessible, and safe for children. These extracts are particularly beneficial for medically compromised children, those under 6 years of age, and children with early childhood caries (ECC), as both chia and fennel seeds are edible and FDA-approved.

Ethical Approval

The study was approved by the Institutional Ethics Committee of SMBT Institute of Dental Sciences and Research, Dhamangaon, Nashik (Approval No. 544/SMBT/04/PG/IEC/247/2023; approved on 13 December 2023).

Acknowledgments

The authors acknowledge the Indian Society of Pediatric and Preventive Dentistry (ISPPD) for providing partial publication support for this manuscript. This research was awarded Prize in the Scientific Paper Presentation at the 20th ISPPD National PG Convention 2024, Dehradun.

Author Contributions

Supraja Ganeshan: Conceptualization, methodology, investigation, data curation, formal analysis, manuscript writing.

Pranav Nayan Dave: Conceptualization, methodology, supervision, manuscript review and editing.

Prasad Musale: Supervision, validation, manuscript review and editing.

Aditi Bulbule: Methodology, laboratory supervision, validation.

Saurabh Mirgal: Formal analysis, data interpretation, manuscript review.

Saloni Gautam: Investigation, data collection, literature review, manuscript editing.

AI Disclosure

No artificial intelligence (AI)-assisted technologies were used in the preparation of this manuscript and research.

Orcid

Supraja Ganeshan https://orcid.org/0009-0007-6153-9367

Data Availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.