An in vitro evaluation of various natural intracanal medicaments for the effect of hardness on tooth structure and antimicrobial efficacy

Abstract

Aim:

This study aimed to evaluate the mechanical and antibacterial properties of therapeutic seed extracts – chia, flax, guava, and papaya – in comparison to calcium hydroxide for potential intracanal application in endodontics.

Methodology:

A total of 150 extracted single-rooted teeth were cleaned, decoronated, and standardized to 12 mm. After biomechanical preparation, samples were sterilized and divided into two groups (A and B), each with five subgroups: calcium hydroxide, chia, papaya, flax, and guava seed extracts. Microbial inoculation was performed using polymicrobial samples from infected root canals, followed by incubation for 7–14 days. Group A samples were sectioned horizontally (3 mm × 3 mm × 2 mm), embedded in acrylic, treated with respective medicaments, and analyzed using confocal microscopy after staining with live/dead bacterial stain. Group B samples were sectioned vertically (4 mm × 2 mm × 2 mm), treated, and subjected to microhardness testing. Seed-based medicaments were prepared by sun-drying, powdering, autoclaving, and mixing with propylene glycol to achieve a homogeneous paste.

Results:

Chia seed extract showed the highest antimicrobial activity and dentin reinforcement, comparable to calcium hydroxide. Papaya and guava seeds exhibited moderate antibacterial efficacy. Flaxseed demonstrated the lowest performance in both aspects. Calcium hydroxide was effective but did not surpass some natural extracts in mechanical enhancement.

Conclusion:

Chia and papaya seed extracts present promising alternatives to calcium hydroxide, offering dual functionality – antibacterial efficacy and mechanical reinforcement. These findings support the integration of natural agents in endodontic treatment. Further in vivo and formulation studies are recommended to validate their clinical use.

INTRODUCTION

The success of endodontic therapy critically relies on the complete elimination of microorganisms from the intricate architecture of the root canal system.[1] Although mechanical instrumentation and irrigation are the foundation for treatment, they often fail to sterilize all canal niches – especially in the presence of complex anatomy – making intracanal medicaments indispensable.[2]

Calcium hydroxide remains the conventional intracanal medicament, prized for its high pH and broad-spectrum antimicrobial action. However, its effectiveness is compromised against resilient, biofilm-forming species like Enterococcus faecalis. Prolonged exposure has also been shown to diminish dentin microhardness and fracture resistance, raising concerns about structural compromise.[3] Notably, a systematic review confirmed that extended contact with calcium hydroxide significantly weakens dentin integrity, particularly when used for more than 30 days.[4] Moreover, residual calcium hydroxide can interfere with sealer bond strength and ultimately affect successful obturation.[5]

Alternative agents such as chlorhexidine, triple antibiotic paste, double antibiotic paste, and mixture of tetracycline isomer, acid, and detergent (MTAD) demonstrate superior antimicrobial efficacy compared to calcium hydroxide.[6,7,8] Nevertheless, their usage is tempered by significant drawbacks, including cytotoxicity, the risk of tooth discoloration, and growing concerns about antibiotic resistance.[9,10]

Consequently, there is growing interest in plant-based medicaments, which combine effective antimicrobial and anti-inflammatory properties with enhanced biocompatibility. These phytotherapeutic agents are rich in bioactive compounds such as phenolics and flavonoids, which disrupt bacterial membranes and can inhibit biofilm formation.[11]

Flaxseed extract, enriched with phenolic compounds, has demonstrated potent antifungal activity against Candida albicans, reportedly outperforming nystatin in experimental settings.[12] Similarly, guava (Psidium guajava) extracts exhibited superior antibiofilm activity when compared to conventional irrigants such as chlorhexidine and sodium hypochlorite.[13] Papaya seed extracts have also revealed significant antimicrobial and antioxidant potential, producing notable inhibition zones against pathogens such as Staphylococcus aureus and Escherichia coli, thereby positioning them as a viable disinfection option.[14] In addition, chia seed extracts have shown promising antibacterial activity, particularly against Gram-positive strains such as S. aureus and Staphylococcus epidermidis.[15]

This study evaluates the mechanical and antibacterial properties of therapeutic seed extracts – chia, flax, guava, and papaya – in comparison to calcium hydroxide for potential intracanal application in endodontics.

METHODOLOGY

A total of 150 freshly extracted single-rooted human teeth with straight canals, extracted for periodontal or orthodontic reasons, were included in the study. The teeth were cleaned using an ultrasonic scaler, and canal patency was confirmed with a #15 K-file. Biomechanical preparation was performed using the ProTaper rotary system up to F3 (Dentsply Maillefer). Irrigation was carried out with 17% ethylenediaminetetraacetic acid followed by 5.25% sodium hypochlorite, and a final rinse with saline and chlorhexidine. All specimens were stored in phosphate-buffered saline and sterilized by autoclaving at 121°C for 15 min.

The samples were randomly divided into two groups (n = 75 each): Group A for antimicrobial evaluation and Group B for microhardness testing. Each group was subdivided into five subgroups (n = 15): calcium hydroxide (control), chia, papaya, flax, and guava seed extracts.

For microbial inoculation, nutrient broth (TM Media) was prepared according to the manufacturer’s instructions. Microbial samples were collected from root canals of patients with symptomatic irreversible pulpitis using sterile paper points and incubated for 28 days to allow biofilm formation. Decoronated teeth were sectioned into standardized blocks (3 mm × mm 3 × 2 mm), embedded in acrylic, inoculated with the microbial suspension, and incubated at 37°C for 21 days.

Herbal medicaments were prepared by sun-drying raw chia, flax, papaya, and guava seeds for 24 h, grinding them into a fine powder, and sterilizing the powders by autoclaving. Each medicament was formulated by mixing 1.2 g of seed powder with 1 mL of propylene glycol until a homogeneous paste was obtained.

In Group A, after 21 days of bacterial incubation, medicaments were applied using a micro-brush and stored in sterile water for 4 days. Antimicrobial efficacy was assessed using Live/Dead bacterial staining (Sigma-Aldrich) and evaluated under confocal laser scanning microscopy.[16]

In Group B, vertically sectioned semicylindrical specimens (4 mm × 2 mm × 2 mm) were embedded in acrylic and polished. Medicaments were placed using a Lentulo spiral and incubated at 37°C for 14 days. Dentin microhardness was evaluated using the Vickers hardness test.[17]

RESULTS

Paired t-test

Chia seeds showed the highest positive antimicrobial effect, making them the most effective among all groups. Guava seeds, calcium hydroxide, and papaya seeds showed significant antimicrobial activity but with negative mean differences, indicating bacterial reduction. Flax seeds had the least effect and were not statistically significant, suggesting limited antimicrobial action. Overall, chia seeds outperformed all other medicaments in reducing microbial load [Figures 1-4].

Figure 1.

Descriptive statistics for hardness for group

Figure 4.

Paired t test : Antimicrobial effect of different medicaments

Figure 2.

Comparison of dentin hardness across groups

Figure 3.

Mean live vs dead bacteria counts per group

ANOVA results

• Live bacteria (L): F = 2.42, P = 0.057 → No statistically significant difference among groups

• Dead bacteria (D): F = 7.14, P < 0.001 → Significant difference among groups.

  1. Group II A – Highest dead bacteria count, indicating the strongest antibacterial effect

  2. Group III A – Slightly lower but still high kill rate

  3. Group V A – Moderate antibacterial effect

  4. Group I A – Lower than Groups II, III, and V

  5. Group IV A – Lowest dead bacteria count, weakest antibacterial effect in this set [Tables 1 and 2].

Table 1.

Descriptive statistics for hardness by group

Group	n	Mean	SD	95% CI lower	95% CI upper	Minimum	Maximum
Calcium hydroxide	15	49.75	10.68	43.83	55.67	32.11	63.00
Chia seeds	15	54.33	7.44	50.21	58.45	39.00	66.00
Papaya seeds	15	41.93	13.42	34.50	49.37	15.00	65.00
Flax seeds	15	39.83	8.67	35.03	44.63	20.00	50.00
Guava seeds	15	50.27	12.23	43.49	57.04	31.00	65.00
Total	75	47.22	11.78	44.51	49.93	15.00	66.00

SD: Standard deviation, CI: Confidence interval

Table 2.

Antimicrobial Group

Group	Pre	Post	AUC (middle)
Calcium hydroxide	60.48	77.17	68.82
Chia seeds	66.66	85.17	75.91
Papaya seeds	64.93	85.01	74.97
Flax seeds	58.85	75.04	65.44
guava seeds	63.95	77.04	70.49

AUC: Area under the curve

Inference

Group II showed the best antibacterial performance, followed by Group III and Group V. Group I was less effective, and Group IV had the weakest effect against bacteria.

DISCUSSION

Antimicrobial efficacy

Chia seed extract had the strongest ability to kill bacteria. This is because it has a lot of alpha-linolenic acid (ALA) and omega-3 fatty acids, which can break down bacterial membranes and lower inflammation.[18] Compared to flax seeds, chia has a better omega-3 profile, which makes it more effective against bacteria. Flax seeds contain ALA and lignans, which have antioxidant properties, but they did not show as strong antimicrobial effects in our study. This might be because the active ingredients in flax, like cyclolinopeptides, are not very soluble or easily absorbed.

Guava seeds had good antimicrobial activity due to their high levels of flavonoids, tannins, and gallic acid, which can disrupt bacterial membranes. These compounds also work with linoleic and oleic acids to help fight bacteria.[19] However, chia seeds were better at killing bacteria because they have more ALA and stronger lipid-based effects. Papaya seed extract also had some antimicrobial potential. This was due to compounds like triterpenoid aldehydes, flavonoids, and alkaloids, which can damage microbial cell walls and proteins.[20] Even though papaya had moderate mechanical results, its antimicrobial effects were still significant.

Calcium hydroxide, which has been used a long time for its strong antimicrobial action due to its high pH, still worked well at reducing bacteria (P < 0.001).

However, it didn’t perform as well as chia and guava in overall antimicrobial ability.

Microhardness analysis

Our study found differences in how strong the dentin was when exposed to various intracanal medicaments, including chia, flax, guava, papaya seed extracts, and calcium hydroxide.

Chia seed extract had the highest microhardness, meaning it provided the best mechanical support. This could be because chia has a lot of mucilage and polysaccharides, which form a gel-like structure that is tough and less likely to break.[21] Flaxseeds, despite having some minerals that help with tooth remineralization, had the lowest microhardness. This might be because it has less calcium and other inorganic elements.[22] Guava seeds had good mechanical performance because they are rich in flavonoids, tannins, and fibrous materials that help make dentin stiffer by strengthening the collagen in the tooth.[23]

Papaya seed extract had lower microhardness. This might be because of papain, an enzyme that helps remove debris but can break down dentin collagen, making the tooth weaker. Furthermor, papaya lacks important reinforcing fibres such as mucilage or lignocellulose, which further explains its weaker performance.[24]

Calcium hydroxide had moderate microhardness but wasn’t as good as some of the natural extracts.

Although it’s commonly used as a standard treatment, long-term use might weaken dentin. This is because the high alkalinity of calcium hydroxide can damage the organic parts of the tooth, especially collagen. Studies show that extended exposure to calcium hydroxide can reduce dentin strength and make it more prone to breaking.[25]

Limitations

This study was done in a lab setting and used a small number of samples. It didn’t test against specific types of micro-organism (polymicrobial), so the results might not apply to all cases. Furthermore, only two factors were checked, suggesting that more research is needed to fully understand how well these materials perform in different ways.

CONCLUSION

This study shows that natural seed-based materials could be useful as intracanal medicaments in root canal treatments. Among the materials tested, chia seeds had the best antimicrobial and mechanical properties. Calcium hydroxide, papaya, and guava seeds had moderate effects, whereas flax seeds were the least effective. Choosing materials that have both antibacterial and structural benefits is important for better treatment outcomes.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.