This in-vitro study was conducted at Saveetha Dental College and Hospitals in the Department of Oral Pathology and Microbiology, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India from December 2021 to July 2022. Ethical clearance and informed consent were not required, as the study did not involve human subjects.

Ripened avocados were purchased from a grocery store and stored at 4 degrees Celsius. A 10 g sample of the edible part of the avocado was added to 200 mL of distilled water and stirred well for 15 minutes using a glass rod. The mixture was then filtered using a double filter. With a heat mantle, the mixture was boiled for five minutes at 60 degrees Celsius. It was filtered again after 24 hours and stored at a low temperature for later use.

A total of 10 cloves were powdered, and 2 g of the powdered cloves were precisely weighed. To this, 100 mL of distilled water was added, and the mixture was boiled for about 10 minutes at 60 degrees Celsius. This process activates the phytochemicals present in the cloves. Using Whatman filter paper No. 1, the mixture was filtered and stored at a low temperature for further use.

A 1 mL sample of the prepared fruit extract and 1 mL of the prepared clove extract were mixed with 9 mL of distilled water. To this mixture, 0.01 g of sodium lauryl sulfate, 0.3 g of sucrose, and 0.001 g of sodium benzoate were added. The resulting mixture was then stored at a low temperature [Table/Fig-1a-f].

[Table/Fig-1]:

a) Photograph showing ripened avocado fruit; b) The extract of the ripened fruit; c) Extraction of clove extraction; d) Prepared extract of cloves; e) Mixture of sodium lauryl sulfate, sucrose and sodium benzoate; and f) Prepared Persea americana and Syzygium aromaticum-based mouthwash.

Assessment of anti-inflammatory activity using bovine serum albumin denaturation assay: The albumin denaturation assay was used to test the anti-inflammatory activity of the mouthwash in the present study. The mouthwash was assessed according to the method described by Mizushima Y and Kobayashi M [18,19]. A total of 0.45 mL of bovine serum albumin (1% aqueous solution) was added to 0.05 mL of the prepared mouthwash at various concentrations (10 μL, 20 μL, 30 μL, 40 μL, 50 μL). The pH of the mixture was adjusted to 6.3 using 1N hydrochloric acid. The samples were incubated at room temperature for about 20 minutes and then boiled in a water bath at 55°C for approximately 30 minutes before being cooled. Spectrophotometric analysis was used to estimate the absorbance at 660 nm, and Diclofenac Sodium was employed as a control.

The following equation was used to calculate the percentage of protein denaturation [18,19]:

% Inhibition=(Absorbance of control-Absorbance of sample)/Absorbance of control×100

Assessment of antioxidant activity using DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay: The antioxidant properties of the formulated mouthwash using P. americana and clove extract were evaluated using the DPPH assay. The mouthwash was diluted to five different concentrations (10 μL, 20 μL, 30 μL, 40 μL, 50 μL). To this, 1 mL of 0.1 mM DPPH in methanol and 450 μL of 50 mM Tris HCl buffer (pH 7.4) were added, and the mixture was then incubated for about 30 minutes. The amount of DPPH free radicals reduced, which depended on the absorbance, was assessed at 517 nm. Butylated Hydroxytoluene (BHT) was used as the standard [20]. The absorbance of the mixture at 517 nm was measured using an Ultraviolet-visible (UV-Vis) spectrophotometer. The formula for calculating the percentage of scavenging activity is:

(Absorbance of control-Absorbance of the test)/Absorbance of control×100.

The data was managed using Microsoft Excel, and statistical analysis was conducted with SPSS software, version 23.0. Independent t-tests were employed to compare the outcomes between the groups, with a p-value of 0.05 considered statistically significant.

Anti-inflammatory activity: The anti-inflammatory activity of the synthesised mouthwash at 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL was measured as 46.68, 60.3, 72.28, 78.62, and 89.62, respectively. In comparison, the activity of the standard diclofenac sodium at the same volumes was 47.14, 60.4, 72.18, 77.96, and 84.82, respectively. The present study demonstrated superior anti-inflammatory activity of the prepared P. americana-based mouthwash at the 50 μL concentration (p-value <0.001) when compared to that of the standard diclofenac sodium [Table/Fig-2,3].

[Table/Fig-3]:

Comparison of the anti-inflammatory activity of the standard mouthwash and the synthesised mouthwash using BSA assay.

Antioxidant activity: The antioxidant activity of the prepared mouthwash was recorded as 60.18, 69.31, 70.946, 74.86, and 85.756 at 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL, respectively. In contrast, the activity of the standard was 76.032, 78.164, 86.046, 88.616, and 93.03 at the same volumes. The study concluded that the antioxidant property of the prepared mouthwash was highest at 50 μL. However, the antioxidant activity was lower for the synthesised mouthwash when compared with that of the standard (p<0.001). The results of the DPPH assay are represented in [Table/Fig-4,5].

[Table/Fig-5]:

Comparison of the anti-antioxidant activity of the standard mouthwash and the synthesised mouthwash using DPPH assay.

The anti-inflammatory and antioxidant properties of the highly nutritious avocado fruit (Persea americana) and clove (Syzygium aromaticum) mouthwash formulation demonstrated a similar anti-inflammatory effect when compared to the standard, diclofenac sodium. However, when compared to the control, the mouthwash’s antioxidant properties were noticeably lower at every tested concentration.

The study conducted by Kristanti CD et al., compared the anti-inflammatory and analgesic properties of infusion and methanolic extracts from avocado seeds on carrageenan-induced paw oedema in mice. The results suggested that both infusion and methanolic extracts of avocado seeds showed a significant reduction in inflammation [21]. However, the present study, which was conducted using the edible part (pulp) of the avocado, resulted in superior anti-inflammatory properties (86.62% zone of inhibition at 50 μL) but demonstrated less activity compared to that of the seeds. The differing scavenging properties may be attributed to the varying methanol and lipid contents between the seeds and the fruit of P. americana. Similarly, Alkhalaf MI et al., conducted a study on the anti-inflammatory activities of both fruit and seed extracts of P. americana, in which the seed extract was reported to have better antioxidant and anti-inflammatory properties, contrasting with the findings of the present study [22]. The varying antioxidant results could be due to the differing amounts of phenolic compounds in the seed, peel, and pulp (64%, 23%, and 13%, respectively). Furthermore, factors that significantly affect the nutritional profiles of avocados include the fertilisers used, soil composition, climate, variety, and ripening stage of the fruit [23]. An earlier study on Wistar rats verified the underlying mechanisms facilitating the healing process, showing increased collagen synthesis linked to the high oleic acid content in the edible portion of avocados, which contributes to the fruit’s anti-inflammatory properties. Hence, the therapeutic benefits of avocado oil are attributed to its high content of fatty acids, particularly oleic acid (47.20%) [24]. Additionally, Avocado-soybean Unsaponifiable (ASU) has demonstrated effectiveness against periodontal disease by altering the expression of Transforming Growth Factor beta 1 (TGF-β1), TGF-β2, and Bone Morphogenetic Protein 2 (BMP-2) [25]. Therefore, avocados possess substantial anti-inflammatory qualities that may have potential applications in the management of oral inflammatory conditions.

In a study on Clove Essential Oil (CEO) by Han X et al., it was found that at a dosage of 0.011%, CEO exhibited better anti-inflammatory properties and strong antiproliferative effects on human dermal fibroblasts. Additionally, proinflammatory biomarkers such as Vascular Cell Adhesion Molecule-1 (VCAM-1), interferon-induced protein 10 (IP-10), interferon-inducible T-cell α Chemoattractant (I-TAC), and Monokine Induced by Gamma Interferon (MIG) were markedly inhibited [26]. Consequently, the anti-inflammatory properties of the bioactive molecule eugenol found in cloves may be responsible for the enhanced anti-inflammatory efficacy of the synthesised mouthwash in the current investigation. This explains why the mouthwash developed in the present study exhibited better anti-inflammatory properties.

In a study comparing the properties of eugenol and cloves, it was identified that both have similar antioxidant effects. The DPPH and ABTS radical scavenging values for clove and eugenol, respectively, were IC50=0.1595 mg/mL and 0.3257 mg/mL, and 0.1696-7 mg/mL. Eugenol, one of the chemical constituents of clove essential oil (CEO), exhibits antioxidant effects, which explains why CEO possesses antioxidant qualities [27]. Additionally, Esmaeili F et al., investigated the anti-inflammatory and antinociceptive effects of Nanogels (NG) prepared from cinnamon and CEO, as nanoemulsion-based gels enhance topical drug delivery and effectiveness. The results showed that cinnamon-NG suppressed paw oedema more than clove-NG [28]. The milder antioxidant property reported in this study could be attributed to the inferior antioxidant capacity of clove compared to cinnamon. Therefore, substituting clove with cinnamon might improve the antioxidant capacity of P. americana, which could be a future recommendation.

The DPPH has been widely used to assess the free radical scavenging potential of desired components. A study assessing the antioxidant activity of avocado peel, which evaluated phenolic contents, resulted in strongest antioxidant activity, following fractionation of the methanolic extract (fraction 8-4.221±0.137 mg/mL) [29]. Hence, fractionation of avocado pulp might influence its antioxidant properties, which are future recommendations. Furthermore, most of these investigations have shown a strong association between the antioxidant potential of avocado extracts and their phenolic components [30,31]. Additionally, these components have been demonstrated to reduce platelet aggregation and oxidation, as well as inflammation [32]. The antioxidant qualities of avocado by-product paste, with and without seeds, were assessed and contrasted, showing that the paste with seeds had less antioxidant activity than the paste without seeds. It was also noted that the antioxidant activity slightly diminished during storage [33]. This implies that the type of formulation and storage conditions also play a role in their activity and should therefore be considered.

Based on the results obtained from the present study, it is suggested that the formulated mouthwash exhibits good anti-inflammatory activity and could be used as an adjunct in treating minor inflammatory diseases. According to the previous studies discussed, this natural derivative-based formulated mouthwash is safer than other chemically manufactured mouthwashes.

The present study failed to assess the oil present in avocado and to compare it with other herbs high in oil, even though oils are known to have significant medicinal properties, particularly in treating inflammatory conditions. Additionally, other avocado components, such as the peel and seeds, were not included in the synthesis of the mouthwash. As a result, the study did not evaluate or compare the properties of these components. Investigating all parts of the avocado could potentially lead to the development of an effective mouthwash with therapeutic value.

The mouthwash synthesised with avocado and clove extract exhibited better anti-inflammatory properties than standard mouthwash, working best at a concentration of 50 μL. Therefore, this formulation could be useful for treating minor inflammatory disorders such as aphthous ulcers. Given that avocado oil has a higher nutritional value than avocado seeds but lower activity levels, experimenting with various other combinations could potentially yield a less toxic yet effective mouthwash, thereby benefiting the medical field. Future in-vivo studies on human subjects could be conducted to validate the efficacy of the mouthwash.