Antimicrobial, anti-inflammatory, and antioxidant effect of onion peel extract (Allium cepa L.) on periodontal pathogen Porphyromonas gingivalis: An in vitro analysis

Abstract

Periodontitis is an inflammatory condition triggered by a host response to pathogenic dental biofilms, particularly Gram-negative anaerobes such as Porphyromonas gingivalis (P gingivalis). Effective management often involves scaling, root planing, and chemical treatments. Herbal remedies, such as onion extracts, have shown potential in combating periodontal pathogens due to their low toxicity and beneficial properties. This interventional study aims to evaluate the antimicrobial, anti-inflammatory, and antioxidant properties of onion peel extract against P gingivalis in an in vitro setting. Onion peels were cleaned, dried, powdered, and extracted using the Soxhlet method. The minimum inhibitory concentration of the extract against P gingivalis was determined. Antioxidant activity was assessed using the 2,2-diphenyl-picryl-hydrazyl assay, and anti-inflammatory effects were evaluated through matrix metalloproteinase (MMP) inhibition assays. The minimum inhibitory concentration for onion peel extract was 0.2 µL/mL, indicating significant antimicrobial activity. The 2,2-diphenyl-picryl-hydrazyl assay revealed 40% antioxidant activity at concentrations ranging from 25 to 100 µL/mL. The anti-inflammatory assay demonstrated a 65% inhibition of MMP-9 and a 72% inhibition of MMP-2, suggesting robust anti-inflammatory effects. The findings suggest that onion peel extract has antibacterial, antioxidant, and anti-inflammatory qualities, implying it could be used therapeutically to treat periodontal diseases. Its effectiveness in lowering P gingivalis growth and inflammation demonstrates its potential as a natural supplement to conventional treatments.

1. Introduction

Chronic periodontitis is associated with the presence of subgingival microbes along with a host response of immuno-inflammatory infiltrate that can eventually cause tooth loss if left untreated.^[1–3] Periodontitis subgingival bacteria are crucial in both the onset and advancement of this condition. Notably, Porphyromonas gingivalis (P gingivalis), a Gram-negative anaerobic bacterium, plays a key role in chronic periodontitis and has been identified in a substantial proportion (85.75%) of subgingival plaque samples from individuals with chronic periodontitis.^[4] P gingivalis is thought to be a major cause of periodontal conditions due to its production of extracellular proteases and other virulence factors that cause periodontal tissue breakdown.^[5] P gingivalis, along with other pathogens that trigger host response, is thought to be an important aspect of the etiology of advanced periodontitis.^[6]

Oral hygiene practices such as chemical interventions like mouthwashes and toothbrushing are effective strategies for periodontitis prevention, but are associated with undesirable effects such as tooth discoloration and taste alterations.^[7] Furthermore, the extended use of antimicrobial and antiplaque mouth rinses is not advised because of potential adverse reactions and the emergence of antimicrobial resistance.^[8–11] Thus, there is a need to search for viable alternative solutions for the treatment and prevention of periodontal diseases. Herbal remedies and alternative medicine have been explored to combat periodontal pathogens with minimal toxicity and with beneficial effects on the host.^[12]

Plants provide a suitable alternative treatment option for the prevention and treatment of periodontal diseases due to relatively safer use, reduced cost, and broad medicinal action.^[13–15] Furthermore, plants contain phytochemicals like alkaloids and flavonoids, which offer antimicrobial, anti-inflammatory, and antioxidant properties, positioning them as potential therapeutic options.^[16] Additionally, the tolerance of gingival tissues to herbal medications is reported to be good.^[17]

One such herbal component is onion. Onion peels contain bioactive phenolics and possess several beneficial biological properties, including antibacterial, antimutagenic, and antioxidant effects.^[18] Onion peels, frequently regarded as trash, have a high concentration of phenolic compounds, which have antibacterial and antioxidant qualities.^[19] Specifically, quercetin, a flavonoid found in onion peels, is well recognized for its antioxidant and anti-inflammatory properties.^[20]

Although onion peel extract has demonstrated promising properties for the treatment of various ailments, the application of onion extract in the context of periodontal diseases remains largely unexplored, to the best of the authors’ knowledge. Consequently, this in vitro study aimed to assess the antimicrobial, anti-inflammatory, and antioxidant effects of onion peel extract against the periodontal pathogen P gingivalis. The null hypothesis was that there would be antimicrobial, anti-inflammatory, and antioxidant effects of onion peel extracts on P gingivalis. The data will offer useful insights into the creation of novel herbal treatments for periodontitis, potentially leading to more accessible and efficient therapeutic approaches.

2. Methods

2.1. Study design

This laboratory-based interventional study was conducted to evaluate the antimicrobial, anti-inflammatory, and antioxidant effects of onion peel extract on the key periodontal pathogen P gingivalis. The study was designed in an in vitro setting, and an appropriate sample size was taken. The experimental design was reviewed and approved by the Institutional Ethical Committee and assigned a reference number (MGV/KBHDC/1002/2023-24). The test bacteria were procured from the American Type Culture Collection.

2.2. Materials

Fresh red onions were obtained from a local nursery in Nasik, Maharashtra, India. Their identification was confirmed by the Pharmacognosy Department, Faculty of Pharmacy, MGV’s Institute, Nashik, India. The outer dry onion peels were separated manually. The onion peels were 1st cleaned with tap water and then dried in the shade for approximately 4 to 5 days at room temperature. The dried peels were coarsely ground by using a domestic grinder (Bajaj GX 8 750-Watt Mixer, India) into a sizable powder. After filtering, the powder with an average particle size of 0.085 mm was utilized for the experimentation. The extracts were stored at 4°C in an amber bottle till further experiments.

2.3. Extract preparation

The Soxhlet extraction method was used for extract preparation.^[21] The crushed plant material was placed into the thimble, which was then inserted into the Soxhlet apparatus. Analytical-grade ethanol was added to a round-bottom flask connected to a condenser and heated to the boiling point of the solvent. The extraction process was continued till the material was entirely extracted; the extract was then kept for cooling. The extract was distilled using a rotary evaporator (Heidolph Instruments GmbH & Co. KG, Germany) to remove any residual solvent, and the semisolid extract was transferred to a China dish and warmed to a low temperature to completely remove the solvent. Lastly, the total weight of the extract was measured.

2.4. Minimum inhibitory concentration test

The minimum inhibitory concentration (MIC) refers to the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. The MIC of the onion peel extract was determined against standard strains of P gingivalis (American Type Culture Collection 33277, Central Research Laboratory, Maratha Mandal, Belgaum, India). Two repetitions of the sample and control groups were performed to obtain the results. For the MIC test, 9 dilutions of each medication were administered along with thioglycollate broth. In the 1st tube, 380 μL of thioglycollate broth was mixed with 20 μL of medication. Furthermore, 200 μL of thioglycollate broth was added individually to each of the next 9 tubes for dilutions. Later, 200 μL of thioglycollate broth was transferred to the 1st tube. This was a 10⁻¹ dilution. 200 μL of the 10⁻¹ diluted tube was moved to the 2nd tube to create the 10⁻² dilution. For every drug, the serial dilution was carried out up to a 10⁻⁹ dilution. Five microliters of P gingivalis maintained stock cultures were obtained and added to 2 mL of thioglycollate broth. The tubes were kept in an anaerobic jar at 37°C for 48 to 72 hours, during which time their turbidity was monitored.^[22]

2.5. Antioxidant activity

Antioxidant activity refers to the ability of a substance to prevent or slow down damage to cells caused by free radicals. Based on the reduction of a stable radical, the 2,2-diphenyl-picryl-hydrazyl(DPPH) radical scavenging activity assay is frequently used to assess antioxidant properties in a reasonably quick manner. A spectrophotometer (Shimadzu UV-1900, Japan) measures the change in color of the solution from purple to yellow as an antioxidant reduces DPPH. As reagents, 0.2 mM DPPH (Sigma-Aldrich, St. Louis) and 0.1 M tris-hydrochloric acid (Tris-HCl) of pH 7.4 were collected. Ten milligrams of the extract was then weighed and dissolved in 1 mL of Dimethyl sulfoxide (Merck Life Science Pvt. Ltd., Mumbai, India). Trolox (Sigma-Aldrich, St. Louis) was used as a DPPH-scavenging chemical, and 3 test tubes were labeled as blank, control, and test. 400 µL of Tris-HCl and 600 µL of ethanol were put into a blank test tube.

One hundred microliters of ethanol, 400 µL of Tris-HCl, and 500 µL of DPPH agent were added to the control, and 100 µL of sample, 400 µL of Tris-HCl, and 500 µL of DPPH agent were added to the test. After mixing each tube and leaving it in the dark for 30 minutes, the absorbance at 490 nm was measured.^[3]

The antioxidant activity was estimated at different concentrations using the formula:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{\%}=\frac{\mathrm{A}\mathrm{c}-\mathrm{A}\mathrm{s}}{\mathrm{A}\mathrm{c}}\times 100}\end{array}}$$

Where, As = sample O.D. and Ac = control O.D.

2.6. Anti-inflammatory assay

The anti-inflammatory properties of a substance refer to its ability to reduce or suppress inflammation in the body, and it was determined using matrix metalloproteinase (MMP) inhibition assays. The electrophoresis device was cleaned, and plates were arranged with a spacer between them, clamped, and screwed tightly. To seal the bottom surface, agarose gel was placed between the 2 glass plates and allowed to sit for 5 to 10 minutes. To prevent bubbles, the proper resolving gel combination was pipetted between the glass plates. The plates were filled to 80%, leaving room for the comb and stacking gel. For the resolving gel and stacking gel to have a perfectly level interface, a small amount of water was laid and permitted to settle down for a period of 45 minutes. The additional water was drained from the area before the stacking gel was applied on top of the resolving gel. After inserting the comb, 30 minutes were given for it to set.

After gathering the MMP samples, the extract was incubated at room temperature for 1 hour. Electrophoresis was carried out, and electrode gels were prepared. After decanting the zymogram renaturing buffer, the gel was incubated for an entire night at 37°C in the zymogram incubation buffer. Coomassie Brilliant Blue R-250 (Sigma-Aldrich, St. Louis) was used for a 1-hour staining period. Positive control (tetracycline hydrochloric acid) and negative control (supernatant of squamous cell carcinoma) were used. The Gel Documentation System (Bio-Rad Gel Doc EZ, Bio-Rad Laboratories, Inc., Hercules, California) captured the gel image, which was then transferred to Total Lab Quant software (Total Lab Ltd., Newcastle, UK) for band quantification based on intensity.^[23,24]

3. Results

3.1. MIC

The antimicrobial activity of onion peel extract against P gingivalis was determined using the MIC assay. A serial dilution method revealed that the extract was effective at progressively lower concentrations. MIC data were categorical (S/R) rather than continuous; a chi-square test for independence was employed to ascertain whether inhibition is substantially correlated with concentration.

Observable turbidity, used as an indicator of bacterial growth, was absent at a concentration of 0.2 µL/mL, indicating complete inhibition of microbial proliferation. This finding was consistent in both replicates of the experiment, confirming the reproducibility and reliability of the antimicrobial effect as shown in Table 1. The test tubes, ranging from 100 to 0.2 µL/mL, consistently showed no visible bacterial growth, whereas subsequent lower dilutions did not maintain this inhibitory effect. This suggests that the threshold concentration for effective bacterial inhibition lies between 0.2 µL/mL and 0.1 µL/mL. At concentrations ≥0.2 µL/mL, the extract reliably inhibited bacterial growth across replicates, confirming both potency and reproducibility. The inhibition of growth was statistically significant (P < .001), confirming that the observed antimicrobial action is not attributable to chance. The onion peel extract thus demonstrates a high degree of antimicrobial potency in vitro against P gingivalis.

Table 1.

Results of MIC against P gingivalis.

Onion peel extract	100 µL/mL	50 µL/mL	25 µL/mL	12.5 µL/mL	6.25 µL/mL	3.12 µL/mL	1.6 µL/mL	0.8 µL/mL	0.4 µL/mL	0.2 µL/mL	B + C	B + O
Pg	S	S	S	S	S	S	S	S	S	S	S	R
Pg	S	S	S	S	S	S	S	S	S	S	S	R

MIC = minimum inhibitory concentrations, Pg = P gingivalis, R = resistant, S = sensitive.

3.2. Antioxidant activity

The antioxidant activity of the onion peel extract was assessed using the DPPH radical scavenging assay, and the results indicated significant potential for scavenging free radicals. The antioxidant activity was estimated at different concentrations according to the previously described formula. A 1-way analysis of variance (ANOVA) was applied to compare the mean % inhibition at 3 concentrations (100, 50, and 25 µL/mL). Results indicated a consistent and concentration-dependent activity across all tested dilutions – 25 µL/mL, 50 µL/mL, and 100 µL/mL. In the 1st trial, the extract demonstrated 43%, 38%, and 41% inhibition at concentrations of 100, 50, and 25 µL/mL, respectively. The 2nd trial was closely aligned, showing inhibition values of 41%, 39%, and 42% at the same concentrations as depicted in Table 2. These values were averaged, indicating approximately 40% inhibition across the tested range as displayed in Figure 1. Overall mean inhibition was 40.7%, and overall standard deviation (SD) was ±1.87. The consistency between replicates affirms the extract’s radical scavenging potential. The purple color of the DPPH solution faded visibly in the presence of the extract, indicating the neutralization of free radicals by the phytochemicals present in the onion peel. For the ANOVA outcome, P > .05 indicated no statistically significant difference in antioxidant activity among the 3 concentrations tested, which signifies a considerable antioxidant ability, even at lower extract concentrations. The colorimetric change was quantitatively verified using spectrophotometric analysis at 490 nm, confirming a significant reduction in optical density relative to the control.

Table 2.

Result of DPPH assay at different concentrations.

Sr. no.	Concentration (µL/mL)	Repeat I (%)	Repeat II (%)	Average inhibition (%)
1.	100	43	41	42.0
2.	50	38	39	38.5
3.	25	41	42	41.5

DPPH = 2,2-diphenyl-picryl-hydrazyl.

Figure 1.

Average antioxidant inhibition percentages at different concentrations.

3.3. Anti-inflammatory test results

The anti-inflammatory potential of the onion peel extract was investigated through its inhibitory effect on MMPs, specifically MMP-2 and MMP-9, using zymographic analysis. ANOVA, accompanied by Tukey’s post hoc test, was employed to compare the mean percentage inhibition across the 3 treatment groups (negative control, onion peel extract, and positive control). Descriptive statistics demonstrated MMP-2 inhibition: mean = (0 + 72 + 100)/3 = 57.3%, SD = 50.1 and MMP-9 inhibition: mean = (0 + 65 + 100)/3 = 55.0%, SD = 50.6.

ANOVA results for both MMP-2 and MMP-9, P < .01, indicated statistically significant differences between treatment groups. Whereas, post hoc Tukey’s test for onion peel extract versus the negative control indicated a significant difference (P < .01), confirming a strong inhibitory effect. Onion peel extract versus positive control showed a significant difference (P < .05), displaying extract is less potent than tetracycline but still effective. Lastly, positive versus negative control showed a highly significant difference (P < .001) (Table 3).

Table 3.

Statistical analysis summary for anti-inflammatory test.

Inhibition	Mean	SD	ANOVA (P-value)	Tukey’s post hoc comparison	Significance
MMP-2	57.3	50.1	P < .01	Onion peel extract vs negative control	P < .01 (significant)
				Onion peel extract vs positive control	P < .05 (significant)
				Positive vs negative control	P < .001 (highly significant)
MMP-9	55.0	50.6	P < .01	Onion peel extract vs negative control	P < .01 (significant)
				Onion peel extract vs positive control	P < .05 (significant)
				Positive vs negative control	P < .001 (highly significant)

ANOVA = a 1-way analysis of variance, MMP = matrix metalloproteinase, SD = standard deviation.

Upon electrophoretic separation and staining with Coomassie Brilliant Blue R-250, distinct bands corresponding to MMP-2 and MMP-9 enzymatic activity were visualized. Band intensity analysis revealed a 72% reduction in MMP-2 activity and a 65% reduction in MMP-9 activity when compared with the negative control (Fig. 2). These measurements were derived using Total Lab software, which provided precise intensity quantification from gel documentation images. The negative control group (squamous cell carcinoma supernatant) showed 100% expression of both MMPs, whereas the positive control group (tetracycline hydrochloride) exhibited complete inhibition. In comparison, the test group with onion peel extract presented substantial attenuation of proteolytic enzyme expression (Table 4). The clarity and density of the gelatinolytic bands were significantly diminished in the extract-treated group, indicating successful suppression of extracellular matrix-degrading enzymes (Fig. 3). This result signifies a biologically meaningful reduction in the activity of enzymes that contribute to connective tissue breakdown in inflammatory conditions. Although its inhibitory strength does not reach the 100% inhibition achieved by tetracycline, the extract’s inhibition represents a biologically and statistically meaningful reduction in MMP activity.

Figure 2.

Result of anti-inflammatory test for onion peel extract. MMP = matrix metalloproteinase.

Table 4.

Results of anti-inflammatory test.

S. no.	Name of mixture	% bands of MMP		% inhibition of MMP
		MMP-2	MMP-9	MMP-2	MMP-9
1.	Onion peel extract	28	35	72	65
2.	Positive control (tetracycline HCl)	00	00	100	100
3.	Negative control (supernatant of squamous cell carcinoma)	100	100	00	00

HCl = hydrochloric acid, MMP = matrix metalloproteinase.

Figure 3.

Anti-inflammatory potential experiment. (A) Gel documentation system, (B) result of anti-inflammatory test. MMP = matrix metalloproteinase.

4. Discussion

This study was conducted to assess the antimicrobial, anti-inflammatory, and antioxidant effects of onion peel extract on the key periodontal pathogen, P gingivalis. The results of the current study revealed that onion peel extract demonstrated significant antimicrobial activity, antioxidant activity, and anti-inflammatory properties. These results can also be used to explore the potential use of onion peel extracts for treating periodontitis.

Further investigation into plant-based interventions is warranted to identify effective alternatives to traditional periodontal disease treatments. Plant-derived chemicals have been shown to possess anti-inflammatory, antimicrobial, and antioxidant properties, thereby demonstrating potential for treating various diseases. Previous research has explored onion peel extracts, indicating their potential in wound treatment due to their capacity to mitigate microbial infections and inflammation, as well as facilitate tissue regeneration.^[25] It has been studied for its potential use in cancer and its antimicrobial effect on pathogens in dental caries.^[19,25] The beneficial attributes of onion peels can be attributed to the presence of phytochemicals. Existing literature has established the presence of health-enhancing phytochemicals in both the edible portions and external layers of onions.^[26,27] Similarly, using gas chromatography-mass spectrometry, different bioflavonoids in onion extracts have also been discovered.^[28] The current study, therefore, substantiates and extends these findings by confirming the therapeutic promise of onion peel extract in the context of periodontal health.

The results of the current study demonstrated that the onion peel extract exhibits inhibitory activity against P gingivalis, with MIC values ranging from 0.2 µL/mL to 6.25 µL/mL. Susila et al reported similar findings, indicating that onion peel extracts exhibit antibacterial activity against both Streptococcus mutans and Enterococcus faecalis.^[29] Similarly, Joković and others found the antimicrobial effects of onion peel extracts against a wide spectrum of gastrointestinal microorganisms in their study.^[30] Further, studies have shown antimicrobial properties of onion peel extracts against several pathogens, such as Gram-positive bacteria and Gram-negative bacteria, wherein the Gram-positive bacteria were found to be more sensitive to Gram-negative bacteria.^[18,31]

While onion peels possess established antimicrobial characteristics, the specific component responsible remains unidentified. Sharma et al^[32] demonstrated that quercetin, extracted from a methanolic onion extract, exhibits antienzyme activity against enzymes found in multidrug-resistant bacteria, including Pseudomonas aeruginosa and Escherichia coli. Quercetin is a compound widely found in onion peels. As a supplement, it reduces inflammation and creates an environment in the oral cavity that is favorable to the symbiotic bacteria linked to health, which in turn promotes a balanced state of periodontal tissue homeostasis.^[33] Onion peel extract enhances macrophage phagocytic activity by augmenting interleukin-12 production. It also inhibits bacterial adhesion by preventing PiLY1 protein binding to calcium in epithelial cells. Furthermore, the extract reduces bacterial motility by altering the bacterial membrane potential, disrupting the primary proton pump, and impeding energy production required for hydrogen ion transport from the cytoplasm to the cell’s surface.^[34]

The current study also demonstrated the anti-inflammatorypotential of onion peel extract, suggesting its possible use for therapeutic applications. The results indicated that onion peel extract exhibited anti-inflammatory activity, inhibiting MMP-9 by 65% and MMP-2 by 72%. Onion peel is known to contain a significantly higher concentration of flavonoids compared to the edible part of the onion. The anti-inflammatory, antibacterial, and antiresorptive properties of flavonoids are significant factors that influence their use in the prevention of periodontal disease. Studies reveal that inflammation plays a significant role in the development of periodontal disease; thus, reducing inflammation aids in the treatment of periodontal disease.^{[35, 20]}

The DPPH assay results indicated peak activity at a concentration of 25%, with subsequent reductions observed at higher concentrations. This finding supports the antioxidant properties of onion peel extract, even at notably low concentrations. The ability of onion peel extracts to demonstrate antioxidant properties can be credited to several phytochemicals that have been identified, such as quercetin, vanillic acid, kaempferol, and p-hydroxybenzoic acid.^[36] Among these, quercetin has been widely studied for its key role in antioxidant effects in onion peels. Quercetin has good antioxidant properties because of free hydroxyl groups, chelation of metal ions, and reduction of oxidase enzymes.^[37] Furthermore, the quercetin content in onion peels has been reported to be approximately 75 times more than that in onion bulbs. Mooney et al found in their study that orally administered quercetin reduces inflammation and encourages the growth of symbiotic bacterial flora.^[34] When used as an irrigant in endodontics, quercetin functions as an antibiofilm and collagen-stabilizing agent.^[29]

Although the current study follows a robust methodology, it has certain limitations. The study was an in vitro study, and a complete clinical setting could not be replicated. Thus, future clinical studies are suggested.

5. Conclusion

In summary, the findings of this investigation suggest that onion peel extract represents a potentially effective natural agent for the management of periodontal diseases, by virtue of its antimicrobial, anti-inflammatory, and antioxidant attributes. Consequently, further research into the application of onion peel extracts for the treatment of periodontitis is justified.

Acknowledgments

The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for supporting this work through the large group project RGP-2/123/46.

Author contributions

Conceptualization: Vedant Bhandari, Swapna Mahale, Shahabe Saquib Abullais.

Funding acquisition: Shahabe Saquib Abullais, Sumedha Thosar, Sultan Alanazi, Suheel Manzoor Baba.

Methodology: Akhil Patil.

Software: Vedant Bhandari, Swapna Mahale.

Supervision: Sultan Alanazi.

Data curation: Vedant Bhandari, Swapna Mahale, Akhil Patil, Suheel Manzoor Baba.

Formal analysis: Vedant Bhandari, Shahabe Saquib Abullais, Sumedha Thosar, Akhil Patil.

Visualization: Vedant Bhandari, Swapna Mahale, Shahabe Saquib Abullais, Sumedha Thosar, Suheel Manzoor Baba.

Writing – original draft: Vedant Bhandari, Swapna Mahale, Shahabe Saquib Abullais, Sumedha Thosar, Akhil Patil, Sultan Alanazi, Suheel Manzoor Baba, Pragati Kaurani.

Writing – review & editing: Vedant Bhandari, Swapna Mahale, Shahabe Saquib Abullais, Suheel Manzoor Baba, Pragati Kaurani.

Resources: Swapna Mahale, Akhil Patil, Pragati Kaurani.

Validation: Swapna Mahale, Shahabe Saquib Abullais, Sultan Alanazi.