Assessment of potential antimicrobial activity of Ocimum basilicum extract and chlorhexidine against Socransky’s complex pathogens of oral cavity: An in vitro study

Abstract

Background and Objective:

Periodontitis is a multifactorial disease initiated by periodontal pathogens and progresses further in destruction of periodontium. Hence, the objective of this study was to test the efficacy of Ocimum basilicum seeds extract on periodontal pathogens.

Materials and Methods:

O. basilicum seeds were authenticated from a recognized taxonomist. They were coarsely powdered; ethanol-based extract preparation was done by the Soxhlet method and aqueous-based extract by hot infusion procedure. Extracts so obtained were assessed for minimum inhibitory concentration, minimum bactericidal concentration, zone of inhibition, and time-kill assay of O. basilicum seeds extract on periodontal pathogens, and comparatively evaluated the effectiveness against 0.12% chlorhexidine (CHX) gluconate in triplicates. Kruskal-Wallis Test was employed wherein the statistical significance was set at P ≤ 0.05.

Results:

The concentration of O. basilicum ethanolic extract against periodontal pathogens was determined to be 10 mg/ml, whereas 4.7 mg/ml of aqueous extract was proven effective against periodontal pathogens. Similarly, aqueous extract of O. basilicum developed a wider zone against periodontal pathogens compared to ethanol-based O. basilicum extract. Statistically significant difference found in the effectiveness between both extract and CHX.

Conclusion:

The antibacterial activity was evident in both the extracts of O. basilicum against anaerobic periodontal pathogens. However, it was more pronounced in aqueous extract, but lower compared to CHX.

INTRODUCTION

Periodontal disease is one of the most crucial issues for dentists and patients. It is identified as a major public health problem throughout the world and is the common reason of tooth loss in adults. Periodontitis is characterized by microbially associated, host-mediated inflammation that results in loss of periodontal attachment.[1] The periodontium is a connective tissue organ, protected by epithelium, that attaches the teeth to the bone of the jaws and provides a continually adapting apparatus for their support during function.[2] A variety of triggering factors like bacterial species, dyscrasias, avitaminosis, etc., cause inflamed gums leading to gingivitis.[3] Salivary tartar has an additive effect to these factors in causing gingivitis.[4] Aggressive periodontitis, chronic periodontitis, and those resulting from conditions such as acquired immunodeficiency syndrome, diabetes, malnutrition, and immunosuppression are the other types of periodontitis.[5] Researchers found that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Porphyromonas gingivalis (P. gingivalis), and Tannerella forsythia (T. forsythia) are likely to cause aggressive periodontitis.[6] Deep pockets are caused by bacteria such as P. gingivalis and A. actinomycetemcomitans, as well as other species, and are linked with resistance to traditional therapies.[7]

Despite major breakthroughs in disease biology and treatment approaches during the last century, the periodontal disease is still unmanageable over the world. Chlorhexidine (CHX) is a commonly prescribed medication for the treatment of dental disease.[8] The proof of its efficacy in plaque control is beyond dispute. It has been used by dental practitioners for nearly three decades. However, the use of CHX for dental disease prevention has been controversial owing to its various side effects such as dry mouth, irritation, teeth staining, and taste disturbances.[9] The use of herbal products offers a valuable alternative to CHX owing to their medicinal values and no side effects.[10,11]

The practice of medicine has evolved over many centuries to reach its current state. India, since time immemorial, is a land where natural herbs and derived products have been used to cure various diseases. Herbal and natural folk medicine products have been utilized for generations for betterment of humankind. The most common herbal ingredients to be incorporated into oral care products (e.g., toothpaste and mouth rinse) are sanguinarine, propolis, Azadirachta indica (neem), charcoal clove, and miswak.[12] Basil, popularly known as the “King of Herbs,” is high in phytochemicals that have substantial nutritional, antioxidant, and health advantages. Basil seeds are authenticated as “Ocimum basilicum” belonging to the family “Lamiaceae,” which is an annual plant.[13,14] Sweet basil seeds contain polyphenolic flavonoids, particularly orientin and vicenin; essential oils such as eugenol, citronellol, linalool, limonene, citral, and terpineol; high levels of beta carotene, lutein, zeaxanthin, Vitamin A, Vitamin C, and Vitamin K; and minerals such as potassium, manganese, copper, calcium, magnesium, and folates.[14] Furthermore, different research has shown that sweet basil seeds provide health benefits such as weight loss, healthy skin, cooling effect, acidity prevention, anti-inflammatory, and anticancer properties.[15,16] Active compounds found in O. basilicum seeds are planteose, mucilage, and polysaccharides. Moreover, secondary metabolites of seeds, including phenolic and flavonoid, have been shown to exhibit pharmacological properties such as antioxidant, antibacterial, antiviral, antidiabetic, anti-inflammatory, antiallergic, anticancer, neurodegenerative, and vasodilatory effects.[17]

A thorough literature search found limited studies reporting the antibacterial activities of diverse species of Ocimum against cariogenic pathogens. Thus, there is an impending need for assessing the antimicrobial properties of a novel O. basilicum extract against selective periodontal pathogens. However, periodontal disease control is urgent, and there are considerable gaps in the study field. Hypothesis states that there is a difference in the effectiveness of O. basilicum seeds extract and 0.12% CHX gluconate against P. gingivalis, A. actinomycetemcomitans, and T. forsythia. Hence, the aim and objective of this study was to assess the potential antimicrobial efficacy of ethanolic and aqueous solvent-based O. basilicum seed extract against periodontal pathogens in comparison with CHX gluconate, so that its formulation can be effectively used to reduce the pathogenic microorganisms in the oral cavity.

MATERIALS AND METHODS

The present study was an in vitro study; it was conducted according to the guidelines of good laboratory practice[18] and The study was approved by the Institutional Research and Ethics Committee of KAHER’s KLE Vishwanath Katti Institute of Dental Sciences (reference number: 1499 and date of approval: 25th October 2021). O. basilicum seeds were authenticated from a recognized taxonomist. The coarse powder of O. basilicum seeds was procured from Ayurveda pharmacy of a recognized institute (AYUSH Approved Central Research Facility and Drug testing laboratory for Ayurveda, Siddha, and Unani Drugs).

The seeds were crushed with a pestle in a mortar to much finer particles, of which 100 g was soaked in 1 L of boiled hot water for 48 h, and the mixture obtained was filtered with Whatman number 1 filter paper (Sigma Aldrich^® Chemicals Pvt. Ltd., Bangalore, India) which was adapted according to hot infusion method on a thermostatically water bath (Labline^® Stock Center, Mumbai, India). The filtrate was concentrated at 40°C in a rotary evaporator (IKA^™), at 190–220 rpm for 48 h and stored in the refrigerator at 4°C in air-tight sterile container till further use [Figure 1].

Figure 1.

Ocimum basilicum extract preparation. (a) Hot infusion method for Ocimum basilicum AE preparation; (b) Filtration of the supernatant O. basilicum AE; (c) Soxhlet method Ocimum basilicum ethanolic extract preparation; (d) Rotary evaporator (IKA^™) to concentrate the filtrate of extract. AE – Aqueous Extract

The Soxhlet method was followed to prepare crude extract of the O. basilicum seeds, where in 99.9% ethanol (Changshu Hongsheng Fine Chemicals Co. Ltd, China) was used as a solvent. The seed powder (100 g) was placed in a muslin cloth bag which was kept in the body of Soxhlet extractor, and to this, ethanol (800 ml) was added. The entire procedure ran for 6 h at a boiling temperature of ethanol 78° C (173° F) completing 24 cycles. Once the process was finished, the ethanol was entirely evaporated using rotary evaporator (IKA^™), at 40°C, leaving a yield of extracted plant material (about 20 mg). The yield was resuspended in dimethyl sulfoxide (DMSO) (Qualigens, Thermo Fisher Scientific Pvt. Ltd, Mumbai, India) in 20 mg/ml ratio to obtain a stock solution. The extracts obtained from both the techniques were subjected to preliminary phytochemical screening for qualitative detection of phytoconstituents using standard procedures as described by Trease and Evans.

The standard fresh strains of P. gingivalis – ATCC 33277, A. actinomycetemcomitans – ATCC 29523, and T. forsythia – ATCC 43037 were procured from LGC Promochem India Pvt. Ltd., Bangalore, India. The strains were cultivated anaerobically in 5 ml sterile brain heart infusion (BHI) broth (Sigma Aldrich^® Chemicals Pvt. Ltd., Bangalore, India) supplemented with 1% horse serum, 0.5 mg/ml of hemin, and 5 mg/ml of Vitamin K, and inoculated in anaerobic chamber (N₂ 80%, H₂ 10%, and CO₂ 10%) at 37°C for 48 h until it achieved the turbidity of 0.5 McFarland standards (1 × 10⁸ colony-forming unit (CFU)/ml).

Twelve mgs of CHX salt was mixed and diluted in 10 ml of distilled water (1.2 mg/ml) in a centrifuge tube (Tarsons Products Ltd., Kolkata, India). The mixture was vortexed vigorously using a MixMate^® Vortex agitator (Eppendorf, Sydney, Australia) for 30 s at 1000 rpm (maximum setting) to obtain a final concentration of 10 ml of 0.12% of CHX solution. Following which 96-well microtiter plate (NEST Biotechnology, Jiangsu, China) were taken, and 10 wells were allotted for each extract in triplicates. A volume o f 100 μl broth was added in all the wells, and 100 μl of each extract was added in the first well; serial doubling dilutions were implied for the extract to requisite concentrations up to the 10^th well. Therefore, the concentration in subsequent wells reduced by 50%. A volume of 10 µl of inoculum was added to all 10 wells and kept for incubation at 37°C in McIntosh and Fildes’ anaerobic jar for 2 days. After 48 h, 10 μl resazurin dye (5 mg/10 ml distilled water) (Hi-Cert^™ HiMedia^® Laboratories, Pvt. Ltd, Mumbai, India) was added and observed for color change from blue/violet to slight pink/magenta except positive control by incubating in anaerobic condition at 37°C for 4 h. The concentration at which resazurin reduces to resafurin compound by color change from blue to pink color was taken as a minimum inhibitory concentration (MIC) value.[19] This procedure was repeated for three periodontal pathogens and two extracts separately. The results recorded in triplicate outcomes and mean value were taken.

The spread plate method was used to determine minimum bactericidal concentration (MBC). The bacterial suspension from alternate wells with dilutions of O. basilicum ethanolic extract (20, 5, 1.25, 0.312, and 0.078 mg/ml), O. basilicum aqueous extract (10, 2.5, 0.625, 0.156, and 0.039 mg/ml), and CHX (0.12, 0.03, 0.0075, 0.0018, and 0.00046 mg/ml) concentrations higher than the MIC value was inoculated on BHI agar plates and incubated for 48 h at 37°C. The concentration at which no bacterial growth was seen, considered bactericidal values. The MBC is defined as the minimum concentration of drug which kills 99.9% of the test microorganisms in the original inoculum.[20]

Agar well diffusion (punch well diffusion) was used to test antimicrobial susceptibility. Lawn culture of test organism was made on respective media. The whole surface of the agar plate was swabbed three times with the cotton swab. Following which, individual 4 mm depth and 6 mm diameter wells were cut in agar plates with sterile borer. These wells were filled with 100 μl of stock solution (20 mg/ml) for each extract and control to determine the antimicrobial activity against the pathogens. CHX gluconate (0.12%) (ICPA Health Products Ltd, Mumbai, India) was used as a positive control and saline (0.90% w/v of NaCl, 308 mOsm/L) (Amanta Healthcare Ltd., Gujarat, India) as a negative control. After 48 h of incubation period, the zone of inhibitions against the extracts was measured using a Scienceware^® Vernier Caliper. The diameters of the clear inhibition zones were measured to the nearest millimeter.

A time-kill assay was performed by the broth microdilution method, according to the Clinical and Laboratory Standards Institute guidelines. MBC values obtained were used for time-kill assay; 48 h grown inoculum was diluted in fresh BHI; and optical density (OD) was adjusted at 540 nm. The increase of value by <0.05 indicates no change in OD. Extract deficient was considered a negative control. Further, these tubes were incubated at 37°C, and aliquot of the cultures was streaked on agar plates at a time interval of 0, 3, 6, 12, and 24 h. After 48 h of incubation, colony formation was observed. The killing time was recorded when the cell density decreased by ≥3 log10 in CFU/ml compared to cell density at 0 h.[21]

The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (HiMedia^® Laboratories, Pvt. Ltd, Mumbai, India) reagent was prepared using 5 mg in 1 ml of phosphate buffered saline – pH 7.4 (HiMedia^® Laboratories, Pvt. Ltd, Mumbai, India), and a cytotoxicity assay was performed on gingival fibroblast.[22,23] In vitro growth inhibition effect of O. basilicum extract was assessed byenzyme-linked immunosorbent assay (ELISA) reader (Epoch, BioTek^® Instruments, Inc., USA) by the determination of conversion of MTT into “formazan blue” by living cells. Fifty microliter of 4000 cells/ml cell suspension was seeded into each well in a 96-well microtiter plate, and the final volume was made up to 150 μl by adding Dulbecco’s Modified Eagle Medium (DMEM) (Gibco^™ Life Technologies, Bangalore, India). One hundred microliter of the O. basilicum extract was added to the wells and incubated for 24 h, in the presence of 5% CO₂, at 37°C in a CO₂ incubator (New Brunswick^™ Galaxy^® 170 R, Eppendorf, Germany). After 24 h, 20 μl of 5 mg/ml MTT reagent was added to the wells. The supernatant was carefully removed without disturbing the precipitated formazan crystals, and 100 μl of DMSO was added to dissolve the crystals formed. The OD was measured at a wavelength of 570 nm.

The experiment was repeated in triplicates for both ethanolic extract and aqueous extract of O. basilicum to ascertain the antimicrobial activity. The collected data were entered in MS Excel and analyzed using IBM-SPSS^® Statistics-Version 21 (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 32.0, Armonk, NY, USA: IBM Corp.). The Kruskal–Wallis test was applied to test the significance among both extracts and CHX for three different organisms. The statistical significance was set at P ≤ 0.05 for the test.

RESULTS

MIC of ethanolic extract and aqueous extract of O. basilicum was determined by resazurin broth dilution method. P. gingivalis was the most sensitive organism to aqueous extract of O. basilicum with the lowest MIC value of 2.5 mg/ml, followed by A. actinomycetemcomitans and T. forsythia with MIC value of 4 mg/ml and 10 mg/ml, respectively. While ethanolic extract showed a significant MIC value of 10 mg/ml for all three periodontal pathogens. A. actinomycetemcomitans and T. forsythia were the most sensitive organisms to CHX gluconate with MIC value of 0.0093 mg/ml, followed by P. gingivalis having MIC value of 0.075 mg/ml [Figure 2].

Figure 2.

MIC for antimicrobial activity using resazurin method. EE = Ethanolic Extract of Ocimum Basilicum, AE – Aqueous Extract of Ocimum basilicum, CHX – Chlorhexidine, MIC – Minimum inhibitory concentration

It was found that concentration of 5 mg/ml, aqueous extract O. basilicum restricted A. actinomycetemcomitans growth on the plate, while MBC value of aqueous extract of O. basilicum against P. gingivalis and T. forsythia was 2.5 mg/ml and 10 mg/ml, respectively. However, ethanolic extract of O. basilicum showed antibacterial property at 12.5 mg/ml against A. actinomycetemcomitans, while the average MBC value for O. basilicum ethanolic extract against P. gingivalis and T. forsythia was 10 mg/ml. MIC and MBC of test sample against periodontal pathogens are depicted in Table 1.

Table 1.

Minimum inhibitory concentration and minimum bactericidal concentration of ethanolic, aqueous-based extract of Ocimum basilicum and 0.12% chlorhexidine against periodontal pathogens

Samples	MIC (mg/mL)			MBC (mg/mL)
	PG	AA	TF	PG	AA	TF
Ethanolic extract of O. basilicum	10	10	10	10	12.5	10
	10	10	10	10	12.5	10
	10	10	10	10	12.5	10
Average	10	10	10	10	12.5	10
Aqueous extract of O. basilicum	2.5	5	5	2.5	5	10
	2.5	2.5	5	2.5	5	10
	2.5	5	10	2.5	5	10
Average	2.5	4	6.7	2.5	5	10
Chlorhexidine	0.075	0.0093	0.0093	0.075	0.0093	0.0093

MIC – Minimum inhibitory concentration; MBC – Minimum bacterial concentration; PG – Porphyromonas gingivalis; AA – Aggregatibacter actinomycetemcomitans; TF – Tannerella forsythia; O. basilicum – Ocimum basilicum

The zone of inhibition for A. actinomycetemcomitans against O. basilicum ethanolic extract was 12 mm which was lower than aqueous extract at 16 mm and CHX at 18 mm. For P. gingivalis, both the extracts had a smaller zone of inhibition compared to CHX. The zone of inhibition was produced by aqueous extract at 13 mm, ethanolic extract at 11 mm, and CHX at 16 mm against P. gingivalis. The zone of inhibition for T. forsythia after 48 h of incubation was broader for CHX with zone of inhibition of 18 mm, followed by aqueous extract 17 mm and ethanolic extract with zone of inhibition of 13 mm, as shown in Table 2. The zone of inhibition of ethanolic, aqueous-based extract of O. basilicum and 0.12% CHX against periodontal pathogens by well diffusion method is represented in Figure 3.

Table 2.

Zone of inhibition of ethanolic, aqueous-based extract of Ocimum basilicum and 0.12% chlorhexidine against periodontal pathogens by well diffusion method

Samples	Zone of inhibition (mm)
	PG	AA	TF
Ethanolic extract of O. basilicum	11	12	13
Aqueous extract of O. basilicum	13	16	17
Chlorhexidine	16	18	18

PG – Porphyromonas gingivalis; AA – Aggregatibacter actinomycetemcomitans; TF – Tannerella forsythia; O. basilicum – Ocimum basilicum

Figure 3.

The zone of inhibition of ethanolic, aqueous-based extract of Ocimum basilicum and 0.12% CHX against periodontal pathogens by well diffusion method; (a) Porphyromonas gingivalis; (b) Aggregatibacter actinomycetemcomitans; (c) Tannerella forsythia. CHX – Chlorhexidine

An almost similar killing profile was observed with O. basilicum aqueous extract and CHX. Both the cases showed a rapid and considerable decrease in the bacterial density after 4 h. At 7 h of exposure, the O. basilicum ethanolic extract also exhibited a similar bactericidal activity. At 24 h, no growth was observed with CHX, while the growth declined up to ~1 log10 with the O. basilicum extracts [Figure 4].

Figure 4.

Time-kill assay of ethanolic, aqueous-based extract and CHX on three different periodontal pathogens at various time intervals. X-axis = Time duration, Y-axis = Concentration of substrate in mg/ml. CHX – Chlorhexidine

Statistical analysis was performed on the results collected. The antibacterial activity of O. basilicum against A. actinomycetemcomitans, P. gingivalis, and T. forsythia was determined using the Kruskal–Wallis test. The P value difference between both extracts and CHX against three periodontal pathogens was found to be statistically significant [Table 3].

Table 3.

Comparison of minimum inhibitory concentration, minimum bactericidal concentration values of ethanolic, aqueous-based extract and chlorhexidine on three different periodontal pathogens

Procedure	Samples	PG		AA		TF
		Mean rank	P	Mean rank	P	Mean rank	P
MIC	Ethanolic extract of O. basilicum	8.00	0.018*	8.00	0.020*	7.50	0.029*
	Aqueous extract of O. basilicum	5.00		5.00		5.50
	Chlorhexidine	2.00		2.00		2.00
MBC	Ethanolic extract of O. basilicum	8.00	0.018*	8.00	0.018*	6.50	0.018*
	Aqueous extract of O. basilicum	5.00		5.00		6.50
	Chlorhexidine	2.00		2.00		2.00

*P≤0.05 – *P-value ≤ 0.05 is considered statistically significant. Test applied – Kruskal–Wallis test; level of significance. O. basilicum – Ocimum basilicum; MIC – Minimum inhibitory concentration; MBC – Minimum bactericidal concentration; PG – Porphyromonas gingivalis; AA – Aggregatibacter actinomycetemcomitans; TF – Tannerella forsythia

The O. basilicum extract was nontoxic to the human gingival fibroblast, against which the cytotoxic activity has been tested [Table 4].

Table 4.

Mean of optical densities of surviving cells of two study groups, namely, chlorhexidine gluconate and Ocimum basilicum extract at a wavelength of 570 nm for different concentrations

Concentration	OD	Mean	Percentage viability(%)	Results as observed
NC	0.164	0.287	100	No lysis
	0.439
	0.259
OBE	0.269	0.274	95.59	No lysis
	0.245
	0.31
CHX	0.154	0.217	75.52	No lysis
	0.264
	0.233

NC – Negative control; O. basilicum – Ocimum basilicum; OBE – O. basilicum extract; CHX – Chlorhexidine; OD – Optical density; nm – Nanometer

DISCUSSION

India is a land of beautiful natural flora, with an abundance of medicinal plants and seeds which are available throughout the country. A few extracts are known to possess antimicrobial activity, especially against bacterial pathogens. O. basilicum (sabja seeds) is one such seed which is predominantly present in the Indian subcontinent. The present study was carried out to test the antibacterial activity of O. basilicum extract against common oral anaerobic organisms, especially periodontal pathogens. Ethanol and water were used as a solvent for extract preparation. Although both the extracts showed promising results, against the various bacterial strains, the effect of aqueous extract was much more pronounced than ethanolic extract. In contrast, Kalra et at.[10] found that O. basilicum oil in aqueous base had less efficacy against periodontal pathogens compared to ethanol-based extract. The active constituent present in Ocimum species is flavonoids and perhaps it is absorbed more in aqueous extract, primarily responsible for the therapeutic potentials of Ocimum species.[17,24]

A few studies done in India have already reported the antibacterial activity of O. basilicum, but there is a marked void in exploring the properties of O. basilicum. The current study thoroughly evaluated the antibacterial properties against periodontal pathogens; similarly, sweet basil essential oils show excellent in vitro anticariogenic bacteria and anti-plaque activities, according to Wiwattanarattanabut et al.,[11] and may be offered as alternative and effective supplements to maintain oral health status.

Analyzing the efficacy of O. basilicum extract against oral periodontal microorganisms, the maximum activity was shown for P. gingivalis, which is supported by Ahmed et al.,[25] concluding that the inhibition zones were always varied and had significantly risen with the concentration of O. basilicum extract, and the growth was entirely prevented at the greatest concentration. On some bacteria, O. basilicum shows an inhibitory action comparable to antibiotics. It would be possible to produce new effective antibiotics using the effective material to be refined from this plant.

As demonstrated in the current investigation, O. basilicum extract had proven to be an efficient antimicrobial against periodontal infections. Another study by Hanif et al.[26] found that Omani basil essential oil exhibited a strong antibacterial activity against all the bacteria tested, except Pseudomonas putida and Pseudomonas aeruginosa. Linalool (69.9%) was identified as the major component present in Omani basil oil which was found highly active against human pathogens. The current evidence obtained from this study shows a significant inhibitory effect of O. basilicum extract on anaerobic microorganisms. The effect produced was equivalent or less than that produced by CHX in the case of anaerobic bacteria. When compared to CHX, O. basilicum is a natural substance with no associated negative effects. It has a better chance of being accepted by the people because it is native to the country and has a religious significance. These findings indicate the possibility of using O. basilicum in oral health care products for reducing microbial load in the oral cavity.

In summary, periodontitis is a multifactorial disease, but periodontal pathogens are primarily believed in initiation and progression of the disease. For a long time, the use of chemical therapeutic agents with the mechanical debridement is considered a treatment of choice with improved results.[27,28] Commonly used therapeutic agents like CHX is chemically derived and is considered the gold-standard therapeutic agent used as an adjunct to mechanical debridement for plaque control. However, certain adverse effects have been reported with the long-term use of CHX. These factors triggered the need for the development of an alternative agent which is equally effective and help in minimizing all the adverse effects seen with the existing agents.[29,30] Herbal medicine is an upcoming alternative and is considered the most acceptable form of therapy by many researchers due to its diverse medicinal properties with a fewer side effects.[31,32] Plants with medicinal properties and therapeutic uses are gaining popularity among the researchers to explore the different uses of plants and their compounds responsible for specific properties.[33]

This study being an in vitro provides just preliminary evidence of antibacterial efficacy of O. basilicum extract. Periodontal disease is a complex illness whose etiology has been linked to a various pathogenic bacteria, but the use of only three organisms is one of the study’s drawbacks. However, predominant periodontal microorganisms were selected in the present study. Future clinical trials will be carried out to demonstrate its effect on the various microorganisms and to ascertain cytotoxicity against the periodontal tissues.

CONCLUSION

Recognizing plants that have medicinal properties becomes important and needs to be developed because it is believed that drugs derived from natural materials are relatively safe and inexpensive. The antibacterial activity was evident in both the extracts of O. basilicum against anaerobic periodontal pathogens. However, it was more pronounced in aqueous-based extract, but its efficacy was lesser compared to CHX. Thus, it can be formulated in the mouthwash and effectively used as an alternative to standard considering its long-term use and lesser side effects. This herbal mouthwash would bring significant changes when used as an integral part in oral health in a community.

Financial support and sponsorship

The sponsorship was obtained from ICPA Health Products Ltd, Mumbai, India, in the form of materialistic support (Chlorhexidine gluconate salt - Batch No: 20BPLS/CHH001).

Conflicts of interest

There are no conflicts of interest.