Antimicrobial and cytotoxicity properties of Plumeria alba flower extract against oral and periodontal pathogens: A comparative in vitro study.

Context: Plumeria alba, commonly known as frangipani or West Indian jasmine, is a traditional and ancient folklore medicine known for its antimicrobial, anti-inflammatory, and antioxidant properties. The extracts from P. alba obtained from the leaves, bark, and flowers, are commonly used to manage bacterial, fungal, and viral infections such as herpes, scabies, and fungal infections. The constituents of the P. alba plant have shown promising antihelmintic, antipyretic, and antirheumatic properties. Although studies have confirmed that extracts from Plumeria species are effective against microbial infections and cancer, its role in managing oral diseases, particularly the chronic inflammatory disease of the gums (gingivitis and periodontitis), has never been explored. Therefore, the current study aimed to explore the antimicrobial and cytotoxic properties of the P. alba flower extract against oral and periodontal pathogens compared to chlorhexidine and doxycycline. Settings and Design: This was an in vitro study. Materials and
Methods: The ethanolic extract was prepared from the freshly plucked P. alba flowers. The antimicrobial properties of the extract were evaluated by testing the minimal inhibitory concentration, minimal bactericidal concentration, and well-diffusion assay against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, Tannerella forsythia, Streptococcus mutans, Actinomyces viscosus, Streptococcus salivarius, and Candida albicans. The results were compared to chlorhexidine and doxycycline. The cytotoxicity was checked by the against human-derived gingival fibroblast and keratinocytes. Statistical Analysis Used: One-way ANOVA for the mean zones of inhibition against all the microorganisms was done.
Results: P. alba extract inhibited the growth for all the tested oral and periodontal pathogens at 25 μg/ml. The well-diffusion assay of P. alba extract was comparable to chlorhexidine but was not statistically significant compared to doxycycline.
Conclusion: P. alba can be used as a promising alternative to chlorhexidine for the management of oral and periodontal infections. Copyright:

INTRODUCTION

Periodontitis is a chronic inflammatory disease of the soft tissues surrounding the teeth.[1] Periodontal disease is caused by the interaction of pathogenic microorganisms to the various cells and receptors in the oral tissues. The host–microbial interaction causes a massive release of various pro-inflammatory chemical mediators and free radicals in the periodontal tissues.[2] Increased production of these free radicals exaggerate the inflammatory response and results in soft-tissue destruction, pocket formation, and alveolar bone loss.[2345] Periodontal microorganisms can even enter the systemic circulation and increase the risk of diabetes mellitus, atherosclerosis, chronic kidney disease, Alzheimer's disease, poor pregnancy outcomes, rheumatoid arthritis, oropharyngeal cancer, and pancreatic cancer.[6] Since the global burden of periodontal disease is rapidly increasing, it is essential to develop more effective therapeutic modalities to control the inevitable periodontal inflammation and its associated systemic complications.[7]

The most common treatment modality to control periodontal diseases is the mechanical debridement of the oral biofilm and calculus from the surface of the teeth. Various adjuncts such as antibiotics, anti-inflammatory, immunomodulatory and other chemical plaque control agents have been used as adjuncts to scaling and root planing to manage gingivitis and periodontitis.[8910] Antibiotics are the most commonly used adjuncts along with scaling and root planing to manage periodontal inflammation.[111213] However, with the widespread use of antibiotics, many oral pathogens have become resistant to the commonly prescribed antibiotics.[14]

Therefore, there is a changing trend of using herbal or plant-based products for the management of gingival and periodontal diseases.[15] Plant-based products are known to possess good anti-inflammatory, astringent, antioxidant, and antimicrobial properties. Green tea, curcumin, cranberry, tulsi, guava, Aloe vera, neem, pomegranate, curry, and mango have shown good antimicrobial properties against oral and periodontal pathogens. The extracts from these herbs have been incorporated into gels, mouthwash, tablets, powders, and local drug delivery agents to treat gingivitis and periodontitis.[16] Recently, Plumeria species have been recognized to have broad-spectrum antibiotic-like effect, however, their effect on oral and periodontal bacteria has never been tested.

Plumeria alba (P. alba), commonly known as frangipani, West Indian jasmine, champa, and temple tree, is used as folklore medicine to treat many inflammatory and infectious diseases.[1718] It is a flowering plant native to Central and South America, Caribbean, Brazil, and India.[17] It belongs to the family Apocynaceae. Various bioactive constituents with potent antimicrobial, anti-inflammatory, anthelmintic, antipyretics, and antirheumatic properties have been extracted from the bark, leaves, and flowers of the P. alba. The stem and leaf of P. alba are used to treat skin disorders such as herpes, scabies, and ulcers.[182122] Radha et al. discovered that P. alba is effective against some common fungi linked with systemic fungal infections such as Aspergillus niger, Penicillium chrysogenum, Microsporum gypseum, and Epidermophyton floccosum.[182324] P. alba also has good antibacterial properties against the Staphylococcus aureus, coagulase-negative Staphylococci, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhi, Staphylococcus saprophyticus, Salmonella paratyphi, and Bacillus anthracis.[2425] The essential oils from the flower of P. alba are effective against S. aureus, Bacillus subtilis, A. niger, Candida albicans (Ca), and P. chrysogenum.[2627] Based on this evidence, it is evident that P. alba is a promising antimicrobial agent for managing infectious diseases, and hence, its role in managing chronic infectious diseases of the oral cavity should be explored. With this background, the present study aimed to evaluate for the first time the antimicrobial efficacy of ethanolic extracts of P. alba flower against oral and periodontal pathogens. The study is of scientific importance as it will cue dentists, clinicians, and researchers to incorporate P. alba into various local drug delivery formulations to manage oral and periodontal diseases.

Aim

The aim of this study was to evaluate and compare the antimicrobial efficacy of ethanolic extract of P. alba flower extract against oral and periodontal pathogens.

Objectives

The objectives of this study were as follows:

To prepare the ethanolic extract from fresh flowers of P. alba Linn.

To evaluate the cytotoxic effects of the P. alba flower extract against human-derived fibroblast and modified keratinocytes gingival fibroblast and modified culture human keratinocyte cell line (HaCaT)

To evaluate and compare the minimal inhibitory concentration and minimal bactericidal concentration (MBC) of the ethanolic extract of P. alba flower extract compared to doxycycline, dimethylformamide (DMF), and chlorhexidine against oral and periodontal pathogens

To evaluate and compare the antimicrobial activity of the P. alba flower extract against common oral and periodontal pathogens compared to doxycycline and chlorhexidine.

MATERIALS AND METHODS

The P. alba flowers were collected the local trees. The flowers were identified and authenticated by a renowned botanist and taxonomist and then deposited in the Department of Pharmacognosy for extraction. The flowers were first washed with distilled water and dried in a hot air oven at 45°C. The dried flowers were coarsely powdered in a grinder, and about 250 g of the P. alba flower powder was obtained. The powder was then macerated with 1000 ml of ethanol for 3 days with occasional shaking. The macerated powder was filtered, and the solvent was removed using a rotatory evaporator.[28] The extract obtained was a semisolid brown color mass with a sweet fragrance. The extract was stored in a desiccator until further investigations.

The following bacterial strains were used for the present study: Porphyromonas gingivalis (Pg): ATCC 33277; Aggregatibacter actinomycetemcomitans (Aa): ATCC 700685; Fusobacterium nucleatum (Fn): ATCC 23726; Prevotella intermedia (Pi): ATCC 25611; Tannerella forsythia (Tf): ATCC 43037; Streptococcus mutans (Sm): ATCC 10449; Actinomyces viscosus (Av): ATCC 15987; Streptococcus salivarius (Ss): ATCC 25975; and Ca. The strains were procured from HiMedia Pvt. Ltd., Mumbai, Maharashtra, India.

The following tests were done to evaluate the antimicrobial properties of extract:

The minimum inhibitory concentration (MIC) was determined by using the “serial dilution technique” with thioglycollate broth (HiMedia Pvt. Ltd., Mumbai, Maharashtra, India) [Figure 1].[2930] In the initial tube, 20 μl of P. alba extract was added into the 380 μl of thioglycolate broth. For further dilutions, 200 μl of thioglycollate broth was added into the following nine tubes separately. From the initial tube, 200 μl was transferred to the first tube containing 200 μl of thioglycollate broth. This was considered as a 10-1 dilution. From a 10-1 diluted tube, 200 μl were transferred to the second tube to make 10-2 dilution. Similarly, the serial dilution was repeated up to 10-9 dilutions. From the maintained stock cultures of the following periodontal pathogens (Pg, Aa, Fn, Pi, Tf, Av, Sm, Ss, Ca), a microliter was taken from each and added into 2 ml of thioglycolate broth. In each serially diluted tube, 200 μl of above-cultured suspension was added. The tubes were then incubated for 48–72 h in an anaerobic jar at 37°C and observed for turbidity. The turbidity was checked using optical density, and the reading was observed at 450 nm. The experiment was done in triplicate for each strain.

Figure 1

Schematic representation of the experimental methodology. MIC: Minimal inhibitory concentration; MBC: Minimal bactericidal concentration; MTT assay: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (created in BioRender)

From the MIC dilutions tubes, the first 3–5 tubes were plated (which was sensitive in MIC) and incubated for 24 h. The next day, the total colony count was recorded. The MBC test was conducted to evaluate the bacteriostatic or bactericidal properties of the P. alba extract against oral and periodontal pathogens. The lowest concentration of the subculture with no growth was considered the MBC.

Agar well diffusion method was then performed by a method briefly described below.[2930] Brain–heart infusion agar (HiMedia Pvt. Ltd., Mumbai, India) was used as the culture media to test the antimicrobial activity of the extract against the pathogens. However, for the antifungal disc diffusion method, the Sabouraud agar medium was used. The facultative anaerobes were incubated in tubes at 37°C for 48–72 h in a carbon dioxide jar. The strict anaerobic tubes were incubated in anaerobic jars (A-35, Don Whitley, Yorkshire, UK) for 48–72 h before the experimentation. The agar plates were brought down to room temperature, and colonies of microorganisms were inoculated on the agar plates using a swab. Within 15 min, the inoculum was adjusted to a McFarland 0.5 turbidity and the suspension was regulated with a photometric device. After the inoculation, a sterile cotton swab was dipped into the inoculum and rotated against the wall of the tube to remove the excess inoculum. The entire surface of the agar plate was then swabbed three times and the inoculum was transferred, while the plates were rotated by approximately 60° between streaks to ensure even distribution. The inoculated culture plates were then allowed to stand for at least 3 min but <15 min. The inoculum preparation and inoculation of culture media were done for the following oral and periodontal pathogens: Pg, Aa, Fn, Tf, Pi, Av, Sm, Ss, and Ca. The stock solution was prepared using about 10 mg of the compound and dissolved in 1 ml of dimethyl sulfoxide (DMSO). A hollow tube of 5 mm diameter was heated and pressed on the inoculated agar plate and removed immediately. A micropipette was used to add 75 μl, 50 μl, 25 μl, 10 μl, and 5 μl in each well. The inoculated plates were then incubated within 15 min of compound application for a period of 24 h at 37°C. After the incubation period, the plates were checked to see if there are any lawns of growth that were confluent or nearly confluent. The diameter of the inhibition zone was measured to the nearest whole millimeter by using a Vernier caliper. The microbiological procedure was repeated three times for each bacterium and the corresponding three values of zones of inhibition for each concentration of P. alba extract and compared to doxycycline, DMF, and chlorhexidine.

The cell proliferation and cytotoxicity of the extract were checked using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [Figure 2]. The cells were exposed to the extract at different concentrations for 48 h. The cell viability was measured using gingival fibroblast and keratinocyte cell line (HaCaT transformed keratinocytes) using the MTT assay.[31] The dose-dependent inhibition of the Plumeria extract on cell proliferation was observed. The details of this assay have been briefly described as follows: the gingival fibroblast/HaCaT transformed keratinocytes were seeded at 1 × 105 cells/mL in 96-well microtiter plates in minimum essential medium with fetal bovine serum. The cells were incubated overnight for attachment. The drug concentrations in serial three-fold dilutions were added in triplicates and incubated for 48 h at 5% CO2 at 37°C. The cells were treated with the extract at varying concentrations 10 ug, 50 ug, 100 ug, 500 ug, 1 mg, 2.5 mg, and 5 mg and compared to 5 μg of positive control (normal saline) for 48 h, respectively. MTT dye (5 mg/mL) was added to each well for at least 4 h of treatment. After that, the cells were treated with MTT (Sigma Chemical Co., St. Louis, MO, USA). After 4 h, all of the media, including MTT solution (5 mg/mL), were aspirated from the wells. The remaining formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm using a 96-well microplate reader (Multiskan™ FC Microplate Photometer). The cytotoxicity index was determined using the untreated cells as a negative control. The percentage of cytotoxicity was calculated using the background-corrected absorbance as follows: % cytotoxicity = (1- adsorbance of the experimental wall)/(adsorbance of the control wall) X 100.

Figure 2

Mean zones of inhibition and serial dilution technique comparing Plumeria alba flower extract to doxycycline, chlorhexidine, and N, N-dimethylformamide for Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, Tannerella forsythia, Streptococcus mutans, Actinomyces viscosus, Streptococcus salivarius, and Candida albicans

The statistical analysis was done by the one-way ANOVA test and results expressed as the mean zones of inhibition against all the microorganisms with statistical significance considered considered at P < 0.001.

RESULTS

The results of the MIC/MBC showed that at 50 μg/ml, all microorganisms were sensitive to the P. alba extract compared to chlorhexidine, doxycycline, and DMF. Av and Sm were most sensitive to the extract at a concentration of 1.6 μg/ml and 3.12 μg/ml, respectively. Pg and Pi required 12.5 μg/ml to inhibit their growth. Whereas, Fn, Ss, and Tf were inhibited at a concentration of 25 μg/ml. It was also noted that Aa and Ca were the least sensitive to the extract and required a higher concentration of 50 μg/ml when compared to the other microorganisms [Figure 2].

P. alba extract also inhibited the colony formation for most of the pathogens (Fn, Pi, Tf, and Ss) at 25 μg/ml, whereas Aa and Ca required a higher concentration of 50 μg/ml. Av was found to be the most sensitive to the P. alba extract at 1.6 μg/ml, followed by Sm at 6.25 μg/ml and Pg at 12.5 μg/ml [Figure 2 and Table 1].

Table 1

Minimal inhibitory concentration/minimal bactericidal concentration of the ethanolic extract of Plumeria alba flower extract compared to chlorhexidine and doxycycline - The results showed that all tested microorganisms were inhibited at 25 µg/ml except Aggregatibacter actinomycetemcomitans which required a higher concentration (50 µg/ml)

Microorganism	Plumeria alba flower extract		Chlorhexidine		Doxycycline		DMF
	MIC (µg/mL)	MBC (µg/mL)	MIC (µg/mL)	MBC (µg/mL)	MIC (µg/mL)	MBC (µg/mL)
Pg	12.5	12.25	8.5	16.6	0.2	0.2	R
Aa	50	50	30.1	15.4	0.4	0.8	R
Pi	12.5	25	16	16	0.2	0.2	R
Fn	25	25	15.6	20.8	0.2	0.2	R
Tf	25	25	8.6	1.25	0.4	0.4	R
Av	1.6	1.6	16	32	0.4	0.2	R
Sm	3.12	6.25	16	16	0.2	0.2	R
Ss	25	25	16	16	0.4	0.4	R
Ca	50	50	16	16	0.2	0.4a	R

a Comparison was done with fluconazole powder. Pg – Porphyromonas gingivalis; Aa – Aggregatibacter actinomycetemcomitans; Fn – Fusobacterium nucleatum; Pi – Prevotella intermedia; Tf – Tannerella forsythia; Sm – Streptococcus mutans; Av – Actinomyces viscosus; Ss – Streptococcus salivarius; Ca – Candida albicans; DMF: N–N-dimethylformamide; MIC – Minimum inhibitory concentration; MBC – Minimal bactericidal concentration; R- resistant; P- P < 0.001, considered as statistical significance

The zone of inhibition as tested by well-diffusion assay showed that the extract of P. alba was compared to an antibiotic (doxycycline) and 0.2% chlorhexidine. The maximum zone of inhibition for P. alba for all the tested microorganisms was less when compared to doxycycline. The zone of inhibition of P. alba extract was comparable to chlorhexidine, which is the gold standard antimicrobial agent against periodontal pathogens (P < 0.001). The Plumeria extract also inhibited Ca (18 mm) and Aa (15 mm), while Av, Sm, and Tf were least inhibited [Table 2 and Figure 2]. Post hoc tests revealed a significant difference in the antimicrobial efficacy of doxycycline, chlorhexidine, and Plumeria extract against different periodontal pathogens.

Table 2

Mean zones of inhibition by Plumeria alba flower extract compared to doxycycline and chlorhexidine as tested by agar diffusion assay: The results showed that zone of inhibition with Plumeria alba flower was comparable to chlorhexidine but the results were not statistically significant when compared to doxycycline

Microorganism	PAFE (mm)	Chlorhexidine (mm)	Doxycycline (mm)	P
Pg	14.2±0.28	16±0.25	41±1.25	<0.05
Aa	15.3±0.15	18±1.35	35±0.11	<0.05
Pi	13±0.25	19±1.82	25±0.25	<0.001
Fn	13±0.18	12±0.41	31±1.22	<0.001
Tf	12±0.25	16±1.31	32±1.45	<0.001
Av	12±0.10	13±1.25	22±1.05	<0.001
Sm	12±0.10	12±0.25	26±1.25	<0.001
Ss	14±0.10	16±0.34	25±1.45	<0.001
Ca	18±0.25	20±0.34	36±1.45	<0.001

Pg – Porphyromonas gingivalis; Aa – Aggregatibacter actinomycetemcomitans; Fn – Fusobacterium nucleatum; Pi – Prevotella intermedia; Tf – Tannerella forsythia; Sm – Streptococcus mutans; Av – Actinomyces viscosus; Ss – Streptococcus salivarius; Ca – Candida albicans; PAFE – Plumeria alba flower extract; P- P<0.001 is considered as statistical significance

Cell proliferation assay also showed that P. alba extract maintained the cell viability of more than 75% of the gingival fibroblast and 80% of the modified culture human keratinocyte cell line (HaCaT) at the concentration of 1 mg/ml and 5 mg/ml, respectively [Figure 3].

Figure 3

Cell proliferation assay via by the MTT assay with Plumeria alba extract against the gingival fibroblasts and cultured human keratinocyte cell line (HaCaT): The results showed that extract maintained the cell viability of more than 75% of the gingival fibroblast and 80% of modified keratinocyte cell line (HaCaT) at a concentration of 1 mg and 5 mg, respectively, compared to control. MTT assay - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

DISCUSSION

The results of the current study confirm that P. alba extract is effective to control and inhibit the growth of Pg, Aa, Fn, Tf, Pi, Av, Sm, Ss, and Ca. Since these microorganisms are associated with the initiation and progression of periodontal disease, their inhibition is could explain the role of P. alba species in management of gingivitis and periodontitis. P. alba was found to inhibit both the early colonizers (Av, Ss, and Pi) and the bridging species (Fn), indicating that it could be used as an effective anti-plaque agent to prevent the colonization and maturation of the dental plaque. The early colonizers in plaque (Av) were inhibited at a concentration of 1.6 μg/ml, whereas the late colonizers were inhibited at a concentration of 25–50 ug/ml. Since the prolonged use of chlorhexidine is associated with the development of various adverse drug reactions, the use of P. alba extract for plaque control is a viable alternative.[32] In addition, the extract can also be tried as an anticariogenic agent as it was found to inhibit the growth of the S. mutans, the main etiological agent for initiation of dental caries.

The antimicrobial properties of P. alba flower can be attributed to the presence of various phytochemical compounds with anti-inflammatory, anti-quorum sensing, and antioxidant properties.[171819202633343536] The key antibacterial constituents in Plumeria species include triterpenes such as a-amyrin, sterols, carbohydrates, tannins, triterpenoids, glycosides, ß-sitosterol, scopoletin, and iridoids such as isoplumericin, plumeride, plumeride coumarate, and plumieride coumarate glucoside.[172023252627] The Plumeria flower extract also contains high amount of antioxidants such as Vitamin C and flavonoids like kaempferol, 3-rhamnoside, and kaempferol 3-rhamno-galactoside,and scopoletin. These antioxidants can lower the inflammatory response in the periodontal tissues and facilitate healing.[35363738394041] The ethanolic extract of the P. alba flower shows more antioxidant capacity compared to the other forms of extract.[41] Kaempferol in P. alba was found to inhibit the lipopolysaccharide-induced nitric oxide production and prevents collagen breakdown by inhibiting collagenase enzyme in the periodontal tissues.[36] P. alba species contain various antimicrobial proteins such as nicotinic acid, mononucleotide adenylyltransferase, beta-ketoacyl-acyl-carrier transferase, and dihydropteroate synthetase that could be responsible for antimicrobial properties. These molecules could be specifically tested against oral and periodontal pathogens in future studies.[38] P. alba is widely known for its analgesic effects. Thirumagal and Geetha (2019) evaluated the anti-inflammatory and analgesic properties of Plumeria species and confirmed that 500 mg of the extract can control the inflammation in both chronic and acute models.[39] A paste made from the bark of P. alba has been used as an analgesic for toothache.[40]

Apart from the antibacterial effect, P. alba is known for its strong antifungal properties.[37] The methanolic extract of P. alba was highly toxic against A. niger, Ca, and P. chrysogenum.[23] Our study also confirms that P. alba flower extract as effective as chlorhexidine to control the growth of Ca, but the results were less significant when compared to fluconazole.[38] Gajapathi et al. (2020) also showed that Plumeria extract can be used as a potential denture cleanser for controlling fungal infections in denture wearers. Their study showed that the extract inhibited the fungal growth to 3.080 × 108 CFU/ml from baseline values of 6.439 × 108 CFU/ml.[42]

CONCLUSION

P. alba is a promising herb with good biocompatibility and antibacterial and antifungal properties against oral and periodontal pathogens. P. alba extract is a promising alternative to chlorhexidine for the management of gingival and periodontal infections. Based on the existing evidence, the antibacterial and antifungal properties of this traditional herb must be clinically applied for benefit of the public and dental fraternity. However, further patient-based comparative clinical studies are needed to confirm its efficacy in managing gingivitis and various forms of periodontitis. It is also important to explore which constituents of P. alba are most effective against oral and periodontal bacteria so that future studies can be based on those specific compounds and more targeted drug delivery can be attempted. Furthermore, clinical studies evaluating the effect of P. alba as an adjunct to non-surgical periodontal therapy and with long-term follow-up are warranted before it can be established as a treatment option to manage gingivitis and periodontitis.

Financial support and sponsorship

MAHE seed fund.

Conflicts of interest

There are no conflicts of interest.