In vitro investigation of antimicrobial activities of ethnomedicinal plants against dental caries pathogens (2025)

Abstract

The study aimed to evaluate the antimicrobial activity of medicinal plant extracts against the bacterial pathogens prominent in dental caries. A total of 20 plant species (herbs, shrubs and trees) belonging to 18 genera and 15 families were documented for dental caries. Antimicrobial activity of solvent extracts and essential oil from plants were determined by zone of inhibition on the growth of Streptococcus mutans (MTCC 497) and Lactobacillus acidophilus (MTCC 10307) using the agar well diffusion method. The results of in vitro antimicrobial assay prove that methanol is more successful in the extraction of phytochemicals from plant samples than aqueous solvent, as methanol extracts show higher antimicrobial activity than aqueous extracts against both the test pathogens. Methanol extracts of Nigella sativa, Psidium guajava and Syzygium aromaticum were the most effective among all 20 plant samples and have potent inhibitory activity against both dental caries pathogens with minimum inhibitory concentration of 0.2mg mL^− 1. N. sativa seed methanol extract was more effective with 22.3mm zone of inhibition at 0.2mg mL^− 1 against S. mutans (MTCC 497), while L. acidophilus (MTCC 10307) was more sensitive to S. aromaticum bud methanol extract at 11.3mm zone of inhibition at concentration 0.1mg mL^− 1. Essential oil extracted from plants also possesses strong antimicrobial activity for both test pathogens, with a minimum inhibitory concentration range of 0.05–0.16mg mL^− 1. Syzygium aromaticum bud essential oil at 0.05mg mL^− 1 was most active against S. mutans (MTCC 497). Plant extracts viewing antimicrobial activity with minimum inhibitory concentration show the efficacy of the plant products that could be considered as a good indicator of prospective plants for discovering new antimicrobial agents against dental caries pathogens. The findings of this study provide a lead to further polyherbal formulations for the treatment of dental caries malaise.

Electronic supplementary material

The online version of this article (10.1007/s13205-018-1283-2) contains supplementary material, which is available to authorized users.

Keywords: Dental caries, Streptococcus mutans, Lactobacillus acidophilus, Antimicrobial activity, Minimum inhibitory concentration

Introduction

Dental caries is one of the widespread oral diseases in humans which causes irreversible damage to the tooth enamel, with difficulties in food intake and hence causes great distress. The occurrence of dental caries is mainly associated with oral microbial pathogens, especially cariogenic bacteria (Kouidhi et al. 2010; Besra and Kumar 2016). The biological nature of the disease is a microbial infection caused primarily by the bacterium Streptococcus mutans (Clarke 1924; Loesche et al. 1975; Loesche 1986; Tanzer et al. 2001). The leading cariogenic pathogen S. mutans carries an enzyme glucosyltransferase (Gtf) that synthesizes water-insoluble glucan from sucrose and forms a layer on the surface of tooth enamel, which serves as an adhesive material for S. mutans itself and other Streptococci sp. (Besra and Kumar 2016). In the second stage, by the accumulation of acidogenic bacteria like Lactobacillus sp. produce acid from sucrose fermentation, create an acidic environment. The colonization and co-aggregation of bacteria able to produce acid (acidogenic) and tolerate the acidic environment (aciduric), final form dental plaque and thus cause local destruction of the tooth because of the acid accumulation and mineral decalcification (Losche 1986; Dhruwet al. 2012). Left untreated, dental caries will gradually lead to tooth loss. Dental caries prevention is preferable to treatment, due to high cost and lack of resources at primary levels. Wide-ranging antimicrobial agents like penicillin, cephalosporin, erythromycin and tetracycline are prescribed by the dental practitioners for dental infections. Exposure to wide range of antibiotics results in evolution of antibiotic resistance in oral pathogens. In addition, allergic reaction of all degrees of severity can occur with most antibiotics (Montqomery and Kroeger 1984). Dental caries is frequent in town residents in comparison to rural inhabitants, as they are less exposed to synthetic antibiotics; they also rely on natural remedies for dental care. In recent years, plant-derived products are gaining popularity in preventing oral illness, due to adaptation of antibiotic resistance in oral microbial pathogens against synthetic antibiotics (Lynch and Robertson 2008). Natural plant products ensure the absence of secondary effects and possible long-term usage in the oral cavity.

Since time immemorial, plants have been used for food, fodder and medicine. The World Health Organization (WHO) suggested that as much as 80% of the world’s people depend on traditional medicine for their primary health care needs (Khan 2002). The primary inhabitants of natural ecosystem or the community people possess a vast amount of traditional knowledge using local biodiversity (Pradhan and Badola 2008). Herbal plant extracts have been used as antimicrobial agents in traditional medicines and have attracted considerable interest in the prevention of dental caries (Lee et al. 2003; Lim et al. 2003; Limsong et al. 2004; Song et al. 2007; Sampaio et al. 2009). In this study, an ethnobotanical survey was conducted in some rural region of Dhanbad district of Jharkhand, India (Supplementary Fig.1). Although Dhanbad has an abundance of coal and minerals, there is an affluent ethnic tradition in terms of medicinal practices also, and only few dedicated ethnomedicinal studies have been published so far. The present study was undertaken to scientifically enumerate medicinal plants used by the rural communities of Dhanbad to treat severe oral ailment dental caries. Moreover, in vitro study of plant extracts was also performed to confine to qualitative and quantitative analysis in small extent.

Materials and methods

Plant sample collection

Frequent field visits were made to different rural regions of Dhanbad district, Jharkhand, India (latitude 23°37′3″N and 24°4′N and longitude 86°50′E). Information about the traditional medicinal plants used to treat dental caries was obtained from experienced traditional healers and local individuals. Total number of plant samples collected for this study, local names of the plants, habit, plant part(s) used and mode of application were recorded. Plants were identified using regional floristic literature.

Preparation of plant-solvent extracts and essential oil

The plant samples were shed-dried at room temperature and ground into powder using a grinder. The powdered plant sample (10g) was extracted using a Soxhlet apparatus with 200mL of solvent for 10h. The solvents used were methanol and distilled water. The solvent from extracts was evaporated under reduced pressure at 40°C using a rotary evaporator. Stock solutions were prepared for each plant extract in 10% aqueous dimethylsulfoxide (DMSO) and preserved at 4°C for further antimicrobial test.

An essential oil from plant samples was extracted by hydro-distillation technique using Clevenger’s apparatus. 100g fresh plant sample was homogenized and extracted with 500mL of distilled water for 12h. Collected essential oils were dried over anhydrous sodium sulphate and kept at 4°C for antimicrobial test.

Bacterial strain and culture condition

The bacterial strains, Streptococcus mutans (MTCC 497) and Lactobacillus acidophilus (MTCC 10307) were requested from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. The growth media employed were, brain heart infusion broth (Himedia, India) for S. mutans and Lactobacilli MRS broth (Himedia, India) for L. acidophilus for the revival of strains. Further strains were preserved on agar plates of their respective growth media.

Antimicrobial assay

In vitro antimicrobial activity of plant extracts and essential oil against S. mutans and L. acidophilus was determined using the agar well diffusion method with modifications. In this method, 0.5 McFarland standard culture (1.5 × 10⁸ cfu mL^− 1) for each test microbe was prepared in Muller Hinton broth (Himedia, India). 100µL inoculum of each test microbe was spread onto Muller Hinton agar (Himedia, India) plates using a sterile cotton swab and allowed to dry and wells were made with a sterile borer in the inoculated agar plates. Different test concentrations, 5.0–0.02mg mL^− 1, of plant extracts and essential oil were prepared in 10% aqueous DMSO. 100µL volumes of each test concentration were propelled into the wells for each test organism. Then the plates were incubated at 37°C for 24h. 10% aqueous DMSO was used as the negative control; ampicillin and tetracycline served as the positive control. Antimicrobial activity of plant extracts and essential oils indicated by a zone of inhibition around well was recorded. The experiments were performed in triplicates and the mean values of the diameter of inhibition zones with standard deviation were calculated (Aneja and Joshi 2009).

The minimum inhibitory concentration (MIC) of plant extracts and essential oil was determined using 96-well microtiter plates (CLSI 2012). An 80µL aliquot of test concentration of each extract, was released into a 96-well microtiter plate. Then each well was inoculated with 100µL microbial inoculums (0.5 McFarland standard culture, 1.5 × 10⁸ cfu mL^− 1) prepared in MHB medium. The microtiter plates were incubated for 18h at 37°C. 10% aqueous DMSO was used as the negative control; ampicillin and tetracycline served as the positive control.

Results and discussion

A total 20 plant species reported by the informants primarily used for dental caries treatment are summarized in supplementary Table1. These plant species belong to 18 genera of 15 different families in which plants from the families Amaranthaceae, Dipterocarpaceae, Euphorbiaceae, Lamiaceae, Meliaceae, Myrtaceae and Sapotaceae were majorly preferred for dental caries. The gathered information includes scientific name, local name, botanical family, plant part used and usage. Usually, the plants were used when fresh or dry, essentially in the form of toothbrush (Azadirachta indica, Jatropha curcas, Jatropha gossypfolia, Shorea robusta, Achyranthes aspera), also as decoction and mouthwash (Allium cepa, Ocimum sanctum, Ocimum basilicum, Psidium guajava, Syzygium aromaticum, Terminalia arjuna, Vitex negundo), sometimes a powder form of seed from Solanum surratense and Mimusop elengi is applied on the caries infected tooth. Essential oil from S. aromaticum dried bud is used to relieve the pain caused by caries. When analyzing the number of citations for the plant parts used to prepare local remedies, a preference for the use of stem becomes noticeable. The inhabitants use the tender stem mostly as toothbrush on a daily routine, because of accessibility and the simplest mode of application. Most of the reported preparations in the area are drawn from a single plant, mixtures are used very rarely. In the current study, the medicinal plants reported were from each habit, 25% of the reported species were herbs, shrub (45%) and the tree (30%).

The most commonly used plant species was A. indica leaf used for malaria, leprosy, skin ulcers, cardiovascular diseases, gingivitis, liver problems and diabetes. P. guajava leaves are medicinally useful and its fruit is a nutritional powerhouse. The leaves are full of antioxidants, antimicrobial and anti-inflammatory agents. Informants present least use reports of Lantana camara, S. surratense and V. negundo, this might be because they are wild plants and are less prominent in human use. Four plants were also reported to have highest fidelity for use in dental caries treatment were P. guajava, A. aspera, S. surratense and M. elengi. Some plants like A. aspera, A. indica, J. gossypifolia, M. elengi, P. guajava, S. surratense and V. negundo were also cited for use against dental pathogens by different workers (Singh et al. 2012).

Antimicrobial activity of plant extracts and essential oil

All plant extracts (methanol and aqueous) show significant antimicrobial activity against both test bacteria S. Mutans (MTCC 497) (Fig.1) and L. acidophilus (MTCC 10307) (Fig.2) with MIC value range from 0.2 to 5.0mg mL^− 1 (Tables1, 2). Methanol extracts of all plants were more active than aqueous extracts. Methanol extracts of A. aspera, A. indica, N. sativa, P. guajava and S. aromaticum show significant inhibitory activity for both pathogens (Fig.3). S. mutans were more sensitive to N. sativa with zone of inhibition 22.3mm and MIC 0.2mg mL^− 1 (Tables3, 1) while, L. acidophilus was more susceptible to S. aromaticum with zone of inhibition 11.3mm and MIC 0.1mg mL^− 1 (Tables4, 2). Essential oil from O. basilicum, O. sanctum, N. sativa, S. aromaticum and V. negundo were also active against S. mutans and L. acidophilus (Table5). It was found that S. mutans was more susceptible to essential oil than L. acidophilus. S. aromaticum bud essential oil shows strong inhibitory activity with MIC 0.05 and 0.08mg mL^− 1 for S. mutans and L. acidophilus, respectively.

Fig. 1.

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Fig. 2.

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Table 2.

Minimum inhibitory concentration value of plant extract against Lactobacillus acidophilus (MTCC 10307)

S. no.	Plants (parts) ↓	MIC (mg mL^− 1)
S. no.	Solvents →	Methanol	Aqueous
1	L. camara (leaves)	0	0
2	S. aromaticum (bud)	0.1	0.5
3	P. guajava (leaves)	0.2	0.3
4	A. cepa (bulb)	0.2	1.0
5	A. indica (leaves)	0.25	0.5
6	N. sativa (seeds)	0.5	0.3
7	A. aspera (root)	0.5	1.5
8	C. longa (rhizome)	0.5	1.5
9	P. nigrum (fruit)	1.5	2.5
10	M. arvensis (aerial parts)	2.0	2.2
11	M. elengi (seeds)	2.0	3.0
12	O. basilicum (leaves)	2.0	4.0
13	O. sanctum (leaves)	2.5	4.5
14	S. robusta (stem)	3.0	3.3
15	M. indica (leaves)	3.0	4.0
16	J. gossypifolia (stem)	3.0	5.0
17	T. arjuna (bark)	3.5	4.2
18	J. curcas (stem)	3.5	4.6
19	S. surratense (seeds)	4.0	0
20	V. negundo (leaves)	4.0	4.5
	Ampicillin^#	0.05
	Tertracycline^#	0.02

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MIC minimum inhibitory concentration

^#Positive control (ampicillin and tetracycline)

Fig. 3.

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Table 3.

Zone of inhibition of plant extract against Streptococcus mutans (MTCC 497)

S. no.	Plants (parts) ↓	ZI (mm)^a
S. no.	Solvents →	Methanol	Aqueous
1	N. sativa (seeds)	22.3 ± 0.57	15.3 ± 0.57
2	A. indica (leaves)	18.3 ± 0.57	11.3 ± 0.57
3	P. guajava (leaves)	18.3 ± 0.57	9.6 ± 0.57
4	S. aromaticum (bud)	16.3 ± 0.57	10.1 ± 0.57
5	A. cepa (bulb)	16.3 ± 0.57	8.3 ± 1.52
6	M. arvensis (aerial parts)	15.6 ± 1.15	7.3 ± 0.57
7	P. nigrum (fruit)	14.6 ± 0.57	4.6 ± 0.57
8	A. aspera (root)	12.3 ± 0.57	5.6 ± 0.57
9	S. surratense (seeds)	11.3 ± 1.15	9.6 ± 0.57
10	C. longa (rhizome)	12.0 ± 0.00	4.6 ± 1.52
11	M. elengi (seeds)	11.3 ± 0.57	7.3 ± 0.57
12	O. basilicum (leaves)	11.0 ± 0.0	9.3 ± 0.57
13	T. arjuna (bark)	9.6 ± 0.57	1.3 ± 1.15
14	O. sanctum (leaves)	8.3 ± 1.52	7.3 ± 0.57
15	S. robusta (stem)	8.9 ± 0.57	2.3 ± 0.57
16	M. indica (leaves)	7.6 ± 0.57	3.3 ± 0.57
17	J. gossypifolia (stem)	5.3 ± 0.57	2.6 ± 1.15
18	J. curcas (stem)	4.6 ± 0.57	2.6 ± 0.57
19	V. negundo (leaves)	3.6 ± 0.57	7.7 ± 0.57
20	L. camara (leaves)	1.7 ± 1.15	0
	Ampicillin^#	27.3 ± 0.57
	Tertracycline ^#	29.3 ± 0.57

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ZI zone of inhibition

^#Positive control (ampicillin and tetracycline)

^aResults are mean ± SD values for three replicates

Table 1.

Minimum inhibitory concentration value of plant extract against Streptococcus mutans (MTCC 497)

S. no.	Plants (parts) ↓	MIC (mg mL^− 1)
S. no.	Solvents →	Methanol	Aqueous
1	S. aromaticum (bud)	0.2	0.25
2	N. sativa (seeds)	0.2	0.3
3	P. guajava (leaves)	0.2	0.5
4	A. indica (leaves)	0.25	0.75
5	A. cepa (bulb)	0.4	1.5
6	A. aspera (root)	0.5	1.0
7	O. basilicum (leaves)	0.7	1.0
8	O. sanctum (leaves)	0.75	1.0
9	C. longa (rhizome)	0.75	1.5
10	M. elengi (seeds)	1.0	3.0
11	P. nigrum (fruit)	1.5	2.5
12	M. arvensis (aerial parts)	2.0	2.5
13	S. robusta (stem)	2.5	3.5
14	J. gossypifolia (stem)	2.5	5.0
15	M. indica (leaves)	3.0	3.5
16	J. curcas (stem)	3.0	4.5
17	T. arjuna (bark)	3.5	4.5
18	L. camara (leaves)	4.0	0
19	S. surratense (seeds)	4.0	5.0
20	V. negundo (leaves)	4.0	6.0
	Ampicillin^#	0.05
	Tertracycline^#	0.02

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MIC minimum inhibitory concentration

^#Positive control (ampicillin and tetracycline)

Table 4.

Zone of inhibition of plant extract against Lactobacillus acidophilus (MTCC 10307)

S. no.	Plants (parts) ↓	ZI (mm)^a
S. no.	Solvents →	Methanol	Aqueous
1	P. guajava (leaves)	22.6 ± 0.57	9.6 ± 0.57
2	N. sativa (seeds)	18.6 ± 0.57	12.3 ± 0.57
3	O. basilicum (leaves)	14.3 ± 0.57	6.3 ± 0.57
4	P. nigrum (fruit)	14.3 ± 0.57	4.6 ± 0.57
5	O. sanctum (leaves)	11.3 ± 1.52	5.6 ± 0.57
6	M. arvensis (aerial parts)	11.6 ± 1.15	7.3 ± 0.57
7	M. elengi (seeds)	11.6 ± 0.57	7.6 ± 0.57
8	J. curcas (stem)	11.6 ± 0.57	2.6 ± 0.57
9	S. aromaticum (bud)	11.3 ± 0.57	9.6 ± 1.15
10	A. indica (leaves)	11.3 ± 0.57	9.3 ± 1.0
11	T. arjuna (bark)	11.3 ± 0.57	1.3 ± 0.57
12	A. cepa (bulb)	10.3 ± 0.57	8.3 ± 0.0
13	A. aspera (root)	8.6 ± 1.15	5.6 ± 0.23
14	V. negundo (leaves)	8.3 ± 0.57	5.6 ± 0.57
15	M. indica (leaves)	8.3 ± 0.57	5.3 ± 0.57
16	C. longa (rhizome)	7.3 ± 0.57	9.6 ± 1.15
17	S. robusta (stem)	6.6 ± 0.57	2.3 ± 0.57
18	J. gossypifolia (stem)	5.6 ± 0.57	2.6 ± 0.57
19	S. surratense (seeds)	4.3 ± 0.57	0
20	L. camara (leaves)	0	0
	Ampicillin^#	30.3 ± 0.57
	Tertracycline^#	29.3 ± 0.57

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ZI zone of inhibition

^#Positive control (ampicillin and tetracycline)

^aResults are mean ± SD values for three replicates

Table 5.

Antimicrobial activity of essential oil against dental caries pathogens

S. no.	Plants (parts)	Streptococcus mutans (MTCC 497)		Lactobacillus acidophilus (MTCC 10307)
S. no.	Plants (parts)	ZI (mm)^a	MIC (mg mL^− 1)	ZI (mm)^a	MIC (mg mL^− 1)
1	S. aromaticum (bud)	16.3 ± 0.57	0.05	12.2 ± 0.57	0.08
3	N. sativa (seeds)	11.6 ± 0.57	0.06	9.5 ± 0.57	0.08
2	O. basilicum (leaves)	9.4 ± 0.57	0.06	9.3 ± 0.57	0.08
4	O. sanctum (leaves)	13.3 ± 0.57	0.08	11.6 ± 0.57	0.1
5	V. negundo (leaves)	3.3 ± 0.57	0.15	5.4 ± 0.57	0.16
	Ampicillin^#	27.3 ± 0.57		30.3 ± 0.57
	Tertracycline^#	29.3 ± 0.57		29.3 ± 0.57

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ZI zone of inhibition, MIC minimum inhibitory concentration

^aResults are mean ± SD values for three replicates

^#Positive control (ampicillin and tetracycline)

This study represents the significant in vitro antimicrobial activity of solvent extracts of collected plant samples against dental caries pathogens and also suggest their efficacy for the treatment of dental caries. The results of our study revealed that the standard strains S. mutans (MTCC 497) and L. acidophilus (MTCC 10307) were more sensitive to plant extracts studied. Our result was also supported by previous workers (Nair and Chanda 2006). It has been proposed that the mechanism of the antimicrobial effects involves the inhibition of various cellular processes, followed by an increase in plasma membrane permeability and finally ion leakage from the cells (Walsh et al. 2003).

Plant species used in this study have previously been tested against both caries pathogens. P. guajava have shown better results against caries pathogens as reported in earlier studies (Palombo 2011; Garode and Waghode 2014; Gurnani et al. 2016). Antimicrobial activity of O. sanctum against S. mutans and L. acidophilus has been published recently by Gadiyar et al. (2017). Susceptibility of dental caries pathogens to S. surratense and M. arvensis were also reported (Raja and Devi 2010). Solvent extracts of A. indica have strong antimicrobial activity against dental caries pathogens (Lekshmi et al. 2012; Lakshmi et al. 2015; Kaushik et al. 2016). Strong anti-Streptococcus mutans activity of P. guajava, P. nigrum and S. aromaticum were reported by Rosas-Pinon et al. (2012). Methanolic extract of A. indica, O. sanctum, M. elengi has considerable antimicrobial activity against S. mutans (Mistry et al. 2014). Freires et al. (2015) also reported S. aromaticum bud essential as most promising species against cariogenic bacteria. Most active antimicrobial plant species with strongest anticaries activity were A. indica, P. guajava, N. sativa and S. aromaticum. The rest of the plant species show concentration dependent activity against both tested bacterial strains with MIC ranges from 0.3 to 6.0mg mL^− 1. This study also reports diverse ethnobotanical taxa as a source of natural alternatives to synthetic antimicrobial agents for the treatment of dental caries.

Conclusion

Dental caries is a pathological condition of the teeth resulting in decalcification of the tooth enamel and disintegration of the remaining organic material. Nature provides diverse plant taxa as a source for antimicrobial agents, which can be a substitute for synthetic antibiotics that generate so many allergic reactions to humans. The present study has revealed significant results on antimicrobial activity of selected medicinal plants against cariogenic pathogens S. mutans and L. acidophilus. The most active plant was P. guajava (leaves), N. sativa (seeds), A. indica (leaves), A. aspera (root), S. aromaticum (bud). These in vitro antimicrobial results suggest the efficacy of these plants in dental caries. Use of more than one plant part, even a combination of plants for the formulation in the treatment of a single ailment can come as a success to complete cure of dental caries.

Electronic supplementary material

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Acknowledgements

The authors are thankful to the Department of Environmental Science and Engineering, Indian Institute of Technology (Indian School of Mines), Dhanbad, India, for providing necessary support for conducting the research. We express deep gratitude to those rural informants of the district, who freely shared the information during the field survey; without their cooperation this work would not have been possible.

Compliance with ethical standards

Conflict of interest

The authors have none to declare.

Footnotes

Electronic supplementary material

The online version of this article (10.1007/s13205-018-1283-2) contains supplementary material, which is available to authorized users.

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