Introduction

The wide variety of microbiota like aerobes, obligate anaerobes, fastidious organisms, and slow growing organisms such as Actinomyces, Bacteroides, Bifidobacterium, Lactobacillus, Candida, Aspergillus, Fusarium, Streptococcus species are present in the oral cavity. Some of them require a surface for adhesion and growth which further forms the sticky matrix of polymerised glucose¹. Dental plaque is the gelatinous mass of bacteria adhering to the tooth surface. First step of plaque formation is pellicle formation which is followed by the formation of sticky film known as plaque which leads to dental caries if not treated. The plaque forming bacteria like Streptococcus mutans, Actinomyces viscosus and Lactobacillus spp. are capable of utilizing the refined carbohydrates for the energy production and generate organic acids as the by-product of the metabolism (Fig 1). Formation of dental plaques follows various mechanisms such as quorum sensing, metabolic communication, co-aggregation².

Dental plaque is one of the most common dental health issues in whole world. This can cause dental caries as well as periodontal diseases if not treated. These effects can further lead to loss of affected teeth. Nearly 20-50% of global population suffer from periodontal diseases which are caused due to chronic plaque formation³. Approximately 13.2% children and 25.9% adults among global population are affected by dental caries which is the major consequence of dental plaque⁴. Mainly children are very prone to dental plaques due to consumption of sweetened products which contains more amount of sugar. Greater amount of dental plaque can be seen in people with biting abnormalities as well as people with improper oral care⁵. Periodontal diseases can lead to various systemic consequences like cardiovascular system, pulmonary system, skeletal system and digestive system. Due to discontinuities in the oral tissues microbiota present in dental plaque may enter in blood stream which leads to various diseases in body. These microbiota can stimulate acute phase proteins as well as pro-inflammatory cytokines which increases the intensity of diseases like diabetes and atherosclerosis. Spread of bacteria to mucosal linings of gut can cause gastric ulcers as well as spread to lungs can cause pneumonia. It can also cause pregnancy complications like low birthweight of infant⁶.

There are various methods used for prevention of formation of plaque like mechanical and chemical methods⁷. The most common and primary treatments include brushing, flossing and cleaning the teeth. Scaling is done for the patients who have accumulated a layer of plaque or tartar on their teeth⁸. Chemical methods like use of anti-adhesive chemicals like fluoride, chlorhexidine, perfluorosulphonamidoalkyl ester, octapinol, delmopinol are effective against S. sanguis and S. mutans⁷. Anti-microbial agents such as antibiotics, enzymes, bisbiguanide antiseptics, phenols, quaternary ammonium compounds, metal ions, natural products, fluorides, oxygenating agents and other antiseptics are used against plaque formation. The most effective plaque controlling agent is dicationic bisbiguanide antiseptic chlorhexidine. The pre-existing plaque can be removed by the enzyme such as dextranase which loosens the plaque adhered to the dental surface⁹. Some biological methods include use of a mutated strain of S. mutans which lacks lactate dehydrogenase gene. Another method specific against S. mutans is by targeted peptides or vaccines which shows bactericidal activity as well as inhibits recolonization of S. mutans to form biofilm².

These chemical agents can show adverse effects on the body like discolouration of teeth and tongue, poor taste, nausea. Besides their side effects, antibiotics also gives rise to the antibiotic resistant bacteria⁹. Various studies are carried out for the search of herbal products for prevention and treatment of plaque for reducing these side effects¹⁰. Herbal medicine shows preventive action against plaque accumulation and it acts on the predisposing factors of oral microorganisms from ancient times due to presence of bioactive components. Herbal mouthwashes are used for prevention for plaque accumulation and have the similar effect as the synthetic and chemical mouthwashes¹¹.

Ocimum basilicum, Azadirachta indica, Syzygium aromaticum, Acacia nilotica and Achyranthes aspera are some medicinal plants containing essential oils which contains bioactive compounds, showing effect against the plaque forming organisms. These bioactive components show various activities such as anti-microbial and antifungal. These activities play a major role as antiplaque agents¹². This review deals with the mechanism of plaque formation, treatment and role of Ocimum basilicum, Azadirachta indica, Syzygium aromaticum, Acacia nilotica and Achyranthes aspera in the prevention of plaque formation.

Main text

Plaque Formation and its mechanism

Dental plaque is the sticky matrix of polymerised sugars, bacteria and salivary proteins. The organic matrix of plaque contains protein-polysaccharide complexes produced by microbes from carbohydrates such as fructans, dextrans, rhamnose, galactose. Small amount of lipids is also present in dental plaque. It also includes the inorganic components such as calcium, phosphorus, magnesium, sodium and potassium¹³. Formation of dental plaques and dental biofilms follows various mechanisms such as quorum sensing, genetic exchange, metabolic communication, co-aggregation². The majorly present organisms in dental plaque are Bacteria such as Actinomyces, Bacteroides, Bifidobacterium, Lactobacillus, Streptococcus species, fungi like Candida and Spirochetes and Mycoplasmas are present in dental biofilm. The first phase of plaque formation is an induction of linking film or conditioning film which is known as pellicle followed by accumulation that is adhesion and growth of bacteria by the mechanism of quorum sensing and then existence phase in which parallel growth and erosion of biofilm takes place. Tannerella forsythus, Treponema denticola, F. nucleatum and streptococci are mainly present organisms in existence phase¹³.

There are three main mechanisms of plaque formation as follows:

Quorum sensing

Many organisms use quorum sensing mechanism for the formation of biofilm. It is also known as biofilm signalling. It is cell to cell communication mechanism observed in prokaryotes. This mechanism is facilitated by the exchange of small molecules known as signals or signalling molecules. It can regulate the genes and operons in the cells¹⁴.

Quorum sensing in bacteria is divided into three classes: viz. 1) LuxI/LuxR-type quorum sensing present in Gram-negative bacteria, which uses acyl-homoserine lactones (AHL) as signalling molecules, 2) oligopeptide-two-component-type quorum sensing present in Gram-positive bacteria, which uses small peptides as signalling molecules and 3) LuxS-encoded autoinducer (AI)-2 quorum sensing is present in Gram-positive as well as Gram-negative bacteria¹⁴.

Co-aggregation

A specific cell-to-cell reaction occurring between different bacterial cells is known as co-aggregation. It is one of the most important mechanisms used by oral bacteria for colonization and formation of dental biofilm. The planktonic bacteria which cannot colonize directly on the dental surface by adhesion can attach to the surface by attaching to specific receptors of other bacteria adhered already to the surface of teeth¹⁵. The early colonizing bacteria of dental biofilm attaches to complementary pellicle receptors via adhesins and the secondary bacteria further attaches to these early colonizers. One of the main bacteria which shows co-aggregation in dental biofilm formation is Fusobacterium nucleatum which co-aggregates with Streptococci and many obligate anaerobes present in oral cavity and hence play an important role as bridging organism between early and late colonizing bacteria¹⁵. The co-aggregation between F. nucleatum and Gram-negative bacteria are facilitated by lectin-carbohydrate¹⁶. Whereas co-aggregation between F. nucleatum and Gram-positive bacteria is rarely inhibited by sugars. Co-aggregation among oral bacteria contribute to bacterial colonization through physico-chemical mechanisms, as well as to genetic exchange and metabolic communication¹⁷.

Metabolic communication

Metabolic communication is the mechanism in which the by-product of one microorganism can be the energy source of another organism¹⁵.  Short-chain fatty acids produced by oral bacteria are an essential carbon source for some other oral bacteria. Symbiosis is observed in oral Streptococcus and Veillonella species as Veillonella use the short chain acids like lactates produced by Streptococci for the production of energy. Similarly P. gingivalis and T. denticola also show metabolic communication by the utilization of succinic acid produced by T. denticola for the production of phospholipids of cell envelope of  P. gingivalis¹⁸.

Medicinal plants that prevent plaque formation

Ocimum basilicum

Sweet basil is one of the plants which is considered for the studies in alternative medicine. Ocimum basilicum L. (O. basilicum) (Fig. 3). It is 20-60 cm long, aromatic, herbaceous, autogamous plant with white-purple flowers. It is an annual and perennial plant. It is cultivated in many countries but the origins of sweet basil are India and other parts of Africa, South America, Philippines, France, Portugal, Italy, Spain, Greece, Malta, Cyprus. In Mediterranean diets such as soup, cheese and pasta dishes Sweet basil is used. In Iran it is used as vegetable and food flavouring¹⁹.

Traditional use of sweet basil is for the treatment of cough, cold, fevers, inflammation, digestive illnesses, bug stings, pain during menstruation, anxiety, headaches, sinusitis, migraines, nerve pain, and variety of neurodegenerative ailments, diabetes, cardiovascular diseases, hypertension. Arial parts of sweet basil are rich in essential oils²⁰. Essential oil of sweet basil also contains anti-microbial, antiviral, antifungal, nematocidal and insecticidal, antioxidant and anti-obesity, carminative, galactogogue, stomachic and antispasmodic properties¹⁹. Sweet basil also contains anticonvulsant, anti-acetylcholinesterase activities which are essential for cure of epilepsy and Alzheimer’s disease as well as helps in anti-aging²¹. Basil is also used as gargles for reduction of bad odour of oral cavity. The essential oils extracted from sweet basil are used in dental and oral hygiene products. There are many bioactive components present in essential oil of sweet basil which shows anti-plaque effect as well as anti-caries effect on teeth²².

O. basilicum is used for extraction of essential oils which are used in various fields like culinary, medicine, cosmetics. These essential oils contain a variety of chemicals which act as therapeutic agents in different diseases. The main chemical components like, phenols, esters, alcohols, oxides. The peculiar aroma of basil plants is due to linalool, methyl cinnamate, 1,8-cineole and estragole²³. The extraction of essential oils is mainly done by gas chromatography and these components of essential oils are identified by mass spectrometry.

Main components in essential oil of the aerial parts of O. basilicum are linalool, eugenol, (Z)-cinnamic acid methyl ester, cyclohexene, alpha-cadinol, 2,4 diisopropenyl-1- methyl-1-vinylcyclohexane, 3,5-pyridine-dicarboxylic acid, 2,6-dimethyl-diethyl ester, beta-cubebene, guaia-1(10),11-diene, cadinene, (E)-cinnamic acid methyl ester, beta-guaiene, estragole and nerol. Many components from them acts as anti-microbial agents. These components act against mainly S. mutans, C. albicans, S. sobrinus, L. casei, L. monocytogenes, P. gingivalis²⁴.

Activity of O. basilicum against dental plaque

Sweet basil contains essential oils which acts against various diseases as well as various microbes. The bioactive compounds present in essential oil acts against dental plaque forming organisms. When essential oil extract of O. basilicum was administered to the patients suffering from dental plaque and purulent gingival diseases in dose of 250mg/ day for 3 weeks it showed reduction of dental plaque and gingival diseases²². Essential oil extract of sweet basil acts against S. mutans when tested using disc diffusion test and MIC in comparison with norfloxacin and ketoconazole. The zone of inhibition observed as 11mm and the MIC was 250μg/ml. When extract is tested against C. albicans it showed zone of inhibition of 12mm and MIC of 500μg/ml²⁴. When essential oil of sweet basil was tested against C. albicans inoculated in brain heart infusion broth it showed MIC of 0.87mg/ml where as a nano emulsion containing sweet basil essential oil showed MIC of 0.41mg/ml which suggests that the nano emulsion is  more effective against dental plaque in less quantity²⁵. When essential oil of sweet basil is tested in vitro against S. mutans and L. casei biofilms using chlorohexidine as standard it showed MIC and MBC of 0.31μl/ml and 1.25μl/ml respectively²⁶. Micro emulsion of sweet basil essential oil containing mouthwash inhibits the biofilm formation of S. mutans as well as it also reduces the adherence of biofilm²⁷. O. basilicum essential oils showed zone of inhibition between 4.0 to 4.8 cm when tested against S. mutans and S. sorbinus²⁸.

Azadirachta indica

Azadirachta indica (Fig. 4) i.e., Neem or Margosa is one of the trees used in traditional medicine for cure of many ailments. It belongs to Meliaceae family. It is tree ranging 40-50 feet in height and straight trunk containing dark brown bark. It contains 5-15 leaflets per leaf. Flowers are small and white in colour. A. indica produces green coloured fruits which turns yellow after ripening.  A. indica is native to East India and Burma and also cultivated in Tropical Africa, South and Central America, Singapore, Malaysia, Philippines, Saudi Arabia²⁹.

In traditional medicine A. indica is used for treatment for many diseases. Extracts of A. indica leaves and bark are used for treatment of leprosy, helminthiesis, rheumatism, respiratory disorders, skin ulcers, anorexia, diabetes, urinary disorders³⁰. Leaves, bark and fruits of A. indica contains essential oils which shows various properties like anti-bacterial, anti-fungal, larvicidal, anti-diabetic, anti-ulcer, anti-inflammatory, immunomodulator, anti-malarial, anti-HIV, anti-tumor, anti-hypertensive, antioxidant²⁹. These essential oils also show anti-dental caries property. Due to this property A. indica oils are used in toothpastes and dental care products as well as in skin care products³¹.

A. indica is one of the medicinal plant used in pharma as well as cosmetic industries. It contains essential oils which shows presence of various compounds. Major compounds present in aqueous extract of A. indica are alkaloids, esters, reducing sugars, carbohydrates, flavanoides, tannins, terpenes, phenolic compounds, isoprenoids, saponins. Ethanolic extract of Azadirachta indica showed presence of alkaloids, reducing sugars, carbohydrates, flavonoids, tannins, phenolic compounds, saponins, glycosides. Apart from these compounds GC-MS analysis of ethanolic extract showed presence of phytol, acetic acid, 4-Cycloocten-1-ol, 8,8’-(iminodi-2,1-phenylene), Hydroxy pivalic acid, 1,3-Diphenyl-2-azafluorene, Germanicol, acetate and Lup-20 (29)-2n-3-ol. These all compounds are extracted and studied by GC-MS³². From all these compounds Nimbin, Azadirachtin, Catechin and Quercetin are major bioactive compounds present in A. indica which acts against dental plaque.

These compounds show anti-microbial activity against various gram-positive and gram-negative microbes. Bioactive compounds present in A. indica acts against S. mutans, S. sobrinus²⁹. These compounds also show effect against fungal pathogens such as C. albicans causing dental diseases³³.

Activity of Azadirachta indica against dental plaque

A. indica contains various compounds in its extracts as well as in essential oils. These compounds acts against various ailments. One of its activity is against dental plaque forming organisms. When n- hexane extract of A. indica was tested against C. albicans with ditchwell diffusion method it showed zone of inhibition of 28mm³⁴. Ethanolic extract of A. indica leaves when tested in vitro by agar diffusion method it showed similar zone of inhibition as 2% sodium hypochlorite against C. albicans and E. faecalis³⁵. When ethanolic extract of neem leaves tested in vivo and further tested for reduction of microbial load in dental plaque sample as well as significant decrease in colony forming units of C. albicans and E. faecalis³⁶. When A. indica extract containing gel was given to patients suffering from baseline plaque for 3-6 weeks of treatment it showed significant decrease in amount of S. mutans and Lactobacilli when compared with treatment using chlorhexidine gluconate³⁷. When aqueous extract of neem was tested using ditch plate method against S. mutans, S. salivarius, S. sanguis and S. mitis showed zone of inhibition of 3.8cm, 2.9cm, 3.4cm and 2.7cm respectively for 50% concentration and 48hr of incubation³⁸. Acetone extract of neem showed zone of inhibition of 22mm against S. mutans whereas for S. salivarius chloroform extract showed zone of inhibition of 18mm. Ethanolic and aqueous extract of neem leaves showed effective inhibition of 16mm against F. nucleatum when tested using disk diffusion method³⁹. Ethanolic extract of A. indica leaves showed minimum bactericidal concentration of 250μg/ml and 500μg/ml for S. mutans and S. mitis respectively whereas for S. salivarius and S. sanguis it is 5mg/ml and 1mg/ml respectively⁴⁰.

Syzygium aromaticum

Syzygium aromaticum (Fig. 5) or clove is an aromatic plant which is used in various industries. It is a member of Myrtaceae family. It has height of about 8-12 m. leaves of this tree are quadrangle in shape and flowers are arranged in clusters. Flower buds are pale in colour initially which turns green and further to red which is an indication that they are ready for harvest⁴¹. Clove is native to Maluka islands in East Indonesia. Major producers of clove are India, Sri-Lanka, Indonesia, Tanzania, Brazil, Pakistan, Madagascar and Malaysia⁴².

S. aromaticum is used traditionally in many industries for flavouring, fragrance, and also in medicine. In many cuisines clove is used as flavoring agent. Clove contains essential oil which is rich in many compounds which shows therapeutic actions against various diseases. Essential oils from clove shows effects like antioxidant, antibacterial, antifungal, antiviral, hepatoprotective, cytotoxic, anesthetic, analgesic, antinociceptive, anti-inflammatory, larvicidal⁴². These essential oils also show effects against dental pathogens which causes dental plaque and periodontal diseases. Due to this property clove oil is used in many dental care products as well as it is also used in traditional medicine⁴³.

S. aromaticum is a very effective plant used in many industries like food and medicine. The essential oils of clove contain many bioactive compounds which are helpful in treating many diseases. Chemical composition of clove essential oil was studied by using GC-MS analysis. Major groups present in clove essential oils are flavonoids, hydroxybenzoic acids, hydroxycinnamic acids, phenolic acids and tannins⁴¹. The essential oils of clove contain β-caryophyllene, eugenol, α-humulene, eugenol acetate, 2-heptanone, ethyl hexanoate, humulenol, calacorene, calamenene, Methyl salicylate, α-pinene, limonene, p-cymene, 2-Heptyl cetate, linalool, (E)-β-Ocimene and many more compounds are present in small amounts. These compounds show potential benefits against various diseases like gastrointestinal diseases, skin infections and also respiratory infections⁴³.

Major components present in Syzygium aromaticum are linalool, eugenol, β-caryophyllene. Bioactive compounds present in clove acts against S. mutans, C. albicans, S. sobrinus, Lactobacillus spp.²⁴. These compounds also show effect against fungal pathogens such as C. albicans causing dental diseases³³.

Activity of Syzygium aromaticum against dental plaque:

S. aromaticum is one of the herbs used for treatment of dental plaques. It contains essential oils and many phytochemicals in its extracts. Aqueous extract of S. aromaticum shows MIC of 31.25 mg/ml against S. mutans⁴⁵. Essential oil extracted from S. aromaticum showed zone of inhibition of 19mm against Lactobacillus spp., 13mm against S. mutans and 9mm against S. salivarius. S. aromaticum extract shows growth inhibition activity against P. gingivalis, S. mutans, P. intermedia and A. viscosus⁴³. S. aromaticum oil shows MIC of 0.2 mg/ml and MBC of 0.8mg/ml against S. mutans and S. sobrinus whereas for S. ratti, S. anginosus and A. actinomycetemcomitans MIC is 0.8mg/ml and MBC 1.6mg/ml. For S. sanguinis and S. criceti MIC is 0.4mg/ml and MBC 1.6mg/ml. apart from these S. gordonii, F. nucleatum, P. intermedia and P. gingivalis are inhibited at MIC of 0.1mg/ml and MBC of 0.2 mg/ml in comparison with ampicillin and gentamycin as standard drugs⁴⁶. Essential oil extracted from S. aromaticum shows MIC of 6.25μg/ml and MBC of 25μg/ml against P. gingivalis⁴⁷.

Acacia nilotica

Acacia nilotica (Fig. 6) commonly known as babool is one of the medicinal plants which is used in traditional medicine. It belongs to Mimosaceae family. It is perennial tree approximately 20 m in height⁴⁸. It has black coloured stem. Flowers are golden yellow in colour and are 1.2-1.5 cm in diameter. It has straight or slightly curved, hairy, thick, gray and are of 5-15 cm in length. Leaves are 30-40 mm long and bipinnate⁴⁹. It is native to India, Pakistan, Iran, Israel, Zambia, Egypt, Kanya, Nepal, Ethiopia⁴⁸.

A. nilotica is used in traditional medicine for treatment of various diseases. Due to its medicinal properties it shows various activities like antibacterial, antioxidant, anti-hypertensive, antifungal, anti-plasmodium, anti-spasmodic, anti-diabetic, anti-acetylcholinesterase, anti-mutagenic⁴⁹. Due to presence of phytochemicals in the extracts and essential oils it also shows activity against dental plaque and dental caries. Babool is also used in many commercial products used for dental care and oral hygiene⁵⁰.

A. nilotica is a very effective plant used in many medicines. The essential oils of A. nilotica contain many bioactive compounds which are helpful in treating many diseases. Chemical composition of A. nilotica essential oil was studied by using GC-MS analysis. Major groups present in A. nilotica essential oils are flavonoids, hydroxybenzoic acids, hydroxycinnamic acids, polyphenols, saponins and tannins⁴⁹.

Major components present in A. nilotica are cinnamaldehyde, eugenol, nerol and phytol. Bioactive compounds present in A. nilotica acts against S. mutans, S. sobrinus⁵¹. These compounds also show effect against fungal pathogens such as C. albicans causing dental diseases⁵².

Activity of Acacia nilotica against dental plaque:

A. nilotica contains essential oils and bioactive compounds in extracts which shows activity against dental plaque. When oil extracted from A. nilotica tested against S. mutans and C. albicans it shows MIC of 9.75μg/ml⁵³. Ethanolic extract of A. nilotica showed MIC of 5mg/ml and zone of inhibition of 31mm against S. mutans whereas petroleum ether extract showed MIC of 10mg/ml and zone of inhibition of 17.5mm⁵⁴. Methanolic extract of A. nilotica twig extract shows zone of inhibition of 40.12 cm and MIC of 0.19mg/ml against S. mutans and zone of inhibition of 42.07cm and MIC of 0.19mg/ml for C. albicans⁵¹. Ethyl acetate extract of A. nilotica shows inhibition zone of 14.67cm against C. albicans whereas methanolic extract showed inhibition zone of 27cm when tested using chlorohexidine diacetate as standard⁵⁵. Aqueous extract of A. nilotica shows zone of inhibition of 15.66mm against S. mutans when incubated for 48hr⁵⁶.

Achyranthes aspera

Achyranthes aspera (Fig. 7) or Devil’s horse whip is an annual or perennial herb used in traditional medicine. It belongs to amaranthaceae family. It is about 1-2 meter in height and have woody base. Leaves are thick, rounded and 6-20 cm in length. Flowers are greenish white and present at axillary or terminal position and are 75 cm long. Its seeds are subcylindrical are reddish brown in colour⁵⁷. Achyranthes aspera is found in India, America, Australia, South Andaman Islands, Baluchistan, Ceylon. It is reported as alien invasive species in northern Bangladesh⁵⁸.

A. aspera is traditionally used in many medicines for cure of various ailments. The essential oils present in it are used as therapeutic agents in traditional medicine for treatment of piles, oedema, snake bites, pneumonia, rheumatism, scabies and many other skin diseases⁵⁷. Volatile compounds present in A. aspera essential oil also show various activities like anti-microbial, antiviral, antifungal, antifertility, anti-inflammatory, antiarthritic, antiparasitic, anticarcinogenic, antioxidant, antiallergic, anti-obesity, antidandruff, antiulcerogenic, antidiarrheal, analgesic, bronchoprotective, antidepressant⁵⁸. A. aspera also shows antiplaque activity and prevents dental problems by inhibiting dental pathogens⁵⁹.

A. aspera is an effective plant against many diseases due to presence of essential oils. These essential oils contain many bioactive compounds which shows therapeutic effects. Chemical composition of A. aspera oil was studied by GC-MS analysis⁶⁰. Major groups present in A. aspera oil are saponins, flavonoids, terpenoids, alkaloids, long chain compounds, aliphatic alcohols, ketones, phenols⁵⁸.

Major bioactive compounds present in A. aspera are Betulin, nerol, eugenol, 1-Hexadecanol and Phytol which are present in essential oils⁵⁷. These compounds show activity against dental plaque forming bacteria. Major activity is seen against S. mutans, C. albicans, L. acidophilus, P. gingivalis²⁹.

Activity of Achyranthes aspera against dental plaque

Anti-plaque activity of A. aspera is due to presence of bioactive phytochemicals present in its extracts. Methanolic extract of A. aspera leaves shows antibacterial activity against S. mutans, L. acidophilus, S. salivarius and S. sanguis⁶¹. Zone of inhibition against S. mutans of Aqueous extract of A. aspera was 13mm whereas it is 18mm, 16mm and 23mm for benzene, petroleum and methanolic extract respectively. MIC of aqueous extract against dental pathogens was 100mg/ml whereas MIC of methanolic extract was 50mg/ml. When aqueous extract is tested against dental pathogen using disk diffusion method it showed zone of inhibition of 7mm and for methanolic extract it was 4mm. Erythromycin and chloramphenicol are used as standard drugs for determining of MIC and zone of inhibition⁶². Zone of inhibition of 21mm against S. mutans was observed by using Aqueous extract of A. aspera roots and stem and chlorohexidine as standard⁶³. MIC of A. aspera aqueous extract against C. albicans was 1.56mg/ml and 0.78mg/ml for acetone extract⁶⁴.  Methanolic extract shows MIC of 125μg/ml and 62.5μg/ml for essential oils of A. aspera against S. mutans. Whereas MBC for essential oils of A. aspera against S. mutans is 125μg/ml⁶⁵.

Bioactive compounds of Ocimum basilicum, Azadirachta indica, Syzygium aromaticum, Acacia nilotica and Achyranthes aspera and its mechanism of action against Dental Plaque: –

Essential oils, alcoholic and aqueous extracts of O. basilicum, A. indica, S. aromaticum, A. nilotica and A. aspera contains bioactive components that are linalool, estragole, methyl-cinnamate, eugenol, nerol, Betulin, 3,12-oleandione, 1-Hexadecanol, Phytol, Cinnamaldehyde, β-caryophyllene, nimbin, azadirachtin, catechin and quercetin which acts against many plaque and biofilm forming organisms.

Linalool (Fig. 8) is mainly found in Lamiaceae, Lauraceae, Apiaceae families. Linalool have various properties such as antibacterial, antifungal and mosquito repellent action. It is found in O. basilicum and S. aromaticum. Anti-microbial activity of linalool is observed by the techniques such as MIC and disc diffusion method. It is also effective against dental plaque forming organisms like S. mutans, Lactobacilli and S. sobrinus⁶⁶. These are the main plaque forming organisms found in dental biofilm. Linalool has the ability to degrade biofilm by penetrating in the extracellular polysaccharides or the slime layer produced by these organisms which results in the killing of bacteria. It also further inhibits the biofilm formation by S. mutans²⁷.

Eugenol (Fig. 8) is one of the bioactive compounds present in O. basilicum, S. aromaticum and A. nilotica. MIC and disc diffusion methods are used for determining the antibacterial and antifungal activity of eugenol. It gives antibacterial activity against P. gingivilis, S. mutans, L. monocytogenes which are major plaque forming microorganisms⁴⁶.  Eugenol also shows antifungal activity against Candida spp. which is one of the oral microorganisms. Eugenol alters fatty acids of cell membrane as well as it changes morphology of cell membrane which leads to disruption of cell membrane. These changes further lead to increase in non-specific permeability of cell membranes. This permeability also affects the transport of ATP and ions in the cell which is followed by cell death. Eugenol also produces intracellular reactive oxygen species which induces cell death and inhibition of Candida spp. as well as inhibition of S. mutans, P. gingivalis, and L. monocytogenes⁶⁷.

Nerol (Fig. 8) is one of the components in the O. basilicum, A. nilotica and S. aromaticum²⁴. Anti-microbial activity is observed by techniques such as MIC and disc diffusion method. It shows the effect against S. mutans which is the major plaque forming microorganism in the oral cavity. Nerol works by a similar mechanism like linalool i.e., degradation of extracellular polysaccharide of dental biofilm which leads to disruption of the plaque²⁷.

Phytol (Fig. 8) is one of the compounds presents in A. nilotica and Achyranthes aspera. It shows anti-microbial activity against C. albicans⁶⁸. MIC was carried out against these organisms and it was found to effective at concentration of 62.5μg/ml of phytol. It inhibits biofilm formation by C. albicans by inhibiting proteins required for biofilm synthesis. This further reduces the microbial growth and plaque formation⁶⁹.

Estragole (Fig. 8) is also known as methyl chavicol. It is the major component of essential oils of O. basilicum. Anti-microbial and antifungal activities of estragole is observed by the technique of disc diffusion method. It has restricted spectrum of action. It shows antifungal effect against C. albicans. As C. albicans is one of the oral pathogens which can act as a biofilm forming organism, estragole inhibits its growth and hence it is effective for the prevention against dental plaque⁷⁰.

Methyl-cinnamate (Fig. 8) is present in the essential oil of O. basilicum. Anti-microbial activity of methyl-cinnamate is observed by the techniques such as MIC and disc diffusion method. It shows antifungal activity against Candida spp. It give antibacterial activity against S. mutans and L. acidophilus which are main organisms of dental plaque formation and eventually dental caries²⁴.

1-Hexadecanol (Fig. 8) from A. aspera shows antibacterial as well as antifungal effect. It acts against S. mutans and C. albicans which are main organisms in plaque formation. Effect of 1-Hexadecanol on these organisms was studied using molecular docking. 1-Hexadecanol inhibits the SAP protein in C. albicans which is responsible for virulence and pathogenesis. It also inhibits spaP gene in S. mutans which is responsible in biofilm and plaque formation. Due to these inhibitions, reduction of growth and further reduction in plaque can be seen⁷¹.

Betulin (Fig. 8) present in A. aspera essential oil. It shows anti-microbial effect against S. mutans, C. albicans. These organisms are major plaque forming organisms present in oral cavity. Betulin is tested against these pathogens by BIC i.e. biofilm inhibiting concentration and it was observed that it inhibits the pathogens at concentration of 240μg/ml. Betulin inhibits the growth of these bacteria by inhibiting the adherence. It targets and inhibits vicRK and gtf genes which are responsible for formation of biofilm. This results in the inhibition of pathogens and further reduction of dental plaque⁷².

Cinnamaldehyde (Fig. 8) is one of the bioactive compounds presents in A. nilotica. These activities are studied by using MIC technique. It shows antibacterial activity against P. gingivilis, S. mutans, S. mitis, L. monocytogenes which are major plaque forming microorganisms⁷³. Cinnamaldehyde also shows antifungal activity against C. albicans. Cinnamaldehyde inhibits the adhesion of the organisms to dental surface which leads to reduction of biofilm formation and plaque formation.

β-caryophyllene (Fig. 8) is present in S. aromaticum essential oil β-caryophyllene shows many properties such as anti-microbial, anti-inflammatory, anaesthetic, anti-cancer. Its anti-microbial activity is observed by the techniques of MIC and disc diffusion method. It gives antibacterial activity against P. gingivilis, A. viscosus, S. mutans, L. monocytogenes, P. intermedia⁴⁶. It also shows activity against Candida spp.  It reduces cell adhesion ability and biofilm formation which further leads to reduction in plaque formation⁴³.

Nimbin (Fig. 8) from A. indica shows antibacterial as well as antifungal activity against dental pathogens. It acts against S. mutans, S. salivarius⁷⁴. Bactericidal activity of nimbin was seen against S. sobrinus with the MIC of 240 μg/ml⁷⁵. In these dental pathogens nimbin inhibits glucan synthesis which further leads to reduction in aggregation of streptococci and their colonization for formation of dental plaque⁷⁶. It also inhibited C. albicans which forms dental biofilm causing plaque. It shows anti-adhesive effect by increasing hydrophobicity of cell surface which reduces the colonization of pathogens⁷⁷.

Azadirachtin (Fig. 8) shows effect against dental plaque forming organisms as well as gingivitis causing organisms. It shows effect against S. mutans, S. sobrinus, C. albicans, L. acidophilus, S. salivarius with MIC of 250 μg/ml in comparison with chlorohexidine mouthwash⁷⁸. Azadirachtin inhibits the cell adhesion to the dental surface and reduces the biofilm formation which further results in inhibition of dental plaque formation⁷⁶.

Catechin (Fig. 8) is one of the bioactive compounds presents in A. indica. It shows anti-plaque activity as well as anti-biofilm forming activity. It shows effect against S. mutans, L. acidophilus, P. gingivalis, A. faecalis, C. albicans, S. sanguis and S. salivarius⁷⁹. Catechin shows anti-microbial effect with concentration of 180μg/ml when studied using MIC. It shows antiplaque activity by inhibiting the biofilm formation by reducing the quorum sensing protein LuxS. It also alters the expression of AHL molecule required for quorum sensing. Catechin also reduces genomic DNA which leads to anti-microbial activity⁷⁵.

One of the bioactive compounds which acts as anti-plaque and anti-quorum sensing agent present in A. indica is quercetin (Fig. 8). It shows inhibitory effect against P. gingivalis, A. faecalis, C. albicans. When tested by disk diffusion method quercetin showed inhibition at concentration of 10mg/ml⁸⁰. It shows MIC 190μg/ml. It inhibits biofilm formation by inhibition of LuxS and AHL in bacteria⁷⁵. In C. albicans quercetin reduces cell adhesion ability and biofilm formation which further leads to reduction in plaque formation⁸¹.

These bioactive compounds present in O. basilicum, A. indica, S. aromaticum, A. nilotica and A. aspera interfere in AMC and signalling. In all the methylation reactions S-adenosylmethionine (SAM) is a major methyl group donor. Autoinducer-2 (AI-2) which is a product of LuxS gene is an important molecule for interspecies communication in gram-positive as well as gram-negative bacteria. However, this is complex due to the dual role of LuxS in signalling and activated methyl cycle (AMC) which is a crucial metabolic pathway⁸². AI-2 is derived from SAM through a sequence of enzymatic reactions. Toxic intermediate S-adenosyl homocysteine (SAH) is formed after donation of a methyl group from SAM. This S-adenosyl homocysteine (SAH) is then hydrolysed to S-ribosyl homocysteine (SRH). LuxS cleaves SRH to produce homocysteine and AI-2 precursors⁸².These compounds lower expression of the LuxS gene. This further leads to reduction in levels of homocysteine and AI-2 precursor as well as accumulation of toxic SRH molecules in cells (Fig. 9). This further acts on disruption as well as prevention of production of biofilm.

70% of cell membrane of C. albicans contains polyunsaturated lipids. Some bioactive compounds induces lipid peroxidation in cell membranes of C. albicans. This further leads to incorporation of lipid peroxides in cell membrane and disruption and deformation⁸³.

Table 1: List of plaque forming microorganisms inhibited by bioactive compound of Ocimum basilicum, Azadirachta indica, Syzygium aromaticum, Acacia nilotica and Achyranthes aspera

Discussion

As an effect of modern lifestyle almost, every individual carries risk of getting affected by dental plaque and then eventually with dental caries which are caused by the microorganisms present as microbiota of the oral cavity. Actinomyces, Bacteroides, Bifidobacterium, Lactobacillus, Streptococcus species, Candida and Spirochetes and Mycoplasmas are some of the main organisms present in the oral cavity¹. These organisms are able to form oral biofilm by the specialized mechanism of quorum sensing, co-aggregation and metabolic communication.

To avoid such effects of dental plaque various techniques like physical, chemical and biological methods are used². Besides these effects it shows side effects like discolouration of teeth, development of resistance against anti-microbials. To avoid these side effects new strategies such as pharmaceutical products containing herbal extracts which are effective as equal as chemical agents are under research¹². Some of the traditional medicinal plants which are used for the treatment of plaque are O. basilicum, A. indica, S. aromaticum, A. nilotica and A. aspera. Various researchers have studied effects of bioactive compounds from these plants and provided data which shows the efficiency of these compounds over chemical agents.

O. basilicum, A. indica, S. aromaticum, A. nilotica and A. aspera has many effects such as anti-microbial, antiviral, antifungal, nematocidal, insecticidal, anti-diabetic, anti-ulcer, anti-inflammatory, immunomodulator, anti-malarial, anti-HIV, anti-tumor, anti-hypertensive, antioxidant, hepatoprotective, cytotoxic, anesthetic, analgesic, antinociceptive, anti-spasmodic, anti-acetylcholinesterase, anti-mutagenic, antifertility, antiarthritic, antiparasitic, anticarcinogenic, antiallergic, anti-obesity, antidandruff, antiulcerogenic, antidiarrheal, bronchoprotective, antidepressant ^{20,29,42,49,58}. Essential oils from these plants are used in culinary, traditional medicine as well as in formulations of dental care products.

Essential oils of O. basilicum contain linalool, methyl-cinnamate, estragole, eugenol and nerol¹⁹. Whereas nimbin, azadirachtin, catechin, quercetin from A. indica³⁷. linalool, eugenol, β-caryophyllene from S. aromaticum⁴³. cinnamaldehyde, eugenol, nerol, phytol from A. nilotica⁵¹. Betulin, nerol, 1-Hexadecanol, Phytol from A. aspera are the main bioactive components acting against dental plaque forming organisms⁶⁰.

The methods which are used in observing anti-microbial activity of these bioactive components are mainly MIC and disc diffusion methods. Linalool from O. basilicum and S. aromaticum acts against S. mutans, S. sobrinus and Lactobacilli⁶⁶. 1-Hexadecanol and Betulin from A. aspera are effective against S. mutans and C. albicans^71,72. Nimbin and Azadirachtin from A. indica is effective against S. mutans, S. sobrinus, S. salivarius, L. acidophilus and C. albicans⁷⁶. These compounds act by interrupting the formation of the slime layer and eventually leads to removal of dental biofilm^{27, 66}. Eugenol from O. basilicum, S. aromaticum and A. nilotica shows good effect against S. mutans, P. gingivalis, and L. monocytogenes as well as C. albicans⁴⁷. Similarly Phytol from A. nilotica and A. aspera as well as Estragole from O. basilicum are also effective against C. albicans⁵⁴. Quercetin from A. indica  shows effect against only P. gingivalis, A. faecalis and C. albicans⁷⁵.these compounds alter the cell membrane and form reactive oxygen species which interrupts in bacterial growth^46,67,70. Nerol from O. basilicum, S. aromaticum and A. nilotica also acts against S. mutans²⁷. Methyl-cinnamate from O. basilicum acts against C. albicans, L. acidophilus, S. mutans⁶⁶. Cinnamaldehyde from A. nilotica acts against C. albicans, L. acidophilus, S. mutans and S. mitis⁷³. β-caryophyllene from S. aromaticum S. mutans, L. monocytogenes, L. acidophillus, P. gingivalis, P. intermedia and Candida species⁴⁵. Catechin from A. indica acts against S. mutans, S. sobrinus, S. salivarius, P. gingivalis, A. faecalis, L. acidophilus and C. albicans⁷⁹. These compounds degrade the biofilm resulting reduction in the plaque^24,27,66.

Discussion

Figure 1: The overall process of the dental caries (original).Click here to view Figure

Figure 2:  Diagrammatic representation of Dental Biofilm formation (original).Click here to view Figure

Figure 3: Structures of bioactive compounds (original)Click here to view Figure

Figure 4: Azadirachta indicaClick here to view Figure

Figure 5: Syzygium aromaticum Click here to view Figure

Figure 6: Acacia niloticaClick here to view Figure

Figure 7: Achyranthes asperaClick here to view Figure

Figure 8: Structures of bioactive compounds (original)Click here to view Figure

Figure 9: Mechanism of action of bioactive compounds from Ocimum basilicum, Azadirachta indica, Syzygium aromaticum, Acacia nilotica and Achyranthes aspera (original).Click here to view Figure

Conclusion

O. basilicum, A. indica, S. aromaticum, A. nilotica and A. aspera contains essential oils contain bioactive compounds such as linalool, estragole, methyl-cinnamate, nerol, eugenol, nimbin, catechin, azadirachtin, Quercetin, β-caryophyllene, cinnamaldehyde, phytol, Betulin and1-Hexadecanol. Main targets of these compounds are S. mutans, C. albicans, L. acidophilus, S. sobrinus, P. gingivalis, A. faecalis, S. mutans and L. monocytogenes which are majorly found microorganisms in dental plaque. Theses bioactive components have similar effects as the chemical anti-plaque agents without any side effects and hence can be used for long term treatment against dental plaque. Hence the herbal products containing bioactive compounds of O. basilicum, A. indica, S. aromaticum, A. nilotica and A. aspera may prove to be more effective in the long run.

Acknowledgment

We acknowledge the funding received from Research Society for the Study of Diabetes in India (RSSDI/HQ/Grants/2017/342).

Conflict of interest

The authors declare no conflicts of interest relevant to this article.

Funding source

Research Society for the Study of Diabetes in India (RSSDI/HQ/Grants/2017/342)

Authors’ Contribution

Conceptualization, Y.P., H.P..D. and A.A.; writing—original draft preparation, Y.P., H.P.D. and A.A.; writing—review and editing,  Y.P., H.P.D. and A.A. ; visualization, Y.P., H.P.D. and A.A. .; supervision, A.A. ; project administration, A.A.; funding acquisition, A.A.. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Not Applicable

Ethics Approval Statement

Not Applicable

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