Antibiofilm and quorum sensing inhibitory activity of Achyranthes aspera on cariogenic Streptococcus mutans: An in vitro and in silico study

Abstract

Context: Traditionally, many cultures use chewing sticks for oral hygiene maintenance. When properly used, these chewing sticks are found to be efficient due to the combined effect of mechanical cleaning, enhanced salivation and the antimicrobial action of leached out plant compounds.

Objective: Achyranthes aspera L. (Amaranthaceae), an ethanomedicinal herb was evaluated for its potential to inhibit growth and biofilm formation by cariogenic isolate Streptococcus mutans as an alternative means of caries management by quorum quenching (QQ).

Materials and methods: Biofilm forming cariogenic isolates were isolated and their susceptibility to the petroleum ether, benzene, methanol, aqueous extracts of A. aspera was evaluated. Gas chromatography–mass spectrometry (GC–MS), phytochemical analyses and structure-based virtual screening for their quorum sensing (QS) inhibitory activities, drug-likeness and bioavailability were also carried out.

Results: The biofilm inhibition percentage obtained for methanol, benzene, petroleum ether and aqueous extracts (125 µg/mL) were ≤94%, ≤74%, ≤62% ≤42%, respectively. GC–MS analyses indicated 61 compounds, of which betulin recorded efficient interaction exhibiting comparable binding energy of −8.72 with S. mutans glycosyltransferase (GTF-SI) whereas 3,12-oleandione exhibited binding energy −5.92 with OmpR subfamily QS regulatory DNA-binding response regulator. Computer-assisted molecular descriptor and Lipinski’s rule violation calculation uncovered the presence of more drug-like compounds.

Discussion and conclusion: Anticaries bioactive compounds of A. aspera with higher QS response regulator binding energy, low toxicity and optimal pharmacokinetic properties were revealed. These compounds with possible QQ ability hold the potential for use as anticaries drug leads and antibiofilm preventative medicine.

• Bioavailability
• biofilm
• caries
• Lipinski’s rule
• molinspiration
• quorum quenching

Introduction

Antibiotic resistance among pathogens has emerged into a most challenging phenomenon of reversing decades progress of therapeutic success since it causes substantially increased rate of infectious disease morbidity and mortality, as well as huge burden of cumulative socioeconomic costs (Chan et al., 2012). The currently employed therapeutic and prophylactic interventions to infectious diseases are limited, however, due to the emergence of resistance to existing therapeutic agents as well as circulation of genetic variants that escape the immune surveillance (Song & Seong, 2007). The scientific approaches of the past are not adequate to address these challenges and consequently there is a need for new research strategies and tactics to minimize these threats and improve global health (Aderem et al., 2011). The observed 2011 World Health Day theme “antimicrobial resistance: no action today and no cure tomorrow” uphold the same.

At present, biofilm-mediated antibiotic resistance has become a major cause of anxiety for many clinical and device associated infections. Further, biofilm tolerance is of major clinical importance since more than 60% of the bacterial infections currently treated by physicians in the developed world are considered to involve biofilm formation (Fux et al., 2005). Since pathogenic bacteria are normally exposed to subinhibitory antibiotic concentrations during antimicrobial therapy, an environmental signal in itself triggering biofilm formation, poses significant problem for the eradication of bacterial infections in patients. Moreover, biofilms also play a vital role in virulence of many pathogenic bacteria (Landini et al., 2010). In this context, detection of biocides targeting biofilm has revolutionized the research approach in medical, pharmaceutical and biosciences (Sihorkar & Vyas, 2001).

Dental caries, otherwise known as tooth decay, is one of the chronic diseases of people worldwide and individuals are susceptible to throughout their life (Selwitz et al., 2007). Dental plaque, a unique microbial biofilm ecosystem comprising a diverse microbial community (Dumitrescu & Kawamura, 2010), has a primary role in its pathogenesis (Sohaibani & Murugan, 2012). Exchange of information among single-cell organisms, the cell-to-cell communication through small chemical molecules, has been termed quorum sensing (QS). Microbial processes like biofilm formation, virulence factor expression and bioluminescence are examples of QS-control. Recently, antivirulence therapies have drawn attention as a new strategy to combat microbial infections, and in that context bacterial QS represents a promising therapeutic target (Kaufmann et al., 2008). Even though toothbrushes and toothpastes have found wide usage, natural methods of teeth-cleaning using chewing sticks from a range of plants have been practiced for thousands of years in Asia, Africa, the Middle East and America. Further, a number of clinical studies confirmed the efficiency of chewing sticks in removing dental plaque due to the collective effect of their mechanical cleaning, salivation enhancement and the action of leached-out antimicrobial substances (Wu et al., 2001). The leaching bioactive compounds exhibit number of reactions in the oral environment including their antimicrobial action on pathogens, mouth refreshment by imparting fragrance in the mouth, bad odor elimination and stimulation of taste buds. Hence, many traditional cultures use only herbal chewing sticks instead of plastic-bristle brushes (Sohaibani & Murugan, 2012).

Since time immemorial, plant wealth is greatly exploited for its therapeutic potential and medicinal efficacy to cure various oral ailments (Hebbar et al., 2004) in India and documented evidence for chewing sticks use in dental health has been found even in ancient Ayurveda texts (Telles et al., 2009). Plants such as Azadirachta indica A. Juss (Meliaceae), Salvadora persica L. (Salvadoraceae), Acacia catechu (L. f.) Willd (Fabaceae), Acacia nilotica L. (Fabaceae), Juglans regia L. (Juglandaceae), Ficus benghalensis L. (Moraceae), Jatropha curcas L. (Euphorbiaceae) (Farooqi et al., 1998) and Achyranthes aspera (Hebbar et al., 2004) are reported as a source for chewing sticks. Many kinds of these chewing sticks were shown to have medicinal and anti-cariogenic properties, which would have been explored for management and even to cure problems such as oral cavity deformities, plaques and infections in ancient India (Venugopal et al., 1998).

Medicinal plants found in the environment harboring high bacterial cell density were long suspected to have protective mechanisms against microbial infections and offer a large and attractive phytochemical repertoire for the discovery of novel microbial disease control agents (Sybiya Vasantha Packiavathy et al., 2012). In spite of the fact that several traditional medicinal plants contain active constituents which are known to have antibacterial activity against various micro-organisms, including dental cary causing bacteria (Kelmanson et al., 2000), earlier studies exclusively focused only on their effects on planktonic bacteria with little emphasis on the highly resistant biofilm (Sandasi et al., 2010).

Achyranthes aspera, an ethanomedicinal herb, is reported to be useful in the indigenous system of medicine, for the treatment of renal dropsy, bronchial affections and leprosy, and it has also been credited with abortifacient, contraceptive, cardiac stimulant, astringent, diuretic, alterative, antiperiodic as well as purgative properties (Tahiliani & Kar, 2000). They are also used as an antidote for snake bites (Siddiqui & Husain, 1990) and cure for women as well as child diseases (Borthakur, 1992), venereal diseases (Hamill et al., 2003), malarial fever, asthma, hypertension and pneumonia (Vasudeva & Sharma, 2006). This ethanomedicinal herb is also reported to have many pronounced activities including hepatoprotective, cancer chemopreventive, anti-inflammatory, anti-arthritic, thyroid-stimulating, anti-peroxidative, reproductive function stimulative, immunomodulatory, wound healing, antioxidant (Edwin et al., 2008), gastroprotective (Das et al., 2012), vaginal contraceptive (Paul et al., 2010) and anxiolytic (Barua et al., 2012) activities. Beneficial effects of this herb are reported to extend to oral hygiene maintenance and caries prevention by folkloric tradition in many parts of India including Dharwad district of Karnataka (Hebbar et al., 2004) and in parts of Tamil Nadu, southern India, where in number of villages, people use its stem and root as teeth cleaning natural toothbrush. In other parts, the ash from the burnt plant often mixed with mustard oil and a pinch of salt is used as a tooth cleaning powder. It is believed to relieve pyorrhea and toothache (Londonkar et al., 2011).

Chlorhexidine is used as a gold standard for comparison and positive control for assessing the anticariogenic potential of other agents. It has been studied extensively and is currently the most potent chemotherapeutic agent against Streptococcus mutans and dental caries. However, the well-known side effect of chlorhexidine, i.e., staining of teeth and restoration, alteration of taste sensation and development of resistant micro-organisms, may limit the long-term use (Agarwal et al., 2010). The medicinal plant’s novel phytochemical repertoire with recognized antibiotic properties may also possess potential antipathogenic activity. These antipathogenic compounds either may not lead to resistance development since they kill bacteria or stop their growth. Alternatively, they act on the expression of pathogenic genes and attenuation by interfering with the bacterial communication system (Sybiya Vasantha Packiavathy et al., 2012), hence providing a solution to antibiotic resistance (Adonizio et al., 2006). Numbers of medicinal plants (Adonizio et al., 2006; Sohaibani & Murugan, 2012; Sybiya Vasantha Packiavathy et al., 2012) are known to have quorum sensing inhibition (QSI) mimics capable of binding to QS response regulators, but fail to activate them. Therefore, the ethanomedicinal A. aspera bioactive compounds can be harnessed for containing the highly resistant biofilm-mediated caries and other problems.

Whereas, to the best of our knowledge, no report is available in the literature on anticariogenic activities of A. aspera, this study is an attempt to find alternative means of dental caries biofilm management by quorum quenching (QQ) with more emphasis on cost-effective prevention than a treatment. Herein, we report for the first time, the growth and biofilm inhibitory potential of A. aspera extracts on a cariogenic isolate of S. mutans. The mode of action of the phytochemical active compounds of this herb on biofilm-specific events is also determined using in vitro as well as in silico methods toward achieving the objective.

Materials and methods

Isolation and characterization of biofilm forming cariogenic organisms

Dental plaque samples (100) were collected from the anterior and molar teeth of both jaws from outpatients undergoing treatment at K.S.R. Institute of Dental Science and Research following standard procedures during the period from January 2010 to December 2010. Among the isolates obtained, those identified as S. mutans (Sohaibani & Murugan, 2012) were selected and used. The slime forming ability of the isolates was evaluated by the tube adherence method (Christensen et al., 1982) and graded as described previously (Murugan et al., 2010). The Congo red agar (CRA) method (Freeman et al., 1989) was employed for assessing the extracellular polysaccharide substance and slime producing ability of the isolates. For the biofilm assay, the microtiter plate assay of Motegi et al. (2006) was employed and efficient strains identified were selected for the biofilm inhibitory study.

Collection and identification of medicinal plant

Whole plant A. aspera was collected from Kolli hills, the preserved mountainous area of the Eastern Ghats, which lies between the geo-coordinates 11° 10′ to 11° 30′ N and 78° 15′ to 78° 30′ E in Tamil Nadu, India. Fresh plant materials (stems and roots) were collected in polythene bags and the specimens were labeled properly using a running number system. Whole plant voucher specimens were also collected. The collected plants were identified using standard taxonomic procedures. The identity of the plant was certified and authenticated by the Joint director of Botanical Survey of India, Ministry of Environment and Forests, Government of India (Certificate no. BSI/SRC/5/23/2010-11/Tech, 1737). Adhering soil debris on the plant specimens was removed by washing and the plant materials were shade-dried and powdered.

Extraction and determination of bioactive compounds antibacterial activity

Bioactive compounds from A. aspera were solvent extracted using a Soxhlet extractor, the thimble of which contained a 20 g powdered plant material in 200 mL of solvents in the order of increasing polarity, namely petroleum ether, benzene, methanol and aqueous for 8 h, filter-sterilized and stored at 4 °C until use. After removing the solvent, the yield of the extracts was calculated and was evaluated for their antimicrobial activities against biofilm-forming S. mutans (Sohaibani & Murugan, 2012) using the Muller–Hinton agar by a well-diffusion method.

Micro-dilution antibacterial assay for minimum inhibitory concentration

The micro-dilution technique described by Song et al. (2007) using 96-well micro-plates was used to determine the minimum inhibitory concentration (MIC) values of extracts against the cariogenic S. mutans. Briefly, 100 µL inoculum suspensions (1.5 × 10⁶ CFU/mL) prepared from 18 h cultures of isolates were added to wells having 100 µL of various concentration of two-fold serially diluted extracts. Equal amounts of trypticase soy broth and the antibiotic triclosan at a concentration of 50 µL/mL were added as negative and positive controls, respectively. After incubation, the MIC values were calculated. The MIC was defined as the lowest concentration that restricted bacterial growth to an absorbance lower than 0.05–550 nm (no visible growth).

Antibiofilm activity of A. aspera on exopolysaccharide production and biofilm formation

The antibiofilm activity of the highly inhibitory A. aspera methanol extract on exopolysaccharide (EPS) was determined by a modified gradient plate technique. Continuous gradient of plant extract on the CRA medium was prepared as explained below. Briefly, an arrow was drawn in the center of the plate indicating concentration gradient, placed on pencil at an angle; 10 mL of the molten CRA was poured and allowed to harden. The plate was then placed flat and 10 mL of the plant extract (250 mg/mL) containing CRA was again poured and allowed to solidify. The selected biofilm forming isolates were inoculated into the center of the plate as a single streak. The organisms were again zigzag streaked perpendicular to the original streak and by crossing it each time. The color and nature of the developed colonies along the line of streak were observed.

The biofilm inhibitory potential of A. aspera methanol extract and the phytochemical betulin 5 µM (Sigma-Aldrich, Steinheim, Germany) were determined by the modified quantitative spectrophotometric microtiter plate method (Pitts et al., 2003). The stability of the caries isolates against the A. aspera methanol extract and the percentage of biofilm inhibition were also calculated as described earlier (Murugan et al., 2011).

Gas chromatography–mass spectrometry determination of phytochemicals

The Clarus 500 Mass Spectrometer (PerkinElmer, Waltham, MA) coupled to a Clarus 500 Mass Spectrometer mass detector with Elite 5 ms (5% phenyl and 95% dimethylpolysiloxane); size, 250 mm; and ID, 0.25 mm 630 m was used to analyze the components of the plant extract. The interface temperature was 280 °C and the scan range 40–450 atomic mass units. The oven temperature was initially held at 70 °C for 2 min, and then programmed 70–280 °C at 10 °C/min where it was held constant for 5 min. Helium was used as carrier gas injected at a constant flow rate of 1 mL/min. The total run time was 40 min. The injection volume was 1 mL. The solvent delay was 2 min and injected in a split ratio of 1:10. Peak analysis was performed by NIST library.

Molecular docking

The three-dimensional crystal structure of bacterial quorum-sensing transcription activator proteins, the DNA-binding response regulator 1NXO (OmpR subfamily, Streptococcus pneumonia) and 3AIC (glycosyltransferase S. mutans) obtained from protein data bank were used for molecular docking. AutoDock 4.0 was used for performing docking simulation. Kollman united atom charges and polar hydrogens were added to the protein PDB using Autodock tools. All rotatable bonds in the ligands were kept as free to allow for flexible docking (Sohaibani & Murugan, 2012). Best conformers were searched using Lamarckian genetic algorithm and 10 independent docking runs were carried out for each ligand. Molinspiration (Ertl et al., 2000) was used to calculate the molecular descriptors and Lipinski’s rule was used to calculate the number of violations for all analyzed ligands (Ertl, 2012).

Result

All the plaque samples collected from human subjects were found to be culture positive and Streptococcus sp. (n = 57) was recorded as the main cariogenic isolate. The frequencies of other suspected caries causatives include Staphylococcus sp. (n = 21) and others (n = 22). Among them, common causative agents implicated in dental caries, S. mutans were selectively isolated and found to have varying biofilm forming ability. Among them, S. mutans KMS 07 recorded higher microtiter biofilm activity and hence were selected for further antibiofilm activity studies.

The results obtained for the antimicrobial activity of A. aspera on biofilm-forming cariogenic S. mutans KMS 07 (Table 1) determined by the agar-well diffusion method revealed that all the four extracts of A. aspera have significant anti-cariogenic growth and biofilm inhibiting activity. The MIC values of the A. aspera extracts ranged from 62.5 to 500 µg/mL; the methanol extract showed minimum MIC value against the cariogenic isolate S. mutans (125 µg/mL), whereas the MIC values of the antibiotics chlorhexidine and triclosan are 2 and 6.25 µg/mL, respectively. The susceptibility profile of these cariogenic isolates to different extracts was observed to vary significantly with respect to the presence of more compounds and their differential extraction. Among the solvents tested, methanol exhibited higher activity indicating potential for extracting the bioactive compounds from anticariogenic A. aspera.

Table 1. The growth and biofilm inhibitory activity of A. aspera extracts on cariogenic isolate S. mutans.

		Growth and biofilm inhibitory activity of the extracts
	Nature of	Growth inhibition	Biofilm inhibition activity
S. No	the extract	(zone of inhibition diameter, mm)	Mean OD	SD	CV	Percentage of reduction
1	Control	–	1.739	–	–	–
2	Aqueous	13 ± 0	1.041	0.09	8.64	42.3
3	Benzene	18 ± 3	0.518	0.44	84.9	74.1
4	Petroleum ether	16 ± 2	0.711	0.048	6.74	62.3
5	Methanol	23 ± 2	0.175	0.077	44.1	94.9

OD: optical density; SD: standard deviation; CV: co-efficient of variation.

The modified original gradient-plate method performed using CRA indicated that A. aspera methanol extracts have a strong growth inhibitory selective action on the cariogenic isolate biofilm formation. The growth of the test cariogenic isolates produced typical black color colonies indicative of biofilm formation in the plant extract less concentrated areas. Whereas the colonies became pinkish red, Bordeaux, white and smooth along with the increasing concentration gradient of the plant extract indicating the concentration-dependent inhibition and interference on the exopolysaccharide synthesis and biofilm formation.

Gas chromatography–mass spectrometry (GC–MS) analyses of the methanol extracts of A. aspera resulted in the identification of 61 components (Figure 1). The relative percentage of identified and isolated phytochemicals, their 2D chemical structure and their contributory important functional groups revealed more bioactive compounds and validated the use of A. aspera as a natural toothbrush. Among the phytochemicals, 1,2-dimethyl piperazine, 4-hydroxy-2,5-dimethylfuran-3-one, 3-acetylthymine, N-methyl-N-nitroso-2-propanamine, (2R,3S,4S)-2,3,4,5-tetrahydroxypentanal, 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one, 5-(hydroxymethyl)furan-2-carbaldehyde, 1-methoxy-3,5-dimethylbenzene, 1-(3-hydroxy-4-methoxyphenyl)ethanone, 2-[2-[(E)-2-[4-(dimethylamino)phenyl]ethenyl]-6-phenylpyran-4-ylidene] propanedinitrile and tetratetracontane were the major compounds. Most were phenolic, and these compounds proved to have varied biological activities. The present study confirmed the potentially rich antibacterial agents in A. aspera and their activity against cariogenic organisms.

Figure 1. GC–MS chromatogram of anticariogenic methanol extract of A. aspera showing the compounds and their percentage of abundance (within parentheses). (1) 3-hydroxyoxolan-2-one (0.19); (2) oxane-2,6-dione (0.91); (3) 1,4-dimethylpiperazine (1.82); (4) methylglyoxal (0.98); (5) 4-hydroxy-2,5-dimethylfuran-3-one (1.39); (6) 4-methoxyphenol (0.32); (7) N-(3-methylbutyl)-N-pentylnitrous amide (0.35); (8) 3,3-diethyl-1-methyldiaziridine (0.28); (9) N-methyl-N-propan-2-ylnitrous amide (1.15); (10) 2,3,4,5-tetrahydroxypentanal (1.20); (11) 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (9.80); (12) 4-hydroxyoxolan-2-one (0.48); (13) 3,5-dihydroxy-2-methylpyran-4-one (0.21); (14) benzene-1,2-diol (0.11); (15) 1-(furan-2-yl)ethane-1,2-diol (0.78); (16) 5-(hydroxymethyl)furan-2-carbaldehyde (60.33); (17) nonanoic acid (0.18); (18) 4-ethenyl-2-methoxyphenol (1.1); (19) hexyl 3-oxobutanoate (0.11); (20) 2,6-dimethoxyphenol (0.05); (21) 3-hydroxy-4-methoxybenzaldehyde (0.02); (22) (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl) oxolan-2-yl] oxy-6-(hydroxymethyl) oxane-3,4,5-triol (0.02); (23) 2-(hydroxymethyl)-2-nitropropane-1,3-diol (0.03); (24) 2-hydroxy-5-methylbenzene-1,3-dicarbaldehyde (0.01); (25) 1-methoxy-3,5-dimethylbenzene (0.12); (26) (1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol (0.12); (27) 1-(3,5-dimethoxyphenyl)ethanone (0.10); (28) oxan-4-ol (0.02); (29) 5,5-diethyl-1-methyl-1,3-diazinane-2,4,6-trione (0.11); (30) 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone (0.03); (31) 4-[(E)-3-hydroxyprop-1-enyl]-2-methoxyphenol (0.05); (32) tetradecanoic acid (0.01); (33) 1-(2,6-dihydroxy-4-methoxyphenyl)butan-1-one (0.01); (34) 3-methyl-1-(4-phenylbenzoyl)-2-propan-2-ylimidazolidin-4-one (0.07); (35) 1-ethyl-2,3,4,5,6-pentamethylbenzene (0.03); (36) ethyl hexadecanoate (0.03); (37) (E)-3-(4-hydroxy-3,5-dimethoxyphenyl) prop-2-enal (0.01); (38) 2-hydroxy-4-methoxy-3,5,6-trimethylbenzoic acid (0.03); (39) henicosan-11-ylbenzene (0.04); (40) 2,7-diphenyl-1H-indole (0.09); (41) 2-[2-[(E)-2-[4-(dimethylamino)phenyl]ethenyl]-6-phenylpyran-4-ylidene]propanedinitrile (0.10); (42) (3S,10S,13R,14R,17R)-17-[(2R,5S)-5,6-dimethylheptan-2-yl]-10,13-dimethyl-2,3,4,7,11,12,14,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3-ol (0.03); (43) 4,7-dimethoxy-1,1,4a-trimethyl-8-propan-2-yl-10,10a-dihydro-9H-phenanthren-2-one (0.12); (44) 1-chloroicosane (0.08); (45) 3-(4-chlorophenyl)-1,2,3,4-tetrahydropyrido[1,2-a]benzimidazole-1,2-dicarboxylic acid (0.06); (46) (E)-3,5-bis(4-hydroxyphenyl)pent-4-ene-1,2-diol (0.04); (47) tetratetracontane (0.02); (48) 2,6-ditert-butyl-4-diazoniophenolate (0.04); (49) (2R,3R,11bS)-2-[(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)methyl]-3-ethyl-9,10-dimethoxy-2,3,4,6,7,11b-hexahydro-1H-benzo[a]quinolizine (0.04); (50) 9-dodecyl-1,2,3,4,4a,5,6,7,8,8a,9,9a,10,10a-tetradecahydroanthracene (0.02); (51) 2-prop-1-en-2-ylpyrazine (0.05); (52) 7-amino-4-chloro-3-(cyclohexylmethoxy)isochromen-1-one (0.06); (53) (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(hydroxymethyl)-5a,5b,8,8,11a-pentamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysen-9-ol (0.05); (54) hexacosane (0.05); (55) 4,4,6a,6b,8a,11,11,14b-octamethyl-1,2,4a,5,6,6a,7,8,9,10,12,12a,14,14a-tetradecahydropicene-3,13-dione (0.05); (56) (4,5-dimethyl-7a-prop-1-en-2-yl-2,3,3a,5,6,7-hexahydro-1H-inden-4-yl)methanol (0.04).

A number of A. aspera phytochemical natural ligands showed the presence of competitive ligands with higher or nearest binding score like that of the natural ligand 3-oxo-C12-HSL and hence they may play a role in QQ, interference on the language of QS (Table 2). The widely used toothpaste anticariogenic agent 5-chloro-2-(2,4-dichlorophenoxy) phenol (triclosan) showed the higher binding energy of −6.42 but it showed violations. Whereas, the antibiotic chlorhexidine showed to bind only with 1NXO having binding energy −3.2. The A. aspera bioactive compound 3,12-oleandione exhibited binding energy of −5.92 without any violations when interacts with S. mutans OmpR subfamily QS regulatory DNA-binding response regulator (PDB; 1NXO). Likewise, A. aspera phytochemicals (3S,10S,13R,14R,17R)-17-[(2R,5S)-5,6-dimethylheptan-2-yl]-10,13-dimethyl-2,3,4,7,11,12,14,15,16,17-decahydro-1H-cyclopenta[a] phenanthren-3-ol natural ligands showed binding energy more than −4.63 when interacts with the same. The compound betulin also showed highest binding energy of −8.72 when interacting with the structure of biofilm exopolymer synthesizing enzyme S. mutans glycosyltransferase (GTF-SI). The bioactive compounds like 1-(4-hydroxy-3,5-dimethoxyphenyl)ethenone, 1-(3,5-dimethoxyphenyl)ethenone, 2,6-dimethoxyphenol, 1-methoxy-3,5-dimethylbenzene as well as 4-methoxyphenol showed higher binding energy with both target proteins (Figure 2). It also revealed the QQ ability of the A. aspera phytochemicals in reducing cariogenic virulence. Hence, the obtained results indicated that the A. aspera has number of bioactive compounds interacting efficiently with QS response regulators and biofilm synthesis mediating the enzyme of cariogenic Streptococcus spp. that may take part in QQ preventing these organisms from expressing cariogenic virulence.

Figure 2. Streptococcus mutans glucansucrase (PDB:3AIC) superimposed structure with A. aspera natural ligand phytochemicals Betulin (2a); (2R,3R,11bS)-2-[(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)methyl]-3-ethyl-9,10-dimethoxy-2,3,4,6,7, 11b-hexahydro-1H-benzo[a]quinolizine (2b); 1,4-dimethylpiperazine (2c); 2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol (2d); 2,3,4,5-tetrahydroxypentanal (2e); 3,5-dihydroxy-2-methylpyran-4-one (2f); 2-hydroxy-5-methylbenzene-1,3-dicarbaldehyde (2g) as well as 1-(4-hydroxy-3,5-dimethoxyphenyl)ethenone (2h). Molecular docking superimposed structures of Streptococcus OmpR subfamily response regulator (1NXO) with A. aspera phytochemical natural ligands (3S,10S,13R,14R,17R)-17-[(2R,5S)-5,6-dimethylheptan-2-yl]-10,13-dimethyl-2,3,4,7,11,12,14,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3-ol (3a); 3-(4-chlorophenyl)-1,2,3,4-tetrahydropyrido[1,2-a]benzimidazole-1,2-dicarboxylic acid (3b); 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (3c); 1-methoxy-3,5-dimethylbenzene (3d); 4,7-dimethoxy-1,1,4a-trimethyl-8-propan-2-yl-10,10a-dihydro-9H-phenanthren-2-one (3e); 5-(hydroxymethyl)furan-2-carbaldehyde (3f); 3-methyl-1-(4-phenylbenzoyl)-2-propan-2-ylimidazolidin-4-one (3g); and 4-hydroxy-2,5-dimethylfuran-3-one (3h).

Table 2. Achyranthes aspera phytochemical natural ligands as well as the antibiotic triclosan docking scores with the active site of S. mutans and S. aureus QS regulators and their predicted bioactivity.

		Docking energy
Compound name	Pub Chem CID	3AIC	1NXO	miLogP	TPSA	N atoms	MW	N violations	N rotb	GPCR	Ion channel modulators	Kinase inhibitors	Nuclear receptor ligand	Protease inhibitors	Enzyme inhibitors
Betulin(1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(hydroxymethyl)-5a,5b,8,8,11a-pentamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysen-9-ol	72326	−8.72	−4.7	7.164	40.456	32	442.728	1	2	0.21	−0.04	−0.41	0.85	0.09	0.51
(2R,3R,11bS)-2-[(6,7-Dimethoxy-3,4-dihydroisoquinolin-1-yl)methyl]-3-ethyl-9,10-dimethoxy-2,3,4,6,7,11b-hexahydro-1H-benzo[a]quinolizine	65033	−8.39	−3.73	4.708	52.538	35	478.633	0	7	0.1	0.02	−0.47	−0.22	0.06	−0.1
1,4-dimethylpiperazine	7818	−6.9	−3.06	0.234	6.476	8	114.192	0	0	−2.85	−2.4	−2.93	−3.44	−3.12	−2.77
(2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-Dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol	5988	−6.81	2.5	−3.745	189.526	23	342.297	2	5	0.45	0.16	0.38	−0.12	0.14	0.71
2,3,4,5-Tetrahydroxypentanal	854	−5.45	0.17	−2.216	97.983	10	150.13	0	4	−1.06	−0.35	−1.35	−1.17	−0.71	−0.01
3,5-Dihydroxy-2-methylpyran-4-one	70627	−5.07	−2.15	0.15	70.667	10	142.11	0	0	−2.03	−1.54	−1.87	−1.97	−1.71	−0.59
2-Hydroxy-5-methylbenzene-1,3-dicarbaldehyde	81744	−5	−2.43	1.603	54.37	12	164.16	0	2	−1.05	−0.48	−1	−0.8	−1.28	−0.54
1-(4-Hydroxy-3,5-dimethoxyphenyl)ethanone	17198	−4.88	−2.29	1.191	55.767	14	196.202	0	3	−0.79	−0.37	−0.88	−0.8	−0.98	−0.35
3,5-Dihydroxy-6-methyl-2,3-dihydropyran-4-one	119838	−4.82	−4.5	−0.457	66.761	10	144.126	0	0	−1.59	−0.96	−2.25	−1.6	−1.53	−0.65
4-Ethenyl-2-methoxyphenol	332	−4.79	−2.77	2.128	29.462	11	150.177	0	2	−0.96	−0.28	−1	−0.77	−1.35	−0.46
4-Hydroxyoxolan-2-one	95652	−4.77	−2.51	−1.486	46.533	7	102.089	0	0	−3.17	−2.98	−3.2	−3.23	−2.68	−2.76
5,5-Diethyl-1-methyl-1,3-diazinane-2,4,6-trione	4099	−4.73	−2.95	1.206	66.478	14	198.222	0	2	−0.57	−0.58	−1.1	−1.09	−0.28	−0.35
1-(3,5-Dimethoxyphenyl)ethanone	95997	−4.72	−3.11	1.877	35.539	13	180.203	0	3	−0.93	−0.52	−1.12	−0.94	−1.13	−0.57
4-Hydroxy-3,5-dimethoxybenzaldehyde	8655	−4.59	−2.35	1.083	55.767	13	182.175	0	3	−0.95	−0.36	−0.8	−0.69	−1.27	−0.39
Benzene-1,2-diol	289	−4.5	−2.85	0.993	40.456	8	110.112	0	0	−3.04	−2.51	−3.1	−2.99	−3.24	−2.68
4-Methoxyphenol	9015	−4.48	−0.96	−3.272	167.901	16	260.135	1	2	0.56	1.11	0.5	0.22	0.37	1.1
3-Hydroxy-4-methoxybenzaldehyde	12127	−4.47	−2.56	1.067	46.533	11	152.149	0	2	−1.2	−0.54	−1.13	−0.91	−1.65	−0.64
2,6-Dimethoxyphenol	7041	−4.35	−2.14	1.341	38.696	11	154.165	0	2	−1.04	−0.45	−0.97	−1.1	−1.25	−0.48
5-(Hydroxymethyl)furan-2-carbaldehyde	237332	−4.33	−4.33	0.561	50.439	9	126.111	0	2	−2.71	−2.19	−2.92	−2.73	−3.21	−2.24
N-Methyl-N-propan-2-ylnitrous amide	92271	−4.21	−3.94	0.815	32.673	7	102.137	0	2	−4.03	−3.85	−4.25	−4.66	−3.92	−3.14
Oxan-4-ol	74956	−4.19	−2.45	0.055	29.462	7	102.133	0	0	−3.22	−3.06	−3.34	−2.98	−3.01	−2.82
4-Hydroxy-2,5-dimethylfuran-3-one	19309	−4.19	−4.08	0.551	46.533	9	128.127	0	0	−2.73	−1.8	−3.43	−2.57	−2.78	−1.61
2-Prop-1-en-2-ylpyrazine	62897	−4.11	−2.97	1.202	25.784	9	120.155	0	1	−1.97	−1.08	−2.29	−1.9	−2.17	−1.25
4-Methoxyphenol	9015	−4.04	−2.68	1.515	29.462	9	124.139	0	1	−2.24	−1.57	−2.31	−2.15	−2.51	−1.73
4,4,6a,6b,8a,11,11,14b-Octamethyl-1,2,4a,5,6,6a,7,8,9,10,12,12a,14,14a-tetradecahydropicene-3,13-dione	565780	−3.18	−5.9	6.921	34.142	32	440.712	1	0	0.01	−0.03	−0.58	0.39	−0.11	0.33
(3S,10S,13R,14R,17R)-17-[(2R,5S)-5,6-Dimethylheptan-2-yl]-10,13-dimethyl-2,3,4,7,11,12,14,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3-ol	22296544	−1.24	−4.63	8.011	20.228	29	398.675	1	5	0.13	−0.01	−0.38	0.85	0.02	0.63
3-(4-Chlorophenyl)-1,2,3,4-tetrahydropyrido[1,2-a]benzimidazole-1,2-dicarboxylic acid	620130	−1.12	−4.58	3.238	92.424	26	370.792	0	3	0.14	−0.13	−0.32	−0.1	−0.03	0.01
1-Methoxy-3,5-dimethylbenzene	70126	−3.69	−4.44	2.819	9.234	10	136.194	0	1	−1.29	−0.8	−1.39	−1.34	−1.53	−0.9
4,7-Dimethoxy-1,1,4a-trimethyl-8-propan-2-yl-10,10a-dihydro-9H-phenanthren-2-one	621440	−2.31	−4.37	4.119	35.539	25	342.479	0	3	0.13	−0.13	−0.43	0.21	−0.15	0.23
2,7-Diphenyl-1H-indole	622897	−2.12	−4.35	5.58	15.791	21	269.347	1	2	0.39	0.15	0.66	0.49	0.07	0.36
3-Methyl-1-(4-phenylbenzoyl)-2-propan-2-ylimidazolidin-4-one	602378	−1.8	−4.17	3.008	40.618	24	322.408	0	3	0.12	0.02	−0.13	−0.36	0.13	0.07
7-Amino-4-chloro-3-(cyclohexylmethoxy)isochromen-1-one	611189	−1.44	−3.93	3.945	65.468	21	307.777	0	3	0.01	−0.08	0.14	−0.04	0.66	0.28
Oxane-2,6-dione	9552079	–	−3.21	0.344	43.376	8	114.1	0	0	−3.04	−2.61	−3.42	−2.87	−2.71	−2.48
(4,5-Dimethyl-7a-prop-1-en-2-yl-2,3,3a,5,6,7-hexahydro-1H-inden-4-yl)methanol	605599	−1.67	−3.92	3.984	20.228	16	222.372	0	2	−0.07	0.07	−0.7	0.28	−0.36	0.29
5-Chloro-2-(2,4-dichlorophenoxy)phenol	5564	−4.69	−6.42	5.133	29.462	17	289.545	1	2	−0.18	−0.18	−0.14	−0.07	−0.43	−0.01

The conducted computational study molinspiration revealing the bioavailability properties, absorption, distribution, metabolism and excretion of the natural A. aspera phytochemicals is presented (Table 2). The obtained values for cell permeability parameters, calculated measure of molecular hydrophobicity (miLogP) and topological polar surface areas (TPSA) given in Table 2 suggest that a number of Andrographis paniculata bioactive compounds should be able to penetrate cell membranes. It was also found that a number of A. aspera bioactive compounds with QS response regulator high binding energy values adhered fully to Lipinski’s rule of five, and hence many compounds should show good absorption or permeability properties through the intestine or biological membrane. The high drug-like molecule activity scores were obtained for GPCR ligands, kinase inhibitors, ion channel modulators, nuclear receptor ligands, protease inhibitors and other enzyme targets (Table 2). Hence, A. aspera bioactive compounds recorded acceptable scores for the calculated molinspiration and Lipinski’s rule violations (Table 2) indicating their lead QSI plant mimics for specifically targeting the biofilm-mediated caries and other infections.

Discussion

Dental caries is an infectious communicable widespread disease all over the world and WHO deem it as a major public health problem (Brighenti et al., 2008). Although dental caries harbors a complex micro-flora, the role of organisms like Streptococcus mitis, Streptococcus sanguis, S. mutans (Sohaibani & Murugan, 2012) and Streptococcus aureus (Kouidhi et al., 2010) is well recognized. They are, however, preventable if the growth and the level of oral cavity cariogenic micro-organisms are reduced using effective practices. Among the several approaches used for biofilm control, the use of anti-biofilm agents having dual functionalities like reducing the cariogenic bacteria viability and controlling their colonization on tooth surface could be more effective (Song et al., 2007). As expected, bacteria in biofilms are encased in a polysaccharide glycocalyx, which provides them protection against host defenses and antimicrobial drugs (Kouidhi et al., 2010); the cariogenic isolates of this study also possess the same. The ability to synthesize virulence-associated biofilm exopolysaccharide was used as a target for the present approach toward the identification of novel drug lead employing the plant phytochemical QQ activity.

From the MIC values obtained, it is evidenced that the anticariogenic activity of the A. aspera extracts are not as potent as that of the antibiotic triclosan, which might be due to their crude and diluted nature. The presences of various bioactive compounds in varying proportions substantiate their extensive regional and international ethanomedical applications. Earlier pharmacological studies on the obtained phytochemical constituents like 3-hydroxy-4-methoxy-acetophenone (Sun et al., 1993) and folkloric tradition also supports some medicobotanical claims of this ethno-medicinal herb. The results obtained in this study revealed their antibacterial activity against the cariogenic isolates for the first time. As the medicinal plant’s traditional usage played a vital role for current plant-based drug and drug lead discovery, the ethanomedicinal herb A. aspera may offer more targeted cariogenic preventive antimicrobials. As Hebbar et al. (2004) suggested, there is scope for screening active compounds having specific effects on oral diseases from this chewing stick plant. Number of traditional medicinal plants having antibacterial activities were shown to have “QS mimics” capable of controlling bacterial QS (Sohaibani & Murugan, 2012; Vattem et al., 2007), an important therapeutic drug target for biofilm-associated infections. Although extracts of this “chewing stick” plant exhibited clear QS inhibitory effect, as the herbal medicine proponents believe, this plant’s enhanced antibiofilm efficacy might be due to the multitude of phytochemicals and the synergistic interactions among them. Since the GC–MS analysis also revealed a mixture of bioactive compounds, the molecular docking and the structure-based virtual screening could contribute to further understanding on their mechanism of action.

Molecular interaction between the bioactive compounds of A. aspera and cariogenic organism’s virulence biofilm QS regulatory proteins and synthesis-mediating glycosyltransferase revealed a possible mechanism of biofilm inhibition through both QQ and enzymatic pathway interference. A number of A. aspera phytochemical natural ligands has shown comparable docking score with low binding energy like that of the natural ligand 3-oxo-C12-HSL and hence they may play a role in QQ, interference on the language of QS. Effective interaction with glucosyltransferase, the key contributor of dental caries associated biofilm formation; structure shown by a number of bioactive compounds widens the possibility of enzymatic inhibition of biofilm synthesis. However, the obtained results validate the traditional use of the plant and give more insight into the effect of its extract on the caries causative S. mutans (Brighenti et al., 2008). As large numbers of bacterial pathogenicity systems are QS controlled ones, their interruption can render them non-virulent, and hence QS interruption is one example of an antipathogenic effect (Adonizio et al., 2006). The in silico studies also the revealed the unsuitability of the known antibiotic triclosan and the principal phytochemical extractive betulin having pharmacological, antiviral activities (Sami et al., 2006) and QS inhibitory activity for internal applications. However, interaction of other A. aspera phytochemicals with QS response regulators makes them potential lead compounds for interfering with caries biofilm formation. The bioactive anti-biofilm agents of plants like this with dual functionalities of both growth inhibition and QS regulator interaction may offer a novel strategy to reduce the development of dental caries by inhibiting the initial adhesion and subsequent biofilm formation (Sohaibani & Murugan, 2012). Hence, the obtained result suggests that A. aspera bioactive compounds natural ligands perhaps deemed the potential leads and further structural modifications might lead to the derivation of prospective QSI mimics.

Conclusion

The obtained results scientifically validates the traditional use of A. aspera as a natural brush for teeth cleaning. Based on the results obtained, it could be concluded that phytochemicals of this traditionally used dental caries preventive natural chewing stick plant could be harnessed for dental caries and other biofilm-mediated disease management. It also leads to exploration of the plant's phytochemical repertoire as a biofilm controlling QSI drug lead.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgements

This project was supported by the Research Center, College of Science, Deanship of Scientific research, King Saud University.