Antibacterial activity of silver nanoparticles-Clinacanthus nutans (AgNP-CN) against Streptococcus mutans

Clinacanthus nutans was found to possess anti-venom, anti-in lammatory, analgesic, antidiabetic, anti-rheumatism, antiviral and antioxidant properties. Silver nanoparticles are nanoparticles between1nm to100nm in size and play signi icant role in medicinal ields. Silver nanoparticles exhibit unique properties, such as excellent conductivity, chemical stability, catalytic and antimicrobial activity. Streptococcus mutans is usually discovered in human oral cavity and the main aetiological of tooth decay. There is no study on antibacterial effect of silver nanoparticles Clinacanthus nutans (AgNP-CN) against Streptococcus mutans reported to date. Therefore, objective of this study is to investigate antibacterial properties of silver nanoparticles Clinacanthus nutans against Streptococcus mutans. Streptococcus mutans was subcultured in brain heart infusion (BHI) broth and agar. AgNP-CN with different concentrations was tested against Streptococcus mutans via disc diffusion assay, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Amoxicillin was used as positive control while DMSO and blank silver nanoparticles were used as negative control. Disc diffusion assay shows AgNP-CN inhibit Streptococcus mutans growth. AgNP-CN shows the greatest inhibition properties (15.3±1.53 mm) in comparison to Clinacanthus nutans leaves extract (6.0±0.01 mm) and blank silver nanoparticles (6.0±0.01mm). MIC value forAgNP-CN is 2.5±0.01mg/mLwhile amoxicillin is 0.007±0.01mg/mL.Whereas MBC values for AgNP-CN is 2.5 mg/mL. Results are concentration dependent, with higher concentration shows better inhibition property.


INTRODUCTION
World Health Organization (WHO) estimated almost half of the world's population is affected by dental caries (WHO, 2017). Dental caries is a multifactorial disease that results from four essential factors which are tooth surfaces, substrate, microorganism on bio ilm and time (Kumar et al., 2015). Microorganisms are one of the main agents responsible to cause dental caries. Countless facultative as well as obligate anaerobic bacteria overshadow the microbiological population of dental caries. How-ever, the greatest crucial aetiological representative of dental caries is Streptococcus mutans (Lemos et al., 2013). Tooth decays occur when a susceptible tooth surface is occupied by cariogenic microbes and nutritional resource of sucrose or processed sugar. Fermentation of sugar will lead to fabrication of lactic acid by the action of bacteria which dissolves the hydroxyapatite crystal formation of the tooth which foundations caries. Streptococcus mutans is one of Gram-positive cocci bacteria and the most common microorganism that caused dental caries (Hana iah et al., 2015). Cavitation produced by Streptococcus mutans may provide an ecological niche where the microorganisms form a protected bio ilm, and enabling caries progression (Rouabhia, 2012). Streptococcus mutans can colonize the oral cavity at any age if the environment is favorable and the amount may increase with age (Köhler and Andréen, 2012).
Silver in colloidal state reveals unique properties such as good conductivity, organic stability, catalytic and antibacterial activity (Ramajo et al., 2009). Silver nanoparticles have tremendous applications in the ield of antimicrobials and therapeutics (Rai et al., 2012). Biosynthetic methods to synthesize AgNP utilizing either one of biological microorganisms or plant extracts have become apparent as a straightforward and sustainable substitute to chemical and physical methods (Ahmed et al., 2016). For example, the use of plant extracts such as Garcinia mangostana and Coptis sinensis in synthesizing AgNP have shown excellent antibacterial activities against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (Veerasamy et al., 2011;Ahmad et al., 2017). Recent study shows silver nanoparticles-Clinacanthus nutans (AgNP-CN) possess anticancer properties against oral carcinoma cell lines (HSC-4) (Yakop et al., 2018).
Clinacanthus nutans (CN) is a small shrub belongs to the family of Acanthaceae, which is well-known to the South East Asian countries primarily Malaysia, Thailand and Indonesia (Zulkipli et al., 2017). Clinacanthus nutans was found to have anti venom, anti-in lammatory, analgesic, anti-diabetic, antirheumatism, anti-viral and antioxidant properties (Arullappan et al., 2014). The lavonoids and phenolic complexes that are produced generally in therapeutic plants can stimulate antibacterial response owing to the existence of carbonyl group (Alam et al., 2016). Methanolic extract of C. nutans leaves possessed antibacterial effect against Staphylococcus aureus, Escherichia coli, Propionibacterium acnes, Staphylococcus epidermidis and Bacillus cereus (Yang et al., 2013). Recent study showed C. nutans chloroform and ethano-lic extracts possessed antibacterial activity against anaerobic periodontal bacteria which were Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitants (Hana iah et al., 2019). Considering vast potentiality of plants as reductants and capping agents in synthesis of silver nanoparticle and medicinal values of C. nutans, this study will be focused on antibacterial activity of AgNP-CN against S. mutans.

Preparation of C. nutans water extract
C. nutans leaves were washed, separated and ovendried at 45C. The leaves were ground into ine powder and drenched into puri ied water at room temperature. Water extracts were accumulated in clean glass bottles and iltered using Whatman no. 1 paper then freeze dried (Hana iah et al., 2019). C. nutans water extract was kept at -20C until further use.

Green synthesis of silver nanoparticle
For production of silver nanoparticle, 1 mM of silver nitrate were combined with C. nutans leaves water isolate and incubated in room temperature until changes of colour to reddish brown pigment which signi ied the development of AgNP-CN (Yakop et al., 2018).

Bacteria Culture
Streptococcus mutans (ATCC 25175 strain) obtained from Microbiology laboratory, Faculty of Dentistry, Universiti Sains Islam Malaysia and subcultured in Brain Heart Infusion (BHI) broth.

Disk diffusion assay
Bacteria were cultured on brain heart infusion (BHI) agar and incubated under 37 • C at anaerobic environment for 24 h. Approximately 1 X 10 8 CFU/mL of bacteria intensities equivalent towards 0.5 McFarland turbidity guideline was employed to inoculate S. mutans into fresh BHI broth then incubated at 37 • C overnight. A sterilized cotton swab was used to disseminate the culture on BHI agar before application of 5 x 6mm paper discs (Whatmann No. 1) infused with 10 µL tested materials (AgNP-CN, AgNP blank (Sigma) and CN) at concentration between 5-40 mg/mL. DMSO 10% was used as negative control and amoxicillin (30U) as positive control. Inhibition zones were measured from the circumference of disks to the circumference of inhibition zone after incubation under 37 • C for 24 h at anaerobic state.    The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was done using two-fold sequential dilution approach as portrayed by (Hana iah et al., 2019). MIC was completed in a sterile 96-well plate. S. mutans (100 µL) at 10 8 CFU/mL was added to several concentrations of AgNP-CN (0.08-40 mg/mL) diluted in fresh BHI broth to a inishing volume of 200 µL/well. DMSO 10% (v/v) was used as negative control and amoxicillin (0.008-0.128 mg/mL) was used as positive control (Ready et al., 2004). Following incubation at 37 • C under anaerobic condition for 24 h, MIC value was de ined as the lowest concentration that impedes the observable growth of bacteria. Minimum bactericidal concentration (MBC) was established from culturing a 5 µL aliquot from well that demonstrated no microbial growth in MIC wells onto sterile BHI agar and incubated overnight at 37 • C and the MBC was demarcated as the lowest concentration stopping microbial growth.

Statistical Analysis
The data was collected and analyzed using twoway ANOVA test where the p value is less than 0.05 (p<0.05).

RESULTS AND DISCUSSION
This study was done to evaluate antibacterial activity of silver nanoparticle-Clinacanthus nutans (AgNP-CN) against S. mutans. AgNP-CN was synthesized via green synthesis method whereby Clinacanthus nutans leaves water extract was used as capping and reducing agents (Yakop et al., 2018). Silver nanoparticles have been known as new emerging antimicrobial agents in the ield of nanomedicine (Awwad et al., 2020). Green synthesis method provides more economical and environment-friendly end products. Recent study shows AgNP-CN was not cytotoxic towards normal cell lines (3T3-L1 cell lines) (Yakop et al., 2018). With green synthesis method, silver nanoparticles produced were reported to exhibit better antimicrobial activities compared to physical and chemical methods (Srikar et al., 2016). This corresponds with present study whereby AgNP-CN shows more antibacterial activity as compared to AgNP blank and CN water extract. AgNP-CN shows antibacterial activity in concentration dependent manner. Disc diffusion assay is the most common technique used to determine antimicrobial resistance worldwide.
It is because this test did not require any special equipment to execute. A vibrant rounded zone of no growth in the immediate part of a disc speci ies susceptibility to that antimicrobial (Sandle, 2016). As shown in disc diffusion assay result (Table 1 ) and Figure 1), with higher concentration, more pronounced inhibition zones were observed.

Figure 3: MBC plate inoculate from MIC well (2.5 mg/mL). No growth of bacteria noted. Thus, MBC= 2.5mg/mL
Determination of MIC is important in research and laboratory diagnostics. It is used to ind new alternative drugs and to con irm microbial resistance such as S. mutans from existing antibiotics. As for personalized medicine, clinicians use MIC value to choose which antibiotics with speci ic dose to administer to patient for certain infections (Hana iah et al., 2019). Study done by Roeslan et al., reported that C. nutans from hexane fraction did not show any MIC value but able to reduce bio ilm formation of S. mutans (Roeslan et al., 2019). This study showed that C. nutans could reduce bio ilm formation but did not kill S. mutans. As proposed by green synthesis method, synthesis of AgNP using plants extract especially C. nutans will enhance its antibacterial activity. This supported present study whereby the MIC value is 2.5 mg/mL (Table 2 ) and (Figure 2). This proved that C. nutans able to enhance AgNP-CN activity against S. mutans. This event could be explained with AgNP is reduced and stabilized by bioactive compounds such as polyphenol, lavonoids, terpenoids, alkaloids in CN water extracts that reported to possess antibacterial activity (Tanase et al., 2020). In this study, AgNP-CN MBC value (2.5 mg/mL) (Figure 3) was less than four times of MIC value (2.5 mg/mL). Meanwhile MIC and MBC values of amoxicillin were 0.007 mg/mL and 0.020 mg/mL, respectively. With the same value of MIC and MBC AgNP-CN is regarded as bactericidal agent against S. mutans.

CONCLUSIONS
AgNP-CN shows potential in inhibiting growth of S. mutans as compared to the CN leaves extract and AgNP blank. The antibacterial effect of AgNP-CN against S. mutans is concentration dependent whereby higher concentration gives larger inhibition zones. AgNP-CN has bactericidal properties against S. mutans.

Con lict of Interest
The authors declare that they have no con lict of interest for this study.