Antibacterial activity of Acmella paniculata extracts against Streptococcus mutans

Acmella paniculata, also known as ’Subang nenek’ in Malaysia, has been used to treat diseases such as toothache and gum infections. People called it a toothache plant, and it has been widely used as traditional medicine. Therefore, this study aims to investigate the antibacterial activities of A. paniculata leaves and lowers extracts towards Streptococcus mutans by using disc diffusion assay, minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) methods. Besides, the anti-bio ilm activity of all the extracts also been determined by using a crystal violet assay. As the results, n-hexane and methanol extracts from leaves showed the highest inhibition zone towards S. mutans when compared to DCM and acetone extracts. Meanwhile, for the lowers extract, n-hexane and DCM showed the highest inhibition zone towards S. mutans compared to methanol and acetone extracts. The best results were then tested for MIC and MBC tests. As for the MIC values of n-hexane and methanol leaves extracts were 25 mg/mL, respectively, and the MBC values were 50 and 100 mg/mL, respectively. Whereas MIC values for n-hexane and DCM lowers extracts were 12.5 mg/mL, respectively, and the MBC values were 50 mg/mL, respectively. Bio ilm formation of S. mutans showed decrement up to 70% after exposure to both leaves extract (n-hexane and methanol) and n-hexane lower extract. Still, it differed when exposing to DCM lower extract, and the result showed that the bio ilm activities of S. mutanswere inhibited at 80%after treatedwithDCM lowers extracts. In conclusion, n-hexane leaves extract, methanol leaves extract, n-hexane lowers extract, and DCM lowers extract of A. paniculata demonstrated bactericidal properties against S. mutans.

This situation is increasingly challenging when other commonly used antibiotics and chemotherapeutics such as penicillin, cephalosporin, erythromycin and tetracycline have begun to be less useful to oral bacteria (Chandad and Grenier, 2007). Besides, (Teles and Teles, 2009) stated that prolonged used of antimicrobials agent as an additive to control plaque mechanism caused two major safety issues which were, development of resistant microorganisms and the risk of oral cancer related to the alcohol content in the mouth rinse formulations. Therefore, the researcher needs to ind new alternative drugs which better and more ef icient as compared to the current medication.
Traditional medicine treatment, such as herb is a common practice in many countries at the healthcare level. Many people believe that herbal remedies are safer as it is a natural product from the plant. Plants rich in secondary metabolites, such as tannins, terpenoids, alkaloids, and lavonoids, which have been found in vitro to have antimicrobial properties (Das et al., 2010). Therefore, the demand of bioactive compound from the plant is continuously increasing to resolve multidrug resistance issue. The genus Acmella Rich (Asteraceae) comprises 39 species that could be found in the tropical and subtropical regions.
Acmella paniculata is an annual herb with 32-60 cm high. It has a lot of glandular and hairy stems with marigold eye lowers (Varghese et al., 2014). In Malaysia, Acmella plant is used as a vegetable, and it is generally known as 'Subang nenek' where the lowers and the leaves have a pungent taste when consumed, caused tingling and numbness (Nomura et al., 2013;Ong et al., 2011).
People believe that this plant is useful for toothache, sore throat and gum infections. Traditionally, the lowers are chewed to get its effect, whereas the pounded plant is used for rheumatism (Estari and Gujjeti, 2013). Due to its growing use, it is vital to determine the antimicrobial activities of A. Paniculata extract against oral bacteria. Therefore, this study aims to identify the antibacterial activity of A.

Sample of A. Paniculata
A. paniculata was collected from TKC Herbal Nursery, Seremban (Malaysia). Botanist con irmed the plant, Dr Mohd Firdaus Ismail from Institute of Bioscience, University Putra Malaysia and the voucher specimen was deposited at the Herbarium of Institute of Bioscience, University Putra Malaysia. The voucher specimen number was MFI 0164/20.

Sample preparation
Flower and leaves of A. paniculata were dried at room temperature and pounded to a ine powder (Model C14, Kesmac Sdn. Bhd., Malaysia) then the samples were stored in black containers. Serial extraction was done to the samples (800 g) with 1 L of n-hexane, dichloromethane (DCM), acetone and methanol (System, Shah Alam, Malaysia) (Shai et al., 2008).
After that, the extracts were iltered by using Whatman No. 1 ilter paper and evaporated by using a rotary evaporator (Laborota 4000, Germany). Each extract was diluted in 10% DMSO (System, Shah Alam, Malaysia). S. mutans strain (ATCC 25175) was used in this study. S. mutans were grown on the brain-heart infusion agar (BHIA) and brain-heart infusion broth (BHIB) (Oxoid Ltd., Basingstoke, UK).

Disc Diffusion assay
S. mutans was cultured on BHIA at 37 • C for 24 hours under anaerobic condition. After that, the bacterial culture was optimized to 0.5 McFarland with Muller Hinton broth (MHB). S. mutans were spread on Muller Hinton agar (MHA) using a sterile cotton swab. Disc infused with the lower and leaves extracts (10-30 mg/mL) were placed on top of MHA. Sodium luoride (1 mg/mL NaF) (Sigma-Aldrich, Missouri, USA) and 10% DMSO were acting as a positive and negative control, respectively. Zones of inhibition were measured after incubation at 37 • C for 24 hours under anaerobic condition. All tests were done in triplicate.

Minimum Inhibition Concentration (MIC) and Minimum bactericidal concentration (MBC)
MIC and MBC assay had been performed by followed method from (Ong et al., 2011). MIC was done by using 96 well plates with two-fold serial dilution method. S. mutans (100 µL) at 10 8 CFU/mL was added to various concentrations of extracts (40.00 to 0.04 mg/mL) diluted in MHB to a inal volume of 200 µL/well. DMSO 10% (v/v) was used as a negative control, and NaF (Sigma-Aldrich, Missouri, USA) was used as a positive control (CLSI, 1999). Following incubation at 37 • C under the anaerobic condition for 24 h, the MIC value was determined as the lowest concentration that inhibits the visible growth of bacteria.
Minimum bactericidal concentration (MBC) was established by culturing a ive µL aliquot from wells that exhibited no bacterial growth in MIC onto sterile MHA and incubated overnight at 37 • C. MBC value was de ined as the lowest concentration preventing bacterial growth. The tests were done in triplicate.

Bio ilm assay
Bio ilm was performed by following the method from Mohamad (Hana iah et al., 2015) with modi ication. Bio ilm formation assay was done by crystalviolet assay (Sigma-Aldrich, Missouri, USA) with a 96-well plate. A total of 1 mL S. mutans have been moved to 10 mL BHIB and been incubated at 37 • C for 24 hours. Later, the culture is diluted 1:100 in BHIB and 150 µL of the bacteria been added into 96well plate followed by 50 µL of the extracts (0.24 to 30 mg/mL) in the same well. NaF (1 mg/mL) and 10% DMSO acted as positive and negative control, respectively.
Then, the microplate well was incubated at 37°C for 24 hours. Next, the bio ilm formation was quanti ied by measuring the absorbance of the solutions (bio ilm and crystal violet) at 595 nm using a microplate reader (Model 680, Bio-Rad, California, USA).

Data analysis
All the data obtained had been analyzed by using Statistical Package for the Social Sciences (SPSS). In the disc diffusion assays, the data were analyzed by using two-way ANOVA as presents of two groups of independent variables which are the four different solvents in ive different concentrations. Next, the anti-adherence and anti-bio ilm test were analyzed by one-way ANOVA. Later, post hoc test was performed by using Turkey analysis for both data.

Disc diffusion assay
Disc diffusion assay of leaves and lower extracts are shown in Tables 1 and 2, respectively. In this experiment, the antibacterial activity was calculated by measuring the inhibition zone that exhibits from each extract by using a metal ruler.
As seen in Table 1, all the values are represented as mean ± standard deviation. Values on the same row with a different (a, b, c) superscript letter were different signi icantly with p<0.05. Next, the values on the same column (*, **, ***) also changed with a signi icant level, p<0.05. The DMSO acted as a negative control, while NaF (1 mg/mL) represented as a positive control. From Table 1, it can be concluded that based on these four solvents, n-hexane and methanol extracts exhibit antibacterial properties as it increased the zone of inhibition against S. mutans on the agar.
Meanwhile, the DCM and acetone extracts did not show any antibacterial properties compared it inhibition zone with the negative control. Next, the data shows that the hexane and methanol extract inhibition zone increased with concentration-dependent manner.
Based on the results in Table 2, we can conclude that from all four solvents tested, only n-hexane and DCM extracts exhibit antibacterial properties as it increased the zone of inhibition against S. mutans on the agar. All the values represented as mean ± standard deviation. Values on the same row with a different (a, b, c, d) superscript letter were different signi icantly with p<0.05. Next, the values on the same column (*, **, ***) were also different from the signi icant level, p<0.05. Meanwhile, the methanol and acetone extracts do not show any inhibition zone. Next, the data indicate that upon an increment of n-hexane and DCM extracts concentration had increased the inhibition zone.
Four solvents were chosen based on their polarity level. N-hexane and DCM were used to extract nonpolar compounds; meanwhile, acetone was used to remove semi-polar compounds, and methanol was used to extract polar compounds. Polar solvents have been used to isolate bioactive compounds that exhibited antibacterial and antiseptic properties such as phenol group (Joffry et al., 2011). However, in this study, n-hexane and methanol leaves extract was found to be the most ef icient to inhibit S. mutans growth. As for the lowers extract nhexane and DCM solvents exhibited more antibacterial activity compared to the acetone and methanol.
As mentioned earlier, even though the polar compound extract generally showed the best effect as it can isolate most of the bioactive compounds in the plant. However, there are many reports stated that the antibacterial activity might not only depend on the solvent used but also depends on the compound structure in the extracts and the strain that been investigated (Padalia and Chanda, 2015).
Besides, (Truong et al., 2019) stated that different organic solvents extracts have different phytochemical constituents in such different amounts, and this has led to differences in the impact of inhibition zone occurs. Therefore, n-hexane and methanol leaves extract as well as n-hexane, and DCM lowers extracts had been chosen to further analyze their antimicrobial activity in this study. Table 3 shows the MIC and MBC values of n-hexane, and methanol leaves extracts and n-hexane and DCM lowers extracts. The samples have been chosen in MIC and MBC analysis because they exhibited antibacterial activities in disc diffusion assay. The MIC value is the lowest concentration that inhibits the bacteria. In the MTT assay, the colour of formazan indicates that the bacteria are living. Meanwhile, the yellow colour indicates that the bacteria are inhibited when it exposed to the extract.

Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC)
Hence, the MIC values for n-hexane and methanol leaves extracts were 25.0 mg/mL, respectively. In the meantime, MIC values for n-hexane and DCM lowers extracts were 12. 5 mg/mL, respectively. MBC value for n-hexane and methanol leaves extract were 50 and 100 mg/mL, respectively. Meanwhile, MBC values for n-hexane and DCM lowers extract were 50 mg/mL, respectively. MIC and MBC values for n-hexane were 1 and 2 mg/m, respectively.
Antibacterial agents were observed as bactericidal if MBC value was less than four times of MIC value. Meanwhile, bacteriostatic activity is de ined when MBC values were more than four-times of MIC value (Clinical and Institute, 1999). In this study, the MBC value of n-hexane and DCM lowers extracts (50 mg/mL) was not more than four times of MIC value (12.5 mg/mL). MBC values of n-hexane and methanol leave also extract less than four-time of the MIC value. Therefore, samples from lowers extracts and leaves extracts were presumed as a bactericidal agent against S. mutans. (Varghese et al., 2014) had suggested that A. Paniculata can be further studied for the production of new antibiotics as they have successfully proven that the crude extracts of this plant have potent antibacterial activity against pathogens.

Anti-bio ilm assay
Reduction pattern in bio ilm formation was a concentration-dependent manner to all samples tested ( Figure 1). More concentration of samples was reducing more bio ilm formation of S. mutans. Samples from lowers extracts (n-hexane and DCM) more potent than the leaves extract. About 60% of bio ilm was inhibited from forming when treated with 50 mg/mL of lowers extract. At 100 mg/mL, bio ilm formation was reduced to 70% of lowers extract compared than leaves extract. Meanwhile, NaF reduced the bio ilm formation up to 84% at 1 mg/mL.
The effect of all samples towards S. mutans bio ilm formation activities were concentration dependentmanner. The highest concentration reduced more bio ilm formation activity. Meanwhile, the lowest concentration decreased less bio ilm formation activity. This indicated that higher concentration comprised more bioactive compounds as compared to lower samples concentration. Previous studies reported that high concentration extracts increased their antibacterial (Ong et al., 2011;Yoshida and Kuramitsu, 2002). This proved that extract concentration is also an essential element in determining the antibacterial activity of various plants.

CONCLUSIONS
In conclusion, n-hexane leaves extract, methanol leaves extract, n-hexane lowers extract, and DCM lowers extract of A. paniculata demonstrated bactericidal properties and exhibited anti-bio ilm activities against S. mutans. All the antibacterial tests (Disc Diffusion assay, MIC and MBC) and antibio ilm effects were concentration-dependent manners. In increasing of samples, concentration makes all the results higher and better.