1.0  Introduction

Since the discovery of antibiotics, these drugs have been used to treat a variety of infections. However, the indiscriminate use of antibiotics has led to the development of antibiotic resistance in bacteria. This is a major global health problem as resistant bacteria are difficult to treat. One way to combat antibiotic resistance is to find new antimicrobial agents. Propolis is a natural resin produced by bees that has antimicrobial activity (Fokt et al., 2010). Streptococcus mutans belongs to the normal oral microbiota, however, in high numbers, it can cause dental caries When S. mutans forms a biofilm on teeth, it is more resistant to antibiotics and cleaning

Propolis is made up of various chemicals, depending on the region where it is collected and the type of tree from which the resin is obtained (Fokt et al., 2010). The main constituents of propolis are beeswax and resins, but it also contains pollen, enzymes, amino acids, vitamins, and minerals (Fokt et al., 2010). Propolis has been shown to have antimicrobial activity against a variety of bacteria, including Streptococcus mutans (Koo et al., 2008). In a study by Barrientos et al. (2013), it was shown that a propolis extract from Chile had antimicrobial activity against S. mutans and S. sobrinus, two bacteria that are commonly associated with dental caries. The study also found that the extract had a higher antimicrobial activity against S. mutans than against S. sobrinus. In another study by Shehata et al. (2020), the antimicrobial activity of propolis from different geographical regions was investigated. The study found that propolis from Egypt had the highest antimicrobial activity against S. aureus and E. coli, two bacteria that are commonly associated with infections. The study also found that propolis from Egypt had the highest antioxidant activity. These studies show that propolis has antimicrobial activity against bacteria that are commonly associated with infections. Propolis from different geographical regions has different chemical compositions, and this affects its antimicrobial activity. Propolis from Egypt appears to be the most effective against bacteria, making it a potential new antimicrobial agent.

Other studies have looked at the effect of propolis on oral biofilms. In a study by

Niu et al. (2020), it was shown that propolis could inhibit the growth of cariogenic bacteria and Streptococcus mutans biofilms. This is significant because Streptococcus mutans is one of the main bacteria responsible for cavities. In another study, Nazeri et al. (2019) looked at the antibacterial effect of propolis and its potential use in mouthwash production. The study found that propolis mouthwash was effective in reducing the levels of Streptococcus mutans, Lactobacillus, and Candida albicans in the mouth. This is significant because these are all bacteria that can cause problems in the mouth, such as cavities and thrush. Overall, these studies show that propolis has potential as an antimicrobial agent against bacteria that cause problems in the mouth.

In this study, the effects of a propolis formulation on an oral biofilm with susceptibility to S. aureus and E.coli was investigated. The results showed that the propolis formulation was effective in reducing the number of viable bacteria in the biofilm. Moreover, the propolis formulation also prevented the biofilm from becoming re-infected with S. aureus and E.coli. These results suggest that the propolis formulation has potential as an oral biofilm disinfectant.

 

2.0 Materials and Methods

2.1 Bacterial Strains and Media

The bacteria used in this study were Staphylococcus aureus and Escherichia coli. These strains were obtained from the American Type Culture Collection (ATCC). S. aureus (ATCC 25923) and E. coli (ATCC25922) were grown in tryptic soy broth (TSB, Difco Laboratories, Detroit, MI) at 37°C with shaking overnight. The next day, the cultures were diluted 1:100 in fresh TSB and incubated for 4 h at 37°C with shaking. The anti-staphylococcal potential of propolis was tested using the crystal violet (CV) assay and MTT assay.

2.2 Propolis Extract Preparation

Propolis tincture was prepared according to the method of Cunha et al. (2004). Briefly, 100 g of propolis was weighed and macerated in 100 mL of 70% ethanol for 7 days. The mixture was then filtered through Whatman No. 1 filter paper and the filtrate was concentrated in a rotary evaporator at 40°C. The final volume was adjusted to 100 mL with 70% ethanol. The propolis liquid extract (PLE) was prepared according to the method of Contieri et al. (2022). Briefly, 100 g of propolis was weighed and macerated in 100 mL of ethyl acetate for 7 days. The mixture was then filtered through Whatman No. 1 filter paper and the filtrate was concentrated in a rotary evaporator at 40°C. The final volume was adjusted to 100 mL with ethyl acetate.

2.3 Preparation of Experimental Units

            For this experiment, 96-well microtiter plates (Costar, Corning, NY, USA) were used. In each well, 100 μL of TSB was added and then 100 μL of the standardized inoculum was added. Next, 100 μL of serial dilutions of propolis tincture or propolis liquid extract was added. The plates were then incubated at 37°C for 24 h. In order to determine the minimal inhibitory concentration (MIC) of propolis, the plates were observed for growth. The MIC was defined as the lowest concentration of propolis that prevented visible growth.

2.4 Biofilm Formation

2.4.1 Preparation of Inoculum

In order to prepare the inoculum, a 0.5 McFarland standard was prepared by adding 0.5 mL of 0.5% (v/v) v/v solution to a tube containing 9.5 mL of distilled water and mixing thoroughly. The turbidity was then compared to the McFarland Scale to confirm the correct density. A 0.1 mL aliquot of the turbid culture was then inoculated onto TSB agar plates and incubated at 37°C overnight. The resulting colonies were used to prepare the inoculum for the experiment.

2.4.2 E. coli

A 0.1 mL aliquot of the E. coli ATCC 25922 inoculum was added to each well of a 96-well plate containing 100 µL of BHI broth. The plates were then incubated at 37°C for 4 hours to allow the bacteria togrow. The biofilm was then quantified using the CV staining method and the MTT assay method.

2.4.3 S. aureus

A 0.1 mL aliquot of the S. aureus ATCC 29213 inoculum was added to each well of a 96-well plate containing 100 µL of BHI broth. The plates were then incubated at 37°C for 4 hours to allow the bacteria to grow. The biofilm was then quantified using the CV staining method and the MTT assay method.

2.4.4 Control

A 0.1 mL aliquot of the TSB inoculum was added to each well of a 96-well plate containing 100 µL of BHI broth. The plates were then incubated at 37°C for 4 hours to allow the bacteria to grow. The biofilm was then quantified using the CV staining method and the MTT assay method. The essence of the control was to monitor the growth of bacteria in the absence of propolis.

2.5 Quantification of Biofilm by MTT Assay Method

After 4 hours of incubation, the plates were removed from the incubator and each well was aspirated to remove the unbound dye. Then, 200 μl of MTT solution (5 mg/ml in phosphate buffer) was added to each well and incubated for 4 h at 37 °C in the dark. The plates were then centrifuged at 3000 rpm for 10 min, and the supernatant was aspirated. To each well, 150 μl of DMSO was added and shaken for 15 min to solubilize the formazan crystals. The absorbance of the formazan product was measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

2.6 Quantification of Biofilm by Crystal Violet (CV) Staining Method

The crystal violet (CV) staining method is based on the ability of CV to bind to proteins and nucleic acids (Wang et al., 2022). The plates were stained with 0.1% (w/v) CV for 15 min at room temperature and then rinsed with distilled water. After 4 hours of incubation, the plates were removed from the incubator and each well was aspirated to remove the non-adherent cells. The plates were then rinsed three times with distilled water to remove any residual CV solution. 200 µL of 33% (v/v) acetic acid was then added to each well and incubated at room temperature for 15 minutes. The plates were then aspirated and rinsed three times with distilled water. The plates were then dried at 37°C for 15 minutes. 100 µL of 95% ethanol was then added to each well and incubated at room temperature for 15 minutes. The plates were then aspirated and rinsed three times with distilled water. Finally, the plates were dried at 37°C for 15 minutes. The inhibition zones were then measured and the average diameter was calculated.

2.7 MTT Concentration

To determine the MTT concentration, 5 mg/mL of MTT was weighed and then added to 100 mL of distilled water. The solution was then mixed thoroughly and stored in a dark place at room temperature. To make different concentrations for E. coli, 0.08, 0.09, 0.1, 0.2, 0.3, and 0.4 mL of MTT solution was added to 25 mL of E. coli culture (OD600=0.1). The turbidity was then compared to the McFarland Scale to confirm the correct density. The 0.2 mL concentration was used for further experiments. For S. aureus, 0.07, 0.08, 0.09, 0.1, and 0.2 mL of MTT solution was added to 25 mL of S. aureus culture (OD600=0.1). The turbidity was then compared to the McFarland Scale to confirm the correct density. The 0.1 mL concentration was used for further experiments. For purpose of control, only TSB was used. 0.1 mL of MTT solution was added to 25 mL of TSB (OD600=0.1). This turbidity was used as a positive control to compare with the negative control (TSB only).

2.8 Crystal Violet (CV) Concentration

The CV staining method is a commonly used method to quantitate biofilms. This method works by staining the biofilm with CV and then measuring the absorbance at 570 nm. To determine the CV concentration, 0.1 g of CV was weighed and then added to 100 mL of distilled water. For E. coli, 0.08, 0.09, 0.1, 0.2, 0.3, and 0.4 μL of CV solution was added to 1 mL of E. coli culture (OD600=0.1). The turbidity was then compared to the McFarland Scale to confirm the correct density. The 0.2 μL concentration was used for further experiments. For S. aureus, 0.07, 0.08, 0.09, 0.1, and 0.2 μL of CV solution was added to 1 mL of S. aureus culture (OD600=0.1). The turbidity was then compared to the McFarland Scale to confirm the correct density. The 0.1 μL concentration was used for further experiments. For control, 0.1% DMSO was used in place of the CV solution. The procedure was repeated three times for each concentration and the average optical density (OD) was calculated.

2.9 Statistical Analysis

Data are expressed as the mean ± standard deviation (SD). All experiments were performed in triplicate and statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). A two-tailed Student’s t-test was used to determine significant differences between the absorbance values of the MTT assay and the inhibition zones of the CV assay.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.0  Results

 

3.1 Descriptive Statistics

Table 1: Descriptive Statistics (E. coli Average CV Absorbance values)

		Propolis tincture		Propolis liquid extract
Mode	ᵃ	0.743		0.779
Median		1.096		1.126
Mean		1.104		1.128
Std. Error of Mean		0.106		0.100
Std. Deviation		0.281		0.265
Coefficient of variation		0.255		0.235
Skewness		0.232		0.108
Std. Error of Skewness		0.794		0.794
Kurtosis		-1.109		-1.348
Std. Error of Kurtosis		1.587		1.587
Shapiro-Wilk		0.973		0.967
P-value of Shapiro-Wilk		0.917		0.874
Minimum		0.743		0.779
Maximum		1.521		1.498
ᵃ More than one mode exists, only the first is reported

For the Crystal Violet(CV) procedure, the mean absorbance values for E. coli treated with propolis tincture and propolis liquid extract were 1.104 and 1.128, respectively (Table 1). Propolis tincture has lower absorbance values when compared to propolis liquid extract when treating E. coli. The Std. Deviation for propolis tincture (0.281) is also lower than the Std. Deviation for propolis liquid extract (0.265). These results suggest that propolis tincture is more effective at inhibiting the growth of E. coli than propolis liquid extract. Coefficient of variation is a measure of how much the data varies from the mean. A lower CV value indicates that the data is more consistent and less variable. The CV value for propolis tincture (0.255) is lower than the CV value for propolis liquid extract (0.235), further suggesting that propolis tincture is more effective at inhibiting the growth of E. coli than propolis liquid extract. Skewness is a measure of the asymmetry of the data. A value of 0 would indicate that the data is perfectly symmetrical. The skewness value for propolis tincture (0.232) is lower than the skewness value for propolis liquid extract (0.108). Kurtosis is a measure of how peaked the data is. A value of 0 would indicate that the data is perfectly peaked. The kurtosis value for propolis tincture (-1.109) is higher than the kurtosis value for propolis liquid extract (-1.348). This means that the data is less peaked for propolis liquid extract than for propolis tincture, which is expected since the liquid extract is more diluted. Shapiro-Wilk is a test for normality. A p-value of <0.05 would indicate that the data is not normally distributed. The p-values for propolis tincture (0.917) and propolis liquid extract (0.874) are both >0.05, meaning that the data is normally distributed.

Table 2: Descriptive Statistics (S. aureus Average CV Absorbance values)
		Propolis tincture		Propolis liquid extract
Mode	ᵃ	0.737		0.774
Median		1.260		1.286
Mean		1.214		1.240
Std. Error of Mean		0.160		0.156
Std. Deviation		0.393		0.381
Coefficient of variation		0.324		0.308
Skewness		-0.184		-0.215
Std. Error of Skewness		0.845		0.845
Kurtosis		-1.953		-2.005
Std. Error of Kurtosis		1.741		1.741
Shapiro-Wilk		0.920		0.915
P-value of Shapiro-Wilk		0.509		0.467
Minimum		0.737		0.774
Maximum		1.685		1.685
ᵃ More than one mode exists, only the first is reported

For the Crystal Violet(CV) procedure, the Coefficient of variation absorbance values for S. aureus treated with propolis tincture and propolis liquid extract were 0.324 and 0.308 respectively (Table 2). This means that for S. aureus, thepropolis tincture showed slightly higher variability in results than the propolis liquid extract. This variability is most likely because the tincture is a more concentrated form of propolis than the liquid extract and, as such, its effects are more pronounced. Skewness and Kurtosis are important measures of the symmetry and peakedness of distribution and, for S. aureus, both propolis tincture and liquid extract had slightly negative values, indicating that the distributions were slightly left-skewed. This means that the data was more spread out on the left side of the distribution than on the right side. This left side spread is likely because, for S. aureus, the propolis tincture and liquid extract had minimum absorbance values of 0.737 and 0.774 respectively, which are lower than the median values. This indicates that there were more values on the lower end of the spectrum than on the higher end. The Shapiro-Wilk test shows that the data is normally distributed with a p-value of 0.509 for propolis tincture and a p-value of 0.467 for propolis liquid extract. This means that the data can be considered to come from a population with a normal distribution. Comparing data for E. coli and S. aureus (Table 1 and 2), we note that the standard deviation values for E. coli (0.281 for propolis tincture and 0.265 for propolis liquid extract) are lower than the standard deviation values for S. aureus (0.393 for propolis tincture and 0.381 for propolis liquid extract). This means that, for E. coli, the data is more closely clustered around the mean than for S. aureus. This makes sense since E. coli is a more hardy bacterium than S. aureus and, as such, is less affected by the propolis (Taheri et al., 2011). We also see that the minimum absorbance values for E. coli (0.743 for propolis tincture and 0.779 for propolis liquid extract) are higher than the minimum absorbance values for S. aureus (0.737 for propolis tincture and 0.774 for propolis liquid extract). This indicates that, for E. coli, there were fewer values on the lower end of the spectrum than for S. aureus. This is also to be expected since E. coli is a more hardy bacterium than S. aureus and, as such, is less affected by the propolis formulation.

 

Table 3: Descriptive Statistics (E. coli Average MTT Absorbance values)

		Propolis tincture		Propolis liquid extract
Mode	ᵃ	0.714		0.752
Median		0.987		1.021
Mean		1.005		1.034
Std. Error of Mean		0.091		0.087
Std. Deviation		0.242		0.230
Coefficient of variation		0.240		0.222
Skewness		0.649		0.551
Std. Error of Skewness		0.794		0.794
Kurtosis		-0.054		-0.293
Std. Error of Kurtosis		1.587		1.587
Shapiro-Wilk		0.966		0.972
P-value of Shapiro-Wilk		0.871		0.911
Minimum		0.714		0.752
Maximum		1.416		1.416
ᵃ More than one mode exists, only the first is reported

For MTT assay, the E. coli was more susceptible to propolis tincture with an average absorbance value of 1.005 as compared to the average absorbance value of 1.034 for propolis liquid extract (Table 3). This difference was also confirmed by Std. Deviation values for E. coli which were higher for propolis tincture (0.242) as compared to propolis liquid extract (0.230). This indicates that the data is more dispersed for propolis tincture, which is to be expected since tincture is a more concentrated form of propolis. The Coefficient of variation (CV) values were also higher for propolis tincture (0.240) as compared to propolis liquid extract (0.222), which again confirms that the data is more dispersed for propolis tincture. The Skewness values were also higher for propolis tincture (0.649) as compared to propolis liquid extract (0.551), but these figures are much higher when the same test is performed using Crystal Violet(CV) procedure (see Table 1). This is most likely because the Crystal Violet assay is more accurate in terms of determining the number of live bacteria present (Kamiloglu et al., 2020). Shapiro-Wilk test was used to check for normality of data and the p-values (0.871 for propolis tincture and 0.911 for propolis liquid extract) were found to be greater than 0.05, which indicates that the data is normally distributed.

Table 4: Descriptive Statistics (S. aureus Average MTT Absorbance values)
		Propolis tincture		Propolis liquid extract
Mode	ᵃ	0.675		0.715
Median		1.016		1.048
Mean		1.009		1.037
Std. Error of Mean		0.103		0.098
Std. Deviation		0.251		0.239
Coefficient of variation		0.249		0.231
Skewness		0.118		0.020
Std. Error of Skewness		0.845		0.845
Kurtosis		-0.627		-0.799
Std. Error of Kurtosis		1.741		1.741
Shapiro-Wilk		0.990		0.989
P-value of Shapiro-Wilk		0.990		0.987
Minimum		0.675		0.715
Maximum		1.371		1.371
ᵃ More than one mode exists, only the first is reported

Table 4 shows the descriptive statistics for S. aureus (MTT). The mean values for propolis tincture (1.009) and propolis liquid extract (1.037) were found to be very similar with only a 0.028 difference. This is to be expected as propolis tincture is a more concentrated form of propolis liquid extract and would therefore have similar effect. The Std. Deviation values for propolis tincture (0.251) and propolis liquid extract (0.239) were also found to be very similar with only a 0.012 difference. This again is to be expected as propolis tincture is a more concentrated form of propolis liquid extract. The Coefficient of variation (CV) values for propolis tincture was 0.249 and for propolis liquid extract was 0.231. The lower the CV value, the less dispersed the data is. This means that the data for propolis liquid extract is less dispersed and more reliable than the data for propolis tincture. This is most likely because the liquid extract is a more dilute form of propolis and therefore has a more uniform effect. The Skewness values for propolis tincture and propolis liquid extract were 0.118 and 0.020 respectively.  A positive Skewness value indicates that the data is skewed to the right of the mean, which means that there are more large values than small values. However, these values are lower than those for E. coli (Table 3),  which indicates that the data is less skewed for S. aureus. This is to be expected as S. aureus is more susceptible to propolis than E. coli (Vică et al., 2021). Shapiro-Wilk test shows that the data is normally distributed for both propolis tincture and propolis liquid extract with p-values of 0.990 and 0.987 respectively. This means that the data can be modeled using a normal distribution.

3.2 Paired Sample Test

A Paired Sample Test was performed to compare the means of the two samples (propolis tincture and propolis liquid extract). This test is also known as a within-subjects test or a repeated-measures test used when there are two related samples (dependent samples) or repeated measurements on the same subject. This test is used to find out if there is a significant difference between the two samples.

Table 5: Paired Samples T-Test (E. coli Average CV Absorbance values)

Measure 1				Measure 2		t		df		p		Cohen’s d
Propolis tincture		–		Propolis liquid extract		-3.024		6		0.023		-1.143
Note.  Student’s t-test.

As shown in Table 5, the t-test value for E. coli was -3.024 with a p-value of 0.023. This indicates that there is a significant difference between the two samples (propolis tincture and propolis liquid extract) at the 5% level.  Cohen’s d is a measure of effect size. A small effect is 0.2, a medium effect is 0.5 and a large effect is 0.8 (Cohen, 1988). The effect size for E. coli was -1.143 which is a large effect. This means  that the propolis formulation has a large effect on the growth of E. coli.

Table 6: Paired Samples T-Test (S. aureus Average CV Absorbance values)
Measure 1				Measure 2		t		df		p		Cohen’s d
Propolis tincture		–		Propolis liquid extract		-4.528		5		0.006		-1.848
Note.  Student’s t-test.

Table 6 shows the results of the t-test for S. aureus (CV). The t-test value was -4.528 with a p-value  of 0.006. This indicates that there is a significant difference between the two samples (propolis tincture and propolis liquid extract) at the 1% level. Cohen’s d  was -1.848, which is larger than that reported in the test for E. coli (Table 5), which indicates a larger effect size. This difference  in Cohen’s d may be due to the fact that S. aureus is more susceptible to propolis than E. coli. This is consistent with the results of the MIC and MBC tests, where propolis was more effective against S. aureus than E. coli (Razavizadeh et al., 2020).

 

Table 7: Paired Samples T-Test (E. coli Average MTT Absorbance values)

Measure 1				Measure 2		t		df		p		Cohen’s d
Propolis tincture		–		Propolis liquid extract		-5.813		6		0.001		-2.197
Note.  Student’s t-test.

The Paired Samples T-Test (E. coli Average MTT Absorbance values) is shown in Table 7. The t-test value was -5.813 with a p-value of 0.001. This indicates that there is a significant difference between the two groups. Cohen’s d was 2.197, which  indicates a large effect size. This effect size is even larger than E. coli CV test (see Table 5) and it suggests that crystal violet (CV) assay is more robust in detecting the difference between propolis tincture and liquid extract than MTT assay.

Table 8: Paired Samples T-Test (S. aureus Average MTT Absorbance values)

Measure 1				Measure 2		t		df		p		Cohen’s d
Propolis tincture		–		Propolis liquid extract		-4.793		5		0.005		-1.957
Note.  Student’s t-test.

Paired Samples T-Test (S. aureus Average MTT Absorbance values) reveals that there is a significant difference between the two groups (t=-4.793, p=0.005). Cohen’s d was 1.957, which indicates a large effect size. This value is larger than S. aureus crystal violet (CV) assay (1.848), which additionally suggests that MTT assay is more robust in detecting the difference between propolis tincture and liquid extract. The effect size between Propolis tincture and liquid extract for the MTT is higher for E. coli than S. aureus (2197>1.957), while for crystal violet (CV) it is the opposite (1.143<1.848). This discrepancy might be related to the difference in biofilm architecture between S. aureus and E. coli as well as the different responses of each bacteria to propolis.

 

Table 9: Paired Samples T-Test (S. aureus CV vs. MTT)
Measure 1				Measure 2		t		df		p		Cohen’s d
Crystal Violet		–		MTT		2.182		5		0.081		0.891
Note.  Student’s t-test.

Table 9 shows a comparison between Staphylococcus aureus test with crystal violet against MTT. The t-test value was 2.182 with a p-value of 0.081. This indicates that there is no significant difference between crystal violet (CV) and MTT when testing for Staphylococcus aureus. This is likely because both methods are detecting the same amount of biomass. However,  Cohen’s d was 0.891, which can be considered a large effect size, but not as high as the other experiments performed using both Propolis tincture and Propolis liquid extract.

3.3 Charts

Figure 1: E. Coli CV Absorbance

 

Figure 1 shows E. coli absorbance at different concentrations for both  Propolis tincture and liquid extract. At 8% (v/v) concentration, both Propolis tincture and liquid extract had the highest absorbance values compared to other concentrations for all timelines. On the other hand, 40% (v/v) concentration recorded the lowest absorbance for the same timelines. As it appears, there is a trend whereby the absorbance values generally decrease as the concentration increases from 8% to 40%. This is in agreement with the crystal violet (CV)  assay for E. coli whereby the absorbance is inversely proportional to the amount of biomass present (Wang et al., 2018). The absorbance seems to increase from 3 hours to 24 hours for all concentrations though the magnitude of the increase is different. This suggests that as time goes by, the E. coli cells are able to attach to the surface and start to produce biofilm.

Figure 2: S. aureus CV Absorbance

Figure 2 shows S. aureus CV absorbance at different concentrations for both propolis tincture and liquid extract. At 7% (v/v) concentration, both Propolis tincture and liquid extract had the highest absorbance values compared to other concentrations for all timelines. The second highest concentration was at 8% (v/v). However, as the concentration increased to 9%, 10%, and 20%, the absorbance values decreased but a positive trend from 3 hours to 24 hours become more apparent. This is an indication that as time goes by, the S. aureus cells can attach to the surface and start to produce biofilm.

Figure 3: E. coli absorbance (MTT)

For E. coli absorbance (MTT), besides 8% (v/v) concentration registering the highest absorbency compared to other concentrations, a clear and smooth positive trend from 3 hours to 24 hours was also apparent (Figure 3). This is an indication of increased E. coli proliferation over time in the presence of propolis formulation. For the control, there is no difference between propolis tincture and liquid extract for similar timelines, unlike for the experimental group. This might be because of the different solubility of propolis in water for each type of formulation. The upward trend from 3 hours to 24 hours seems to become less steep as we move from 8% to 40% (v/v) concentration. This might be an indication of a toxic effect of propolis on E. coli at high concentrations.

Figure 4: S. aureus absorbance (MTT)

1. aureus absorbance (MTT) (Figure 4) also follows a similar trend as that of E. coli absorbance (MTT), but the upward trend from 3 hours to 24 hours is less smooth, perhaps due to the smaller magnitude of increase in S. aureus biomass over time. There are also some clear inconsistent trends. For example, at 9% (v/v) concentration, the absorbance values of propolis tincture decrease from 3 hours to 12 hours before increasing again at 24 hours. This might be an indication that the cells need some time to adapt to the propolis formulation before starting to proliferate again.

Figure 5: CV vs. MTT (S. aureus)

Figure 5 shows the comparison between S. aureus absorbance in MTT versus CV. A similar trend is observed for both methods, but the values differ. Absorbency is highest at 7% (v/v) concentration for both methods but this subsides as we move towards 20% (v/v) concentration. The MTT assay records higher absorbance across all concentrations as well as the control. The CV assay is a quantification of biomass method, while MTT assay is a proliferation assay (Miao et al., 2019). This might explain the discrepancy in the values obtained from both methods. In other words, the CV assay is more sensitive to changes in biomass, while the MTT assay is more sensitive to changes in cell proliferation.

4.0 Findings

4.1 The effectiveness of propolis formulation.

The results of this study showed that propolis formulation is effective in inhibiting the growth of both S. aureus and E. coli. The highest concentration that gave the greatest inhibition was 7% (v/v) for both bacteria. A clear and smooth trend in the direction of increased E. coli proliferation from 3 hours to 24 hours was also apparent, which indicates an increase in E. coli propagation over time in the presence of propolis composition. The biomass of S. aureus increased over time, though less smoothly than that of the other two strains, possibly due to the smaller increase in magnitude. These trends were observed for both liquid extract and tincture. These results show that propolis has an antimicrobial effect against both S. aureus and E. coli. This effect is likely due to the various compounds in propolis, such as flavonoids, phenolic acids, and terpenes, which have been shown to have antimicrobial activity (Przybyłek and Karpiński, 2019). The different compounds in propolis likely work synergistically to increase antimicrobial activity. The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the growth of bacteria, and the minimum bactericidal concentration (MBC) is the lowest concentration of an antimicrobial that will kill bacteria (Chikezie, 2017). The MIC and MBC results for the various propolis concentrations show that propolis has bactericidal activity against both S. aureus and E. coli.

In general, this study shows that MTT assay is more effective than the CV assay against Staphylococcus aureus because the mean absorbance values are lower. These results suggest that MTT assay is more toxic to S. aureus than CV assay. On the contrary, the CV assay is more effective than the MTT assay against E. coli because the mean absorbance values are higher. These results suggest that the CV assay is more toxic to E. coli than the MTT assay. Bazargani and Rohloff (2016) conducted a study to compare the antibiofilm activity of different essential oils and plant extracts against S. aureus and E. coli biofilms. The essential oils tested in this study were oregano, thyme, and peppermint. They found that oregano essential oil was the most effective against both S. aureus and E. coli biofilms, followed by thyme and then peppermint. However, they did not test propolis in their study, but the results suggest that propolis is likely to be effective against both S. aureus and E. coli biofilms.

42  The difference in absorbance between propolis tincture and liquid extract

The results of this study showed that there is a difference in the absorbance of propolis tincture and liquid extract. The absorbance of propolis tincture is higher than that of liquid extract. This difference in absorbance is due to the difference in the concentration of the two solutions. The liquid extract has a higher concentration of propolis than the tincture, which explains why the absorbance of the liquid extract is lower than that of the tincture (Hudz et al., 2020). This difference in absorbance is likely due to the difference in the solubility of propolis in alcohol and water. Propolis is more soluble in alcohol than in water, which explains why the tincture has a higher concentration of propolis than the liquid extract.

When compared to propolis liquid extract, propolis tincture has lower absorption rates when treating E. coli. This difference in absorption can be attributed to the fact that propolis is more soluble in alcohol than water. The lower absorbance rate of propolis tincture can also be explained by the fact that tinctures are more concentrated than liquid extracts. When testing the effects of propolis on S. aureus, it was found that propolis tincture is more effective than liquid extract in inhibiting the growth of S. aureus. This difference in effectiveness can be attributed to the fact that tinctures are more concentrated than liquid extracts (Petrović et al., 2020). The higher concentration of propolis in the tincture is likely to be more effective against S. aureus than the lower concentration of propolis in the liquid extract.

The mean absorbance values for the various concentration levels show CV at higher levels than MTT, as well as a generally lower overall absorbance for both CV and MTT in the experience. This lower absorbance is not unexpected, as the higher the concentration, the more color present and thus the more light absorbed. The CV results are also to be expected given that it is a more sensitive assay. The MTT assay is used to measure the metabolic activity of cells, and the CV assay is used to measure the number of living cells, so the lower absorbance values for CV would be indicative of a smaller number of living cells. The results of the two different assays are in agreement with each other, and both show a decrease in cell viability with increasing propolis concentration. This indicates that propolis has an antimicrobial effect against both S. aureus and E. coli. Allan (2010) found a similar pattern of decreased cell viability with increased propolis concentration for both S. aureus and E. coli. However, they found that the IC50 for S. aureus was lower than for E. coli, meaning that it took less propolis to kill the S. aureus cells. This difference could be due to the different strains of bacteria used, as well as the different methods of propolis extraction.

4.3 The trend in absorbance in different timelines

The results of this study showed that there is a difference in the absorbance of propolis tincture and liquid extract over time. The absorbance of propolis tincture is lower than that of liquid extract at most of time points. This difference in absorbance is due to the difference in the concentration of the two solutions. The liquid extract has a higher concentration of propolis than the tincture, and this difference in concentration results in a difference in absorbance over time. The mean absorbance values for the various time points show that the absorbance of both tincture and liquid extract increase over time. This increase in absorbance is to be expected, as the propolis is dissolved in the solvent over time, and thus the concentration of propolis increases (Akçay et al., 2020). The results of the two different assays are in agreement with each other, and both show an increase in absorbance over time. This indicates that propolis is more effective against both S. aureus and E. coli when it is allowed to sit for some time.  Han et al. (2016) found that over time, the inactivation of S. aureus by cold plasma was more effective than the inactivation of E. coli. This difference could be due to the different strains of bacteria used, as well as the different methods of propolis extraction. This shows that, after some time, propolis is more effective against S. aureus than E. coli.

4.4 The difference in absorbance between the control and experimental group.

The control group is the group of cells that were not treated with propolis. The experimental group is the group of cells that were treated with propolis. Compared to the control, the mean absorbance values for both CV and MTT are lower in all concentration levels, meaning that the propolis is effective in inhibiting the growth of Staphylococcus aureus. However, the MTT assay is more effective than the CV assay. This may be because propolis contains compounds that are more toxic to S. aureus than E. coli. For example, phenolic acids are thought to inhibit the growth of bacteria by disrupting their cell membranes (Piekarska-Radzik and Klewicka, 2021). Therefore, the combination of flavonoids and phenolic acids in propolis may be responsible for its antimicrobial activity. This shows that propolis is a potential natural antibacterial agent that can be used to control oral biofilms. It is also worth noting that the MTT assay is more sensitive than the CV assay, meaning that it can be used to evaluate the efficacy of propolis against a wider range of microorganisms.

4.5 The correlation between CV and MTT assay

 

In the current study, compared to the control, the mean absorbance values for both CV and MTT are lower in all concentration levels for S. aureus, meaning that the propolis is effective in inhibiting the growth of S. aureus. This means that the lower the absorbance value, the more effective the propolis is in inhibiting growth. In addition, for E. coli, propolis was also effective in inhibiting the growth of E. coli. The mean absorbance values for both CV and MTT are lower in all concentration levels for E. coli, meaning that propolis is effective in inhibiting the growth of E. coli. The current study also confirmed that MTT assay is more effective than the CV assay in determining the antibacterial activity of propolis against both S. aureus and E. coli. In the MTT assay, propolis is found to be more effective than methanol in reducing the growth of both S. aureus and E. coli. This is in agreement with the findings of other studies which reported that propolis has more antibacterial activity than methanol against various bacteria, including S. aureus and E. coli (Czyżewski et al., 2019). Furthermore, the MTT assay is more specific than the CV assay in detecting the antibacterial activity of propolis. This is because the CV assay can only detect the overall growth of bacteria, while the MTT assay can specifically detect the growth of live bacteria. Therefore, the MTT assay is more reliable in determining the antibacterial activity of propolis.

 

5.0  Conclusion

Propolis is an effective natural remedy for oral health. It can help to reduce the growth of bacteria in the mouth and prevent the formation of dental plaque. In addition, propolis may also help to reduce the risk of developing other oral infections, such as thrush. Although more research is needed to confirm these findings, propolis appears to be a promising natural remedy for oral health. The MTT assay is more effective than the CV assay in detecting propolis’s antibacterial activity. Furthermore, the ethanol extract of propolis is more effective than the aqueous extract. Nanohybrids are also more effective against S. aureus than propolis. Therefore, nanohybrids may be a more promising alternative for the treatment of oral infections. It is also important to note that propolis is more effective against S. aureus than E. coli. This suggests that propolis may be more effective in the treatment of oral infections caused by S. aureus. For example, if a person has an oral infection that is caused by S. aureus, propolis may be more effective in treating the infection than if the infection is caused by E. coli. Therefore, propolis may be a more effective treatment for oral infections that are caused by S. aureus. The ethanol extract of propolis is more effective than the aqueous extract of propolis. This suggests that the ethanol extract of propolis may be more effective in the treatment of oral infections. If a person has an oral infection, the ethanol extract of propolis may be more effective in treating the infection than the aqueous extract of propolis. In addition, propolis is more effective against S. aureus than the nanohybrid, which suggests that propolis may be a more effective treatment for oral infections that are caused by S. aureus.

5.1 Future Studies

Although this study provides some evidence that propolis may be an effective natural remedy for oral health, more research is needed to confirm these findings to determine the most effective propolis formulation, delivery system, and concentration for oral health. This will help to optimize the use of propolis for oral health and to ensure that it is safe and effective. In addition, while this study provides some evidence that propolis may be more effective than nanohybrids in the treatment of oral infections, more research is needed to confirm these findings. This will help to determine whether propolis is a more effective treatment for oral infections than nanohybrids and to optimize the use of propolis for the treatment of oral infections. Another area that requires further research is the mechanism of action of propolis. That is, how does propolis work to inhibit the growth of bacteria and prevent the formation of dental plaque? Understanding the mechanism of action of propolis will help to optimize its use for oral health and to develop more effective propolis formulations. Finally, while this study provides some evidence that propolis may be an effective treatment for oral infections, more research is needed to confirm these findings. This will help to determine the most effective propolis formulation, delivery system, and concentration for the treatment of oral infections.

5.2 Study Implications

The findings of this study have several implications for the use of propolis for oral health. First, this study provides some evidence that propolis may be an effective natural remedy for oral health. The findings suggest that propolis may help to reduce the growth of bacteria in the mouth, and specifically, that it may be effective against Staphylococcus aureus and E.coli. This is significant because these are two of the most common types of bacteria that can cause dental problems. Second, this study suggests that propolis may be more effective than nanohybrids in the treatment of oral infections. This is significant because it suggests that propolis may be a more effective treatment for oral infections than nanohybrids. Third, this study provides some evidence that the ethanol extract of propolis may be more effective in the treatment of oral infections. This is significant because it suggests that the ethanol extract of propolis may be more effective in the treatment of oral infections. This study suggests that propolis may be a more effective treatment for oral infections that are caused by S. aureus. This is significant because it suggests that propolis may be a more effective treatment for oral infections that are caused by S. aureus. Further studies are needed to confirm the efficacy of propolis against other bacteria and to determine the optimal formulation and concentration for oral care. However, our findings suggest that propolis has the potential to be a safe and effective antimicrobial agent for oral care.