Hibiscus sabdariffa L. Flower Distilled Water Extract Antibacterial Activity towards Streptococcus mutans: An in vitro Study

 

Ida Bagus Narmada1,2*, Muhammad Fulan Ardhani1, Sonya Liani Ramadhani1,

Inggit Dwi Virgianti1, Putri Pramita Larasati1, Ervina Restiwulan Winoto1,2,

Alexander Patera Nugraha1,2, Tengku Natasha Eleena binti Tengku Ahmad Noor3,4

1Department of Orthodontic, Faculty of Dental Medicine - Universitas Airlangga, Surabaya.

2Dental Regenerative Research Group, Faculty of Dental Medicine, Airlangga University, Surabaya, Indonesia.

3Military Dental Officer of Royal Medical and Dental Corps, Malaysian Armed Forces.

4Membership of Faculty of Dental Surgery, Royal Collage of Surgeon, Edinburgh University, United Kingdom.

*Corresponding Author E-mail: ida-b-n@fkg.unair.ac.id

 

ABSTRACT:

Dental and oral health is an essential factor that must be considered because it affects general body health. Streptococcus mutans is known to be a cause of dental plaque formation. Dental plaque is a biofilm formed by microorganisms from solid substrate bonds exopolymer matrix. Fixed orthodontic treatment in its use impacts changes in the environment and composition of the oral flora, as well as increases the amount of plaque accumulation that causes damage to hard tissue and periodontal tissue. The aim of this study is to investigate the minimum concentration of compounds in Hibiscus Sabdariffa L. extract in inhibiting metabolic activity and killing S. mutans in vitro. Methods: This study was an in vitro, the experimental study with post-test only control group design. First, H. Sabdariffa L. flower extraction was carried out with the help of distilled water, and then serial dilution was carried out to obtain various concentrations of H. Sabdariffa L. flower extract. Several S. mutans cultures were then exposed to various concentrations of H. Sabdariffa L. flower, and then a colony count was performed in each culture to identify Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). It was found that S. mutans had 0% growth at a concentration between 25% and 100%, and S. mutans had a growth of 7.1% / 92.9% growth inhibition of S. mutans at a concentration of 12.5%. MBC of roselle flower extract against S. mutans is 25% and MIC of roselle flower extract against S. mutans is 12.5%.

 

KEYWORDS: Antibacterial, Hibiscus Sabdariffa, Communicable disease, Medicine, Dentistry.

 

 


INTRODUCTION: 

Dental caries is a localized dental disease caused by damage to the hard tissue of the teeth due to the interaction of several factors, such as the host (tooth) bacteria, substrate (diet), and time. One of the triggers for caries is inadequate oral hygiene, which is indicated by plaque formation. Dental plaque is a thin film that adheres tightly to the tooth surface and contains a collection of bacteria1. Streptococcus mutans bacteria are known to cause dental plaque formation2.

 

Dental plaque is a biofilm formed by microorganisms from solid substrate bonds exopolymer matrix3. Plaque that forms on the teeth causes demineralization of tooth enamel. Demineralization happens because the bacteria in the plaque form organic acids through sugar metabolism4. Plaque is also a growing place for pathogenic bacteria that cause periodontitis3,5.

 

Orthodontic fixed appliance is used to tidy up the arrangement of teeth. However, oral hygiene for orthodontic fixed appliance (OFA) users should be more noticed. Bad oral hygiene causes the existence of the OFA to become a place for bacteria to attach, which will induce plaque formation6. Brackets and archwires, as components of orthodontic fixed appliances, act as barriers to brush bristles in cleaning teeth resulting in excessive plaque accumulation, especially around the tooth surface of the brackets or under the arches of braces7. Using brackets and ligatures has been shown to increase gingival inflammation and the risk of demineralization. Demineralizing the tooth surface causes white spots or carious lesions to appear1.

 

Various antimicrobial agents have been developed in recent years. Various medicinal plants have been studied, and one of the plants that can be used as a potent phytochemical agent in medical therapy is Hibiscus sabdariffa L. plants, primarily petals, contain polyphenols, organic acids, and polysaccharides8. Polyphenols in H. sabdariffa flower petals, among others: anthocyanins (delphinidin 3-sambubioside, cyanidin 3-sambubioside, delphinidin 3-glucoside, and cyanidin 3-glucoside), protocatechuic acid, and gossypetin9.  Phytochemical components in H. sabdariffa include phenols, alkaloids, tannins, flavonoids, saponins, organic acids, anthocyanins, and polysaccharides10. Flavonoid content inhibits and kills microorganisms that can cause human disease11.

 

The antimicrobial potency of H. sabdariffa flower extract is worthy of investigation on S. mutans, a "normal" oral microorganism. This study was concerned with the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) percentage concentration of H. sabdariffa flower extract that can inhibit metabolic activity and kill S. mutans bacteria that cause gingival inflammation and tooth surface decay. MIC is minimum concentration a compound needs to inhibit bacterial growth, meaning that the number of bacterial colonies present does not increase. MBC is the minimum concentration required for the compound to kill bacteria, meaning the number of bacterial colonies decreases. MIC has a condition that the bacterial colonies formed in exposure to compounds must be <10% compared to when bacterial colonies are formed without exposure to compounds. MBC has a condition that the bacterial colonies formed with exposure to compounds are close to 0% compared to when bacterial colonies are formed without exposure to compounds. Furthermore, this study aims to investigate MIC and MBC of H. sabdariffa flower extract antibacterial activity towards S. mutans in vitro.

 

MATERIALS AND METHODS:

Study setting:

This research is considered an experimental laboratory with a post-test-only control group design and simple random sampling. Samples used S. mutans in Brain Hearth Infusion Broth (BHIB) medium (Merck 1.10493.0500, Darmstadt) at 37°C for 24hours. Bacterial isolates were obtained from  S. mutans ATCC 25175 (ATCC, UK) stock from the Research Center of the Faculty of Dentistry, Airlangga University. S. mutans bacterial suspension was prepared according to standard 0.5 McFarland (1.5 x 10-8 CFU/mL). The total sample is

32 and divided into eight groups, each with four samples. The group was divided into positive control group (2% NaF product Merck 1.06449.0250, Darmstadt), negative control (without the addition of H. sabdariffa flower extract), treatment 1(100% H. sabdariffa flower extract), treatment 2(50% H. sabdariffa flower extract), treatment 3(25% H. sabdariffa flower extract), treatment 4(12.5% H. sabdariffa flower extract), treatment 5(6.25% H. sabdariffa flower extract), and treatment 6(3.125% H. sabdariffa flower extract). 

 

H. sabdariffa flower extract process:

H. sabdariffa flower extract is extracted using the maceration method with distilled water as solvent (Water One 5 Liter, Sidoarjo). The extract was made by drying H. sabdariffa flower in an oven for 24hours at 60°C. Then, the dried flower petals are ground to a H. sabdariffa flower simplicia powder. 500g of H. sabdariffa flower simplicia powder was put into an Erlenmeyer, and 1 L of distilled water was added, then the Erlenmeyer was stored for 24hours. After 24hours, the extraction results were filtered to obtain the first extraction. Then the residue is added to the Erlenmeyer, given distilled water, and left for 24hours for the second extraction. After 24hours, filter again and get the second extract. The results of the first filter (filtrate 1) and the second filter (filtrate 2) were concentrated separately by vacuum using a vacuum evaporator (B-One Vertical Rotary Evaporator RE-2000 VN, China). The total extract results of filtrates one and two were then mixed and stored at cold temperatures12. The phytochemical content of H. sabdariffa flower extract was tested, including alkaloids, flavonoids, saponins, and tannins.

 

MIC and MBC of H. sabdariffa flower extract:

The research began with all the tools for research sterilized using an autoclave (Hirayama HICLAVE HVE-50, Saitama) for 15 minutes at 121°C and 1.5 atm pressure. Then, isolating and culturing S. mutans carried out by inoculating one osse of pure S. mutans into BHIB medium, then incubating at 37°C for 24 hours in an incubator. MIC and MBC tests were carried out using the microdilution method. The H. sabdariffa flower extract was serially diluted with 100%, 50%, 25%, 12.5%, 6.25%, and 3.125% in the BHIB medium. The lowest concentration of H. sabdariffa flower extract suspension, which produces an inhibitory (bacteriostatic) effect on S. mutans bacteria and is characterized by the clarity of the suspension, is recorded as the MIC. Tubes that looked clear were taken 0.1ml each and then planted on Tryptic Soy Agar (TSA) medium (Merck 1.05458.0500, Darmstadt) using the spreading technique. Then incubate at 37°C for 24 hours. The lowest concentration of H. sabdariffa flower extract, which produced a bactericidal effect on S. mutans bacteria, was indicated by the absence of bacterial colonies growing on the agar medium, which was recorded as MBC. The result data are the MIC and MBC values in each treatment group. The test method was to visually observe the presence of bacterial colonies using the Total Plate Count (TPC) method, which formed in the TSA after serial dilution of H. sabdariffa flower extract.

 

Statistical analysis:

The results of MIC and MBC measurements were analyzed using the Shapiro-Wilk test for normality test and the Lavene test for homogeneity test (p>0.05). The Kruskal-Wallis’s test for significance, and the Mann-Whitney test to compare the results between treatment groups (p<0.05).

 

RESULT:

Phytochemical contents analysis of H. sabdariffa flower extract:

This study examined the content in H. sabdariffa flower extract summarized in Table 1. The result of the effectiveness of roselle flower extract in inhibiting the growth of S. mutans bacteria from determining MIC and MBC was carried out using the serial dilution method. Variations in the concentrations used include 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. In addition, a positive control was also made where the bacterial suspension in the medium was given 2% NaF and a negative control where there was medium and bacterial suspension without being given H. sabdariffa flower extract or 2% NaF. After the extract was diluted in a test tube containing BHIB medium, the bacteria were incubated in each test tube, as shown in Figure 1. The result of bacterial growth examination after the incubation period was completed using the TPC method using the spreading technique, which can be seen in Figure 2.

 

Table 1: Results of analysis of the phytochemical contents of roselle flower extract

Content

%

Flavonoids

4.95%

Alkaloids

9.82%

Tannins

0.98%

Saponins

5.11%

 

Figure 1. Reduction of concentration using the serial dilution method in test tubes containing BHIB medium.


 

Figure 2: The results of bacterial colony culture in various groups with 4 replications (a-d).

 


The results of counting colonies with the TPC method that formed on the medium showed the performance of H. sabdariffa flower extract in inhibiting the growth of S. mutans bacteria, which can be seen in Table 2.

 

Table 2: Result of colony count/TPC of S. mutants after treated with H. sabdariffa flower extract

Treatment Group

Colony Number (CFU/ml)

% Growth

%

Resistance

1st Replication

2nd Replication

3rd Replication

4th Replication

T1

0

0

0

0

0.0%

100.0%

T2

0

0

0

0

0.0%

100.0%

T3

0

0

0

0

0.0%

100.0%

T4

13

13

12

15

7.1%

92.9%

T5

40

36

34

35

21.1%

78.9%

T6

66

81

88

71

43.1%

56.9%

C (+)

0

0

0

0

0.0%

100.0%

C (-)

177

184

172

189

100.0%

0.0%

Min

0.00

 

Max

189.00

Mean

38.53

SD

60.33

 

The concentrations at 100%, 50%, and 25% of the treatment, there was no colony formation on the agar medium. Meanwhile, colonies were still growing at treatment 12.5%, 6.25%, and 3.125%. Roselle flower extracts 12.5% in treatment four can be expressed as the MIC because, with this concentration, the growth of bacterial colonies can be suppressed by as much as 92.9% (> 90%). While MBC is determined as a minor concentration that can kill bacteria so that no growing colonies are obtained, a concentration of 25% is obtained in treatment three, as MBC can be seen in Figure 3.

 

Figure 3: Percentage of inhibition of bacterial growth in each treatment group.

 

The results of the normality test using the Shapiro Wilk test showed that the data of negative control, 12.5%, 6.25%, and 3.125% were typically distributed (p>0.05), while the data of positive control, 100%, 50%, and 25% were not normally distributed (p< 0.05). Furthermore, the data is the direct result of the Kruskal Wallis non-parametric statistical test (p<0.05) because there are abnormally distributed data. The Kruskal Wallis test results obtained a p <0.05, which means that there are groups that have significant differences compared to other groups in this study. Finally, the Post Hoc statistical test using the Mann-Whitney test obtained the significance level between treatment groups, which can be seen in Table 3.

 


Table 3: Post hoc analysis results with the Mann-Whitney test

Sig.

C (+)

C (-)

T1

T2

T3

T4

T5

T6

 C (+)

 

 

 

 

 

 

 

 

C (-)

0.014*

 

 

 

 

 

 

 

T1

1.000

0.014*

 

 

 

 

 

 

T2

1.000

0.014*

1.000

 

 

 

 

 

T3

1.000

0.014*

1.000

1.000

 

 

 

 

T4

0.013*

0.020*

0.013*

0.013*

0.013*

 

 

 

T5

0.014*

0.021*

0.014*

0.014*

0.014*

0.020*

 

 

T6

0.014*

0.021*

0.014*

0.014*

0.014*

0.020*

0.021*

*Information: significant different between groups at p<0.05.


 

DISCUSSION:

Orthodontic fixed appliances consist of wires and brackets made of metal or ceramic, which are attached to the buccal, labial or lingual surfaces of the teeth using a bonding material13. Insertion of OFA is associated with an increased incidence of white spot lesions, caries, and gingival inflammation in patients14. This problem is probably because orthodontic patients have difficulty brushing their teeth. After all, many components and bracket surfaces can become a place for plaque to accumulate, so patients experience decreased oral hygiene15.

 

S. mutans is one of the dominant bacteria in supragingival plaque formation. The increase in S. mutans correlates with the increase in plaque accumulation16. S. mutans correlate with increased white spot lesions, caries, and gingival inflammation. An increase in plaque accumulation, especially in patients with OFA, can ultimately increase the incidence of white spot lesion problems, caries, and gingival inflammation17. Previous study investigated that the accumulation of S. mutans bacteria could increase after the installation of OFA in the early stages after three months of use. Bacterial accumulation increases and poses a risk of demineralization with the appearance of white spot lesions at the same time18.

 

The use of antibacterial substances is given as an effort to reduce the incidence of various kinds of oral problems caused or related to bacteria. One substance that has antibacterial potential is extracted from the roselle flower, which has the Latin name H. sabdariffa19. H. sabdariffa have high numbers and are easy to cultivate in Indonesia20. H. sabdariffa is a one-year-old shrub and grows to a maximum height of 2.4m. Stems red, round, and hairy; alternate leaves grow to 3–5 strands with a length of 7.5–12.5cm. Leaves are generally green, with a reddish midrib and short petioles21.

 

S. mutans was observed by measuring the MIC and MBC concentrations after H. sabdariffa flower extract application. The results of the S. mutans colony count test that formed in the medium at each concentration of H. sabdariffa flower extract showed that the MIC was obtained at a concentration of 12.5% which was indicated by stagnant bacterial growth with an inhibition value of 92.9%. MBC for H. sabdariffa flower extract in this study at a concentration of 25% was shown to be a minimum concentration to make 0% bacterial growth. The results of this calculation indicate that the H. sabdariffa flower extract obtained with assistance has bactericidal properties. The criterion for a bactericidal compound is that the ratio between MBC to MIC is ≤ 4. The compound is bacteriostatic if the ratio between MBC and MIC is > 4.19 The bactericidal properties indicate that H. sabdariffa flower extract has excellent potential as an antibacterial compound.

 

The ability of H. sabdariffa flower extract to inhibit the growth of S. mutans is based on the content of 9.82% alkaloid compounds, 5.11% saponins, 4.95% flavonoids, and 0.98% tannins. Alkaloid compounds can be inhibitors of DNA synthesis. Alkaloid compounds contain alkaline groups, which are essential and can react with acidic S. mutans DNA. Alkaline groups disrupt DNA synthesis, inhibiting S. mutans protein synthesis, thereby reducing the rate of S. mutans proliferation21. Saponin compounds can cause the release of proteins and enzymes from S. mutans cells because these compounds have properties resembling surfactants or detergents. Surfactants are a type of compound that can dissolve lipids. The cell membrane of S. mutans is composed of lipids so that saponins can dissolve the S. mutans cell membrane, thereby reducing the bacterial cell wall's surface tension and damaging the membrane's permeability. The damage suffered by S. mutans has a significant impact on the survival of the bacteria22,23. Flavonoid compounds can form extracellular complexes that dissolve the lipid and protein layers of S. mutans cell membranes. Soluble lipid and protein layers cause an increase in cell membrane permeability so that the homeostasis of the cell will be disrupted, and intracellular components can move out of the cell. The released intracellular components cause S. mutans cells to lose their metabolic function so that the growth of S. mutans will decrease or even kill S. mutans23,24.

 

Tannin compounds inhibit the action of S. mutans enzymes, which play a role in the attachment and formation of biofilms on tooth surfaces. S. mutans produces glucosyltransferase enzymes that help these bacteria form biofilms and ultimately form plaque on the teeth. Glucosyltransferases work by forming glucan compounds from sucrose. Tannins are reported to inhibit the action of the glucosyltransferase enzyme on S. mutans25. The content of H. sabdariffa flower extract, which has the highest activity in providing antibacterial properties, is anthocyanin. Anthocyanins are pigment compounds that are an example of a class of flavonoids, anthocyanins can damage bacterial cell membranes and cause cell death26. The highest content of flavonoids in H. sabdariffa flower extract is desoxyflavonoid and flavonol which can reduce the amount of plaque attachment to the tooth surface after rinsing12.

 

CONCLUSION:

From this study result can be concluded that MBC of H. sabdariffa flower extract against S. mutans is 25% and MIC of H. sabdariffa flower extract against S. mutans is 12.5% in vitro. Suggestions for future research is necessary to carry out in vivo research regarding the effect of H. sabdariffa flower extract on the growth of S. mutans bacteria on the surface of the teeth and gingiva of experimental animals. Then, it is necessary to carry out clinical trials of H. sabdariffa flower extract in patients who use orthodontics fixed appliance on the incidence of white spot lesions, caries, and gingivitis.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

This research was funded by Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) fiscal year  of 2022 by Ministry of Education Indonesia with appointment number 1004/UN3/2022 and 189/E5/PG.02.00.PT/2022, 851//UN3.15/PT/2022.

 

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Received on 26.03.2023           Modified on 03.05.2023

Accepted on 06.06.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):25-30.

DOI: 10.52711/0974-360X.2024.00005