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 Table of Contents  
ORIGINAL RESEARCH
Year : 2022  |  Volume : 14  |  Issue : 2  |  Page : 144-150

Antibacterial effect of guava leaves extract mouthwash versus chlorhexidine mouthwash in high caries risk patients: A randomized clinical trial


Conservative Dentistry Department, Faculty of Dentistry, Cairo University, Cairo, Egypt

Date of Submission15-Dec-2021
Date of Decision06-Mar-2022
Date of Acceptance09-Mar-2022
Date of Web Publication26-Apr-2022

Correspondence Address:
Dr. Engy Magdy Kassem
Conservative Dentistry Department, Faculty of Dentistry, Cairo University, 11 Al Saraya, Old Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_348_21

Rights and Permissions
  Abstract 

Aim: To evaluate the effect of guava leaves extract mouthwash on antibacterial activity against streptococcus mutans, salivary pH, and patient satisfaction to taste compared with chlorhexidine mouthwash in high caries risk patients. Materials and Methods: Eighty participants were randomly allocated into two groups based on the mouthwash used: A1 (guava leaves extract) and A2 (Chlorhexidine). The participants were instructed to use the mouthwash once a day for one week and then repeatedly on a monthly basis for three months. Bacterial count, salivary pH, and patient satisfaction to taste were assessed after one week, one month, and three months. Statistical analysis of data using MedCalc software was performed for normality by using Kolmogrov-Smirnov test and Shapiro-Wilk test. Repeated-measures ANOVA and two-way ANOVA were used to assess the interaction of variables for intragroup comparison, and the significance level was set at P ≤ 0.05. Results: By taking into consideration the bacterial count, both mouthwashes showed significant antibacterial effects. However, no statistically significant difference was found between them against the streptococcus mutans during the follow-up periods: one week, one month, and three months, respectively (P = 0.3568, P = 0.0673, and P = 0.0601). Regarding the salivary pH, no statistically significant difference was found between the tested groups during the follow-up periods. Regarding the taste, no statistically significant difference between both mouthwashes was found (P = 0.4254). However, Chlorhexidine showed a statistically significant taste alteration (P < 0.0001) and prolonged taste in the mouth (P = 0.0001). Conclusion: Guava leaves extract mouthwash exhibits promising antibacterial activity without causing taste disturbances.

Keywords: Chlorhexidine, Guava, pH, Streptococcus Mutans


How to cite this article:
Kassem EM, Khairy MA, Abouauf EA. Antibacterial effect of guava leaves extract mouthwash versus chlorhexidine mouthwash in high caries risk patients: A randomized clinical trial. J Int Oral Health 2022;14:144-50

How to cite this URL:
Kassem EM, Khairy MA, Abouauf EA. Antibacterial effect of guava leaves extract mouthwash versus chlorhexidine mouthwash in high caries risk patients: A randomized clinical trial. J Int Oral Health [serial online] 2022 [cited 2022 Sep 27];14:144-50. Available from: https://www.jioh.org/text.asp?2022/14/2/144/344068


  Introduction Top


Dental caries remains a major public health problem and is considered the most prevalent lifestyle-dependent chronic disease affecting a large number of people in many countries in the world. It is a multifactorial disease that can occur at any age, and it affects primary or permanent teeth.[1] Oral biofilm formation is a daily natural process; however, its presence is an important step for caries formation. It was found that streptococcus mutans is not the only basic bacterium involved in the development of plaque, but it is also involved in the initiation of caries.[2] Although toothbrushing and dental flossing are the most dependable used mechanical plaque removal methods, some people lack proper plaque control. As a result, several chemical anti-plaque agents have been introduced as mouthwashes to improve oral health and to maintain good oral hygiene.[3]

Chlorhexidine is a broad-spectrum cationic antimicrobial mouthwash that can be adsorbed to oral tissues for long periods and released when its concentration in the oral cavity is decreased.[4] However, side effects such as taste alteration, staining of teeth and oral mucosa, and increased bacterial resistance are seen when chlorhexidine is used for long durations.[5] This has resulted in a reoccurrence of interest in traditional herbal medicine due to cultural familiarity, inexpensiveness, and convenience, and oral hygiene has been affected by it. Natural herbs have been used alone or in a combination with other products and have been scientifically proven to be safe against various oral health problems such as bleeding gums, halitosis, oral ulcers, and caries.[6],[7] Guava (Psidium Guajava) is a plant that has been used to treat several systemic conditions. Guava leaves are used in the United States as an antibiotic for wounds, ulcers, and toothache in the form of dressing or decoction. Also, the leaves are used to cure malaria, gastroenteritis, vomiting, diarrhea, coughs, sore throat, and inflamed gums in Nigeria, South Africa, and Kenya.[8] Guava sticks have been used since ancient times for effective teeth cleaning. In addition, they have been used as a toothpaste in folkloric practices to preserve oral hygiene.[9]

It is useful to test the null hypothesis that guava leaves extract mouthwash has a similar effect on antibacterial activity, salivary pH, and patient satisfaction to taste as does chlorhexidine mouthwash in high caries risk patients.


  Materials and Methods Top


Study setting

The randomized clinical trial was carried out in the Faculty of Dentistry, Cairo University, Egypt. The protocol of the study was registered in www.clinicaltrials.gov, and it was given an identification number NCT0377309. The study was approved by the Research Ethics Committee (CREC), Faculty of Dentistry.

Sample size calculation

Sample size was calculated by using G*power version 3.1.9.2 Software, University of Kiel, Germany. The effect size was 0.6767 fluorescence calibrating numerical value or unit (FCU). Overall, 72 subjects were divided into two equal groups to be able to reject the null hypothesis that the population means of the experimental and control groups are equal with a power of 0.8. This was increased to 40 in each group to compensate for losses during the follow-up. The Type I error probability associated with this test of this null hypothesis is 0.05.

Materials

  1. Guava leaves extract mouthwash (Nawah Scientific, Mokattam, Egypt).


  2. Chlorhexidine mouthwash (Hexitol, Arab Drug Company “ADCO” for pharmaceutical and chemical industries, Cairo, Egypt).


Armamentarium used in the study

Digital pH Meter: A calibrated digital pH meter (AD12 pH tester, Adwa Instruments, Szeged, Hungary)

Recruitment, study design, and grouping

Screening was done until the target population (80 subjects) met the inclusion criteria, which were high caries risk using the CAMBRA caries risk assessment form, having a minimum of two cavitated or non-cavitated carious lesions, and a baseline DMF score of 3 to 5.[10],[11] However, the exclusion criteria were subjects taking any drug or having a disease that can affect salivary secretion or pH, being medically compromised, and the use of antibiotics or mouthwashes within the past three months.[12],[13] The study, which extended for three months, was a triple-blinded and randomized, two parallel-arms clinical study with a 1:1 allocation ratio. The eligible subjects were randomly allocated into two equal groups (n = 40) based on the tested mouthwash, where (A1) represents the participants who received guava leaves extract mouthwash and (A2) represents the control group, where the participants received chlorhexidine mouthwash. According to the time related to the mouthwash usage (T), (T0) represents the pH and bacterial culture before using the mouthwash; (T1) represents pH, bacterial count, and patient satisfaction to taste after seven days of using the mouthwash daily; (T2) represents pH, bacterial count, and patient satisfaction to taste after one month; (T3) represents pH, bacterial count, and patient satisfaction to taste after three months of using the mouthwash seven days each month.

Randomization, sequence generation, and blinding

Randomization was done by using (www.randomization.com), including the number of subjects divided into two groups marked with letters A and B. The primary investigator, participants, and all the outcome assessors were blinded to the study. The mouthwashes were poured into opaque brown glass bottles, and they were labeled with nonidentifiable numbers to ensure that the primary investigator and participants were blinded.

Intervention

Preparation of guava leaves extract

The ethanolic extract of the guava leaves was prepared in Nawah Scientific, Egypt. The leaves were washed and left to dry in the air for three days away from the sun to prevent over dryness of the leaves. The dried leaves were grounded to form a powder, and the net weight of the guava leaves powder was 2 kg. Six liters of 70% ethanol was added to the powder and stirred for 24 h with an electrical stirrer for three days to prepare the extract. Afterward, Whatman No. 4 filter paper was used to filter the extract; then, it was dried in a rotary evaporator (Rotavapor R-114, BÜCHI Labortechnik AG, Flawil, Switzerland) at 60°C to obtain a dry extract. The dried extract was powdered; used for the preparation of chosen concentrations of the extract; and stored in a securely sealed vessel at 4°C as a means of protection from any contamination, degradation, or disintegration.[14]

Determination of the total phenolic and flavonoid content

Total polyphenol content in the guava leaves extracts was determined by Folin-Ciocalteau method using Gallic acid standard.[15] Rutin standards were used to determine the total flavonoid content.[16] It was found that total phenolics (Gallic Acid Equivalent) = 647.89 mg /g extract and the total flavonoids (Rutin Equivalent) =81.44 mg / g extract.

Determination of minimum inhibitory concentration (MIC)

The disk diffusion method was used to assess streptococcus mutans sensitivity to the guava leaves extract mouthwash on blood agar. Into sterile glass petri dishes, 30 ml of freshly prepared and autoclaved brain heart infusion (BHI) agar was poured. Three to five well isolated colonies of the same morphological type were selected from a blood agar plate culture and transferred with a sterile loop into a tube containing 5 mL of BHI broth, which was next incubated at 37°C for 24 h. Then, the disks were saturated with 20 µL of each of the experimental mouthwashes. A sequence of twofold dilution of the extract ranging from 20 mg / ml to 1.25 mg / ml was used. Guava leaves mouthwash disks were placed immediately over the plates. The plates were incubated at 37°C for 24 h in a candle extinction jar (5% CO2). The plates were then observed for uniform culture growth and the formation of millimeter-sized inhibition zones around the disks. The diameter of each inhibition zone for each disk was calculated for the remaining measurements. The zone of inhibition was first shown as a concentration 5 mg / ml with a diameter of 11 mm and it was then increased gradually by increasing the concentration, considering that 5 mg/ml guava leaves extract was used in the study.[17],[18]

Preparation of mouthwash

The required concentration of 0.5% guava leaves extract mouthwash was prepared by adding 0.5 gm of extract to 100 ml distilled water.[19] Each mouthwash was poured in an opaque and numbered glass bottle. Each bottle contained enough for a week’s worth of use. The bottles were refilled with freshly prepared mouthwash each month to be used for another week.

Mouthwash and regimen

Each participant was given a bottle of the randomly chosen mouthwash. The participants were asked to rinse once a day with 15ml of the mouthwash for one week each month.[20] This process was repeated for three months. The participants were asked to retain the mouthwash in their mouth for 1 min before spitting it, and not to consume any food or drink for at least 30 min after the use of mouthwash.[21] The participants were told to store the mouthwash in the refrigerator to extend its shelf life. Then, the participants presented to the clinic and salivary samples were collected.

Outcome assessment

1. Streptococcus mutans count (Primary outcome) Microbiological analysis

The saliva samples were transferred immediately to the Microbiology Department, Cairo University and they were labeled with nonidentifiable numbers to ensure blinding of the microbiologist. Mitis Salivarius (MS) agar was used. A tenfold (1:10 or 101) dilution was obtained by adding 1 ml of saliva to a test tube containing 9 ml of sterile phosphate buffer saline at pH 7. With a sterile glass spreader, 0.1 ml of the 104 dilution was flecked uniformly on the agar surface to obtain homogeneous bacterial growth. MS Agar plates were located in an anaerobic atmosphere for 48 h at 37°C.[22],[23] The colony counting was done by multiplying a number of colony forming units (CFU) by the number of times the original milliliters of sample was diluted and expressed as the number of colony forming units per milliliter (CFU/ml).[24]

2. Evaluation of salivary pH (Secondary outcome)

The salivary pH was measured immediately to avoid any changes in pH with time. The pH meter was calibrated before measurement by using the supplied standard buffering solutions of pH 4 and 7. The salivary pH was measured in a small beaker. The electrode was immersed completely in the saliva to ensure accurate measurement, and then pH was recorded.

3. Patient satisfaction to taste (Tertiary outcome)

Patient satisfaction to taste was assessed through a questionnaire handed to the participants to evaluate the difference in the response of participants toward the used mouthwashes.[25] It was presented to the participants in both languages: English and Arabic. The participants were asked to rank the mouthwash according to the taste perception, duration, and alteration of taste. They ranked every item from very bad, to bad, neutral, good, and very good.

Statistical analysis

Data were analyzed by using Medcalc software, version 19 for windows (MedCalc Software Ltd, Ostend, Belgium). The data were checked for normality by Kolmogrov-Smirnov and Shapiro-Wilk tests. Continuous data were described by using the mean and standard deviation. To compare continuous data between groups, the T-test was used, whereas repeated-measures ANOVA and two-way ANOVA were used to test the interaction between variables in the intragroup comparison. Categorical data were described as numbers and percentages. Comparisons between categorical variables were done by using the chi-square test. A p-value less than or equal to 0.05 was considered statistically significant, and all tests were two-tailed.


  Results Top


Effect of tested mouthwashes on salivary bacterial count

The intergroup comparison showed no statistically significant difference during the follow-up periods: one week, one month, and three months, respectively (P = 0.3568, P = 0.0673, and P = 0.0601). The intragroup comparison between guava and chlorhexidine mouthwashes revealed statistically significant differences between follow-up periods (P < 0.001). Results are presented in [Table 1].
Table 1: Effect of mouthwashes and follow-up periods on salivary bacterial count

Click here to view


Effect of tested mouthwashes on salivary pH

The intergroup comparison showed a statistically insignificant difference during the follow-up periods: baseline, one week, one month, and three months, respectively (P = 0.1745, P = 0.1564, P = 0.3285, and P = 0.2188). The intragroup comparison within the guava mouthwash group showed a statistically insignificant difference between different follow-up periods (P = 0.0568), whereas the chlorhexidine mouthwash group showed a statistically significant difference between the follow-up periods (P = 0.0133). Results are presented in [Table 2].
Table 2: Effect of mouthwashes and follow-up periods on salivary pH

Click here to view


Patient satisfaction to taste

Regarding the taste of the mouthwashes, the intergroup comparison showed no significant difference between both mouthwashes (P = 0.4254). However, the duration of the taste remaining in the mouth within an intergroup comparison revealed a statistically significant difference between both mouthwashes (P = 0.0001). As for the taste alteration, the intergroup comparison revealed statistically significant differences between both mouthwashes (P < 0.0001). Considering the continuation of using the mouthwash, the intergroup comparison revealed no statistically significant difference between the mouthwashes (P = 0.8277). Results are presented in [Table 3].
Table 3: Frequency and percentage of answers for patient satisfaction survey

Click here to view



  Discussion Top


Dental caries is considered the major oral health problem in industrialized countries, and bacteria in dental plaque are considered one of the key factors for its development.[26] So, mouthwashes are incorporated in dental practice to be used with toothbrushing to control the cariogenic plaque effectively.[27],[28] According to literature, chlorhexidine is one of the most effective antiplaque agents to date, and it is commonly used in high-risk groups of patients.[29] However, modern trends have witnessed a popular preference toward the use of naturally occurring herbal products in the field of medicine and dentistry mainly to avoid the side effects that might occur from the long-term use of chemical agents.[30]

Guava is a medicinal plant that is shown to have positive effects on health, as it is proved to have hepato-protection, antioxidant, antimicrobial, antispasmodic, anticancer, antihyperglycemic, analgesic, and anti-stomach-ache effects.[31] The phytochemical analysis showed that guava leaves were rich in several bioactive compounds that were responsible for the antibacterial activity.[32] Moreover, guava has been shown to be effective in treating oral diseases such as caries, ulcers, gingivitis, and toothache.[33]

Many in vitro studies[19],[34],[35],[36] have reported the potent antibacterial effect of guava leaves on streptococcus mutans; however, few in vivo studies[37] assessed the antibacterial activity and effect on salivary pH, especially in comparison to chlorhexidine, and no studies tested the effect of guava leaves on taste perception. Therefore, this study was conducted to evaluate the effect of guava leaves extract mouthwash on patient satisfaction to taste, antibacterial activity against streptococcus mutans, and salivary pH versus chlorhexidine mouthwash in high caries risk patients.

Ethanol was used as a solvent in guava extract preparation, because studies have shown that ethanolic-based crude extracts have better antibacterial activity compared with aqueous extracts.[38],[39] The phytochemicals in guava show a higher solubility in ethanol than water due to its polar nature, which allows a better dissolving capacity and results in high effectiveness in leaching out of more bioactive compounds during extraction.[14],[40]

The participants were told to use the mouthwashes once daily for a week each month according to the CAMBRA program that recommends the use of antibacterial agents to reduce the level of cariogenic bacteria. Studies showed that this regime proved to be effective in reducing caries incidence in thousands of patients.[41] Regarding the results of antibacterial activity, both mouthwashes showed a significant reduction in the colony counts of streptococcus mutans between the follow-up periods. However, no statistically significant difference between both mouthwashes was found. Regarding the effective antibacterial potential of guava leaves extract mouthwash, the results were in agreement with those of several studies.[14],[34],[39] Singla et al. (2018)[42] found that guava extract mouthwashes caused a significant reduction in the colony counts of streptococcus mutans after seven days of using it. The antibacterial activity of guava leaves extract is attributed to several bioactive compounds such as flavonoids, polyphenols, and tannins. Guaijeverin and quercitin are flavonoids found in guava leaves that make complexes with extracellular proteins, soluble in the bacterial cell wall, and stop the bacterial cell cycle.[43],[44],[45],[46] Guava contain tannins that are poly-phenolic compounds that bind to proline-rich proteins, inhibiting protein synthesis and causing protein denaturation, which will damage the metabolism of bacteria.[47] Also, they effectively dissolve the fatty layer of the bacterial cell wall that causes cell fluid leak out of the cell and destroys it.[48]

As for chlorhexidine, many studies have proved that it is a gold standard antimicrobial product due to different properties. Its antibacterial activity depends on the attraction of opposite charges. As chlorhexidine is cationic (positively charged) in nature, it binds to negatively charged bacteria, and it destabilizes the cell wall, thus interfering with osmosis. The antibacterial effectiveness is found to be dose dependent and at low concentrations it modifies the bacterial osmotic balance, resulting in the release of cytoplasmic components and causing cell death. However, at high concentrations, the molecule leads to coagulation and solidification of cytoplasmic proteins.[49],[50] It was found that rinsing with 10 ml of 0.2% chlorhexidine mouthwash once a day can cause a reduction of 30 to 50% in streptococcus mutans.[51]

Regarding salivary pH, both mouthwashes showed a rise in salivary pH from the first week of using it. This is in accordance with Gupta and Rawal (2021),[52] who stated the fact that the rinsing mouthwash may increase salivary flow and sequentially stimulate salivation, which increases the salivary bicarbonate concentration and thus increases salivary pH. It was found that natural products have a remarkable effect on increasing the pH of saliva and dental plaque due to phenolic and flavonoid compounds, especially catechins.[53] Also, chlorhexidine mouthwash proves to have a positive prolonged effect on the salivary pH. This is evidence of the unique substantivity of chlorhexidine mouthwash.[54],[55]

Regarding the results of taste perception and acceptability, none reported taste alteration nor bitter taste after using guava leaves extract mouthwash. This could be due to the fact that it is a natural product that is believed to be safe and nontoxic. Almost no studies have assessed taste satisfaction and alteration of guava leaves extract, but a study[42] did mention that the guava leaves extract mouthwash had an acceptable taste. On the other hand, the majority of participants in the chlorhexidine group had an altered taste, and they felt an aftertaste while using the mouthwash. The taste changed, with bitter taste sensation increasing, and salty taste decreasing. It was clarified that chlorhexidine effectively blocks the pathway required for the perception of saltiness by binding to taste pores and taste buds with its strong cationic charge.[56] However, for the reduced bitter taste perception, it might be due to taste buds adapting to the bitter taste after treatment with bitter amphiphilic chlorhexidine, which leads to a reduction in the perception of other bitter amphiphilic compounds.[57] Accordingly, chlorhexidine is found to have the highest mean value of taste alteration.

The results suggest the acceptance of the null hypothesis and confirm the effect of the tested mouthwashes on taste alteration, salivary pH, and an antimicrobial agent. The limitations of this study include the mouthwash preparation as it had to be freshly prepared each time and determining the proper administration method of such extracts is considered a challenge. Patient compliance to properly follow the mouthwash protocol is considered a factor that might affect the results. Proposing various benefits such as low cost, ease of availability, and antibacterial activity with safe long-term use, natural herbal mouthwashes could be used on a daily basis as an anticaries agent. More clinical studies are recommended to confirm these results and to find out the proper protocol to be followed, such as dose and how long these herbal extracts could be used for effective caries management. Further investigations are needed on how to improve the taste and increase the shelf life of guava mouthwash without affecting its antibacterial effect.


  Conclusion Top


Regarding the limitations of this study, it can be concluded that both mouthwashes exhibit similar antibacterial activity against streptococcus mutans. Accordingly, guava leaves extract mouthwash could be a suitable alternative to chemical agents such as chlorhexidine in the management of high caries patients.

Acknowledgment

Nil.

Financial support and sponsorship

This study was self-funded by the authors.

Conflicts of interest

Nil.

Author contributions

All authors had contributed equally and given approval for publication of the article.

Ethical policy and institutional review board statement

The trial protocol was registered in the Clinical Trials Registry (clinical trails.gov) with I.D.: NCT0377309. The Research Ethics Committee (CREC) of Faculty of

Dentistry, Cairo University accepted the trial with approval number 18-12-30 (Date: 25 December 2018).

Patient declaration of consent

The authors have obtained all appropriate patient consent forms. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. This randomized clinical trial was held in the Faculty of Dentistry, Cairo University, Egypt.

Data availability statement

Data are available from the corresponding author on reasonable request.

List of Abbreviations

FCU (fluorescence calibrating numerical unit),

CAMBRA (caries management by risk assessment),

DMF (decayed, filled, missed),

BHI (brain heart infusion),

MS (Mitis Salivarius),

CFU (colony forming units).

 
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