|Year : 2014 | Volume
| Issue : 3 | Page : 232-236
In vitro activity of ethanolic and water extract of guava leaves at various concentrations against Lactobacillus acidophilus
Deepika Jain, Pralhad Dasar, Sandesh Nagarajappa, Sandeep Kumar, Bhuvnesh Airen, Shilpa Warhekar
Department of Public Health Dentistry, Sri Aurobindo College of Dentistry, Indore, Madhya Pradesh, India
|Date of Web Publication||15-Nov-2014|
343, Vyanktesh Nagar, Aerodrome Road, Indore 452 005, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: Chemical substances used for prevention of dental caries are known to have many side-effects. Thus, natural products should be explored for their anticaries action. Objectives: To prepare 5% and 20% concentrations of ethanolic and water extracts of guava leaves and to assess their activity against Lactobacillus acidophilus. Materials and Methods: In vitro experimental study was conducted in Department of Biosciences. Ethanolic and water extracts of guava leaves were prepared using Soxhlet extractor. Two concentrations 5% and 20% weight/volume of both extracts were prepared. Test organism L. acidophilus Microbial Type Culture Collection 447 was obtained in lyophillized form. After revival in nutrient broth, bacteria were grown on Lactobacilli de Man, Rogosa, Sharpe agar for further experiment. Antimicrobial testing of extracts was done using Agar well-diffusion method. Ten plates each were prepared for both extracts. Chlorhexidine (0.2%) served as a positive control and distilled water as a negative control. Results: Mean zone of inhibition produced by 5% and 20% ethanolic extract was 11.2 mm and 14.1 mm respectively and by 5% and 20% water extract was 1.6 mm and 5.1 mm respectively. Statistical analysis of results using one-way ANOVA and post-hoc Tukey's test revealed that activity of 5% ethanolic extract and 5%, 20% water extract was significantly less than that of 0.2% chlorhexidine. There was no statistical difference in efficacy of 20% ethanolic extract of guava and 0.2% chlorhexidine (P = 0.270). Conclusion: Ethanolic and water extracts of guava leaves possess antibacterial activity against L. acidophilus with 20% ethanolic extract being as efficacious as 0.2% chlorhexidine.
Keywords: Dental caries, guava leaves, Lactobacillus acidophilus
|How to cite this article:|
Jain D, Dasar P, Nagarajappa S, Kumar S, Airen B, Warhekar S. In vitro activity of ethanolic and water extract of guava leaves at various concentrations against Lactobacillus acidophilus. J Indian Assoc Public Health Dent 2014;12:232-6
|How to cite this URL:|
Jain D, Dasar P, Nagarajappa S, Kumar S, Airen B, Warhekar S. In vitro activity of ethanolic and water extract of guava leaves at various concentrations against Lactobacillus acidophilus. J Indian Assoc Public Health Dent [serial online] 2014 [cited 2018 Dec 16];12:232-6. Available from: http://www.jiaphd.org/text.asp?2014/12/3/232/144809
| Introduction|| |
Dental caries is a global oral health problem  having a high morbidity potential. It causes pain, discomfort as well as financial burden. The prevention of dental caries is the responsibility of health care professionals. 
One of the main predisposing factors for the onset of dental caries is the presence of bacterial species that can lower the plaque pH to or below critical pH.  It is well established that dental caries causing microorganisms inhabit the dental plaque. Out of 300-or-more species of microorganisms inhabiting the dental plaque  lactobacilli have been found to be present in high numbers in superficial and deep carious lesions. 30% of the clones belong to Lactobacillus acidophilus group.  Lactobacilli act on carbohydrate and produce lactic acid which may lead to demineralization of tooth.  Agents having activity against L. acidophilus can cause a reduction in the occurrence of dental caries. Many products such as antibiotics including penicillin, ampicillin, tetracycline, erythromycin and vancomycin are very effective in preventing dental caries in vitro as well as in vivo. However, excessive use of these anticariogenic agents can result in alterations of the oral and intestinal flora and can cause undesirable side-effects such as the development of bacterial tolerance, vomiting, diarrhea.  Gold standard for the prevention of plaque accumulation and caries activity is Chlorhexidine, which has shown antibacterial activity against L. acidophilus. , Chlorhexidine usage is associated with many side-effects such as staining of teeth, altered taste sensation, stenosis of the parotid duct. ,, Therefore, there is a need to search antimicrobial agents that are specifically active against oral pathogens and also safe for human beings.
Psidium guajava is a phytotherapic plant commonly known as Guava. It is also known as poor man's apple.  It belongs to the Family Myrtaceae which contains at least 133 genera and more than 3800 plant species.  It is believed to have active components that help to treat and manage various diseases.  The leaves of the plant P. guajava Linn are reported to possess antioxidant, hepatoprotective, antiallergy, antimicrobial, antigenotoxic, antiplasmodial, cytotoxic, antispasmodic, cardioactive, anticough, antidiabetic, antiinflammatory and antinociceptive activities.  It is also used for the treatment of sore throats, vomiting menstrual complications, skin sores and wound. The infusion decoction made from the leaves and/or bark of the tree has been used for treatment of diarrhea, malaria and dysentery. These therapeutic uses of guava are expected to be due to the presence of some effective components. 
The leaves of guava have been reported to be used for the maintenance of oral hygiene.  People chew tender leaves of guava to prevent bleeding from gums and bad breath. Guava contains tannins, phenols, triterpenes, flavonoids, essential oils, saponins, carotenoids, lectins, vitamins, fibres and fatty acids. However, lot of pharmacological activities are attributed due to the presence of flavonoids, lutein, zeaxanthin and lycopene. Leaves of the guava tree are a rich source of flavonoids, especially quercetin, which is mainly responsible for the antibacterial activity.  It contains tannins, which are found to be effective against many bacteria such as Escherichia coli, Staphylococcus aureus, etc. 
A number of studies have investigated the activity of various parts of guava plant extracts against specific oral pathogens mainly associated with periodontal diseases and oral malodor while others have focused on the ability of the products to inhibit the formation of dental biofilms by reducing the adhesion of microbial pathogens to the tooth surface, which is a primary event in the formation of dental plaque and the progression to tooth decay and periodontal diseases.
The present study was carried out with the research hypothesis that guava leaves possess antimicrobial efficacy against cariogenic bacteria such as L. acidophilus. The aim of the study was to assess the antibacterial efficacy of ethanolic and water extracts of Guava leaves at 5% and 20% weight/volume (w/v) concentrations against L. acidophilus.
| Materials and Method|| |
Study setting and study design
The present in vitro experimental study was conducted in the laboratory of Department of Biosciences, Maharaja Ranjit Singh College, Indore (M.P.). The ethical clearance had been obtained from the Ethical review committee of Sri Aurobindo College of Dentistry, Indore.
Test organism was L. acidophilus (Microbial Type Culture Collection [MTCC] 447), which was obtained from MTCC gene bank, Institute of Microbial Technology, Chandigarh, India. L. acidophilus was obtained in lyophilized form in a glass vial. The revival of bacteria was done using nutrient broth having a pH of 7.4. The nutrient broth containing bacteria was kept in an incubator at 37°C for 48 hours.
Preparation of extract
This preliminary study considered only the qualitative analysis of activity of Guava leaves extract against L. acidophilus. Therefore, the two higher concentrations, that is, 5% and 20% w/v were tested for antibacterial activity. The plant specimen (Leaves of P. guajava Linn.) for the proposed study was collected from the botanical garden of Devi Ahilya Vishwavidyalaya in the month of May 2013. Leaves of P. guajava L. (Myrteceaae) were cleaned and dried in an oven at 60°C for 5 hours. The dried leaves were then grounded to powdered form. Preparation of the extract was done using Soxhlet extractor. The extracts were filtered using Whatman no. 4 filter paper and then dried in a rotary evaporator for 5-6 hours at 60°C. The dried extract was converted into a powder form which was utilized for the preparation of desired concentrations of the extracts. The required concentrations of 5% and 20% ethanolic extract were prepared by adding 0.5 g and 2.0 g of powder respectively in 10 ml of ethanol. Similarly for the water extract the same amount was added in the distilled water. The extracts were stored at 4°C in sterile bottles.
The antibacterial activity of guava extracts was checked by agar well-diffusion method which was performed on the next day of preparation of the extract. Antibacterial testing was carried out in laminar airflow to avoid contamination by other organisms. A measured amount, that is, 0.5 ml of suspension of inoculums having 3 × 10 8 L. acidophilus/ml (estimated using Mc Farland standard) was streaked on de Man, Rogosa, Sharpe (MRS) agar. Two groups of plates were prepared: Ethanolic extract group and water extract group. In each group, there were 10 plates. In all the plates, 4 wells were punctured in agar with the help of well borer.
The 4 wells prepared in ethanolic extract group were filled carefully with 0.08 ml of 5% ethanolic extract of guava, 20% ethanolic extract of guava, 0.2% chlorhexidine (positive control) and sterile distilled water (negative control). Similarly in water extract group, the wells were filled with 5% water extract of guava, 20% water extract of guava, 0.2% chlorhexidine and distilled water. All the plates were kept in an incubator at 37°C for 48 hours. After 48 hours zones of inhibition were measured.
Statistical analysis was performed using Statistical Package for the Social Sciences (IBM SPSS Statistics 20.0, Chicago). ANOVA test was applied to know the difference between the groups such as 5%, 20% ethanolic extract and 0.2% chlorhexidine and 5%, 20% water extract and 0.2% chlorhexidine. Post-hoc analysis was employed to specifically find that between which groups did significant difference existed. P < 0.05 was considered as statistically significant.
| Results|| |
The present study was conducted to assess the efficacy of Guava leaves extracts on L. acidophilus using agar well-diffusion method. Mean zone of inhibition exhibited by 20% and 5% ethanolic extract was 14.1 mm and 11.2 mm [Table 1], [Figure 1]. Water extract at similar concentrations exhibited 5.1 mm and 1.6 mm zones of inhibition [Table 1], [Figure 2]. Highest mean zone of inhibition was exhibited by 0.2% chlorhexidine (15.4 mm) [Table 1], [Figure 1] and [Figure 2].
|Table 1: Mean inhibition zones of varying concentrations of ethanolic and water extracts of guava leaves and chlorhexidine |
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|Figure 1: Demonstrating the zones of inhibition produced by 20% ethanolic extract (well no. 1), 5% ethanolic extract (well no. 2), 0.2% chlorhexidine (well no. 3) and distilled water (well no. 4) against Lactobacillus acidophilus|
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|Figure 2: Demonstrating the zones of inhibition produced by 5% water extract (well no. 1), 20% water extract (well no. 2), distilled water (well no. 3) and 0.2% chlorhexidine (well no. 4) against Lactobacillus acidophilus|
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The antibacterial efficacy of ethanolic extract of guava leaves at 5%, 20% concentration and 0.2% chlorhexidine was compared using one-way ANOVA (F = 11.390, P = 0.000) [Table 2]. Results that showed a significant difference were further analyzed for statistical significance between specific groups using Tukey post-hoc analysis [Table 3]. There was a significant difference between the efficacy of 5% (11.2 ± 2.29 mm) and 20% (14.1 ± 2.50 mm) ethanolic extract and 0.2% chlorhexidine (15.4 ± 1.034 mm) (F = 11.390, P = 0.000). On post-hoc analysis, it was revealed that the efficacy of 20% ethanolic extract (14.1 ± 2.50 mm) was not significantly different than 0.2% chlorhexidine (15.4 ± 1.034 mm) P = 0.271. However, the efficacy of 5% ethanolic extract (11.2 ± 2.29 mm) was significantly lower than 0.2% chlorhexidine (15.4 ± 1.034 mm) (P =0.012).
|Table 2: Comparison of antibacterial efficacy (inhibition zone) of 5% and 20% ethanolic extract of guava leaves against L. acidophilus|
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There was a significant difference in the activity of 5% (1.6 ± 1.83 mm) and 20% (5.1 ± 2.14 mm) water extract and 0.2% chlorhexidine (15.7 ± 1.034 mm) (F = 132.993, P = 0.000) [Table 4]. Post-hoc analysis revealed that the activity of 0.2% chlorhexidine (15.7 ± 1.034 mm) was significantly higher than 5% water extract (1.6 ± 1.83 mm) as well as 20% water extract (5.1 ± 2.14 mm) of guava leaves. The effect of water and ethanolic extract of guava were found to be dependent on the concentration. 20% concentration of water and ethanolic extracts was significantly higher than 5% concentration [Table 4] and [Table 5].
|Table 3: Comparison of efficacy of 5% ethanolic extract of guava, 20% ethanolic extract of guava and 0.2% chlorhexidine against L. acidophilus|
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|Table 4: Comparison of antibacterial efficacy (inhibition zone) of 5%, 20% water extract of guava against L. acidophilus|
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|Table 5: Comparison of efficacy of 5% water extract of guava, 20% water extract of guava and 0.2% chlorhexidine against L. acidophilus using post-hoc Tukey test|
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| Discussion|| |
In this study extracts of guava leaves were tested against L. acidophilus. The guava leaves had been reported to contain essential oils, flavonoids, saponins, nerolidiol, β-sitosterol, ursolic, crategolic and guayavolic acid. These substances were reported to have strong antibacterial action.  Prabu et al., have demonstrated Guajaverin, a flavonoid in the methanolic extract of leaves of guava that exhibited strong antibacterial activity against caries causing Streptococcus mutans. Thus, this study attempted to evaluate the efficacy of guava leaves, a rich source of antibacterial substances against caries causing L. acidophilus.
In the present study, agar well-diffusion method was employed for microbiological assay that had been reported to be more sensitive than other methods like disc diffusion method.  L. acidophilus was cultured on MRS agar as per the recommendations of MTCC Chandigarh. Results of the present study demonstrated almost similar efficacy of 20% ethanolic extract of guava leaves and 0.2% chlorhexidine. Apart from leaves, extracts from other parts of guava had also been found to possess antibacterial activity. Ngoroyemoto et al., studied the ethanolic and methanolic extracts from roots of guava and found these to be effective against L. acidophilus.
The efficacy of 5% and 20% ethanolic extract was found to be better than 5% and 20% water extract. The water and ethanolic extracts differ in their composition. Ethanolic extract contains tannins as well as flavonoids, whereas water extract contains tannins but not flavonoids. This difference in composition of ethanolic and water extract can be attributed to the difference in solubility of various components of guava leaves in water and organic solvents.  Flavonoids had been reported to exhibit good antibacterial activity. 
Similarly, the difference in the activity of 5% and 20% water extract of guava leaves against L. acidophilus can be attributed to the difference in the concentration of different antibacterial compounds present in the 5% and 20% concentrations of water extract. The present study evaluated qualitatively the antimicrobial potential of guava leaves extract against L. acidophilus. However, further quantitative research is needed to know the minimum inhibitory concentration and to evaluate the effectiveness and safety of guava extracts in vivo.
| Conclusion|| |
The ethanolic and water extract of guava leaves possess antibacterial activity against L. acidophilus with 20% ethanolic extract being as efficacious as 0.2% chlorhexidine.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]