Effects of Malus domestica Fruit Extracts Against Clinically Isolated Dental Pathogens

MATERIALS AND METHODS

Collection and authentication: The fruits of M. domestica (Fuji apple) were purchased from local market, Kota Seriemas, Nilai, Malaysia in the month of March, 2011. The collected fruits were authenticated by a Botanist at KPJ Healthcare University College, Kota Seriemas, Nilai, Malaysia.

Preparation of extracts: The purchased fruits M. domestica were washed thoroughly with water and sliced into small pieces and pulverized. The pulverized fruits (1 kg) were extracted successively with petroleum ether, ethanol and water by cold maceration technique for 7 days. The mixture was filtered separately and the excessive solvents were evaporated. The extracts were concentrated using Rotary vacuum evaporator under reduced pressure. The colour, consistency and percent yield of the extracts are depicted in Table 1. All the extracts were stored in desiccators until use¹².

Isolation of dental pathogens: Five pathogenic microorganisms viz., Staphylococcus aureus, Streptococcus mutans, Lactobacillus subtilis, Klebsiella sp. and Pseudomonas sp., were isolated from the dental cavities of 24 patients (6 males and 18 females) (Table 2). The isolates were identified by standard morphological analysis and bio-chemical tests, respectively¹³. The identity of the microorganisms was further confirmed by growing in specific media as per the method developed by Cheesborough¹⁴.

Screening of antibacterial activity of various extracts against the isolated dental pathogens: The antibacterial activity of the extracts of M. domestica fruits was assessed by cup-plate method¹⁵ with modifications.

Table 1:	Colour, consistency and yield (%) of Malus domestica fruit extracts

Table 2:	Clinically isolated dental pathogens

Table 3:	Antibacterial activity of M. domestica fruit extracts against dental pathogens
ap<0.05 , aap<0.01, aaap<0.001, radial zone of inhibition of M. domestica fruit extracts against bacteria vs. the normal diameter of cup (8 mm)

Twenty milliliters of the Muller Hinton agar media was poured into sterile petri dishes and left to solidify. The isolated bacterial suspensions (10⁸ CFU mL^–1) were streaked uniformly in two directions at 90° by using sterile swabs on the surface of Muller Hinton agar medium. In each of these plates, 5 cups (8 mm in diameter) were made using a sterile cork borer. Cups were filled with 0.1 mL of each extract of M. domestica (100 mg mL^–1), standard drug, chlorhexidine (0.2% w/v)¹⁶ and normal saline. The plates were then incubated in the upright position at 37°C for 18-24 h. After incubation, the radial zone of inhibition in millimetre was measured¹⁷. Each sample was assessed in triplicate and the data is expressed as Mean±SEM. The results of bacterial sensitivity testing on M. domestica fruit extracts were compared with reference standard and tabulated in Table 3.

Preliminary phytochemical analysis: The highly active extract of M. domestica fruits was subjected to preliminary phytochemical screening by standard methods to identify the presence of secondary metabolites such as alkaloids, amino acids, carbohydrates and glycosides, fixed oils and fats, flavones and flavanones, gums and mucilage, phenolic compounds and tannins, proteins, saponins, sterols and terpenoids¹⁸. The results are tabulated in Table 4.

Statistical analysis: The values are represented as Mean±SEM and the data obtained from this study was subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s t-test.

RESULTS

The percentage yield of extracts of M. domestica fruits was in the range of 3.2-30.3. Aqueous extract had the highest percentage yield followed by ethanol extract and showed the presence of more secondary metabolites which may be soluble in high polarity solvents. The pathogens, gram-positive bacteria such as Staphylococcus aureus, Streptococcus mutans, Lactobacillus subtilis, Gram-negative bacteria such as Klebsiella sp. and Pseudomonas sp. were isolated and identified from 24 patients having dental cavities (Table 2).

Table 3 represents antibacterial activity of all the extracts of M. domestica fruits at concentration of 100 mg mL^–1 against each of the isolated organism. Anti-bacterial activity against the isolated pathogens was assessed by cup plate agar diffusion method and the extracts showed varying degree of inhibitory effect (Table 3). The results showed that the ethanol extract (100 mg mL^–1) has more potent antibacterial activity against S. aureus, S. mutans and Pseudomonas sp., followed by aqueous extract (100 mg mL^–1) whereas, petroleum ether extract (100 mg mL^–1) has not shown any zone of inhibition against the tested microorganisms. The study revealed that the ethanol extract exhibited maximum zone of inhibition compared to the other extracts. All the results were compared with chlorhexidine 0.2% w/v. Also ethanol extract exhibited high significant (p<0.001) antibacterial activity against S. aureus than aqueous extract (p<0.01).

DISCUSSION

The earlier study by Ahmad et al.¹⁹ supported the potent antibacterial activity of ethanol extract (100 mg mL^–1) than aqueous extract. Ahmad et al.¹⁹ screened medicinal plants to detect antimicrobial activity and demonstrated that alcohol is a better solvent as compared to water. Successful prediction of bioactive compounds from plant material is largely dependent on the type of solvents used in the extraction procedure. The pure bioactive compound from active extract could be a good lead antibacterial compound which on further modification can be safe and effective antibiotic. Consequently for a continuous search of new effective and affordable antimicrobial drugs, phytochemical tests on active ethanol extract of M. domestica fruit was carried out which showed the presence of terpenoids, flavonoids, phenolic compounds and tannins (Table 4). The antibacterial activity may be due to the individual phytoconstituents present or in the combination.

Table 4:	Preliminary phytochemical tests of active ethanol extract of Malus domestica fruit
+: Positive, -: Negative

Literature has shown that flavonoids²⁰, phenolic compounds²¹, tannins²² and terpenoids²³ show most of the antibacterial activity. Staphylococcus aureus is specifically more susceptible to phenolic compounds²¹. Subedi et al.²⁴. reported that a type of flavonoid, furocoumarins inhibit bacterial growth by reacting with DNA and disrupting DNA replication. The hydrophobic character of phenolic compounds potentially impairs the cellular function and membrane integrity²⁵. Tannins are responsible for the inactivation of adhesions, enzymes and cell-enveloped proteins of microbes, in conjunction with the ability to bind to the extracellular and soluble proteins²⁶. Anticariogenic activity of nine labdane type-diterpenes and four sesquiterpenes against the microorganisms responsible for dental caries was reported by Souza et al.²⁷. The presence of hydroxyl group in sesquiterpenes, which is an efficient uncoupler of the bacterial plasma membrane creates instability and breaks the membrane’s phospholipid-sterol interactions and is often lethal to microorganisms²⁸. All these observations explain the growth inhibitory activity of extracts of M. domestica fruits and provide good evidence that the fruit of M. domestica has antibacterial effect against the bacterial isolates tested in this study. The results of the present study corroborate the common belief that the fruits of M. domestica are efficacious against dental infections and would justify its further investigation to isolate potential bioactive herbal metabolites for the treatment of tooth decay.

CONCLUSION

This is a preliminary study to evaluate the antibacterial activity of M. domestica fruits. The crude extracts demonstrating antibacterial activity against the isolated pathogens from dental caries could result in the discovery of new chemical classes of antibiotics. Before declaring M. domestica fruit as a potent antimicrobial drug, further research is required on isolation of specific bioactive constituents responsible for antibacterial activity and testing the efficacy at various doses which is under process of our investigation. The study emphasizes the accuracy and efficacy of traditional remedies and inspires people to realize the importance of natural resources for their potent medicinal values.

ACKNOWLEDGMENTS

The authors are grateful to the Management of KPJ Healthcare University College, Kota Seriemas, Nilai, Malaysia, for their funding, continuous encouragement and support.