Subscribe Now Subscribe Today
Research Article
 

Effects of Malus domestica Fruit Extracts Against Clinically Isolated Dental Pathogens



J. Anbu Jeba Sunilson, A.V. Anita Gnana Kumari, Abdullah Khan and K. Anandarajagopal
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

Background and Objective: Medicinal plants have a vital role in the development of novel antimicrobial agents. The fruit Malus domestica is known as apple and commonly used to prevent tooth decay by killing dental pathogens. However, there were no scientific reports about the fruits of Malus domestica having antimicrobial activity against dental pathogens. The aim of this study was to extract the active constituents present in the fruits of Malus domestica by cold maceration method using petroleum ether, ethanol and water. Methodology: The antibacterial activity was assessed by cup plate method against five isolated dental pathogens such as Staphylococcus aureus, Streptococcus mutans, Lactobacillus subtilis, Klebsiella sp. and Pseudomonas sp. and the radial zone of inhibition was measured. Results: The results revealed 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. All the results were compared with standard drug chlorhexidine 0.2% w/v. Conclusion: Hence, the study concludes that Malus domestica fruit extracts possess significant (p<0.001, p<0.01, p<0.05) antibacterial activity against dental pathogens. The phyto chemical tests on ethanol extract showed the presence of bioactive constituents such as terpenoids, flavonoids, phenolic compounds and tannins which could be responsible for the antibacterial activity of M. domestica fruit.

Services
Related Articles in ASCI
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

J. Anbu Jeba Sunilson, A.V. Anita Gnana Kumari, Abdullah Khan and K. Anandarajagopal, 2016. Effects of Malus domestica Fruit Extracts Against Clinically Isolated Dental Pathogens. European Journal of Dentistry and Medicine, 8: 12-16.

DOI: 10.3923/ejdm.2016.12.16

URL: https://scialert.net/abstract/?doi=ejdm.2016.12.16
 
Received: December 30, 2015; Accepted: March 25, 2016; Published: June 15, 2016



INTRODUCTION

Tooth decay also known as dental caries is one of the most common infectious diseases throughout the world and lead to damage the hard tooth structure and causes infections. This is caused by the bacterium that lives in the mouth1. Bacteria, staphylococci and streptococci ferment sugars and produce acids. These acids affect primary decalcification of enamel which leads to total destruction and the decalcification of dentin. Major end products of fermentation are lactic acid, dextrans and levans which are found to cause dental caries2,3.

In recent years many types of toothpaste, mouthwash and antibiotics are introduced, still tooth decay and its related complications continue to be a major medical problem. Over the last decade, the awareness about the ways to prevent the microbial infections has been increasing. Natural products are the fundamental source of the development of new pharmaceuticals search for novel antimicrobials from natural products against pathogens continues to discover more effective and less toxic antimicrobial agents4. Due to the identification of medicinal plants from indigenous pharmacopoeias and the recognition of the value of traditional medical systems, particularly of Asian origin, plants still make an important contribution to health care in modern medicine5. Medicinal plants produce a definite physiological action on the human body due to the presence of some secondary metabolites in the plant tissues which determines the medicinal value of plants6.

Now a days, screening of antimicrobial activity of several medicinal plants has been increased due to the increasing failures of chemotherapeutic agents and antibiotic resistance exhibited by pathogenic microbes7. Malus domestica which belongs to the family, Rosaceae is commonly known as apple. The plant is widely distributed in China, United States, Iran, Turkey, Russia, Italy, Malaysia and India. The fruit of Malus domestica is traditionally used to prevent the tooth decay by killing the dental pathogens. The fruits are consumed worldwide in different forms such as fresh juices and cider8. Their beneficial properties to human health are related to the high content of phenolic compounds, dietary fibre, sugar and vitamins9 which are responsible for curing cancer, cardiovascular diseases, asthma and diabetes10.

Apples contain many types of phenolic derivatives and flavonoids (flavan-3-ols, flavonols, procyanidins, chalcones and anthocyanins). Malus domestica exhibit efficient antioxidant property owing to the presence of its phytoconstituents which are also well known to have anti-inflammatory, antiviral and antimicrobial properties11. However, there was no scientific report for the antimicrobial activity of Malus domestica fruits against dental pathogens. Hence the present work was undertaken to scientifically evaluate the antibacterial activity of M. domestica fruit 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
Image for - Effects of Malus domestica Fruit Extracts Against Clinically Isolated Dental Pathogens

Table 2: Clinically isolated dental pathogens
Image for - Effects of Malus domestica Fruit Extracts Against Clinically Isolated Dental Pathogens

Table 3: Antibacterial activity of M. domestica fruit extracts against dental pathogens
Image for - Effects of Malus domestica Fruit Extracts Against Clinically Isolated 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
Image for - Effects of Malus domestica Fruit Extracts Against Clinically Isolated Dental Pathogens
+: 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.

REFERENCES

1:  Janine, B., 2010. The Clinical Use of Probiotics. In: The Use of Probiotics for Oral Health, Barlow, J. (Ed.). Probiotics International Ltd., United Kingdom, pp: 36-39

2:  Kornman, K.S. and W.J. Robertson, 1985. Clinical and microbiological evaluation of therapy for juvenile periodontitis. J. Periodontol., 56: 443-446.
CrossRef  |  PubMed  |  Direct Link  |  

3:  Vermani, A., Navneet and Prabhat, 2009. Screening of Quercus infectoria gall extracts as anti-bacterial agents against dental pathogens. Indian J. Dent. Res., 20: 337-339.
CrossRef  |  PubMed  |  Direct Link  |  

4:  Saleem, M., M. Nazir, M.S. Ali, H. Hussain, Y.S. Lee, N. Riaz and A. Jabbar, 2010. Antimicrobial natural products: An update on future antibiotic drug candidates. Nat. Prod. Rep., 27: 238-254.
CrossRef  |  PubMed  |  Direct Link  |  

5:  Adebolu, T.T. and S.A. Oladimeji, 2005. Antimicrobial activity of leaf extracts of Ocimum gratissimum on selected diarrhoea causing bacteria in Southwestern Nigeria. Afr. J. Biotechnol., 4: 682-684.
Direct Link  |  

6:  Sheeba, E., 2010. Antibacterial activity of Solanum surattense Burm. F. Kathmandu Univ. J. Sci. Eng. Technol., 6: 1-4.
CrossRef  |  Direct Link  |  

7:  Elizabeth, K.M., 2005. Antimicrobial activity of Terminalia bellerica. Indian J. Clin. Biochem., 20: 150-153.
CrossRef  |  Direct Link  |  

8:  Alberto, M.R., M.A.R. Canavosio and M.C.M. de Nadra, 2006. Antimicrobial effect of polyphenols from apple skins on human bacterial pathogens. Electron. J. Biotechnol., 9: 205-209.
CrossRef  |  Direct Link  |  

9:  Hagen, S.F., G.I.A. Borge, G.B. Bengtsson, W. Bilger, A. Berge, K. Haffner and K.A. Solhaug, 2007. Phenolic contents and other health and sensory related properties of apple fruit (Malus domestica Borkh., cv. Aroma): Effect of postharvest UV-B irradiation. Postharvest Biol. Technol., 45: 1-10.
CrossRef  |  Direct Link  |  

10:  Boyer, J. and R.H. Liu, 2004. Apple phytochemicals and their health benefits. Nutr. J., Vol. 3.
CrossRef  |  Direct Link  |  

11:  Fratianni, F., A. Sada, L. Cipriano, A. Masucci and F. Nazzaro, 2007. Biochemical characteristics, antimicrobial and mutagenic activity in organically and conventionally produced Malus domestica, Annurca. Open Food Sci. J., 1: 10-16.
CrossRef  |  Direct Link  |  

12:  Sunilson, J.A.J., K. Anandarajagopal, A.V.A.G. Kumari and S. Mohan, 2009. Antidiarrhoeal activity of leaves of Melastoma malabathricum Linn. Indian J. Pharmaceut. Sci., 71: 691-695.
CrossRef  |  PubMed  |  Direct Link  |  

13:  Sakazaki, R.S.T., 1986. Vibro Species as Causative Agent of Food-Borne Infection. In: Development of Food Microbiology, Robinson, R.K. (Ed.). Elsevier, London, pp: 123-151

14:  Cheesborough, M., 2006. Biochemical Test to Identify Bacteria. In: District Laboratory Practice in Tropical Countries, Volume 2, Cheesborough, M. (Ed.). 2nd Edn. Cambridge University Press, Cambridge, ISBN: 9780521676311, pp: 63-87

15:  Rashmika, P.P., 2011. Evaluation of antibacterial activity of methanol extracts of leaves and aerial parts of Corchorus aestuans Linn. Int. Res. J. Pharm., 2: 228-230.
Direct Link  |  

16:  Phatak, A.A., R.D. Patankar, U.C. Galgatte, S.Y. Paranjape, A.S. Deshpande, A.K. Pande and R. Thombre, 2011. Antimicrobial activity of a poly-herbal extract against dental micro flora. Res. J. Pharmaceut. Biol. Chem. Sci., 2: 533-539.
Direct Link  |  

17:  Iroha, I.R., A.E. Oji, O.K. Nwosu, E.S. Amadi 2011. Antimicrobial activity of Savlon®, Izal® and Z-Germicide® against clinical isolates of Pseudomonas aeruginosa from hospital wards. Eur. J. Dentistry Med., 3: 32-35.
CrossRef  |  Direct Link  |  

18:  Krishnaiah, D., T. Devi, A. Bono and R. Sarbatly, 2009. Studies on phytochemical constituents of six Malaysian medicinal plants. J. Med. Plants Res., 3: 67-72.
Direct Link  |  

19:  Ahmad, I., Z. Mehmood and F. Mohammad, 1998. Screening of some Indian medicinal plants for their antimicrobial properties. J. Ethnopharmacol., 62: 183-193.
CrossRef  |  PubMed  |  Direct Link  |  

20:  Cushnie, T.P.T. and A.J. Lamb, 2005. Antimicrobial activity of flavonoids. Int. J. Antimicrob. Agents, 26: 343-356.
CrossRef  |  PubMed  |  Direct Link  |  

21:  Subedi, A., M.P. Amatya, T.M. Shrestha, S.K. Mishra and B.M. Pokhrel, 2012. Antioxidant and antibacterial activity of methanolic extract of Machilus odoratissima. Kathmandu Univ. J. Sci. Eng. Technol., 8: 73-80.
Direct Link  |  

22:  Akiyama, H., K. Fujii, O. Yamasaki, T. Oono and K. Iwatsuki, 2001. Antibacterial action of several tannins against Staphylococcus aureus. J. Antimicrob. Chemother., 48: 487-491.
CrossRef  |  Direct Link  |  

23:  Chung, P.Y., P. Navaratnam and L.Y. Chung, 2011. Synergistic antimicrobial activity between pentacyclic triterpenoids and antibiotics against Staphylococcus aureus strains. Ann. Clin. Microbiol. Antimicrob. Vol. 10.
CrossRef  |  

24:  Seyyednejad, S.M., S. Maleki, N.M. Damabi and H. Motamedi, 2008. Antibacterial activity of Prunus mahaleb and Parsley (Petroselinum crispum) against some pathogen. Asian. J. Biol. Sci., 1: 51-55.
CrossRef  |  Direct Link  |  

25:  Safary, A., H. Motamedi, S. Maleki and S.M. Seyyednejad, 2009. A preliminary study on the antibacterial activity of Quercus brantii against bacterial pathogens, particularly enteric pathogens. Int. J. Bot., 5: 176-180.
CrossRef  |  Direct Link  |  

26:  Rao, A.S., A.K. Nayanatara, S.R. Kaup, S. Arjun and B.A. Kumar et al., 2011. Potential antibacterial and antinfungal activity of aqueous extract of Cynodon dactylon. Int. J. Pharmaceut. Sci. Res., 2: 2889-2893.
Direct Link  |  

27:  Souza, A.B., C.H. Martins, M.G.M. Souza, N.A.J.C. Furtado and V.C.G. Heleno et al., 2011. Antimicrobial activity of terpenoids from Copaifera langsdorffii Desf. against cariogenic bacteria. Phytother. Res., 25: 215-220.
CrossRef  |  PubMed  |  Direct Link  |  

28:  Solis, C., J. Becerra, C. Flores, J. Robledo and M. Silva, 2004. Antibacterial and antifungal terpenes from Pilgerodendron uviferum (D. Don) Florin. J. Chilean Chem. Soc., 49: 157-161.
CrossRef  |  Direct Link  |  

©  2021 Science Alert. All Rights Reserved