Antimicrobial Evaluation of Some Dental remedial Plant Extracts from Pakistan  

Ammara Aassan1 , Quratulain Syed1 , I. Amjad2 , Abida Hassan3
1 Pakistan Council of Scientific & Industrial Research, Lahore, Pakistan
2 Haleebfoods Pvt Ltd, Pakistan
3 GCU Faisalabad, Pakistan
Author    Correspondence author
Medicinal Plant Research, 2012, Vol. 2, No. 3   doi: 10.5376/mpr.2012.02.0003
Received: 09 Sep., 2012    Accepted: 14 Sep., 2012    Published: 26 Sep., 2012
© 2012 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hassan et al., 2012, Antimicrobial Evaluation of Some Dental remedial Plant Extracts from Pakistan, Vol.2, No.3 11-18 (doi: 10.5376/mpr.2012.02.0003)


The study was conducted to determine antimicrobial activity of stem extracts popularly used in folk medicine to treat dental plague and caries in human. The sampling was done during the months of May, June and July 2011. Two methods were employed for the determination of antimicrobial activities, an agar well diffusion method and determination of MIC. The aqueous, ethanolic, and hexane extracts were assayed for antimicrobial activities. The following bacterial strains were employed in the screening studies: Streptococcus mutans, Enterococcus faecalis, Prophyromonas gingivalis, Streptococcs sobrinus, Lactobacillus acidophilus, Lactobacillus plantum, Streptococcus sanguis, Actinomyces viscosus, Lactobacillus casei, Streptococcus salivarius, Staphylococcus aureus, Bacillus sublitis, Streptococcus viridians, Escherichia coli, Aspergillus niger, Penicillum notatum and Candida albicans. The results has revealed significant antibacterial effect of the ethanol extract. The study thus justifies ethanolic medicinal use of the plants as a dental plague remedy. Key words: aqueous, ethanolic, hexane extracts, antimicrobial organisms, minimum inhibitory concentration (MIC)

Aqueous; Ethanolic; Hexane extracts; Antimicrobial organisms; Minimum inhibitory concentration (MIC)

Herbal medicine is an integral part of traditional medicines. There are usually simple botanicals employed in their more or less crude form and sometimes as food supplements (Shaw et al., 1997). Herbal dentifrices are gaining popularity and high patronage in the western countries (Harmmer–Been et al., 2006). Many studies have been shown that the herbal dentrifices are as good as the conventional ones (in controlling oral bacterial load) (Van der weijden et al., 1998; Mullally et al., 1995; Tanner and Still Man, 1993).

Dental plague, a microbial film on the tooth surface, plays an important part in the development of caries and periodontal diseases (Marsh, 1992). Mutans streptococci can colonize the tooth surface and initiate plague formation by their ability to synthesize extracellular polysaccharides from sucrose, mainly water-soluble glucan, using glycosyltransferase (Gibbons et al., 1975; Hamada and Slade 1980; Jacquelin 1995). De novo synthesis of water-insoluble glucan is essential for the adherence of Streptococcus mutans and other oral microorganisms to the tooth surface, forming a barrier that prevents the diffusion of acids produced by the bacteria. The acids accumulate in situ and decalcify minerals in the enamel. This sucrose-dependent adherence and accumulation of carcinogenic streptococci is critical to the development of pathogenic plague (Schu¨pbach et al., 1995; Slots and Rams1992). To avoid dental caries due to the carcinogenic bacteria, inhibition of glucosyltransferase activity by the specific enzyme inhibitors (Roga 1982; Yanagida 2000), inhibition of intial cell adherence of S. mutans by polyclonal and monoclonal antibodies (Raamsdonk et al., 1995), and inhibition of cell growth of S. mutans by antibacterial agents have been investigated. The third line of research has attracted a great deal of attraction, and effective antimicrobial agents against these oral pathogens could play an important part in the prevention of dental caries and periodontal diseases, particularly those that affect plague formation (Kubo et al., 1992; kubo et al., 1993; Tsutiya et al., 1994; Watanabe, 2000).

The present study has undertaken to establish the scientific bases for the traditional use of the stem of Mangifera indica (mango), Jacaranda Mimosifolia (sukh Azhardicta indica (Neem chan), Salvadora persica (Pilu), Acacia nilotica (kikar), which are used as a dental remedy and to identify the possible active principles and to appraise its clinical potential.

1 Results and Discussion
1.1 Antimicrobial activity of extracts
The antimicrobial activities of stems of the different plant extracts are indications of either difference in nature of the constituents or in concentration. Overall, the ethanol extracts showed higher anti microbial activity as well its potency (MIC). In the case of Mangifera indica, there is no indication of anti microbial activity of aqueous extract against fungus while hexane extract showed slight activity against Aspergillus niger. The ethanolic extract of Jacaranda Mimosifolia has showed moderate antifungal activity while the hexane and aqueous extract were non-active. The aqueous extract has showed good activity against streptococcs sobrinus while hexane extract against streptococcus salivarius and Staphylococcus aureus. The results of Salvadora persica (Pilu) showed that again the ethanolic extract is more active than others. The aqueous extract is more active against streptococcs sobrinus but not for fungus, on the other hand the hexane extract has showed moderate activity against Aspergillus niger and Penicillum notatum. The aqueous extract of Acacia nilotica (kikar) has showed good result against streptococcs sobrinus while moderate against all microorganisms for the hexane extract. The overall activity of Azhardicta indica (Neem) extracts were found more active than others, it also showed good results against fungal strains in the case of aqueous extracts, but overall the ethanolic extract showed its good potential against microbes.

1.2 MIC determination
The MIC results of M. indica has showed significant MIC against Aspergillus niger, Penicillum notatum and Candida albicans at 5.0, 10, and 15 while no results were found for J. mimosifolia and S. persica against fungus. A .nilotica and A. indica have high potency for Aspergillus niger, Penicillum notatum but no results were found for Candida albicans. Among the all five plant extracts the A. indica is found better against bacterial activity but M. indica has showed better results against fungus (Table 1; Table 2; Table 3; Table 4; Table 5; Figure 1; Figure 2; Figure 3; Figure 4; Figure 5).

 Table 1 Comparative Zones of inhibition (mm) for Mangifera indica (mango)

 Figure 1 Comparative zones of inhibition (mm) for Mangifera indica (mango)

 Table 2 Comparative zones of inhibition (mm) for Jacaranda Mimosifolia

 Figure 2 Comparative zones of inhibition (mm) for Jacaranda Mimosifolia

 Table 3 Comparative zones of inhibition (mm) for Salvadora persica (Pilu)

 Figure 3 Comparative zones of inhibition (mm) for Salvadora persica (Pilu)

 Table 4 Comparative zone of inhibition for Acacia nilotica (kikar)

 Figure 4 Comparative zone of inhibition for Acacia nilotica (kikar)

 Table 5 Comparative zones of inhibition for Azhardicta indica (Neem)

 Figure 5 Comparative zones of inhibition for Azhardicta indica (Neem)

It shows good clinical significance. Its traditional use as a chewing stick, remedy for dental and oral infection two major purposes of maintaining good oral hygiene and preventing formation of biofilm.

The findings from this studies revealed that the herbal remedy under investigation has shown some good scientific bases for its use as a herbal dental remedy due to its significant antimicrobial activities. These plant extracts can be used in the toothpastes or toothpowders in order to prevent and control the dental biofilm formation.

2 Conclusion
The study was able to produce scientific bases for the traditional use of stems of Mangifera indica (mango), Jacaranda Mimosifolia (sukh Azhardicta indica (Neem chan), Salvadora persica (Pilu), Acacia nilotica (kikar) as a dental remedy. However the clinical potential could not be appraised due to the lacking of toxicological data. Additional research is needed to improve the safety of traditional herbal remedy. More research work is required to enhance its stability, bioavailability, and pharmacology of the product. Further study is needed for determination of active anti-microbial constituents and structural relationship which can help in the synthesis of effective and safe drugs; using spectroscopic techniques such as Nuclear magnetic resonance NMR, infra red spectrophotometry IR, mass spectrometery, UV and elemental analysis.

3 Materials and Methods
3.1 Collection and identification of plant material
A large quantity of the stem of was collected from the locally available resources (Pakistan) between May to July 2011. The plants were identified and authorized by Dr.( Retd) Saber of the Department of Botany, Lahore. The material was sun dried for one week. This was further subjected to air-drying for another week.

3.2 Extraction and preparation of extracts
The dried stem material was then reduced to very fine powder using a mechanical grinder. The pulverized material (500g) of each plant was transferred into the soxhlet extractor and subjected to different solvents (ethanol, and hexane) and autoclaved distilled water. The extracts were concentrated to dryness using rotary evaporator at reduced pressure. The extracts were further dried to semi-solids with the aid of an oven at 20℃. the dried extract was stored in the refrigerator at a temperature of -4℃ until use.

3.3 Antimicrobial activity
Microbial strain of Streptococcus mutans, Enterococcus faecalis, Prophyromonas gingivalis, streptococcs sobrinus, Lactobacillus acidophilus, lactobacillus plantum, streptococcus sanguis, Actinomyces viscosus, lactobacillus casei, streptococcus salivarius, Staphylococcus aureus, Bacillus sublitis, Streptococcus viridians, Escherichia coli, Aspergillus niger, Penicillum notatum and Candida albicans were all isolated from the patients of dental caries/plague of local dental cilinics and hospitals.

Antimicrobial screening –Agar well diffusion method was used (NCCLS, 1999; Bailey and Elvyn, 1970; Barry and Thornsberry, 1985; cruicksbank et al., 1975; Silva et al., 1996). Inocula of test organisms obtained from sources were obtained by growing each pure isolates in nutrient broth (Oxoid) for 18 h at 35℃. 0.2 mL was then used to seed a molten nutrient agar medium cooled to 45℃. This was poured unto sterile Petri dishes and used for analysis.

The fungal isolates were treated in the different way. They were first grown in Sabourand dextrose broth (Oxoid) and assayed using Sabourand agar. The fungal isolates were incubated at 25℃ for 72 hours.

Extracts were tested at 100 mg concentration. This was prepared by dissolving 1g of crude extract in 2.5 mL of autoclaved distilled water to pour 400 mg/mL 0.25 mL was then delivered into wells (8 mm in diameter) bored unto the surface of nutrient agar plates.

Commercial antibiotics prepared with ciprofloxacin 25 µg and amoxicillin 25 µg were used in parallel in the plates. The plated were incubated at 37℃ for 24 h and the zones of inhibition were measured in millimeter diameter and recorded.

3.4 Determination of Minimum Inhibitory concentration (MIC)
The agar-well diffusion method was also used using method of Rajbhandari and Schöpke (1999). The extracts were incorporated into molten nutrient agar at concentrations of 2.0 mg/mL, 2.5 mg/mL, 5.0 mg/mL, 7.5 mg/mL, 10 mg/mL, 15 mg/mL, and 20 mg/mL (Table 6). A loopful of the test isolates diluted 106 cfu mL was used to streak the plates and incubated. The minimum inhibitory concentration of the extract was regarded as the lowest concentration that did not permit growth of the test organism.

 Table 6 Minimum inhibitory concentration (MIC) (mg/mL)

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