|Year : 2015 | Volume
| Issue : 2 | Page : 74-78
An exploration of the effects of Commiphora glileadenis on a Streptococcus mutans biofilm
Hossam A Eid1, Yhya H Assiri2, Manea M Musleh3, Tarek H Taha4, Nehal M El-Deeb5, Gamal M Hamad6
1 Department of Oral Medicine & Periodontology, Faculty of Dentistry, Suez Canal University, Egypt, Department of Periodontics, College of Dentistry, Gulf Medical University, Ajman, United Arab Emirates
2 Intern Dentist, King Khalid University, Saudi Arabia
3 Department of Dentistry, Ministry of Health, Saudi Arabia
4 Environmental Biotechnology Department; Genetic Engineering and Biotechnology Research Institute (GEBRI); City of Scientific Research and Technology Applications (SRTA- CITY), New Borg El-Arab, Alexandria city, Egypt
5 Biopharmaceutical Product Research Department; Genetic Engineering and Biotechnology Research Institute (GEBRI); City of Scientific Research and Technology Applications (SRTA- CITY), New Borg El-Arab, Alexandria city, Egypt
6 Food Technology Department, Arid Land Cultivation Research Institute (ALCRI); City of Scientific Research and Technology Applications (SRTA- CITY), New Borg El-Arab, Alexandria city, Egypt
|Date of Web Publication||14-Jul-2015|
Hossam A Eid
Department of Oral Medicine and Periodontology. Faculty of Dentistry, Suez Canal University, Ismailia
United Arab Emirates
Source of Support: None, Conflict of Interest: None
Streptococcus mutans (S. mutans) is an important bacterium involved in the first steps of dental biofilm formation with subsequent initiation of several oral diseases.
Aim: To evaluate the influence of Myrrh plant extract on S. mutans and its biofilm formation.
Material and Methods: The antibacterial activity of the Myrrh prepared plant extract was tested against S. mutans using disc diffusion method. The ability of the Myrrh plant extract to inhibit biofilm formation of S. mutans was also determined.
Results: showed the ability of the tested Myrrh oil plant extract to cease the bacterial growth. The recorded clear zone is 11 mm with extract concentration 42.5%. Myrrh extract was able to inhibit the S. mutans biofilm formation in 78.5% inhibition percentage on fibroblast cellular viability.
Conclusion: Myrrh plant extract has promising antibacterial effect as well as on biofilm inhibition of S. mutans. The significant antimicrobial effect of the Myrrh plant extract indicates about its promise in controlling S. mutans biofilm, which has suspected role in the etiology of dental caries and periodontal diseases.
Keywords: Dental biofilm, myrrh extract, Streptococcus mutans, Southwestern Saudi Arabia
|How to cite this article:|
Eid HA, Assiri YH, Musleh MM, Taha TH, El-Deeb NM, Hamad GM. An exploration of the effects of Commiphora glileadenis on a Streptococcus mutans biofilm. Saudi J Oral Sci 2015;2:74-8
|How to cite this URL:|
Eid HA, Assiri YH, Musleh MM, Taha TH, El-Deeb NM, Hamad GM. An exploration of the effects of Commiphora glileadenis on a Streptococcus mutans biofilm. Saudi J Oral Sci [serial online] 2015 [cited 2019 Dec 15];2:74-8. Available from: http://www.saudijos.org/text.asp?2015/2/2/74/160767
| Introduction|| |
Bacterial biofilm-dependent oral diseases such as periodontal, dental caries and endodontic diseases are major worldwide medical, economic and public health issues.  The specific conditions of the oral cavity may develop infectious foci that could affect other human body systems. Dental plaque is an archetypical biofilm found naturally on teeth composed of a complex microbial biota represents a highly diverse biofilm. , Recent advances in molecular microbiology have improved the understanding of dental plaque biofilm, which considered the etiological agent for most of the dental diseases such as dental caries and periodontal disease. ,
Periodontal diseases affect the soft tissues and bone (periodontium) that support the teeth. Caries is a unique infection of the dental hard tissues (enamel, dentin and cementum). , The initiation of both diseases is marked by an increase in the complexity of the microbiome. , In periodontitis, a keystone pathogen, Porphyromonas gingivalis impairs host immune responses and appears necessary but not sufficient to cause periodontitis. ,, However, dental caries had been linked to S. mutans. Contemporary microbiome studies now indicate that singular pathogens are not obvious in either caries or periodontitis. ,, Both diseases appear to result from a perturbation among relatively minor constituents in local microbial communities resulting in dysbiosis, as they act synergistically to stress the ability of the host to respond and protect. ,,
Streptococci are commensal bacteria that pioneer oral film colonization and may be involved in severe systemic diseases, such as infective endocarditis  and atherosclerosis.,, S. mutans is an important bacterium involved in the first step of biofilm formation with subsequent initiation of some oral diseases ,,,
Myrrh is routinely used as a home remedy among the local Saudi population due its antibacterial and analgesic effects especially in Southwestern area of Saudi Arabia. ,,, It is an oleo-gum resin obtained from the tree Commiphora molmol, and the shrub-like tree Balsamodendron myrrh, which grow in the northern and eastern parts of Africa and Arabia. Myrrh consists of 7-17% volatile oil, 25-40% resin, 57-61% gum, and 3-4% impurities. ,, Several researchers have discussed the use of myrrh in the treatment of ulcers, schistosoma, respiratory catarrh, diabetes, and topically for wounds and abrasions. , A study by Azizah Al-Mobeeriek 2011  stated that the toxicity of myrrh may, therefore, preclude its use in the raw form, but it could be used in the solution. A controlled and low concentration of myrrh solution could aid in faster healing as an adjunct or as a replacement for the current therapies available. Hence, the aim of this work was directed to test the possibility of using the myrrh plant extract against cariogenic S. mutans bacteria. This to stop and/or prevent the synergistic effect between S. mutans and P. gingivalis hence, we can stop dental caries, and periodontal diseases process progression.
| Materials and Method|| |
Bacterial strain and preservation
Streptococcus mutans ATCC 25175, DSM No: 20523 reference strain was obtained from MERCIN, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. The bacterial strain was preserved by adding 250 μL of 60% glycerol to 750 μL Luria broth (LB) culture and kept at 80°C.
Preparation of plant extract
The suggested probable myrrh plant [Figure 1] Commiphora glileadenis was collected from local herbal area from mountain at Aseer region, Southwestern of Saudi Arabia and were submitted to the standard extraction procedures according to Borenfreund and Puerner (1985)  with some modifications. The obtained plant was washed three times using tape water, dried and 10 g of the plant was submitted to extraction using 100 ml of ethanol/water (3:1). The plant/solvent mixtures were shaken at 200 rpm and 30°C for 18 h. The mixture was then spinning down at 3000 rpm for 30 min and the obtained supernatant was evaporated at 50°C for 3 days. The obtained pellet was weighed and dissolved in sterile distilled water and kept at 4°C till use.
Antibacterial activity against Streptococcus mutans
The antibacterial activity of the myrrh (C. glileadenis) prepared plant extract was tested for their antibacterial activity against S. mutans using disc diffusion method according to (Duraipandiyan and Ignacimuthu, 2007)  with some modifications. LB agar media was prepared according to the manufacture instructions (Oxoid, England), sterilized and poured into sterile Petri plates. Overnight culture (18 h) of S. mutans was freshly prepared using sterile LB broth. The bacterial strain was diluted with sterile saline solution to reach 0.5 McFarland standard and was then spread over the LB plates using sterile cotton swabs. After dryness of the plate's surfaces, sterile filter paper discs were added to the plates where 25 μl of the plant extract was loaded to the filter discs and the plates were then kept at 4°C for 30 min. The plates were then incubated at 30°C for 18 h and were checked for clear zones formation. The formed clear zones were recorded and measured in millimeter.
Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was determined using descending concentrations of the myrrh plate extract. The highest plant concentration was diluted using sterile saline and was tested for their antibacterial activity against S. mutans. The MIC was determined using different liquid cultures of S. mutans submitted to the prepared dilutions of myrrh plant extract. Using 96-well flat-bottom microtiter polystyrene plate; 150 μl of an overnight culture of the strain was mixed with the prepared extract dilutions and were incubated at 30°C for 48 h. After incubation, the optical density (OD) of the bacterial growth was measured at 600 nm using (Spectrophotometer).
Quantitative assay of biofilm inhibition
The ability of the myrrh plant extract to inhibit biofilm formation of S. mutans was determined according to Ahsan et al.,  with some modifications. In brief, triplicates of 100 μL of a previously prepared overnight bacterial culture in LB were inoculated in 96-well flat-bottom microtiter polystyrene plate with 50 μL of the nontoxic dose of the treatments. The plate was incubated for 48°C at 30°C without shaking. The plate was decanted once and followed by washing for three times with 200 μL sterile phosphate buffered saline buffer. The plate was then dried at 60°C for 1 h. The remaining biofilm was stained with 0.1% crystal violet for 15 min, decanted and washed three times with 200 μL of sterile distilled water. The plate was air dried for 15 min followed by the addition of 150 μL of 95% ethanol. The absorbance was measured at 570 nm using ELISA assay plate reader. S. mutans ATCC 25175 strain was used as the positive control and un-inoculated LB broth as negative control.
Human fibroblast cell line was washed twice with Dulbecco's modified eagle's medium (DMEM) media supplemented with 200 μM L-glutamine and 25 μM hydroxyethyl-piperazine ethanesulafonic buffer; (all chemicals and media, Cambrex). About 2 × 10 5 cells were suspended in DMEM culture media with 10% fetal bovine serum (Gibco-BRL). The cells were left to adhere on the polystyrene 6 well plates for 4 h in an incubator (37°C, 5% CO 2 , 95% humidity). The cells were washed twice from debris and dead cells by using RPM1 supplemented media.
Cytotoxicity and proliferation assays.
To detect the nontoxic or recommended dose of the plant extract that does not exert any toxic effect, Human fibroblast cell was used as normal cell model. The cytotoxicity assay was performed according to the protocol described by Borenfreund and Puerner (1985)  To that end, about 6 × 10 4 cell/ml cell suspension was seeded in 96-well plates, after cellular reached a semi confluences, 100 μl of different plant extract concentrations was added. After 3 days of incubation, cells were stained with 100 μl of neutral red stain for 3 h. Only living cells have ability to accumulate neutral red into liposomes providing a quantitative assay to the cytotoxic effects. The stain intensity was assayed using automated enzyme-linked immunosorbent assay microplate reader adjusted at 540 nm (reference filters 620 nm).
| Results|| |
Antibacterial activity against Streptococcus mutans
The ability of myrrh plant extract to inhibit the growth of S. mutans was detected using disc diffusion assay. The results revealed that myrrh extract at 20% concentration was able to stop and cease the bacterial growth by 11 mm clear zone.
As depicted in [Figure 2], the S. mutans strain fail to grow at the presence of diffused components of myrrh extract.
|Figure 2: Detected clear zone of Streptococcus mutans at the presence of myrrh plant extract|
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Determination of minimum inhibitory concentration
The estimation of the lowest myrrh extract that able to inhibit the growth of S. mutans was achieved spectrophotometrically. Different dilutions of the plant extract were added to the bacterial strain broth culture for 48 h and the OD was measured at 600 nm. As shown in [Figure 3], the recorded MIC that able to stop the bacterial growth was 5%. This result could be confirmed by the failure of 2.5% to inhibit the bacterial growth.
|Figure 3: Minimum inhibitory concentration of myrrh extract against Streptococcus mutans at 600 nm optical density|
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The myrrh plant extract was tested for its ability to inhibit the biofilm formation of S. mutans. The obtained results revealed the success of the extract to inhibit the biofilm formation compared with the positive control (S. mutans with no additions) as shown in [Figure 4]. The myrrh extract was able to partially stop biofilm formation by 42.5%.
|Figure 4: Biofilm inhibition of Streptococcus mutans by the myrrh extract|
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Cytotoxicity of the plant extract
The cytotoxicity results of the extracts on fibroblast cells concluded that, generally, the maximum herbal concentration (200 mg/ml) recorded 78.8% inhibition percentage on cellular viability as shown in [Figure 5]. In addition, the IC50 of the extract on cells reached 25 mg/ml. The general outlook indicated that, the nontoxic and recommended dose of the extract that record 90% cellular viability was between 12.5-6.25 mg/ml.
|Figure 5: Cytotoxicity assay of myrrh plant extract against human fibroblast cell line|
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| Discussion|| |
Efforts have been made to develop the new treatment of natural sources for the management of oral diseases, and, therefore, for the prevention of subsequent systemic illnesses. New drugs and materials have been challenged to prevent and treat these conditions, especially by means of bacteria elimination either by its destruction or prevent the formation of the biofilm, which is crucial for its existence. , Besides, The serious increase in the nonsensitivity of microbes to conventional therapeutic agents may be open a door to search for alternative approaches for exploring of new drugs from available local natural resources. The use of myrrh is common among Saudis, but few studies have been conducted on its clinical and general use, side-effects, and interactions with other drugs.  According to results of this study myrrh (C. glileadenis) plant extract, was able to inhibit the growth of S. mutans at 20% concentration to stop and cease the bacterial growth by 11 mm clear zone. As depicted in [Figure 2], the S. mutans strain fail to grow at the presence of diffused components of myrrh C. glileadenis extract.
As well as myrrh C. glileadenis plant extract was tested for its ability to inhibit the biofilm formation of S. mutans. The obtained results revealed the success of the extract to inhibit the biofilm formation compared with the positive control (S. mutans with no additions) as shown in [Figure 4]. The myrrh extract was able to partially stop biofilm formation by 42.5%. The cytotoxicity results of the extracts on fibroblast cells concluded that, generally, the maximum herbal concentration (200 mg/ml) recorded 78.8% inhibition percentage on cellular viability as shown in [Figure 5]. In addition, the IC50 of the extract on cells reached 25 mg/ml. The general outlook indicated that, the nontoxic and recommended dose of the extract that record 90% cellular viability was between 12.5-6.25 mg/ml. These results share the previous studies, which indicated that myrrh acts as an antiseptic, anti-inflammatory, and a circulatory and immune system stimulant , It has also been shown to stimulate plasma cell production and induce angiogenesis and, therefore, remodeling within 7 days of an injury occurring. , Accordingly, The investigation demonstrated that myrrh is biocompatible with soft and even hard tissues to some extent since it induced a severe inflammatory reaction in the rats (rather than an autoimmune reaction). In addition, the oleo-resin present in myrrh has been shown to be more toxic than its oil. It can, therefore, be assumed that its toxicity is dose dependent, , However, Azizah et al., 2011  study indicated that the response to a concentrated dose of myrrh can be very toxic locally, causing pronounced inflammation (especially if the wound is open). The toxicity of myrrh may, therefore, preclude its use in the raw form, but it could be used in the solution.
Since the presence of dental plaque biofilm is mandatory for initiation of oral diseases that affects both soft and hard tissues with subsequent dental caries and periodontal diseases establishment. The success of myrrh C. glileadenis plant extract in ceasing and inhibiting S. mutans growth. Besides, inhibition of biofilm formation can provide a new method by using myrrh C. glileadenis plant extract in different forms to control the progression of dental caries and periodontal diseases.
Further research is required to clarify the various properties of the myrrh C. glileadenis plant herbs, to determine their optimal dose, forms, and its adverse effects and toxicity. Other factors should be also considered, such as the type and location of the oral diseases. The absence of control group is a limitation of this study, which should be considered in the future investigation for myrrh C. glileadenis plant herbs.
| Conclusion|| |
Results of this study revealed that the myrrh plant extract has promising antibacterial effect as well as on biofilm inhibition of S. mutans.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]