Studying the Affects of Salvia officinalis and Commiphora myrrha Extracts on Poly Methyl Methacrylate Acrylic (PMMA) and Flexible Acrylic Materials Exposed to Escherichia coli

 

Basim Shareef Ensaif¹, Mohammed Ridha H. Al-Rubaie², Dawood Salim Edan³

¹Dentistry Department, Usoul Aldeen University College, Baghdad –Iraq

²Prosthodontic Department, College of Dentisy. Albian University. Baghdad –Iraq

³Experimental Therapy Department, Iraqi Center For Cancer and Medical Genetics Research, Baghdad –Iraq

 

ABSTRACT:

Myrrha and Sage extract solutions are promising prophylactic and disinfectant agents against microorganisms, suggesting that these solutions might be useful as antimicrobial topical solutions for dental contamination. Three different concentrations of Myrrha and Sage extract solutions 2%, 5% and 10% were separately exposed to the E.coli and chlorohexidine solution that used as control to treat the PMMA and Flexible Acrylic plates, the percentage of colonies reductions were calculated. In case of using Sage extract solutions, the colonies reductions were 82.70%, 90.65% and 94.61% respectively for PMMA plates compared with 97.86% reduction for Chlorohexidine with mean p-value 0.0023, 0.0025, 0.0026 and 0.0009 respectively. As regard Myrrha extract solutions, the colonies reductions for Acrylic flexible plates were 91.75%, 96.15%, 97.08% respectively, compared with 97.91% reduction for Chlorohexidine with mean p-value (0.0017, 0.0010, 0.0005 and 0.0006) respectively. This research considers both Sage and Myrrha and extract solutions of different plants are promising solutions and capable of using them as alternative anti plaque solutions for either PMMA and Flexible Acrylic plates.

 

 

INTRODUCTION:

E. coli is a Gram-negative, facultative aerobic, coliform bacterium of the genus Escherichia that is generally found in the lower intestine of warm-blooded organisms (endotherms). Tenaillon et al. (2010) Most E. coli strains are useful, but others can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination. Vogt RL, et al. (2005).

 

 

 

 

 

 

And if found in contamination is often found together with staphylococci And may produce staphylococcus proteins therefore it may responsible for colonization and virulence, Curtiss et al. (1983) but the harmless strains are part of the normal microbiota of the gut, and can benefit their hosts by producing vitamin K₂, and preventing colonization of the intestine with pathogenic bacteria, having a symbiotic relationship. Bentley R, et al. (1982), Hudault S, et al.(2001) and Reid G, et al. (2001).

 

E. coli and other anaerobes constitute about 0.9% of gut microbes, and fecal–oral transmission is the essential route through which pathogenic strains of the bacterium can cause disease. Cells can be able to survive outside the body for a limited time, which makes them potential indicator organisms to test environmental samples for  contamination in feces. Eckburg PB, et al.(2005) and Feng P et al. (2002) E. coli has been studied and previously associated in maxillary osteomyelitis in the jaw. Padhiary, S. K. et al.(2013).

Some Plant extracts are used in past as prophylactic and disinfectant agents against micro-organisms especially bacterial and fungal organisms, and it was recommended in previous time as anti-microorganisms, Replacing the requirements in current time to the antibiotics, chemical detergents or anti fungal drugs. Denture composition are made of either thermally activated poly-methyl methacrylate Acrylic [PMMA] or flexible acrylic which is composed of thermoplastic flexible polymer. The suggested solutions of plant extract are derived here of different plants: Salvia officinalis and Commiphora myrrha.

 

Salvia officinalis has numerous common names. One of the best-known name is Sage, it has been used since ancient times as a diuretic and used as a local anesthetic for the skin, and for other uses (Watters, L. L. (1901). Commiphora myrrha or called as Myrrha has also been recommended as an analgesic for toothaches and can be used in liniments for bruises, aches, and sprains. Myrrh is a common ingredient of tooth powders. Myrrh and borax in tincture can be used as a mouth-wash. A compound tincture, or horse tincture, using myrrh is used in veterinary practice for healing wounds (Al Faraj, S (2005).

 

The purpose of this study is to evaluate the antimicrobial activity of Salvia officinalis and Commifora myrrha plant extract solutions on Poly Methyl Methacrylate Acrylic [PMMA] and flexible Acrylic dental materials which contaminated with very normal and common microbe called E.coli compared to chemical detergents such as chlorohexidine.

 

MATERIAL AND METHODS:

Preparation of samples:

The Sage and Myrrha powders were purchased from formal Iraqi agriculture market. Then were prepared 2%, 5% and 10% by dissolving 2 gm, 5 gm and 10 gm respectively of each above one in 100 mL of D.W then vortex until complete dissolving and left in 4 °C after filtrations by (Millipore filter 40µmiter).

 

Preparation and construction of Poly Methyl Metha Acrylate (PMMA) plates:

According to (Williams BL, et al. 1983), acrylic plates are constructed as bellow from the poly methyl methacrylate [PMMA], which are supplied in form of powder (polymer) and liquid (monomer).

 

Pieces of modeling wax sheets are prepared to the dimensions: length: 7 mm, width : 30 mm, and thickness :2 mm. The wax pieces were adapted and butting them in a flask filled with plaster gypsum material then the upper and lower flasks were closed. The flask was put in hot water path about 100 C^o temperature for 15 minutes then cooled and put hot water to clean the flask from melted wax ( wax elimination ). The acrylic resin was put in the space of eliminated wax, which is prepared by powder- liquid ratio of 2.5:1 by weight, then heated in hot water bath (100^oC) for (30) min. to activate the polymerization of PMMA. This is a simple molding processing technique. the separation of the flasks is started "Deflasking". Then removing the acrylic plates and start finishing the products which means removing the excess fragments of acrylic material, smoothing the plastic and polishing the surface to increase penetration surface resistance.

 

Preparation and construction of Flexible acrylic plates:

Flexible acrylic resin is a thermoplastic flexible polymer composition offered for use in a wide range of applications, one of them is the fabrication of removable dental prosthesis (Cotrim LEF, etal. 2001).

 

Stone gypsum material is used for flaking which can resist the high temperatures that needed for polymerization of the flexible acrylic resin. First, heating degree should reach to 237 ^oC under high pressure for 5 minutes. and then cooling period started then: deflasking, removing of the plates, finishing, smoothing and polishing the surface of flexible acrylic plate.

 

Growing of microorganisms:

E.coli was grew first on the Nutrient broth and incubated over night in the 40 specimen containers which divided (20) containers for PMMA plates and other (20) containers for Acrylic plates.

 

Exposing samples on microorganism:

 The antimicrobial activity was determined in the following steps, after incubating the microorganism over night, 0.5 mL from each tube was taken and put in the Eppendorf tube ( Before treatment). After that, the media was discarded from all tubes and added 4 mL from (Sage or Myrrha, D.W and Chlorohexidine as control) with different concentrations and left it for 30 minutes with mixing at each 2 minutes. After the standing time, the tubes were rinsed with D.W and then addition 4 mL from D.W for each tube and stand for 2 minutes. After that, 0.5 mL from each tube was taken and put it on the Eppendorf tube.

 

The efficiency of samples on microorganisms:

MacConkey broth media was used to identify the E.coli bacteria. Specific dilution was used in this experiment, D.W : Stock as (1000:1 ) was prepared from each tube then 50 µL form each dilution was taken and spread on the media using L. Shape loop, and then incubate for 12 -18 hrs for determining the efficiency of samples on microorganisms.

 

Inhibition percentage:

The inhibition percentage of each sample was calculated according to the comparison between (Non-treated bacteria) and (treated bacteria) by calculating the colonies that formed in petri-dishes containing MacConkey agar.

 

FTR measurements:

Using Fourier transform infrared (FTIR) spectroscopy processing technique:

The raw data compounds that measured for Myrrha and Sage extract solutions were converted by this technique to the desired result: The sample's spectrum (Light output) as a function of infrared wave length or equivalently wave number. Figure (5)

 

RESULTS AND DISCUSSION:

Plant extract are promising prophylactic and disinfectant agents against bacterial organisms due to its natural organic and safety, suggesting that these extracts might be useful as antimicrobial agent and disinfectant solution for dental prosthesis. Dental plates that made of either thermally activated poly methyl methacrylate acrylic [PMMA] or flexible acrylic which is thermoplastic flexible polymer composition, these materials are widely used in construction of removable dental prosthesis and some maxillofacial prosthesis.

 

In figure (1 and 2) Showed the number of colonies and percentage of colonies reductions by using chlorohexidine 2% solution as control and the following Sage extract solutions 2%, 5% and 10%, that were separately treated the PMMA plates exposed to the E.coli.

 

In Sage extract solutions, the colonies reductions were 82.70%, 90.65% and 94.61% respectively for PMMA plates compared with 97.86% reduction for Chlorohexidine with mean p-value 0.0023, 0.0025, 0.0026 and 0.0009 respectively.

 

A

 

 

B

 

C

 

Figure (1) Showing the colonies growth number as mentioned in Petri dishes (A) table (B) and draft (C), were grown on MacConkey agar that previously and separately treated with Sage extract solutions on the PMMA plates.Colonies growth were tested by using Chlorohexidine 2% solution as control and the following Sage extract solutions 2%, 5% and 10%.

In figure (3 and 4) Showed the number of colonies and percentage of colonies reductions by using chlorohexidine 2% solution as control (chemical detergent) and the following Myrrah extract solutions 2%, 5% and 10%, that were separately treated the Acrylic flexible plates exposed to the E.coli.

 

When the Myrrah extract solutions 2%, 5% and 10% exposed to Flexible Acrylic, the colonies reductions were 91.75%, 96.15%, 97.08% respectively, compared with 97.91% reduction for Chlorohexidine with mean p-value (0.0017, 0.0010, 0.0005 and 0.0006) respectively

 

A

Concentration	2%	5%	10%	Chloro hydroxyl
Mean	82.70	90.65	94.61	97.86
P value	0.0023	0.0025	0.0026	0.0009
Significant different	Yes	Yes	Yes	Yes

 

B

 

Figure (2) Showing the percentage of colonies reductions as mentioned in table (A) and draft (B), were grown on MacConkey agar that previously and separately treated with Sage extract solutions on the PMMA plates. Colonies growth were tested by using Chlorohexidine 2% solution as control and the following Sage extract solutions 2%, 5% and 10%.

 

 

A

 

B

 

C

 

Figure (3) Showing the colonies growth number as mentioned in Petri dishes (A) table (B) and draft (C), were grown on MacConkey agar that previously and separately treated with Myrrha extract solutions on Flexible Acrylic plates.Colonies growth were tested by using Chlorohexidine 2% solution as control and the following Sage extract solutions 2%, 5% and 10%.

 

 

A

Concentration	2%	5%	10%	Chloro hydroxyl
Mean	91.75	96.15	97.08	97.91
P value	0.0017	0.0010	0.0005	0.0006
Significant different	Yes	Yes	Yes	Yes

 

B

 

Figure (4) Showing the percentage of colonies reductions as mentioned in table (A) and draft (B), were grown on MacConkey agar that previously and separately treated with Myrrha extract solutions on the Flexible Acrylic plates.Colonies growth were tested by using Chlorohexidine 2% solution as control and the following Sage extract solutions 2%, 5% and 10%.

 

 

(Figure 5) FTRI diagrams showing the spectrum ranges of (A) Myrrah (B)Sage, Reflecting the extracted compounds by reading the wave-lengths.

 

Previous study has been reported a case of E.coli associated osteomyelitis in a diabetic individual who had presented with very aggressive bilateral maxillary necrosis. After extensive literature search, to the best of our knowledge, this is the first case of maxillary osteomyelitis associated with E.coli which we have come across. Padhiary, et al. (2013)

 

Another study showed E.coli trained producing Streptococcus mutants proteins responsible for colonization and virulence. Curtiss, et al. (1983).

 

Although, Cotrim, et al. (2001) said that 52% of dentists didn't believe in the possibility of cross-infection between the dental – office and dental laboratories, while the truth is the dental office-laboratory connection may be represented a potential cross-infection pathway if no effective disinfection procedures are taken. The dentists in their clinics receive patients need to repair their fractured or unfilled dentures and already these dentures are contaminated with any types of micro-organisms. Thus, American Dental Association (ADA) stated that every dental patient should be treated as a potential source of infection.

 

Also, the dental laboratory technicians deal and handle these materials during steps of prosthesis fabrication. They may expose the products to be contaminated with various microbial and fungal organisms.

 

Every denture wearer, also, is subjected to microbial and fungal contamination, especially, if the wearer has bad oral hygiene where his mouth, normally, rich in microorganism, fungal and bacteria.

 

Mean while, less than 60% of the denture wearers use any one disinfectant due to the cost of the product Al Faraj, S (2005).Therefore denture disinfection should be of low cost to be affordable to most of the denture wearers.

 

These microorganisms can cause endogenous infections and form biofilms which are defined as community of microorganisms adhering to a prosthesis surfaces and surrounded by complex matrix of extra-polymeric substances (Williams BL, etal.1983), and these different micro-organisms live together in dental prosthesis surfaces forming a multicellular community (Haake SK.1996).

 

A previous studies stated that new dentures must be disinfected before replacement of the prosthesis in patient's mouth and before and after adjustment procedures Al Faraj, S (2005), because a large variety of disease are caused by many micro-organisms which are found in patient's mouth such as Streptococci pneumonia, Staphylococcus auras, Escherichia coli, and Mycobacterium bovis (Haake SK.). Also, denture induced Stomatitis was reported to be an inflammatory reaction of the denture - bearing mucosa, being characterized by different degrees of erythema and affects many complete upper denture wearers (Williams, etal.1983).

 

Currently a known method of disinfection is the use of chemical disinfectant solutions, which may interfere, with frequent exposure with the properties of PMMA or the flexible acrylic because the long-term immersion of the prosthesis in chemical disinfectant may cause deterioration of denture base material by changing mechanical properties of acrylic resin Al Faraj, S (2005) Therefore, there are several drawbacks in the use of such chemical agents for disinfection of prosthesis.

 

Chlorohexidine 2% solution is considered the best choice among chemical antiseptics for dental biform control Al Faraj, S (2005). It is a broad- spectrum disinfectant that works by destroying the cell membrane and precipitating in the cell cytoplasm. So, chlorohexidine 2% solution is convenient to use it as control antiseptic agent versus plant extract solutions for comparison.

 

Thus, research is necessary to find an alternative method of disinfection by using solutions which do not cause any significant alteration in the structure of prosthesis.

 

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Received on 06.10.2018          Modified on 11.11.2018

Accepted on 20.12.2018        © RJPT All right reserved