CHAPTER-1 INTRODUCTION

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1 CHAPTER-1 INTRODUCTION PHOTODYNAMIC THERAPY Photodynamic therapy (PDT) is necessary for an efficient treatment in curing oral cavity disease and it is also extremely important to develop new technique that are potent, cost effective and non invasive with minimal, compact New Approaches have been experimentally realize to inactivation of food pathogen bacteria with different photosensitizer using laser treatment (Bertoloni, et al., 2000). NATURAL PHOTOSENSITIZER The photodynamic therapy has been used as natural photosensitizer (Oryza sativa) is an edge over the chemical phtosensitizer because antibiotics used in the procedure arises with side effects to human being and reduces the food quality. Natural photosensitizer as Oryza sativa is not used in previous work leads to a non toxic, safe, cheaper, and locally available substance which used with the combination of photodynamic therapy SIGNIFICANCE OF PHOTODYNAMIC THERAPY Bacteria have an important role in human and animal morbidity, and a great number of them have developed antibiotic resistance. In the past recent years photodynamic inactivation has been studied by using experimental report on photodynamic inactivation of bacteria have been using with different coherent and non- coherent laser light source. (Calin, and Parasca, 2009). Most modern PDT applications involve three key components: a) A photosensitizer b) Light source c) Oxygen The basis of PDT is the interaction of light with photosensitive agents to produce an energy transfer and a local chemical effect (Fan, et al., 1997). This is broadly similar to what is seen in photosynthesis, although in these cases many photosensitizers that have been used in PDT, each has its own unique excitation properties. Photodynamic killing of Streptococcus mutant bacteria alternative to the prominant application of antibacterial drugs used in the treatment of periodontal diseases (Meisel, and Kocher, 2005). 1

2 The mechanism of bacterial inactivation has not yet been reported. The idea is that inactivation of the bacteria by coherent light radiation has been produced, in principle, as follows: after the absorption of the photon by the photosensitizer it passes from the ground state, S 1, to its first excited singlet state, S 1 *. From this state, the photosensitizer can return again to the ground state (by radiative or non-radiative transitions) or it can pass into a triplet excited state S 3 * (intersystem crossing). From this excited state, the photosensitizer can return to the ground state. The photosensitizer in the triplet state, S 3, is extremely reactive, due to its long life (10-3 ). For the singlet state, S 1, the life time is short (10-9, 10-7 ), so the photosensitizer cannot react with other molecules before returning to the ground state. Bacteria Used for Photodynamic Therapy Streptococcus sanguinis formerly known as Streptococcus sanguis, is a Gram-positive facultative aerobic coccus species of bacteria and a member of the viridians Streptococcus and lactic acid bacteria group. S. sanguinis is a normal inhabitant of the healthy human mouth where it is particularly found in dental plaque. Table.1 Streptococcus species with their causing symptom S.NO. Streptococcus sps. INFECTION 1. S. agalactiae Pneumonia 2. S. bovis Endocarditis 3. S. sanguinis Oral cavity 4. S. oralis Valvular heart disease 5. S. viridans Meningitis Table.2 Classifiction of Streptococcus CLASSIFICATION Prokaryotae Bacteria Division Firmicutes Class Bacilli Order Lactobacillales Family Streptococcaceae SUBTYPE OF STREPTOCOCCI Genus Species Streptococcus S. sanguinis Streptococcal infections are caused by species of the genus Streptococcus, which includes Streptococcus pyogenes (Group A), S. agalactiae (Group B), S. faecalis (Group D), S. Pneumoniae (Pneumococcus) and the viridians Streptococci group. Group A beta- hemolytic streptococci (for example S. pyogenes) cause nosopharyngeal and cutaneous lesions, different strains infecting these sites. Group B streptococci produced infections of the new born Group C, G and other streptococci are responsible for respiratory infections. 2

3 Alfa (green) hemolytic streptococci (e.g. enterococci) are an important cause of infections of the urinary tract, endocarditis, postsurgical infections. Group D streptococci (e.g enterococci) are an important cause of infections of the urinary tract, endocarditis, postsurgical infections. Pneumococci (Streptococcus pneumonia) produce primary bacterial pneumonia, septicemia, meningitis, and other infections. It is well known that the accumulation of bacterial biofilms on tooth surfaces results in some of the most prevalent bacterial-induced human diseases, caries and inflammatory periodontal diseases ((Kristoffersson, et al., 1985; Sbordone, and Bortolaia, 2003) Adhesion, colonization, and growth of oral bacteria on tooth surfaces lead to a multispecies biofilm, called dental plaque (Rosan, and Lamont, 2000; Hicks, et al., 2003; Marsh, 2004). It is the cause of common oral infectious diseases such as dental caries, gingivitis, periodontitis, and peri-implantitis. Plaque is not only present on natural teeth but also on dental materials and implant components as well. Application of photodynamic inactivation of bacteria by light radiation and photosensitizer has some unknown fields: which are the bacteria with sensibility to light radiation; the direct effect on the microbial population; the type of photosensitizer which is selectively fixed in different bacterial species; the way it should be administered; how to prepare the photosensitizer; what is the therapeutic concentration; how much time must pass from the photosensitizer administration to the exposure to light; which type of source for the radiation is better (continuous or pulse operation); the parameters of the light (wavelength, energy, pulse duration, frequency, time of exposure); how to monitor the biologic response and the treatment. Until now there have been some studies aimed at analysing some of these factors. The most important results regarding the type and the parameters of light radiation sources for photodynamic inactivation are summarised in the following pages. In this context, modern research in microbiology and related domains (biophysics, chemistry, medicine, physics) were focused on the development of new methods of antimicrobial therapy, more efficient and faster, non-invasive and non-toxic, which would not lead to microbial resistance (Sommer et al., 2000). One of these non-invasive methods, based on the use of light sources, is the photodynamic inactivation of bacteria. 3

4 TECHNIQUE USE IN PHOTODYNAMIC THERAPY LASER: - The laser is an acronym for light amplification by stimulates the emission of radiation. It is a mechanism for emitting, electromagnetic radiation, typically light or visible light, via the process of stimulated emission. The emitted laser light is a spatially coherent, narrow low divergence bean that can be manipulated with lenses. In laser emits light of in-step waves of identical frequency, phase and polarization. Laser consists gain medium inside a highly reflective optical cavity, as well as a means to supply energy to a gain medium. The gain substance is a compound with properties that allow it to amplify light by stimulated emission. In its simplest form, a cavity consist of two minors arranged such that light bounces back and forth, in the time duration through the gain substance. The typically one of the two minors, the output coupler, is partially transparent. The output laser beam is emitted through the minors. The principle of supplying the energy required for the amplification is called pumping. The energy is typically supplied as an electrical current or as light at a different wavelength. Such lightn may be provided by a flash lamp or another laser. 4

5 REVIEW OF LITERATURE A) Efficacy of chemical photosensitizer with laser radiation on microbes Wilson et al., (1993) reported that 80 S exposures to light from an He-Ne laser was unable to kill Strep. sanguis, F. nucleatum or Actinomycetem comitans using an energy dose of 584 mj energy density 44 J cm. Burns et al., (1993) reported the viabilities of the cariogenic organisms Streptococcus. mutans, Streptococcus. sobrinus, Lactobacillus. casei and Actinomycetes. viscosus were unaffected by a 90 S exposure to light from either an He-Ne laser energy dose 657 mj, energy density 50 J cm or a Gas laser energy dose 990 mj, energy density 1.5 J cm. Bertoloni et al., (2000) reported the present invention relates to the use of photosensitising compounds and laser irradiation to kill the microbes involved in a number of oral diseases including inflammatory periodontal disease and caries or in wound infections and in disinfectioning or sterilising wounds and lesions in the oral cavity, New approach to inactivation food pathogen bacteria after photosensitization. Maish Tim (2000) reported the use of photosensitizer and light as an anti-microbial agent against periodontal microbial biofilms should also represent an attractive method of eliminating oral bacteria. Stringer et al., (2000) reported PAD does not give rise to deleterious thermal effects, and adjacent tissues are not subjected to by stander thermal injury. Photoactivated dye treatment does not cause sensitization and killing of adjacent human cells such as fibroblasts and keratinocytes. neither Clinical studies have reported positive results for PDT treatment of carcinoma-in-situ and squamous cell carcinoma in the oral cavity, with response rates approximating 90 percent. The treated sites characteristically show erythema and oedema, followed by necrosis and frank ulceration. Komerik et al., (2003) Presented in vivo experimental studies showed that toluidine blue-pdt can selectively kill P. gingivalis in the oral cavity and significantly decrease the level of alveolar bone loss in rats affected by periodontitis, Nakanishi H, Mac Robert A. J, Henderson B, Speight P, Wilson M. In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photosensitisation in an animal model. Michael Wilson (2004) reported Lethal photosensitisation of a wide range of bacteria responsible for caries, periodontal diseases and root canal infections has been demonstrated using red light in conjunction with a number of photosensitisers, including Toluidine Blue, 5

6 phthalocyanines and chlorins. The advantages of this approach are that bacteria can be eradicated in very short periods of time (seconds or minutes). Luksiene and Zukauskas (2009) reported Modern biophotonic technology based on photosensitization, which is successfully used to cure cancer and infectious diseases (photodynamic therapy), might serve as a promising tool to decontaminate food and food-related surfaces from different pathogens. Pinheiro et al., (2009) reported Radicular canal cultures were conducted (n = 10): the first one was performed right after access and location of the radicular canal; the second was performed after the conclusion of chemical mechanical instrumentation, and the last one after photodynamic therapy. The photodynamic therapy was performed with 4 J/cm energy lowintensity diode together with toluidine blue. B) Efficacy of natural photosensitizer with laser radiation on microbes Pathak (1986) reported Phytophotodermatitis, evoked by several furocoumarins (psoralens) produced by such plant families as Umbelliferae, Leguminosae, Rutaceae, occurs only after contact with a plant (sap, leaves, etc.) and subsequent exposure of the skin to long-wave ultraviolet radiation. There is a considerable overlap because phytodermatitis may result from mechanical injury, chemical irritation, or sensitizing substances. Schuitmaker et al., (1990) reported that bacteriochlorin, a new photosensitizer in photodynamic therapy. The photosensitizing properties of bacteriochlorin a (BCA), a nontoxic derivative of bacteriochlorophyll were investigated in vivo, BCA has an absorption band at a wavelength at which tissue is penetration is optimal (760nm), and is show preferential tumor retention in green melanoma implanted in the anterior chamber of rabbit eyes. Menezes et al., (1990) reported the He-Ne/TBO combination appeared to be more effective than the GaAs/AlPcS 2 combination, achieving log 10 reductions of 2 95, 5 40 and 3 34 in the total anaerobic count, streptococci and actinomyces respectively with a light energy dose of 1 31 J. If effective in vivo, lethal photosensitization may be useful as a means of eliminating plaque bacteria from a carious lesion prior to its restoration. Komerik et al., (2000) showed that LPS from Escherichia coli and proteases of Pseudomonas aeruginosa were inactivated after exposure to red light and toluidine blue o. 6

7 Demidova and Hamblin (2005) recently demonstrated that several types of Bacillus spores can be inactivated by red light irradiation in the presence of phenothiazinium dyes under mild experimental conditions. Iwunze and Ekiko (2004) Soybean oil (SBO) is used as a medium to solubilize zinc phthalocyanine (ZnPc), a known photosensitizer, which has been found to be of potential use in photodynamic therapy. It absorbs red light in this medium at 670 nm with good molar absorptivity comparable to its value in most organic solvents (2.45 x 10(5) M (-1) cm (-1)). The wavelength of the fluorescence emission band peaked at 700 nm when excited at 591 nm, and when excited at 560 nm, the emission band was observed at 680 nm. Ulatowska-Jarza et al., (2006) reported antimicrobial PDT with chlorophyll-derived photosensitizer and semiconductor laser. The study was carried out on Escherichia coli strains isolated poultry and cows. The bacteria were incubated and treated by photolon, a plant photosensibilizator, and exposed after that to laser light at 662 nm. Olson and Allan (2010) reported that Salmonella choleraesuis, Escherichia.coli 0157:H7, Staphylococcus aureus, Cmphylobacter jejuni and Listeria monocytogenes, the test compound was Turmeric, prepared as 0.01% by weight in 30% muller hinton broth in phosphate buffered saline. Gasparetto et al., (2010) reported extracts from Alternanthera maritime as natural photosensitizers in photodynamic antimicrobial chemotherapy (PACT) against Candida dubliniensis. Crude hexane and ethanol extracts of A. maritima were produced. The prepared extracts presented absorption at nm. The contents were irradiated with a 685 nm diode. 7

8 MOTIVATION Oryza sativa (Rice):- In addition to being a rich source of dietary energy, rice is a good source of thiamine, riboflavin and niacin. Unmilled rice contains a significant amount of dietary fibre. The amino acid profile of rice shows that it is high in glutamic and aspartic acid, while lysine is the limiting amino acid. Rice alone cannot supply all of the nutrients necessary for adequate nutrition. Rice is rich in genetic diversity, with thousands of varieties grown throughout the world. In its natural unmilled state rice comes in many different colours, including brown, red, purple and even black. These colourful rice varieties are often prized for their health properties. Unmilled rice has a higher nutrient content than milled or polished white rice. Rice is used as photosensitizer in our work was not reported before in other work. Rice is the predominant staple food for 17 countries in Asia and the Pacific, nine countries in North and South America and eight countries in Africa Rice provides 20 percent of the world s dietary energy supply. Oryza sativa (Natural Photosensitizer) has been used with an effective low power laser radiation red diode laser (635 nm) and blue laser (405 nm) with different exposure of time that was give effective control over the growth of bacteria. Photodynamic therapy was used because it is cost effective, potent and best way to optimize the bacterial colony with different laser radiation. The bacteria from the junk food regard as remove while packaging process in food industries by applying photodynamic therapy. 8