Antimicrobial susceptibility of some plant extracts against human dental caries pathogen

Document Type:Thesis

Subject Area:Biology

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This is because some plants apart from containing some better medicinal value to the human beings, it is also important to note that there are some plant extracts that contain some rather dangerous substances which may be hazardous to the human life. Therefore, the plant extracts should be done with extreme care and this will enable the production of high-end medicinal substances that will benefit the people and deal with pathogens. One of the key pathogens that the plants can work well in dealing with human dental carriers’ pathogens. The aim of the current investigation was to study the antimicrobial activities of some essential oils to know they have antibacterial or antifungal activity, also to determine beneficial effect of those essential oils that used. This was done through tow techniques: the agar diffusion assay as well as the agar dilution method was used for determination of Minimum Inhibitory Concentration (MIC). The objective that must dependably be remembered is that the antimicrobial causes less harm to the host as compared to the pathogen. Chapter One 1. Antimicrobial Activity 1. Introduction An antimicrobial is an agent that destroys microorganisms or stops their growth. Antimicrobial pharmaceuticals can be assembled by the microorganisms they act principally against. For instance, anti-infection agents are utilized against microscopic organisms and antifungals are utilized against growths. They can likewise be characterized by their capacity. Specialists that murder bacteria are called microbicidal, while those that just repress their development are called biostatic. The utilization of antimicrobial pharmaceuticals to regard contagion is recognized as antimicrobial chemotherapy, whereas the consumption of antimicrobial medications to avert disease is known as antimicrobial prophylaxis.

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The objective that must dependably be remembered is that the antimicrobial causes less harm to the host as compared to the pathogen. This is called specific danger. We have had our most prominent accomplishment with antibacterial anti-microbials, since there are various things that are diverse in procaryotic bacterial cells from the eucaryotic cells of the host. These distinctions make it moderately simple to focus on the pathogen and extra the host. Assaulting the eucaryotic cells of organisms, helminths and protozoa, and the host cells that infections have attacked is more troublesome. Antimicrobials may work against only certain microbes. The well-being of an individual is affected by dental wellbeing. Oral wellbeing affects general wellbeing as it causes significant torment and enduring. It affects a man's discourse, choice of nourishment, personal satisfaction, and prosperity.

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In perspective of the pervasiveness of oral illnesses, their effect on people and society, and the cost of their treatment, oral infections might be viewed as a noteworthy general medical issue and they are recorded among the most well-known of the constant sicknesses that influence humanity. Oral infections are the fourth most costly sicknesses to treat in specific nations. This is especially legitimate in common regions. Antimicrobial mouth flushes have furthermore been proposed as assistants for mechanical plaque control strategies. The most normally used antiplaque administrator is chlorhexidine gluconate. The use of chlorhexidine has some potential disservices like changed flavor sensation, teeth recoluring, and change of safe infinitesimal creatures that challenged person its application on whole deal start. It is important to develop some imaginative strategies that exhibition against both dental caries and periodontal afflictions in the meantime.

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Therapeutic plants have been discovered valuable in the cure of various sicknesses including bacterial ailments attributable to a rich wellspring of antimicrobial specialists. With the learning of corrective properties of the restorative plants against oral microorganisms and their consolidation in clinical practice we can mean to lessen if not expel this infection substance. Because of a fast increment in the rate of contaminations, anti-microbial protection in microorganisms and because of symptoms of manufactured anti-microbials, restorative plants are picking up notoriety over the medications. It has been all around reported that restorative plants present antimicrobial movement towards oral microbes. The writing review of the fables prescription uncovers the utilization of Psidium guajava leaves to keep up oral cleanliness, a dried product of Terminalia chebula as an anticaries operator; stem of Achyranthes aspera for the treatment of toothache and stem of Mimusops elengi, fortifies the gums.

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They have been exposed to be lively not in favor of oral pathogens. The growth of numerous cariogenic bacteria was found to be inhibited by flavonone phytolexins from Sopphora exigua (Leguminosae) with, 4’-tetrahydroxy-8lavandulylflavanone being most active. Many cariogenic bacteria together with oral streptococci and other mutans, actinomyces and lactobacilli were inhibited by two active isoprenylflavonones namely Artocarpin and art ocarpesin which were isolated from Artocarpushet erophyllus (Moraceae). It has been shown that Erycrystagallin totally concealed the integration of radio marked thymidine and glucose in S. mutans, signifying that compound tamper with bacterial uptake of metabolites. Combinations of plant extracts, extracts were mixed in a ratio 1:1 and study their effects. Identify and quantify some active molecules in the two plants extracts. The aim of the study The aim of the current investigation was to study the antimicrobial activities of some essential oils to know they have antibacterial or antifungal activity, also to determine beneficial effect of those essential oils that used.

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This was done through tow techniques: the agar diffusion assay, as well as the agar dilution method, was used for determination of Minimum Inhibitory Concentration (MIC). Chapter Two Literature Review 2. silica gel plates) and afterward overlaying with agar containing pointer life forms. Measuring the antimicrobial action includes techniques practically equivalent to those used to decide the base inhibitory centralization of antimicrobial synthetic specialists, for example, antimicrobials or disinfectants. This includes uncovering the test creature to serially weakened concentrate/compound (either in fluid or strong media) and deciding the base focus that restrains development. These strategies are portrayed in ISO 20776 (Clinical research facility testing and in vitro analytic test frameworks), which expresses that "Weakening techniques are utilized to decide the base inhibitory fixations (MICs) of antimicrobial operators and are the reference strategy for antimicrobial helplessness testing.

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In weakening tests, smaller scale life forms are tried for their capacity to create noticeable development on a progression of agar plates (agar weakening) or in soup (juices weakening) containing serial weakening of the antimicrobial specialist. As it were, rosemary was gotten from the Latin words "dew of the ocean. The plant can likewise be alluded to as anthros. This plant has a stringy root system (Wood, 2011). The essential oils have recently begun to receive much attention as possible sources of safe and natural alternative medicines because they have been known to possess various medicinal activities. Consequently, studies on essential oils to evaluate the pharmacological properties in order to find possible alternative medicines have become active in recent years. Tripathi, 2013). The tests performed in Mueller Hinton Broth (MHB; OXOID-CM405) with the exception of the yeasts (sabouraud dextrose broth-SDB; DIFCO).

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Bacterial strains are cultured overnight at 37°C in Mueller Hinton Agar (MHA) whereas for yeasts they are cultured overnight at 30°C in Sabouraud Dextrose Agar (SDA) (Alim et al. Persistent dental disease is painful, and most importantly, it has also been suggestively linked to diabetes, high blood pressure, heart disease, and multiple sclerosis later in life. The pain can be worsened by heat, cold, or sweet foods and drinks [9]. aspera and M. elangai, the study is focused on assessing the plant extracts with different solvents. Hence, for the present investigation, S. mutans, C. albicans are the bacterial and fungal strains selected as target organisms from infected patients and screened using hexane, ethyl acetate, ethanol and methanol extracts of leaves of P. For example, bearberry (Arctostaphylos uva-ursi) and cranberry juice (Vaccinium macrocarpon) have been reported in different manuals of phytotherapy to treat urinary tract infections while species such as lemon balm (Melissa officinalis), garlic (Allium sativum) and tea tree (Melaleuca alternifolia) are described as broad-spectrum antimicrobial agents (Heinrich et al.

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In South Africa and Uganda, many individuals in the rural areas receive their health education and health care from practitioners of traditional medicine (Jager et al. Light et al. Lewis, 2014). In the Eastern Cape Province, of South Africa, Elephantorrhiza elephantina, Hermannia incana, Pelargonium reniforme, Alepidea amatymbica, and Bulbine latifolia are the plants recommended most frequently for the treatment of diarrhoea by both traditional healers and rural dwellers. Other parts of plants that can be extracted for therapeutic compounds are roots, aerial parts, flowers, seeds, stem barks, etc. Chan et al. Plant secondary metabolites are used as the basis for the production of valuable synthetic compounds such as pharmaceuticals, cosmetics, or more recently nutraceuticals (Bourgaud et al. These secondary metabolites are largely viewed as potential sources of new drugs, antibiotics, insecticides, and herbicides (Crozieret al.

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This is because of their biological significance and potential health effects, such as antioxidant, anticancer, anti-aging, anti-atherosclerotic, antimicrobial and anti-inflammatory activities. Rosemary removes and furthermore, unstable oil from rosemary have been critical in advancing the menstrual stream and utilized as abortives. The concentrates of the rosemary plant are generally gotten as corrective fixings. The moisturizer of the rosemary plant is utilized to forestall hairlessness and to animate hair development. It is likewise used to enhance the absorption procedure and improving the blood flow in our bodies particularly in the head and brain (Reppert, 2015). Rosemary is likewise utilized as an antimicrobial operator. The plant is topically connected or even expended in some sort of balm of the rosemary abandons, and it has comparable impacts. Fragrant healing additionally uses rosemary oil for the same purpose (Wood, 2011).

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Mint, on the other hand, is used in the procedure known as aromatherapy, a medical form of therapy used to induce relaxation. It has however not been proven to heal any form of illness. Additionally, mint has been proven to be an effective pesticide. Many of these essential oils are used for aromatherapy used to create a sense of body relaxation The examples of some common aromatic plants and their uses [29]. Table 1: Some common essential oil producing aromatic plants Common name Scientific name Parts used Medicinal uses Almond Prunus communis Nut Flavoring Clove Eugenia caryophyllus Bud Dentistry & flavoring Cinnamon Cinnamomum zeylanicum Leaves Flavoring Coriander Coriandrum sativum Seed Flavoring Eucalyptus Eucalyptus globules Leaves Decongestant Lavender Lavendula officinalis Flower Perfumery Sandalwood Santalum album Wood Perfumery Lemongrass Cymbopogon flexuosus leaves Flavoring & medicines Geranium Pelargonium graveolens Leaves & shoots Perfumery & cosmetics Cardamom Elettaria cardamomum Fruit Perfumery & flavoring Patchouli Pogostemon patchouli leaves Perfumery & flavoring Chamomile Matricaria chamomilla herb Flavoring & cosmetics Mentha Mentha arvensis Leaves and stem Pharmaceutical, food flavour & cosmetics Vetiver Vetiveria zizanioides roots Perfumery Citronella (java) Cymbopogon winterianus leaves Pharmaceutical, flavoring & cosmetics Jasmine Jasminum officinale flower Perfumery Rose Rosa damascene flower Perfumery & cosmetics Physio-Chemical Properties- Essential oils have several common physical properties like characteristic fragrances and high refractive indices.

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They are mostly optically active and immiscible with water, but sufficiently soluble to impart their characteristic fragrance to water. Factually, the aromatic waters are dependable on this slight solubility. Essential oils are however soluble in ether, alcohol and organic solvents [30]. This is also the official standard method for extracting essential oils for quality control. In this standard method the distillation procedure is closely defined vegetable products, according to the reference method indicated in the standard is done with steam distillation, collecting the distillation product in xylene. During hydrodistillation the essential oil components form an azeotropic mixture with water. Most of the essential oils do not mix well with water in the liquid phase so after condensation, they are separated by decantation. The distillation period can take from 15 to 30 minutes or longer.

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Hydrodistillation at reduced pressure is better because lower temperatures are used. Figure 3. Schematic diagram of a steam distillation apparatus. with organic solvents at ambient pressure. Extraction with organic solvents is principally used in the perfumery and cosmetic industries. aliphatic hydrocarbons: propane, butane, hexane 2. alcohols: methanol, ethanol, 2-propanol 3. hydrocarbons with a carbonyl group: acetone, methyl acetate 4. halogen derivation: dichloro methane, dichloroethane, freons. The main disadvantages of solvent extraction are : 1. except where stated, carbon dioxide is the solvent and the process is used commercially). Liquid CO2 has several advantages over most conventional organic solvents, namely: • non flammability; • because it is gaseous under ambient conditions, it is easily removable; • colorless and without odor or taste; • Extracts obtained by this method cover a broader spectrum of volatilities than do those obtained by hydrodistillation, which confers to the oil the absolute character; • Extracts are free of residues from the extraction solvent.

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n the subcritical state, liquid CO2, behaves as a non-polar solvent, with temperatures between -55ºC and 31ºC, and pressure between 5 and 74 bar. Under normal working conditions (temperature 0º-10ºC, pressure 50-80 bar) it is a selective solvent, dissolving mainly non polar and slightly polar components with molecular weights up to about 400 [32]. Components such as chlorophyll, carotenoids and alkaloids are virtually insoluble in liquid CO2. Esters 6. Monoterpenes 7. Sesquiterpenes Higher molecular weight compounds: 1. Aliphatic hydrocarbons 2. Alcohols 3. The methods that are applied in the determination and success of this research are analyzed fully below: 3. Sampling Sampling is the process of picking experimental subjects from a group of organisms for the purpose of research. In this case, the different types of bacteria were picked from the teeth of the patients with dental problems. The samples were then taken to the microbial labs for testing using the antimicrobials such as the rosemary and the mentha essential oils.

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Sample collecting is the first step in any researches which related to identification or any microbiology fields; in this study the samples were collected from those patients that they had a dental plaque from teeth. Rosemary plant belongs to the family of Lamiaceae. It’s an herb found mostly in the Asia and the mediterranean places. Traditionally, this plant has been used in traditional medicines and even in the cosmetics. Further, this plant extract has been used in the flavouring of food. Essential oil extracted from the plant has been used in the past as a powerful antibacterial, anti-mutagenic and antioxidant. Research indicates that there are very few species of the plant left, estimated to be between 16 and 18 species. Mints are known to be perennial herbs and are also aromatic. The species are widely distributed across the planet but are however known to grow well in wet environments especially in moist soils.

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They are known to grow to between 10-120 cm tall and prefer distributing themselves on an indeterminate area. Wild mint (Mentha longifolia) has peppermint-scented aroma and is usually used for the treatment of mouth, throat irritation and sore throat. This bacteria has been identified as not only responsive for the metabolization of the lactic acid found in the dental enamel but is also responsible for in the synthesizing the extracellular polysaccharides, this is mainly a dextran and forms a significant component of plagues. Thus it further aids in reinforcing the carcinogenicity of the carbohydrates that has been taken in. distributed of carriers in the cavities ins not only due to the increased intake of sweets, sweetened drinks and beverages and fast food, studies have established that there exists resistance of the bacteria S.

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mutans to the antibacterial agents that is used mainly as oral care products. With a high number of intake of oral care products, it is not surprises therefore that there is also a surge in the dental carriers amongst the individuals. Further, the cloves and cinnamon has been used by some individuals and its influences are well documented. With different studies done on the effects of cloves, garlic, liquorice, lavender and sage, it would be expected that a combination of this herbs can be utilized to produce the most efficient product. Gram Stain and chemical test The gram staining, also called Gram's technique, is a technique of staining used to differentiate and classify bacterial species into two large sets (gram-negative and gram-positive). Gram-positive bacteria need a dense mesh-like cell wall prepared of peptidoglycan (50–90% of cell cover), and as a consequence are stained purple by crystal violet, while gram-negative bacteria have a thinner layer (10% of cell envelope), so do not recall the purple stain and are counter-stained pink by safranin.

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There is four steps to determine the gram stain: 1. Substrates can be used to measure various metabolic activities such as increase or decline in acidity or alkalinity, the growth of substances that impairs growth, and enzyme hydrolysis. Oxygen transmission is guaranteed in the system, this is through an optically clear film that is situated on both sides of the card. Using an optically clear film aids making sure that it’s sealed, this ensures that there no any contact with organism-substrate. The 64 cards used in the experiment has pre-inserted tube that is used for the process of inoculation. This cards has unique bar codes that contains individual product type information, the lot number, a unique identifier, used to connect with the sample either before the experimentation begins or after. Oil floats on the water, which for its part is gradually returned to the heated flask through the diagonal conduit.

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After 2 hours of extraction, the oil volume collected in the burette can be directly measured. Sometimes, the Clevenger apparatus gives higher yields than hydrodiffusion. Several factors can explain this phenomenon. In particular, the packing of the plant material in the hydrodiffusion column sometimes creates pockets that are poorly exposed to steam, and therefore not properly extracted. McFarland standard) and the optical density (OD) of each microbial was at which is equal to which is used as the inoculum for performing agar well diffusion assay. A inoculum of each test organism was spread onto the MLA so as to achieve a confluent growth. The agar plates were allowed to dry and wells of were made with a sterile borer in the inoculated agar plates. The extracts were dissolving in 2% tween 80 for the bioassay analysis.

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A 100μl of each extract was propelled directly into the wells (in triplicates) of the inoculated specific media agar plates for each test organism. Further, the reviews have led to the acceptance of plant extracts in providing a solution to the current challenges that are being experienced in the dentistry industry. The oral disease challenge, which led to the ultimate rise of this discussion happen to be experienced in the whole world. The challenges include; Increased failure of antibiotics in getting rid of bacteria, financial problems amongst patients in most of the developing countries and unexpected reactions on patients by the antibacterial agents that are administered to the patients. Dental wellbeing plays a very crucial role in the success of a person. A person who is orally okay has got very high chances of succeeding in life.

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As a result of the above, well dissemination was put into practice in this test of determining the base inhibitory centralization of synthetic antimicrobial specialists. Well, dissemination was deemed to be appropriate for this exercise because of some reasons. The reasons include: The process is an inexpensive one, minimal time required for the accomplishment of the task and finally the provision of a clean and safe environment to conduct the test. The clean and secure testing environment is made possible via either immunization that is done to the agar plates or the pouring of a liquid to the petri dish which has to be vaccinated. In the disc diffusion method that was settled on, bacteria are usually put on an agar plate that is already loaded with bacteria. Water has always been considered as the main solvent during the extraction of the botanical compounds from the sample plant extracts.

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Of recent, there has been widespread use of a solution that is composed of methanol and ethanol. The usage of the ethanol-methanol solvent is as a result of the ability of the solution to give a consistent and reliable antimicrobial activity. As per the current investigation, the plant extracts under study were all of them prepared via water and a solution of organic solvents which would act as the solvents. The reason for settling on water and the organic solution was because of the need to ensure that all the plant extracts that were being tested in this case dissolve on the solvents as per their polarities. Furthermore, the possibility of the extracts to act as an adjuvant in a biofilm which is usually considered as the natural locality for the bacteria that are responsible for the formation of caries.

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The above coupled with appropriate oral hygiene and the control in the usage of fluorides when making dentifrices and other products that are meant to take care of peoples’ oral system for instance mouthwashes. The results that were obtained have got some contradictory bits within them. For instance, the results of T. chebula indicated a wide and easily noticeable difference when contrasted with what is existing in the available literature. Food and Drug Administration. Also, when the data that is available is closely looked into, concerning the methodology that is used during the studying of the anti-microbial activities that take place I plants, there can be an achievement of the most diverse outcomes. Though the information from the clinical studies available does not possess entire relevance towards the control of caries, the safe usage of the plant extracts towards the curing of varied human diseases show that their application will be appropriate for the caries control.

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It's also important to note that both the toxic and allergic potentialities of the plant extracts were not included in this study. This automatically implies that no final verdict can be made in this study as regards to the toxic and allergy levels of the extracts. Inhibition zone Table1: Results of Agar well Diffusion method are expressed in mm of inhibition zone The table illustrates the effect on the bacteria S mutant of both rosemary and Mentha essential oils. When 100 ml of the two oils is used, there is no effect on the bacteria. In all instances, the result is 0, hence the mean is 0. Nevertheless, when more than 100 ml oil is used, there is a significant change in the figures. Another key factor noted from the experiment is, the more the amount of oil used, the higher the effect on the bacteria.

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gm/ml 7 7 7. gm/ml 6 6. gm/ml 4. gm/ml 0 0 0 0 0 0 0 0 Table2: Results of Agar well Diffusion method are expressed in mm of inhibition zone The table shows that Mentha has more effect on the bacteria S aures as compared to rosemary essential oil. Similar to the experiment done before, when more than 100 ml oil is used, there is a significant change in the figures. gm/ml 6. gm/ml 5 4. gm/ml 3. gm/ml 0 0 0 0 0 0 0 0 The table shows the effect of different concentrations of the essential oils of the microbials against the growth levels of the bacteria Rothia mucilaginosa. Adding more of the essential oil inhibits more of the bacteria and eventually resulting in its death. mg/ml or less of both essential oils do not affect the inhibition zone of the bacteria. th table (E.

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coli) Rosemary Mentha E. coli 1st 2nd 3rd Mean 1st 2nd 3rd Mean 800gm/ml n/a n/a n/a n/a 4 4. gm/ml n/a 4 n/a 4 3. sanguis 1st 2nd 3rd Mean 1st 2nd 3rd Mean 800gm/ml 6 6. gm/ml 4. gm/ml 3 3. gm/ml 2 2. gm/ml 0 0 0 0 0 0 0 0 The table shows the comparison between the different concentration of the essential oils and the inhibition zone of the bacteria S snguis. Both rosemary and mentha essential oil increase the inhibition zone for the bacteria Candida albicance. The inhibition zone increases with an increase in the amount of the essential oil used. The mean values of the inhibition zones indicate that the bacteria will eventually die with the addition of rosemary essential oil. mg/ml or less of both essential oils have no effect on the inhibition zone of the bacteria.

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Minimum inhibitory concentration (MIC) The minimum inhibitory concentration of a microbial is determined by the lowest concentration of the antimicrobial essential oil that inhibits or deters the growth of a microorganism, in this case the different types of bacteria. This was done through the agar diffusion assay. Antimicrobials such as rosemary have a pungent, pine-like aroma. The smell resembles that of eucalyptus or rather the tea tree. The inhibition zone of these essential oils varies based on the different types of bacteria used in the test. For instance from the above tests, we can determine that rosemary essential oil had the most effect on the bacteria S. The results were tabulated and charts are drawn from them. The test uses different levels of concentration of the essential oils. The results show that with the addition of the essential oils, the inhibition zones of the different bacteria increases.

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For instance, when 100mg/ml of rosemary essential oil is used, the inhibition zone of S. sanguis is not affected. The table and the charts also show that the mentha essential oil had more effect on the different types of bacteria as compared to the rosemary essential oils. Mentha essential oil had the most effect on the bacteria S aures with a mean value of 11. This was an indication that the continuous addition of the mentha essential oil would eventually lead to the death of the bacteria S aures. Moreover, the essential oil caused an inhibitory effect on the bacteria E coli. On the other hand, the rosemary essential oil displayed no results when applied to the bacteria E coli. P. Growing & using rosemary. New York: Wiley. Tehon, A. Growing rosemary herbs.

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Industrial Crops and Products 62, 250–264. Alim A, Goze I, Cetin A, Atas AD, Vural N, Donmez E. Antimicrobial activity of the essential oil of Cyclotrichium niveum (Boiss. Manden. Et Scheng. BARRETT, B. and D. KIEFFER. Medicinal plants, science, and health care. Journal of Herbs, Spices and Medicinal Plants, vol. J. BARNES, S. GIBBONS and E. M. WILLIAMSON. Journal of Ethnopharmacology, vol. pp. Lewis, N. African traditional herbal research clinic. Respiratory Diseases 2013, Newsletter, vol. Pakistan Journal of Biological Sciences, vol. no. pp. NOUMI, E. and A. BORIES, F. GRELLIER, F. FRAPPIER, A. LAURENS and R. HOCQUEMILLER. pp. SAMUELSSON, G. Drugs of natural origin: a textbook of pharmacognosy. th ed. Stockholm: Swedish Pharmaceutical Press. International Journal of Pharmaceutical Sciences Review and Research, vol. no. pp 106-110. CHAN, C. G. GONTIER. Production of plant secondary metabolites: a historical perspective.

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Plant Science, vol. pp. CROZIER, A. Essential oils in food preservation: mode of action, synergies, and interactions with food matrix components. Frontiers in Microbiology 3, 12, doi: 10. fmicb. Kumar R, Tripathi YC. Training manual on, extraction technology of natural dye & aromatherapy and cultivation value addition of medicinal plants. Journal of American Science 6(10), 820-826. Moyler, D. A. Extraction of essential oils with carbon dioxide.  Flavour and fragrance journal, 8(5), 235-247.

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