Study on the potential antimicrobial activities of spp. against skin infecting microbes and detection of their possible mutagenic effect using molecular methods

A Thesis Submitted In Fulfillment of the Requirements for The Degree of Doctor of Philosophy In Pharmaceutical Sciences (Microbiology and Immunology)

By

Ashraf Osman Abdellatif Mohamed M.Sc. of Microbiology and Immunology (2014) A Pharmacist at the Medical Services Administration (Sudan)

Under Supervision of

Dr. Mohammed A. M. Ramadan Dr. Alaa El-Dein M. S. Hosny

Professor of Microbiology and Immunology Professor of Microbiology and Immunology Faculty of Pharmacy Faculty of Pharmacy Cairo University Cairo University

Dr. Mohamed Abd El-Aaty M. Rabeh

Asst. Professor of Pharmacognosy Faculty of Pharmacy Cairo University

Microbiology and Immunology Department Faculty of Pharmacy Cairo University 2019 Abstract

Introduction: The extracts and oils from Melaleuca spp. ( family) have been traditionally used as a topical therapeutic agent for the treatment of various skin infections. The current study aimed to evaluate the potential antimicrobial activities of the different active components of Melaleuca spp. plant as topical agents against some medically important pathogens related to the common skin infections and detection of their possible mutagenic effect. Methods: The M. alternifolia leaves were subjected to successive fractionation using different solvents with different polarities. The TTO was extracted from plant leaves using steam hydro-distillation and then subjected to GC/MS analysis to characterize its main components. The aqueous extract fraction was used to synthesize silver nanoparticles (AgNPs) via green nanotechnology. The different extracted fractions, TTO, and AgNPs were evaluated for their antimicrobial activities against representative skin-infecting pathogen including bacteria, fungi and viruses. Furthermore, the Transmission electron microscope examination was used to characterize the synthesized AgNPs and to visualize the treated microbial cells to figure out the basic changes that may be referred to a certain mode of action. Moreover, the possible mutagenic effect of TTO and AgNPs was investigated against S. typhimurium TA89 and TA100 strains using the Ames test. Additionally, to outline the basic molecular mechanism involved in apoptosis induction in malignant melanoma (A-375) and squamous cell carcinoma (HEp-2) cell lines, the Annexin V/PI staining for apoptosis detection and cell cycle analysis were monitored using flow cytometry and mRNA expression levels of the apoptosis-regulatory genes P53, BAX, and BCL-2 were determined by real-time PCR and western blot after treatment with TTO. An anticandidal oral emulgel preparation and a nanoemulsion containing AgNPs have been developed using the TTO. The oral emulgel was assessed against C. albicans and compared with two oral anticandidal preparations obtained from the local market. On the other hand, the TTO-AgNPs nanoemulsion was assessed against S. aureus, P. aeruginosa, and C. albicans as representatives for Gram positive, Gram negative, and yeast fungi, respectively. Results: The major oil components characterized by GC/MS analysis were: terpinen- 4-ol, limonene, γ-terpinene, α-terpinene, cineol, and α-terpinolene. The characterization of AgNPs by transmission electron microscopy showed them to be homogenous and spherical with an average size of 13.3 nm. The bioassay results showed that both TTO and AgNPs possess potent antimicrobial properties against tested strains, producing marked inhibition zones (14.8-24.7 mm). Transmission electron microscope observations showed that non-treated cells were intact and showed a smooth surface, while TTO-treated and AgNPs-treated cells underwent considerable structural changes. Additionally, tests against HSV-1 and HSV-2 showed that AgNPs had the strongest antiviral activity, causing 44.0% and 45.04% reduction of the cytopathic effect for HSV-1 and HSV-2, respectively. The mutagenesis assay findings obtained by the Ames test clearly demonstrate that both TTO and AgNPs have no mutagenic activity either in the S. typhimurium T98 or TA100 strains. The results also showed that TTO exhibited strong cytotoxicity towards A-375 and HEp-2 cell lines, with IC50 values of 0.038% (v/v) and 0.024% (v/v) respectively. This cytotoxicity resulted from TTO induced apoptosis in both A- 375 and HEp-2 cell lines as evidenced by morphological features of apoptosis and Annexin V/PI staining results in addition to the activation of caspase- 3/7 and -9, upregulation of pro-apoptotic genes (P53 and BAX) and downregulation of the anti- apoptotic gene BCL-2. Additionally, cell cycle analysis showed that TTO caused cell cycle arrest mainly at G2/M phase. When compared with miconazole gel and nystatin suspension, the developed TTO oral emulgel showed good antimicrobial activity against C. albicans and appeared to be equally effective as the miconazole gel and interestingly, it was superior to nystatin. Conclusion: In conclusion, the data from this study suggest that both TTO and AgNPs obtained by using M. alternifolia leaves have a broad spectrum of activity against the selected skin pathogens. The study also indicates that treatment with TTO induced cellular death by mitochondrial apoptosis through a P53-dependent pathway that involves target proteins of the BCL-2 family. Moreover, the results confirm that TTO is an effective apoptosis inducer in A-375 and HEp-2 cancer cell lines. Furthermore, this investigation as a whole has proven the efficacy of TTO and AgNPs as agents, acting singly and in combination, against microorganisms from different groups (Gram-positive, Gram-negative, and yeast). These results provide a basis for combined preparations to be explored as a mean of enhancing efficacy whilst minimizing the rapid development of resistance to single antimicrobial agents. However, further, in vivo experiments are warranted.

Keywords: Tea oil, silver nanoparticles, GC/MS, antimicrobial, skin infections, Transmission Electron Microscope, malignant melanoma, squamous cell carcinoma, cell cycle analysis, apoptosis, chemoprevention, emulgel, nanoemulsion.

Introduction

Infectious diseases, particularly skin and mucosal infections, are common in many communities due to lack of sanitation, potable water and awareness of hygienic habits (Bartram and Cairncross, 2010). It has been estimated that skin diseases account for 34% of all occupational diseases (Njoroge and Bussmann, 2007).

As the primary interface between the body and external environment, the skin provides the first line of defense against broad injury by microbial and chemical agents (Chanda and Baravalia, 2010). The most damaging consequence of disruption to the skin is the invasion by pathogenic microorganisms (Grice and Segre, 2011).

In skin and soft tissue infections, the most common bacteria are; Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, and the Bacteroides group. Other bacterial includes Mycobacterium tuberculosis, Mycobacterium leprae, Neisseria gonorrhoeae, Pasteurella tularensis, Bacillus anthracis and Pseudomonas aeruginosa (Stulberg et al., 2002).

On the other hand, the common fungi which cause skin infections are; Candida albicans, Candida neoformans, Epidermophyton floccosum, Trichophyton tonsurans, Malassezia furfur (Crawford and Hollis, 2007). As well, viral infections occur when a virus penetrates the stratum corneum and infects the inner layers of the skin. Examples of viral skin infections include herpes simplex, herpes zoster, and warts. Additionally, some systemic viral infections, such as chicken pox and measles, may also affect the skin (Chanda and Baravalia, 2010; Sterling, 2016).

Natural products from the are gaining popularity because of several advantages such as; often having fewer side-effects, better patient tolerance, being relatively less expensive, and acceptable due to a long history of use and provide rational means for the treatment of many diseases. For these reasons, several plants have been investigated for treatment of skin diseases ranging from itching to skin cancer (Tabassum and Hamdani, 2014).

The leaves extracts and oils from Melaleuca spp., from the family Myrtaceae, have been traditionally used as a topical therapeutic agent for abscess, sores, athlete's foot, wart, insect bites, rashes, minor wounds, and skin irritation as well as chicken pox and measles. Moreover, the essential oils of Melaleuca spp., especially Melaleuca alternifolia is recently used in many cosmetics for its antiseptic activities. Many of these antimicrobial activities are attributed to monoterpenes, sesquiterpenes and their related alcohols, which are the main components in the of Melaleuca spp. (Carson et al., 2006).

However, based on pharmacological studies, their use was recommended to be for topical applications to avoid their unexpected side effects if used systematically (Hammer et al., 2006)

Despite preliminary findings of therapeutic advantages of medicinal plants, some of their constituents may be potentially toxic, mutagenic, carcinogenic, or teratogenic. Therefore, medicinal plants must be tested with regard to quality, safety, and efficiency, like conventional drugs (Eren and Özata, 2014).

Aim of the work

The aim of the current study was to evaluate the potential antimicrobial activities of the different active components of Melaleuca spp. plant as a topical agent against skin-infecting microorganisms and detection of their possible mutagenic effect. The study was also focused on the application of these components in a drug form for the treatment of skin infections.

Plan of the work

1. Assessment of different antimicrobial activities of extracts and oils from Melaleuca spp., against representative skin-infecting pathogens, including bacteria, fungi, and viruses. 2. Qualitative and quantitative analysis to characterize and identify the different active components in the extracts and oils. 3. Transmission electron microscope examination of treated microbial cells to visualize the basic changes that may be referred to a certain mode of action. 4. Molecular study to evaluate the cytotoxic and mutagenic effect of the active components on human malignant melanoma and squamous cell carcinoma cells proliferation through quantifying the expression of p53, BAX, and Bcl-2 as pro-apoptotic and anti-apoptotic gene markers, respectively. 5. Enhancement of the antimicrobial activities of the active components through micronized and nanoforms.

2. Review of literature

2.1. Skin infections

Human skin, the outer covering of the body, is the largest organ in the body and constitutes the first line of defense against infections, temperature changes, and other challenges to homeostasis (Madison, 2003). This defense may be compromised in the event of skin trauma from either physical or chemical injuries, allowing for microbial invasion, and may lead to severe skin infections which can be a major cause of death (Boer et al., 2016; Church et al., 2006).

Skin infections are widespread and are among the most prevalent and disabling diseases. The Global Burden of Disease found that skin infections were the fourth leading cause of non-fatal health burden, expressed as years lost to disability, and noted an ‘urgent need for the inclusion of skin disease prevention and treatment in national and global health policies’ (Hay et al., 2014).

Skin infections are caused by a big range of the microbes including bacteria, fungi, and viruses. Breaks in the skin integrity, particularly those that inoculate pathogens into the dermis, frequently cause or exacerbate skin infections (May et al., 2009). The most common bacterial strains associated with skin infections are; Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, and Bacteroides group. Other bacterial agents includes Mycobacterium tuberculosis, Mycobacterium leprae, Neisseria gonorrhoeae, Pasteurella tularensis, Bacillus anthracis and Pseudomonas aeruginosa (Stulberg et al., 2002).

On the other hand, many fungal strains are associated with skin infections such as; Candida albicans, Candida neoformans, Epidermophyton floccosum, Trichophyton tonsurans, and Malassezia furfur (Crawford and Hollis, 2007). Also, many viruses are associated with skin infections such as herpes simplex virus (HSV), varicella zoster, measles and rubella and human papillomavirus (Sterling, 2016).

2.2. Urgent need for new and novel antimicrobial agents

Due to the overuse and incorrect prescribing of the currently available conventional antimicrobial drugs, resistance has emerged among common skin pathogens (DeRyke et al., 2005; Fair and Tor, 2014). Multidrug-resistant bacteria, including nosocomial pathogens, have become an important cause for higher skin care costs; therefore, a pre-antibiotic antimicrobial agent with broad-spectrum activity and a different mode of action against resistant microbes is urgently needed (Borges et al., 2016; Xiao, 2014). Candidate agents include the plant-derived extracts, essential oils, and metal nanoparticles which have been recently introduced as a promising antimicrobial agent with unique properties and potencies (Hajipour et al., 2012; Rai and Kon, 2013).

2.3. Plants as a source of new and novel antimicrobial compounds

The search for new antimicrobial agents has continued to be concentrated on higher plants as sustainable sources and the ways to identify new bacterial targets for the discovery of novel inhibitors are a high research priority (Barrett and Barrett, 2003). The use of medicinal plants and preparations taken from them for the treatment of infectious diseases caused by microbial pathogens have a long history in almost all cultures (Karou et al., 2007) and compounds such as emetine, quinine, berberine, and artemisinin are well-known examples of plant-derived antimicrobial compounds that remain highly effective agents in the fight against microbial infections (Osbourn and Lanzotti, 2009).

Plants defend themselves against attack by insect predators, herbivores, pathogenic fungi, bacteria and viruses by certain structural and chemical defense strategies such as the production of secondary metabolites. Various secondary metabolites have been reported to apparently function as a defense against these organisms and are thus important for the plants' survival and reproducible fitness (Wallace, 2004). Plants have evolved a fast array of secondary metabolites that have been subjected to natural selection during evolution. They are present in all higher plants, usually in a high structural diversity (Grayer and Kokubun, 2001; Wink, 2003).

2.6. Traditional uses of Melaleuca spp.

Members of the genus Melaleuca have long been utilized in the folk medicines in various parts of the world. M. alternifolia is the most well-known that has been reported with a wide range of traditional uses in Asia, Africa, America, and (Sharifi‐ Rad et al., 2017; Southwell and Lowe, 2003).

For instance, M. alternifolia has been a widely practiced herbal remedy through topical application to treat bruises, colds and flu, insect bites, insect repellant, and for skin infections (Carson et al., 2006). The traditional use of M. alternifolia against skin infections such as acne, herpes, and scabies has also been mentioned by Sharifi‐ Rad et al., (2017). The local uses of its essential oil against microbial infections, e.g. tinea pedis were documented in a number of clinical studies (Satchell et al., 2002; Tong et al., 1992).

The bark and leaves of M. leucadendron have been reported to be used as tranquilizer, sedative, and for pain-relieving purposes in Taiwanese folk medicine (Sharifi‐ Rad et al., 2017), while it was utilized for the treatment of gout and related symptoms in Vietnamese (Nguyen et al., 2004). The leaves of M. quinquenervia are recorded in Thai traditional medicine to treat gastrointestinal disorders and to repel insects (Moharram et al., 2003). Also, the Melaleuca cajuputi is one of the Melaleuca species used against microbial infections in folk medicines (Al-Abd et al., 2015).

2.7. Melaleuca alternifolia and Melaleuca leucadendron

2.7.1. Scientific classification of Melaleuca alternifolia

Kingdom: Plantae Clade: Angiosperms Clade: Clade: Order: Family: Myrtaceae Genus: Melaleuca Species: M. alternifolia

Figure (1): Photo of Melaleuca alternifolia leaves

2.7.2. Scientific classification of Melaleuca leucadendron

Kingdom: Plantae Clade: Angiosperms Clade: Eudicots Clade: Rosids Order: Myrtales Family: Myrtaceae Genus: Melaleuca Species: M. leucadendron

Figure (2): Photo of Melaleuca leucadendron leaves

M. alternifolia is commonly known as the tea tree, it has attracted the attention of many scientists due to its unique biological and chemical properties. Essential oil or crushed leaves obtained from M. alternifolia are traditionally used to treat coughs and colds, and as an antiseptic for wound and skin treatment or throat infections. Many studies provided information about the broad spectrum of antimicrobial activity of M. alternifolia oil in vitro (Carson et al., 2006; Hammer et al., 1998; Sharifi‐ Rad et al., 2017).

On the other hand, The M. leucadendron essential oil activity was tested against B. subtilis, E. coli, A. niger, and C. albicans by measuring the inhibition zone (Fernández-Calienes Valdés et al., 2008). In particular, M. leucadendron extract showed slight antifungal activity against M. canis (Fernández-Calienes Valdés et al., 2008; Pujiarti et al., 2011; Rini et al., 2012).

2.9. (TTO)

Tea tree oil (TTO) is the essential oil steam distilled from M. alternifolia leaves. Traditionally, the oil was used for acne, eczema, skin infections like herpes, wounds, warts, burns, insect bites and nail mycosis. Other uses mentioned are colds, sore throat and gingival infections, hemorrhoids and vaginal infections (Bursch et al., 1992; Carson et al., 2006; Hammer et al., 1998; Tong et al., 1992). TTO has widely been used as a topical antiseptic, and it became increasingly more popular as a safe, natural, and effective antiseptic. The high efficacy and efficiency of tea tree oil in treating topical infections has stimulated considerable interest, and its incorporation into preparations, especially in cosmetics, is increasing at a rapid rate (Aburjai and Natsheh, 2003).

2.11. Mutations and the molecular basis of cancer

Genes that contribute to cancer include oncogenes and tumor suppressor genes (Bouck, 1990). The oncogenes change a normal healthy cell into a cancerous cell and by contrast, tumor suppressor genes protect a cell from becoming cancerous. The tumor suppressor proteins control cell growth by monitoring cell division, repairing base mismatches in DNA and controlling cell death (apoptosis) (Levine and Puzio-Kuter, 2010; Maiuri et al., 2009).

Over the past several decades, attention has been focused on understanding the molecular basis of carcinogenesis (Weinberg, 1996a). Studies revealed that several factors and mechanisms are involved in the control of cancer and one of these mechanisms is apoptosis (Kam and Ferch, 2000). Apoptosis or programmed cell death is a key regulator of physiological growth control and tissue homeostasis (Elmore, 2007). Cell death, mostly by apoptosis, is crucially involved in the regulation of tumor formation and also determines the treatment response (Fulda and Debatin, 2006). The apoptotic process is modulated by various tumor suppressor genes, including P53 and proto-oncogenes (Oren, 1992). After DNA damage, some cellular responses are triggered by transcriptional activation of P53 and BCL-2 family proteins in order to maintain the integrity of healthy cells. The activation of P53 leads to either DNA repair and recovery or to apoptosis (Elmore, 2007; Norbury and Zhivotovsky, 2004).

The p53 protein prevents the multiplication of damaged cells that are more likely to contain mutations and exhibit abnormal cellular growth than undamaged cells. Hence, p53 protein is the guardian of the genome preventing cancer formation and the mechanisms by which p53 accomplishes its tumor suppressor activity are still not completely understood. The best-described mechanism is its ability to modulate gene expression (Levine et al., 1991; Prives and Hall, 1999).

Recently, several p53-inducible genes that encode for proteins with apoptotic potential have been identified. However, the tumor suppressor p53 can trigger cue all death independently of its transcriptional activity through sub- cellular translocation and activation of pro-apoptotic Bcl-2 family members (Reed, 1995).

The BCL-2 family of proteins consists of both pro-apoptotic and anti- apoptotic members such as BAX and BCL-2 which are important mediators of the mitochondrial outer membrane permeabilization that is accompanied by apoptosis (Ola et al., 2011). They also have been reported to play a central role in regulating cytochrome c release from mitochondria (Martinou and Youle, 2011).

The anti-apoptotic protein BCL-2 is located in the outer mitochondrial membrane and plays an essential role in promoting the survival of cells and inhibiting the effects of pro-apoptotic proteins (Youle and Strasser, 2008). Overexpression of BCL-2 has been demonstrated to inhibit cell death induced by many stimuli, including growth factor deprivation, hypoxia, and oxidative stress (Yip and Reed, 2008). On the other hand, the pro-apoptotic protein BAX controls cell death through its participation in the disruption of mitochondria, and its expression is regulated by the tumor suppressor gene P53 (Korsmeyer, 1999). Upregulation of BAX enhances opening of the mitochondrial voltage-dependent anion channel, resulting in loss of membrane potential with subsequent release of cytochrome c (Gogvadze et al., 2006).

By apoptosis, unwanted or damaged cells are eliminated from the system. Thus, induction of tumor cell apoptosis would be considered a protective mechanism against the development and progression of cancer (Bursch et al., 1992). Compounds that suppress the proliferation of malignant cells by inducing apoptosis may represent a useful mechanistic approach to cancer chemoprevention (Sporn and Suh, 2002).

Chemoprevention is a pharmacological approach using natural, synthetic, or biological agents that can prevent, inhibit, and reverse carcinogenic progression. It has been regarded as a new, hopeful, safe, and efficient strategy for cancer treatment (Gullett et al., 2010; Mehta et al., 2010; Sporn and Suh, 2002).

2.12. Development of herbal-based formulations for topical use

Topical administration is the favored route for local delivery of therapeutic agents due to its convenience and affordability. However, conventional topical drug delivery systems suffer from drawbacks such as poor retention and low bioavailability (Singh Malik et al., 2016). The specific challenge of designing a therapeutic system is to achieve an optimal concentration of a certain drug at its site of action for an appropriate duration. The successful formulation of topical delivery products requires the careful manipulation of defensive barriers and selection of a suitable drug carrier (Paudel et al., 2010; Singh Malik et al., 2016).

The skin forms a barrier to the external environment and is impermeable to the drugs due to epidermal cell cohesion and stratum corneum lipids (Figure 4). Extensive researches are carried to develop newer topical drug delivery systems with improved efficacy and least side effects on skin barrier function (Naik et al., 2000; Pandey et al., 2013).

Figure (4): Skin structure (Adapted from; www. courses.lumenlearning.com)

2.12.1. Nano-scale formulations as advances in topical drug delivery systems

Nanotechnology can be used to modify the drug permeation/penetration by controlling the release of active substances and increasing the period of permanence on the skin, besides ensuring direct contact with the stratum corneum and skin appendages and protecting the drug against chemical or physical instability (Gupta et al., 2013; Neubert, 2011). Nanoparticles based on lipid systems are the most common type of nanoparticles studied for nanostructured topical formulations. Solid lipid nanoparticles, nanoemulsions, and nanostructured lipid carriers are the main types of matrix nanoparticles while liposomes are the main type of vesicular particles evaluated in permeation studies (Gupta et al., 2013).

These nanostructure systems are an upcoming option for drug delivery because of their advantages over the conventional formulations. These colloidal particulate systems with size ranging from 10 nm to 1000 nm offer targeted drug delivery, sustained release, protection of labile groups from degradation, and drug adhesivity to the skin. Therefore, the use of nano-scaled formulations in drug delivery is expected to increase the specificity of drugs and thus reduce the expected side effects in addition to decreasing the dose of the administered drugs (Gupta et al., 2013; Schwarz et al., 2012).

Due to their unique size and composition-dependent properties, nanoemulsions have a lipophilic interior, they are efficient at transporting hydrophobic substances in aqueous environments. They present a higher capacity to penetrate and/or permeate the skin, probably due to their flexibility and can allow for deeper penetration of water-immiscible active ingredients and increase their effective concentration in target tissues. They are being exploited as cutaneous delivery vehicles for many products (Debnath and Satayanarayana Kumar, 2011; Gupta et al., 2013; Thakur et al., 2012).

Nanoemulsions are being created as drug delivery vehicles for topical use, intranasal, and intratracheal use, as well as for ingestion and parenteral use (Shah et al., 2010). They form an ideal vehicle to be used in treatment for many skin infections as they are stable for the transport of lipophilic compounds into the skin. Nanoemulsions also do not have the problem of creaming, flocculation, coalescence, and sedimentation, which are commonly associated with macroemulsions, thus ensuring better stability of the formulation (Shah et al., 2010; Thakur et al., 2012).

2.13. Inorganic nanoparticles

Nanoparticles, which are defined as particles with size up to 100 nm (Nalwa, 2005), show completely new and improved properties. They provide specific characteristics such as particle size, distribution properties, and morphology, as the surface area of nanoparticles increased, their biological activity increases (Jahn, 1999).

Recent advances have shifted to inorganic nanoparticles for specific targeting and control of their cellular actions. Inorganic nanoparticles generally possess versatile properties suitable for cellular delivery, including wide availability, rich functionality, good biocompatibility, potential capability of targeted delivery and controlled release of carried drugs and high stability for long periods (ud Din et al., 2017).

2.13.1. Inorganic nanoparticles and their uses in topical formulations

Nano-sized particles of ZnO and titanium dioxide (TiO2) used in sunscreens are prime examples of inorganic nanoparticles. They are not only transparent but also cosmetically desirable. The TiO2 is more effective in UVB and ZnO in the UVA range, the combination of these particles assures a broad- band UV protection (Singh and Nanda, 2014; Smijs and Pavel, 2011).

Recently, silver nanoparticles (AgNPs) have received much attention due to their extraordinary antimicrobial effects against a wide range of microorganisms, including antibiotic-resistant strains (Abbasi et al., 2016). Their small sizes and large surface areas provide good contact with microorganisms, confer enhanced bioactivity and bioavailability of Ag+ and allow better penetration into microbial cells (Hajipour et al., 2012; Rai et al., 2012).

2.13.2. Nanoparticles synthesis using plants (Green synthesis)

Various methods are applied to synthesize silver nanoparticles (AgNPs); among them is the green synthesis method, in which an aqueous plant extract is used as reducing and capping agent. The green synthesis method provides a simple, cheap, fast, energy-efficient, and eco-friendly alternative to the traditional chemical and physical methods of nanoparticles synthesis (Hebbalalu et al., 2013; Kulkarni and Muddapur, 2014).

Plant extracts offer many advantage when being used for nanoparticle synthesis such as; they are safe and nontoxic, easily available, and in most cases, have a broad variety of metabolites that can aid in the reduction of silver ions, and being quicker than microbes in the synthesis (Prabhu and Poulose, 2012), and can also be suitably scaled up for large-scale synthesis (Shankar et al., 2003).

2.13.3. Applications of silver nanoparticles as antimicrobial agent

The use of AgNPs as an antimicrobial agent is relatively new. Because of their high reactivity due to the large surface to volume ratios, nanoparticles play a great role in inhibiting bacterial growth in solid and aqueous mediums (Ravishankar Rai and Jamuna Bai, 2011).

The silver has long been recognized as an inhibitor towards many bacterial strains and microorganisms commonly present in the medical and industrial processes (Murphy, 2008). It is a non-toxic, safe inorganic antibacterial agent being used for centuries and is capable of killing many microorganisms (Jeong et al., 2005).

Silver has been described as being oligo-dynamic and is capable of causing a bacteriostatic or even a bactericidal effect at minute concentration. They are highly toxic to microorganism exhibiting a strong biocidal effect on many species of bacteria but have low toxicity towards animal cells. It has a significant potential for a wide range of biological applications such as antibacterial agents for antibiotic-resistant bacteria, preventing infections and healing wounds (Percival et al., 2005).

There are many applications of Ag+ in the pharmaceutical industry such as topical skin ointments and creams containing silver to avoid the infections of wounds and burns (Schultz, 2003). Furthermore, silver is used in the formulation of dental resin composites, bone cement, ion exchange fibers and coatings for medical devices as it exhibits a good antimicrobial activity (Panáček et al., 2006). This advantages applied in the medicine to reduce infections and to prevent bacterial colonization on human skin (Durán et al., 2005).

Summary and Recommendations

The outcomes of this study could be summarized in the following points:

 Among the different tested extracts, tea tree oil (TTO) and silver nanoparticles (AgNPs) were the most active, while the aqueous extract was totally inactive against the selected microbes at the tested concentrations.  The TTO was obtained from the leaves of M. alternifolia leaves by steam hydro-distillation using Clevenger apparatus, while the AgNPs was obtained by green synthesis method using the aqueous extract of M. alternifolia leaves as better alternative synthesis method with many advantages over other conventional methods such as; hazardless, ease of applicability for large scale production, economically feasible, and eco- friendly.  The characterization of TTO using GC/MS analysis allowed the identification of 40 components in the extracted TTO. The major characterized components were; terpinen-4-ol, limonene, γ-terpinene, α- terpinene, cineol, and α-terpinolene.  The characterization of AgNPs using a JOEL transmission electron microscope showed spherical and homogenous AgNPs with an average size of 13.3 nm.  The TTO and AgNPs showed significant activity against Pseudomonas aeruginosa with a zone of inhibition 13.8 mm and 15.3 respectively.  Interestingly, TTO showed promising antifungal activities against Candida albicans better than the standard amphotericin B.  The antibacterial and antifungal activities studies showed a great improvement in the biological activities of the aqueous extracts after AgNPs green synthesis with inhibition zones ranging from 15.3 to 24.2 mm compared to the controls.  Antiviral activities of TTO and AgNPs showed that AgNPs had stronger antiviral activity compared to TTO, causing 44.0% and 45.04% reduction of the cytopathic effect for HSV-1 and HSV-2, respectively. Additionally, HSV-2 was found to be more susceptible.  The interaction between TTO and AgNPs with bacterial cells, using Staph. aureus and E. coli as models by transmission electron microscope showed that, non-treated cells were intact and showed a smooth surface, while treated cells underwent considerable structural changes.  Transmission electron microscope observations confirmed the damage to the structural integrity of the cells and considerable morphological alteration to tested S. aureus and E. coli bacteria; TTO caused pores on the outer membrane of bacterial cells which enabled the cell constituents to pass easily through these and also caused collapsing of the cell.  Silver nanoparticles-treated cells were severely distorted and became disrupted and the AgNPs aggregated and localized non-specifically on the cell wall, and were seen within the cell wall and cytoplasm. Moreover, TEM analysis showed that the cell wall was severely damaged and the cytoplasmic membrane became separated from the cell wall. Therefore, the outer membrane or the cell wall of the bacteria is most likely to be the cellular target for both TTO and AgNPs due to the formation of perforations.  Mutagenesis assay findings obtained by the Ames test clearly demonstrate that both TTO and AgNPs have no mutagenic activity either in the S. typhimurium T98 or TA100 strains.  The TTO exhibited strong cytotoxicity towards A-375 and HEp-2 cell

lines, with IC50 values of 0.038% (v/v) and 0.024% (v/v) respectively.  Morphological changes in TTO-treated cells showed that untreated A-375 and HEp-2 cells were regular, hyperchromatic, and have condensed nuclei with no evidence of folding in the cellular and nuclear membranes. In contrast, TTO-treated cells showed the early apoptotic features of peripheral condensation of chromatin and nucleolar segregation.  The TTO induced apoptosis in both A-375 and HEp-2 cell lines as evidenced by morphological features of apoptosis and Annexin V/PI staining results in addition to the activation of caspase- 3/7 and -9, upregulation of pro-apoptotic genes (P53 and BAX) and downregulation of the anti-apoptotic gene BCL-2. Additionally, cell cycle analysis showed that TTO caused cell cycle arrest mainly at G2/M phase.  The developed TTO 2% emulgel showed good antimicrobial activity against C. albicans when compared with miconazole gel and nystatin suspension. It appears to be equally effective as the miconazole gel and interestingly, it was superior to nystatin. Moreover, it showed good stability and homogeneity without phase separation during stability studies period.  TTO/AgNPs nanoemulsion preparation showed good stability and promising antimicrobial activities with synergistic effects on S. aureus and P. aeruginosa as well as, an additional effect on C. albicans.

In conclusion, the data from this study suggest that both TTO and AgNPs obtained by using M. alternifolia leaves have a broad spectrum of activity against the selected skin pathogens acting singly and in combination, against microorganisms from different groups (Gram-positive, Gram-negative, and yeast). These results provide a basis for combined preparations to be explored as a mean of enhancing efficacy whilst minimizing the rapid development of resistance to single antimicrobial agents.

The study also indicates that TTO is an effective apoptosis inducer in malignant melanoma and squamous cell carcinoma cancer causing cellular death by mitochondrial apoptosis through a P53-dependent pathway that involves target proteins of the BCL-2 family, which make TTO a good chemopreventive candidate against these types of skin cancers, potentially able to restore the apoptotic machinery and sensitize these tumors to chemo- and radiotherapies.

Together with previously published data, the lipophilic nature of tea tree oil, and the unique properties of AgNPs, these promising results suggest that both TTO and AgNPs may be suitable candidates for topical use in the treatment of skin infections related to the tested microbes, however, further in vivo experiments are warranted.

Recommendations:

 Further researches are needed to investigate the active components in vivo using different diseased models and develop their application for pharmaceutical industries.  The absence of a mutagenic response by TTO and AgNPs against S. typhimurium TA98 and TA100 strains in the Ames test requires to further confirm the results with other mutagenesis assay methods such as micronucleus assay, comet assay, chromosome aberration assay.  Further studies are required to elucidate the precise molecular mechanisms and targets for cell growth inhibition which will allow the rationale design for more effective molecules for the eventual use as a cancer chemopreventive and/or therapeutic agents.  As the field of nanotechnology continues to develop, the studies on the cellular uptake of nanoparticles, with respect to their size and shape, are required in order to advance nanotechnology for medical applications.  Similarly, it would be of interest to assess nanoparticles toxicity so as to use them for intracellular applications because detailed studies on uptake kinetics of nanoparticles by cells have not been well characterized and quantified.