*2.4. Runs of Homozygosity Estimation*

For ROH calculation, we used a window-free method for consecutive SNP-based detection [29] implemented in the R package "detectRUNS" [30]. One SNP with a missing genotype and up to one possible heterozygous genotype was allowed in the run. The minimum ROH length was 1000 kb.

ROH were estimated for each animal and then categorized in the corresponding ROH length classes: (1–2 Mb, 2–4 Mb, 4–8 Mb, 8–16 Mb, >16 Mb). The total number of identified ROH was calculated for each length category in each of the individuals of each breed. The mean sum of ROH was computed by adding up the length of all ROH for each individual in the sheep populations and then the results were averaged per breed population.

**Table 2.** Mean ROH length and mean ROH number in Russian sheep populations.

1 *n*—number of individuals; 2 Min—minimum values of estimations observed in individual animals within breeds; 3 Max—maximum values of estimations observed in individual animals within breeds.


**NEURODEGENERATIVE DISEASES**

#### **Neurodegenerative Diseases**

http://dx.doi.org/10.5772/45957 Edited by Uday Kishore

#### **Contributors**

Alexander Gow, Ferihan Cetin, George E. Barreto, Ricardo Cabezas, Marco Fidel Ávila, Janneth Gonzalez, Ramon El-Bachá, Ludis Morales, Daniel Torrente, Juan Andres Orellana, Masahiro Kawahara, Tomohiro Chiba, Yan Zhang, Luca Lovrecic, Stavros J. Baloyannis, Cenk Suphioglu, Justin Read, Yang, Ivana Delalle, Manuel J Rojas, Camilo Orozco Sanabria, Francisco Olea, Christian Lefebvre D'Hellencourt, Avinash Parimisetty, Rana Awada, Antonio Ibarra, Humberto Mestre, Daniel Zajarias-Fainsod, Yael Cohen-Minian, Eyer, Eva Babusikova, Marisol Herrera-Rivero, Yusuf Tutar, Aykut Ozgur, Lütfi Tutar, Weili Xu, Anna Marseglia, Camilla Ferrari, Hui-Xin Wang, Iuliana Nicola-Antoniu, Analava Mitra, Faraz Farooq, Martin Holcik, Alex MacKenzie, Uday Kishore

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*2.4. Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy*

The samples were observed without any pre-treatment with a FEI/Philips XL30 ESEM (low vacuum mode—1 torr, 20 kV, BSE detector). The possibility of working in "low vacuum mode" made it possible not to cover the samples with a conductive layer of carbon or gold. Depending on the size of the collected fragments, they were either observed whole or broken up to obtain a significant portion that was compatible with the size of the sample holder. The elemental analyses were carried out using an X-ray energy dispersive spectrometer, EDAX AMETEK Element, coupled to SEM.

A selection of fragments was also embedded in an epoxy resin, cross-cut with a diamond saw and then mechanically polished. Polished cross sections were then analyzed by SEM-EDX, too.


**5. Conclusions**

Based on Gronwall–Bellman inequality, we have proved the Ulam stability of *n*th order delay integro-differential equations. By applying Lipschitz conditions and the Banach contraction principle, the existence and uniqueness theorem of a solution was given. In addition, the expression of the solution played a great role in the proof of the main theorems. The Ulam stability of the *n*-th order delay integro-differential equation is related to many applications, such as the effect of tire dynamics on vehicle shimmy and optimal control of a size-structured population, and the research in this field is still open. In future work, we recommend that interested scholars extend their work to the Ulam stability of a fractional delay integro-differential equation with a Caputo derivative.

**Author Contributions:** Conceptualization, S.W.; writing—original draft preparation, S.W.; writing—review and editing, S.W. and F.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank the reviewers for their helpful suggestions and comments.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.


**3.1. PLM process model**

The company's product development process mainly comprises two wide-ranging processes; the New Technology Introduction (NTI) process and the New Product Introduction (NPI) process. The general research and development exercise occurs through New Technology Induction (NTI) process in their research and Development (R&D) department before the NPI process starts. Emission-related legislation is a key driver in technology development for this company. New technology, for instance, an after-treatment system that will reduce engine emission, would be developed in the NTI stage, and this system would be integrated with the engine through NPI process. This chapter focuses on the NPI process as most directly aligned with generic product development and PLM. However, it is noted that the background processes of NTI critical in product lifecycle especially as new technologies come on stream during life, enabling redesign and retrofitting of new components and subsystems.

As shown in **Figure 6**, this NPI process in the company has seven stages starting from the identification of market needs to the review of a product's performance in the field, i.e. "Requirements" to the "Review of Market Performance" (see Tahera [4] for further details of the company processes). Each stage leads to a formal gate review. Based on prescribed criteria, a product must pass through review at the gateways (GW1, GW2,…GW7) before the product development project proceeds to the next stage.

identifies high risks in design decisions, CAE analysis and design changes are undertaken until the risk is reduced to an acceptable level to proceed with the project. These CAE analyses typically fall into three main areas: structural analysis, mechanism or dynamic analysis and thermo-fluid-flow dynamics. They result in the determination of parameters like material properties, geometric idealisation, and physics, which help to define the scope of the design activity. When the overall risks are assessed, design verification and validation actions are decided and planned to mitigate risk. Verification and validation activities can range from

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Ideally, most of the development related testing should start after the requirements have been identified i.e. after the Gateway 1 (GW1) and continue till the product is validated, i.e. until Gateway 5 (GW5), after which the engine is released to production. However, as depicted in **Figure 6**, these testing activities can spread further across subsequent stages of the process. At each stage, functional tests include the performance and emission (P&E) tests and mechanical tests for durability and reliability. Performance testing measures engines properties. For example, power and fuel consumption of an engine may be measured given a regulated fuel and air intake into the engine cylinder under steady state conditions of constant speed and load. While ensuring the performance, engines need to satisfy legislative conditions, for instance, the chemical constitution of the exhaust gases. The durability and reliability tests are conducted in peak harshness and tougher condition for a reasonably short period of time, called accelerated tests, forcing components or engine to fail/pass. For example, a gross thermal test procedure specifies the test cycle for determining the thermal fatigue resistance of

design changes, further CAE analysis to testing.

**Figure 6.** Stage-gate process for new product introduction in a company study.

Testing and the key activities of design, computer aided design and engineering (CAE), and procurement of prototypes are considered in this study. The latter is a major activity since these need specialist design and manufacturing expertise, often involving new manufacturing processes, materials and technologies. A more detailed flow diagram of these stages is presented in **Figure 6** to show the integration of the key activities.

As the diesel engine is a mature product and design changes happen incrementally, engineers in the company start with an existing analysis of the previous generation of products. For a new product introduction (NPI) programme, product objectives are checked against a current product issues (CPI) database. The CPI database provides information about failure modes and effects of current products, which will need special attention for next generation products. This process is carried out by lead team members who are the technical specialists, component owners, design owners and the verification and validation managers.

The NPI process starts in the requirement gathering phase, and should be finished before Gateway 1 (GW1), i.e. before the concept demonstration phase, however spread across the SD phase. Initially, the design alternatives are included in the analysis, because selection of a design is made based on the risk with that particular design and the associated time and cost of its validation program. All design options are considered during this phase. These help to analyse the trade-offs that can be made across different design options. If this analysis Testing and PLM: Connecting Process and Product Models in Product Development http://dx.doi.org/10.5772/intechopen.80364 81

**Figure 6.** Stage-gate process for new product introduction in a company study.

development. Although, this chapter will only discuss the product design and development activities but it is important to highlight that development of the company's product support starts in parallel with product design and development. Further, to aid support, the product

The company's product development process mainly comprises two wide-ranging processes; the New Technology Introduction (NTI) process and the New Product Introduction (NPI) process. The general research and development exercise occurs through New Technology Induction (NTI) process in their research and Development (R&D) department before the NPI process starts. Emission-related legislation is a key driver in technology development for this company. New technology, for instance, an after-treatment system that will reduce engine emission, would be developed in the NTI stage, and this system would be integrated with the engine through NPI process. This chapter focuses on the NPI process as most directly aligned with generic product development and PLM. However, it is noted that the background processes of NTI critical in product lifecycle especially as new technologies come on stream dur-

As shown in **Figure 6**, this NPI process in the company has seven stages starting from the identification of market needs to the review of a product's performance in the field, i.e. "Requirements" to the "Review of Market Performance" (see Tahera [4] for further details of the company processes). Each stage leads to a formal gate review. Based on prescribed criteria, a product must pass through review at the gateways (GW1, GW2,…GW7) before the

Testing and the key activities of design, computer aided design and engineering (CAE), and procurement of prototypes are considered in this study. The latter is a major activity since these need specialist design and manufacturing expertise, often involving new manufacturing processes, materials and technologies. A more detailed flow diagram of these stages is

As the diesel engine is a mature product and design changes happen incrementally, engineers in the company start with an existing analysis of the previous generation of products. For a new product introduction (NPI) programme, product objectives are checked against a current product issues (CPI) database. The CPI database provides information about failure modes and effects of current products, which will need special attention for next generation products. This process is carried out by lead team members who are the technical specialists,

The NPI process starts in the requirement gathering phase, and should be finished before Gateway 1 (GW1), i.e. before the concept demonstration phase, however spread across the SD phase. Initially, the design alternatives are included in the analysis, because selection of a design is made based on the risk with that particular design and the associated time and cost of its validation program. All design options are considered during this phase. These help to analyse the trade-offs that can be made across different design options. If this analysis

component owners, design owners and the verification and validation managers.

is monitored by the company during its operation and up to its disposal.

ing life, enabling redesign and retrofitting of new components and subsystems.

product development project proceeds to the next stage.

presented in **Figure 6** to show the integration of the key activities.

**3.1. PLM process model**

80 Product Lifecycle Management - Terminology and Applications

identifies high risks in design decisions, CAE analysis and design changes are undertaken until the risk is reduced to an acceptable level to proceed with the project. These CAE analyses typically fall into three main areas: structural analysis, mechanism or dynamic analysis and thermo-fluid-flow dynamics. They result in the determination of parameters like material properties, geometric idealisation, and physics, which help to define the scope of the design activity. When the overall risks are assessed, design verification and validation actions are decided and planned to mitigate risk. Verification and validation activities can range from design changes, further CAE analysis to testing.

Ideally, most of the development related testing should start after the requirements have been identified i.e. after the Gateway 1 (GW1) and continue till the product is validated, i.e. until Gateway 5 (GW5), after which the engine is released to production. However, as depicted in **Figure 6**, these testing activities can spread further across subsequent stages of the process. At each stage, functional tests include the performance and emission (P&E) tests and mechanical tests for durability and reliability. Performance testing measures engines properties. For example, power and fuel consumption of an engine may be measured given a regulated fuel and air intake into the engine cylinder under steady state conditions of constant speed and load. While ensuring the performance, engines need to satisfy legislative conditions, for instance, the chemical constitution of the exhaust gases. The durability and reliability tests are conducted in peak harshness and tougher condition for a reasonably short period of time, called accelerated tests, forcing components or engine to fail/pass. For example, a gross thermal test procedure specifies the test cycle for determining the thermal fatigue resistance of core engine components. Typically, performance and emission related tests are performed before the mechanical durability and reliability testing.

Testing occurs at different levels of the product. Component level testing happens primarily at suppliers of components, although the case study company also carries out testing to investigate areas of design concern. Engine level testing involves standalone engines on a test bed. Machine level testing involves engines mounted in a machine or vehicle to reproduce expected conditions of use. **Figure 7** indicates how engine level and machine level testing are mainly conducted in parallel in the three consecutive stages, for different purposes in the product development and PLM. The stages are characterised by the type of testing activity. Stage 2 has Concept/System Demonstration (SD), stage 3 has Design Verification (DV), stage 4 has Product Validation (PV) and stages 5 and 6 focus on Certification.

*Concept/system demonstration (SD)* testing is primarily to demonstrate 'performance capability'. It shows that the technology can deliver the required performance. Alternative concepts are analysed and evaluated at this phase. A combination of old and new parts are built into an engine called a MULE. This MULE engine is tested to verify the performance of new parts.

*Design verification (DV)* is primarily to develop optimal performance and validate hardware at the optimised performance. The aim is to ensure that design outputs meet the given requirements under different use conditions. At this stage, testing focuses on the verification of a chosen design, through detailed analysis and testing of stress, strength, heat transfer and thermodynamics etc. This stage validates the hardware prior to commitment to expensive production tooling.

*Product validation (PV)* checks the effect of production variability on performance and any remaining hardware variation. This phase performs hardware testing which is limited to late design changes and emissions conformance testing. In this phase, detailed testing for reliability and durability occurs and the intended product is validated. The mandatory tests required for compliance usually occur during PV phases.

Testing for certification happens in stages 5 and 6 before product is released to customers. Global emission regulations for diesel engine manufacturers provide requirements for the testing and evaluation of new components and new engine designs. It is an imperative for certification that the company follows the standard regulations during product development in terms of how a product needs to build and tested during validation for certification. For instance, to meet the in-use compliance, the company needs to demonstrate that the engine will meet specific levels of particulate emissions that will be detected and measured at the end of the useful life of the product.

characteristics identification data, usually the unique numbers, operating data (engine on/off and physical variables), event data (failure and maintenance history) and environmental and

**Figure 7.** Flow diagram of testing and associated activities in diesel engine design and manufacture (adapted from [4]).

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**1.** to monitor: how a product is used by a specific customer groups to identify any inappro-

**2.** to monitor the current health of the product to plan and design their aftermarket service,

**3.** to feedback to the beginning of the lifecycle as the product health monitoring data are the key input for designing the next generation of the product. This enables a reliable specifi-

cation for the design phase and the development of a product description.

condition data. These field data are useful for several reasons:

priate and misuse,

i.e. repairs and maintenance services.

The case study company considers that testing of their product continues into use. As one senior engineer in the company remarked "*in fact, real tests start when products are in use*". Their engines are equipped with a remote monitoring system that allows them to capture and collect field data. They have special user groups and they have established close relationship with consumers who help to collect more reliable data. The data consists of equipment Testing and PLM: Connecting Process and Product Models in Product Development http://dx.doi.org/10.5772/intechopen.80364 83

core engine components. Typically, performance and emission related tests are performed

Testing occurs at different levels of the product. Component level testing happens primarily at suppliers of components, although the case study company also carries out testing to investigate areas of design concern. Engine level testing involves standalone engines on a test bed. Machine level testing involves engines mounted in a machine or vehicle to reproduce expected conditions of use. **Figure 7** indicates how engine level and machine level testing are mainly conducted in parallel in the three consecutive stages, for different purposes in the product development and PLM. The stages are characterised by the type of testing activity. Stage 2 has Concept/System Demonstration (SD), stage 3 has Design Verification (DV), stage

*Concept/system demonstration (SD)* testing is primarily to demonstrate 'performance capability'. It shows that the technology can deliver the required performance. Alternative concepts are analysed and evaluated at this phase. A combination of old and new parts are built into an engine called a MULE. This MULE engine is tested to verify the performance

*Design verification (DV)* is primarily to develop optimal performance and validate hardware at the optimised performance. The aim is to ensure that design outputs meet the given requirements under different use conditions. At this stage, testing focuses on the verification of a chosen design, through detailed analysis and testing of stress, strength, heat transfer and thermodynamics etc. This stage validates the hardware prior to commitment to expensive

*Product validation (PV)* checks the effect of production variability on performance and any remaining hardware variation. This phase performs hardware testing which is limited to late design changes and emissions conformance testing. In this phase, detailed testing for reliability and durability occurs and the intended product is validated. The mandatory tests required

Testing for certification happens in stages 5 and 6 before product is released to customers. Global emission regulations for diesel engine manufacturers provide requirements for the testing and evaluation of new components and new engine designs. It is an imperative for certification that the company follows the standard regulations during product development in terms of how a product needs to build and tested during validation for certification. For instance, to meet the in-use compliance, the company needs to demonstrate that the engine will meet specific levels of particulate emissions that will be detected and measured at the end

The case study company considers that testing of their product continues into use. As one senior engineer in the company remarked "*in fact, real tests start when products are in use*". Their engines are equipped with a remote monitoring system that allows them to capture and collect field data. They have special user groups and they have established close relationship with consumers who help to collect more reliable data. The data consists of equipment

before the mechanical durability and reliability testing.

82 Product Lifecycle Management - Terminology and Applications

of new parts.

production tooling.

for compliance usually occur during PV phases.

of the useful life of the product.

4 has Product Validation (PV) and stages 5 and 6 focus on Certification.

**Figure 7.** Flow diagram of testing and associated activities in diesel engine design and manufacture (adapted from [4]).

characteristics identification data, usually the unique numbers, operating data (engine on/off and physical variables), event data (failure and maintenance history) and environmental and condition data. These field data are useful for several reasons:

To help deliver these benefits the company creates two descriptions for monitoring and new product development in addition to the PLM descriptions mentioned in Section 2 for product, design and performance:

of the accessibility and reparability of the inherent systems and their related equipment in the event of failure, as well as of integrated systems shutdown during planned maintenance" [25]. Maintainability procedures and techniques not only avoid and fix failures they also consider how a system might fail. Three types of maintenance can be distinguished: breakdown maintenance (corrective maintenance), preventive maintenance, and predictive maintenance

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Condition monitoring and fault diagnosis techniques are used for predictive maintenance [26]. Product health monitoring is a research area that covers failure detection, current health assessment and remaining useful life prediction [26, 27]. According to Fu et al. [28], most failures do not occur instantaneously. There is degradation and associated symptoms before the actual failure. The main objective of the predictive maintenance is to reliably identify these degradation processes so that maintenance can be affected before the actual breakdown. Predictive maintenance is based on the product's performance and condition monitoring data. For example, in well-established methods, vibration data is analysed to find the frequency

At the design and development design stage the main characteristics of a product are determined and product performance is evaluated. Therefore, design for maintainability should be considered during the product development. However, according to Coulibaly et al. [29], there is lack of an efficient tool for considering maintainability and serviceability at the early design. Also, there is limited research on how information from design, CAE and tests can

Kiritsis et al. [30] have commented that clear definition of the information for maintenance is required if appropriate and adequate information is to be collected. Usually, data collected during Middle of Life (MOL) phase of product is for maintenance management purposes and may not be appropriate for feeding back to the Beginning of Life (BOL) phase to redesign or improve a product. Although people involved in this process often have a clear understanding of the required information, it is not straightforward to define or determine exactly what

A baseline performance description would allow degradation over a period of use to be assessed. As mentioned before, advanced engineering products such as the diesel engines studied here are equipped with instruments such as sensors, meters, controllers, and computational devices and have the processing capacity to self-detect/ predict certain problems. Next section proposes a conceptual model to facilitate this process. Design and testing data from the EOL stages can be a useful reference point for comparing with monitoring data for predictive maintenance. Also, this model can help to clearly define the information required

**4. Extending the proposition: testing data for predictive maintenance**

This section extends a method for managing the iteration of design and testing during the product development stage [3] to predictive maintenance during the product use phase. First,

responses to identify the type of fault present in the equipment [27].

(condition-based maintenance).

support product maintenance.

information will be required.

to be collected to comparison.


*Article* **Niosomal Nanocarriers for Enhanced Dermal Delivery of Epigallocatechin Gallate for Protection against Oxidative Stress of the Skin**

**Danhui Li 1 , Nataly Martini 1 , Zimei Wu 1 , Shuo Chen 1 , James Robert Falconer 2 , Michelle Locke 3 , Zhiwen Zhang 4 and Jingyuan Wen 1,\***

**Abstract:** Among green tea catechins, epigallocatechin gallate (EGCG) is the most abundant and has the highest biological activities. This study aims to develop and statistically optimise an EGCGloaded niosomal system to overcome the cutaneous barriers and provide an antioxidant effect. EGCG-niosomes were prepared by thin film hydration method and statistically optimised. The niosomes were characterised for size, zeta potential, morphology and entrapment efficiency. Ex vivo permeation and deposition studies were conducted using full-thickness human skin. Cell viability, lipid peroxidation, antioxidant enzyme activities after UVA-irradiation and cellular uptake were determined. The optimised niosomes were spherical and had a relatively uniform size of 235.4 ± 15.64 nm, with a zeta potential of −45.2 ± 0.03 mV and an EE of 53.05 ± 4.46%. The niosomes effectively prolonged drug release and demonstrated much greater skin penetration and deposition than free EGCG. They also increased cell survival after UVA-irradiation, reduced lipid peroxidation, and increased the antioxidant enzymes' activities in human dermal fibroblasts (Fbs) compared to free EGCG. Finally, the uptake of niosomes was via energy-dependent endocytosis. The optimised niosomes have the potential to be used as a dermal carrier for antioxidants and other therapeutic compounds in the pharmaceutical and cosmetic industries.

**Keywords:** niosomes; catechin; dermal delivery; antioxidant activity; oxidative stress; skin barrier; penetration; cellular uptake

## **1. Introduction**

The skin is the largest organ of the human body, which makes it the direct target of oxidative stress due to the exposure to reactive oxygen species (ROS) from the surrounding environment. The most important function of human skin is protection by providing a barrier from pathogens, and physical and chemical damages. It also plays a crucial role in thermoregulation and endocrine function such as vitamin D synthesis [1,2]. The skin comprises three layers: the epidermis, which consists of keratinocytes; the dermis consisting of connective tissue, and the subcutaneous layer [3]. The epidermis can be divided into four layers, including the stratum corneum (SC), stratum granulosum, stratum spinosum and stratum basale. The dermis is composed of connective tissues, which are also rich in glands, white blood cells and blood vessels [4,5]. SC is the highly hydrophobic surface layer that contains 18 to 21 cell layers and is composed of corneocytes that are

**Citation:** Li, D.; Martini, N.; Wu, Z.; Chen, S.; Falconer, J.R.; Locke, M.; Zhang, Z.; Wen, J. Niosomal Nanocarriers for Enhanced Dermal Delivery of Epigallocatechin Gallate for Protection against Oxidative Stress of the Skin. *Pharmaceutics* **2022**, *14*, 726. https://doi.org/10.3390/ pharmaceutics14040726

Academic Editors: Alyssa Panitch and Montse Mitjans Arnal

Received: 30 January 2022 Accepted: 23 March 2022 Published: 28 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

terminally differentiated keratinocytes anchored in a lipophilic matrix [3,4]. The 'bricks and mortar' model is often employed to describe the structure of SC, in which intercellular lipid accounts for 10% of the dry weight of this layer, and the rest is an intracellular protein (mainly keratin). Keratins are a family of alpha-helical polypeptides with a molecular weight ranging from 40,000 to 70,000 Daltons, making the corneocyte layers dense and relatively impervious to external compounds [6].

The skin is continuously exposed to environmental threats such as UV radiation, pollution, micro-organisms, and viruses, which lead to ROS production. ROS are also formed during the normal cellular metabolism and immune reactions. More than 80% of environmental ROS that damage the skin is produced by UV [7]. Antioxidants such as glutathione, ubiquinol and thiols inhibit oxidation reactions by donating electrons to free radicals [7]. Our bodies also produce enzymatic antioxidants, such as superoxide dismutase and glutathione peroxidases. Other non-enzymatic antioxidants such as vitamin E (alpha-tocopherol) and vitamin C (ascorbic acid) are obtained from the diet [8]. However, the antioxidants produced by our bodies are inadequate to protect against oxidative stress, and antioxidants are often used as dietary supplements to replenish the level of endogenous antioxidants and hence help to delay the onset of aging or diseases [8].

Catechins are a group of powerful antioxidants with health-promoting effects, and epigallocatechin gallate (EGCG) is one of the catechins found in green tea. EGCG has several beneficial effects on the skin, including anti-aging, anti-inflammatory, and anticancer properties. According to a study, treating normal human epidermal keratinocytes with EGCG prevented UVB-induced intracellular release of hydrogen peroxide while also inhibiting UVB-induced oxidative stress-mediated skin damage [9]. EGCG has been shown to inhibit UV-induced collagen production and collagenase transcription in human dermal fibroblasts [10]. In addition, on the human model, catechins were shown to have anti-aging functions [9]. A double-blind, placebo-controlled experiment of adult women found that catechins can reduce total sun damage when given as oral catechins supplements [11]. The oral administration route is generally the most accepted for drug administration, particularly for long-term prevention purposes. However, when administered orally, catechins readily undergo several metabolic transformations by intestinal microflora and enzymes; therefore, they are poorly bioavailable [12]. The application of EGCG is also limited by its unstable physiochemical properties, which can be degraded quickly. Many studies have reported that green tea catechins were vulnerable to degradation caused by the elevation of temperature, pH, and metal ions of incubation media [13]. The instability is part of the reason for the poor bioavailability and also presents as an issue in the manufacturing process. Therefore, the oral bioavailability of catechin represents a big challenge. Topical application of these bioactive compounds may be able to overcome the problem, as this route bypasses metabolism by the liver and gastrointestinal track with relatively low enzymatic degradation. However, the skin barrier, which is due to the SC layer, impedes the transport of exogenous compounds into the skin and restricts diffusion of external substances into the deeper dermis layer. Therefore, we hypothesise that loading EGCG into a drug carrier would help overcome the skin barrier, improve penetration into the deeper skin layers and improve their stability. The compound can therefore exert its beneficial effect at the site of interest.

Niosomes are versatile drug carrier systems that have been administered via various routes; they are surfactant-based nanocarriers that are mainly composed of non-ionic surfactant and cholesterol [14–17]. Niosomes have been extensively studied in topical drug delivery due to their ability to significantly improve penetration across the skin barrier and deposition in the dermis layer [18–24]. Drugs with a variety of physicochemical properties have been investigated for topical and transdermal delivery using niosomes, for example, diacerein [25], itraconazole [26], tretinoin [27], salidroside [28] and finasteride [29], demonstrating their advantages in topical delivery. In addition, a range of bioactive compounds has also been loaded into niosomes, such as curcumin, rutin, and Ginkgo

biloba extract [30–32]. Silymarin-loaded niosomes demonstrated superior antioxidant activity over silymarin suspension in vitro [33].

In formulation development, various factors might impact the final product's performance. Design of Experiment (DOE) is a planned set-up of experiments to acquire information efficiently and precisely. It applies to any process that has quantifiable inputs and outputs. DOE was first designed for agricultural applications, but it has since become a frequently used technique in process sectors, including the chemical, food and pharmaceutical industries [34]. It may be used to explore the effect of multiple variables on responses by altering them all at once in a small number of tests. By this strategy, the costs and time involved with the research and production of medicine may be significantly decreased [34,35]. Furthermore, it aids in the creation of the "best possible" formulation composition and gives a comprehensive knowledge of the process and product behaviors [36]. This study aimed to develop an optimal niosome formulation using the DOE methodology and evaluate the formulation for topical administration of EGCG for protecting the skin from external oxidative stress. An ex vivo investigation on human skin was carried out to evaluate drug deposition and the antioxidant activity of the EGCG-niosomes. The uptake of niosomes by human skin fibroblasts was also investigated.

#### **2. Materials and Methods**

#### *2.1. Materials*

Span® 60, EGCG ≥ 98% (HPLC), cholesterol (CH), Triton™ X-100, dihexadecyl phosphate (DCP), Fluorescein 5(6)-isothiocyanate (FITC), Sulforhodamine B (SRB), trichloroacetic acid (TCA), dimethyl sulphoxide (DMSO), methanol and acetonitrile (ACN) were purchased from Merck (Merck, Kenilworth, NJ, USA). Dulbecco's Modified Eagle Medium (DMEM) with high glucose and L-glutamine, Phosphate-Buffered Saline (PBS), Hank's Balanced Salt Solution (HBSS), penicillin-streptomycin, fetal bovine serum of New Zealand origin (FBS), trypsin-EDTA, DAPI and CellTracker were purchased from Thermo Fisher Scientific (Auckland, New Zealand). Malondialdehyde (MDA), glutathione peroxidase (GSH-px) and superoxide dismutase (SOD) kits were purchased from Biovision (Biovision Inc., Milpitas, CA, USA). Trifluoroacetic acid (TFA) was purchased from Fluka (Fluka, Darmstadt, Germany). Distilled, deionised water was used throughout and was obtained from a Millipore water purifier.

#### *2.2. High-Pressure Liquid Chromatography Method for Quantification of EGCG*

An Angilent Technologies 1100 series high pressure liquid chromatography (HPLC) system equipped with a vacuum degasser, autosampler, thermostatted column compartment and photodiode-array detector (PDA) was used. A C18 column (Jupiter, 250 × 4.6 mm, 5 mm, Phenomenex, Torrance, CA, USA) was used for HPLC method development for EGCG. The mobile phase consisted of Milli Q water (0.1% TFA) and methanol at 75:25 ratio. EGCG was analysed at flow rate of 0.8 mL/min, with an injection volume of 20 µL and wavelength of 280 nm at 25 ◦C.

#### *2.3. Preparation of EGCG Loaded Niosomes*

A total of 150 mmol of surfactant, cholesterol and DCP (2 µmol) was dissolved in organic solvents (methanol/chloroform, 4:1, *v*/*v*) and then the mixture rotatory evaporated to form a thin lipid film on the wall at 45 ◦C. The the lipid film was purged with nitrogen to remove any organic solvents. The thin film was then hydrated with PBS (pH 7.4, 10% ethanol) containing 2 mg of EGCG at 58 ◦C to form EGCG-niosomes. The niosome suspension was then extruded through a 400 nm polyester membrane with an ER-1 extruder (Eastern Scientific, Rockville, MD, USA) for 10 cycles and then stored at 20–25 ◦C for the niosome membrane to anneal.

#### *2.4. Optimisation of Formulation with Design of Experiment*


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**Chapter 14**

© 2013 Siracusano and Girasole, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

**The Role of Altered Lipid Metabolism**

Despite the novel pathogenetic and therapeutic acquisitions sepsis remains the main cause of death in the critically ill patient (p.). The incidence of sepsis is increasing, with more than 750 000 cases occurring in the United States every year; sepsis is complicated by organ dysfunction giving rise to severe sepsis which causes more than 200 000 deaths each year [1]. The cardiovascular syndrome is the cause of the major part of the fatalities leading to the picture of the septic shock with 90,000 deaths annually. The myocardial dysfunction in sepsis has been widely studied and many causes of heart dysfunction have been described. The presence of a metabolic compromise is well known in chronic heart failure and has been considered as a basis for contractile failure, interestingly similar alterations have been recently described also in sepsis. The purpose of this study is to examine the current

Sepsis has been defined as the systemic host reaction to an infection [2]. The typical immune response in sepsis presents a first stage, characterized by an increased production of proinflammatory interleukins (ILs) by monocytes, macrophages. lymphocytes and endothelial cells in response to a few molecules, common to all virulent pathogens, called pathogenassociated molecular patterns (PAMPs) [3]. They are recognized by receptors on the surface of the immune cells, named Pattern Recognition Receptors (PRP) the most important being the Toll-like Receptors (TLRs), existing in 13 different types each recognizing a different microbial constituent. TLR are an important component of immune activity programmed to respond quickly to the infectious challenges by recognizing PAMP. Lipopolysaccharide (LPS) in particular is recognized by the complex TLR4-CD14 in the plasma membrane and the inflammatory signal is transduced by the recruitment of the adaptor molecules that associate with the intracellular Toll/interleukin-1 receptor (TIR) domain of the TLR to initiate signal transduction. Of these adaptor molecules myeloid differentiation primary- response protein 88 (MyD88) is associated with all TLRs except TLR3 while the TIR-domain containing adaptor

**in Septic Myocardial Dysfunction**

Luca Siracusano and Viviana Girasole

http://dx.doi.org/10.5772/51001

available data on this topic.

**1. Introduction**

Additional information is available at the end of the chapter

**Chapter 14**


*2.3. Reverse Phase-Liquid Chromatography Mass Spectrometry Analysis (RP-LC-MS*/*MS)*

The digested protein samples (above) were resuspended in 10 µL of 0.1% formic acid and analyzed by RP-LC-MS/MS in an Easy-nLC II system coupled to an ion trap LTQ-Orbitrap-Velos-Pro hybrid mass spectrometer (Thermo Fisher Scientific). The peptides were concentrated (on-line) by reverse phase chromatography using a 0.1 × 20 mm C18 RP precolumn (Thermo Fisher Scientific) and then separated using a 0.075 × 250 mm C18 RP column (Thermo Fisher Scientific) operating at 0.3 µL/min. Peptides were eluted using a 180-min dual gradient. The gradient profile was set as follows: 5−25% solvent B for 135 min, 25−40% solvent B for 45 min, 40−100% solvent B for 2 min, and 100% solvent B for 18 min (Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid and 80% acetonitrile in water). ElectroSpray ionization (ESI) was done using a nano-bore emitter stainless steel ID 30 µm (Proxeon) interface. The Orbitrap resolution was set at 30,000. Peptides were detected in survey scans from 400 to 1600 amu (1 µscan), followed by twenty data-dependent MS/MS scans (Top 20) using an isolation width of 2 u (in mass-to-charge ratio units), a normalized collision energy of 35%, and a dynamic exclusion that was applied during 60 s periods.


**14. Pedicle ILM transposition**

Hu [89] suggested a non-inverted flap provides a more physiological scaffold for healing. The pedicle is created by circular ILM peeling in the macular area but leaving a hinge attached superiorly to pivot the mobilised ILM to cover the hole. This method reduces flap loss during fluid-air exchange observed using free flaps [90].


*4.1. Qualitative Traits*

All qualitative traits are found to have significant variations except leaf hairs, corolla color, fruit cross-section, and fruit position (Tables 2 and 3). Some other previous reports also published a similar type of fruit curvature [42–44]. Eggplant is a herbaceous plant, mostly upright in nature [45]. Our results demonstrated that 17.69% upright, 49.23% intermediate, and 33.08% prostrate growth habit among all the studied genotypes

(Table 2). Shekar et al. [46] clustered the eggplant plants into upright and intermediate. Islam et al. [42] saw 48% intermediate, 45% upright, and 7% prostrate growth habit at vegetative stage among the studied genotypes. They suggested that the plant growth habit is used for the identification of eggplant varieties.

As per leaf blade length, eggplant genotypes were divided into two groups, viz., intermediate and short [47]. They reported that 30 genotypes had intermediate leaf blade length while five genotypes showed short leaf blade length. Similarly, based on leaf blade width, the genotypes were divided into three types, viz., wide, intermediate, and narrow [45]. Osei et al. [13] found significant variation in leaf blade length and width in eggplant. In the current study, we distributed eggplant genotypes into three types, viz., very strong (5 genotypes), strong, intermediate (43 genotypes), and weak (59 genotypes), based on leaf blade lobing (Table 2). Sunseri et al. [48] reported 60% weak, 37% intermediate, and 3% strong leaf blade lobbing in eggplant. In our investigation, 6 genotypes had very acute, 28 had acute leaf blade tip angle followed by intermediate leaf blade tip angle, which includes 64 genotypes, while 32 genotypes each had obtuse. Sunseri et al. [48] observed 62% acute, 33% very acute, and 5% intermediate leaf blade tip angle in eggplant. Dash et al. [49] noticed acute leaf blade tip angle in many eggplant genotypes. A much higher variation was observed in leaf prickle (Table 2), where most of the genotype (72 genotypes) had intermediate prickle on the leaf. Sunseri et al. [48] noted that leaf prickles were absent in 25% of genotypes, whereas the remaining genotypes produced 40% very few, 25% few, and 5% both intermediate and many prickles. Tiwari et al. [50] noted prickles in stem, petiole, calyx including peduncle, and leaf including veins in eggplant. Many leaf hairs (98.46%) are found in most of the genotypes. Light violet (31.54%), pale violet (64.62%), and white (3.85%) colors were noted among the studied genotypes for corolla Color (Table 2).

In the present study, we described eight distinctive traits of eggplant fruits (Table 3). For traits related to the fruit calyx prickles, fruit color distribution, fruit color at ripening, and fruit flesh density much higher variation were observed in the cultivated eggplant (Table 3). Similar kinds of fruit calyx prickle distribution were also reported by [45]. It was noticed that 68 genotypes had no curvature on fruit; 37 genotypes showed slightly curved fruit and 25 genotypes had curved fruit. Sunseri et al. [48,51] reported sickleshaped, snaked-shaped, curved, and U-shaped eggplant fruits along with fruits with no curvature. Eggplant genotypes were distributed into two types: rounded (39 genotypes) and depressed (91 genotypes), on the basis of fruit apex shape. Sunseri et al. [48] reported that 38, 34, and 28% of eggplant genotypes had depressed, rounded, and protruded types of fruit apex shape, respectively. Eggplant fruits traits variations are important in protection from UV irradiation, insect attack in plants as well as socio-economic value. More fruit color variation was noted by [52] and [53], which support our present study. Solaimana et al. [51] found uniform and stripped fruit color distribution. The variations in fruits color were also described by [51,54,55]. Tiwari et al. [50] divided the eggplant fruits into six color groups i.e., green (37.27%), purple (25.45%), milky white (13.62%), purple-black (12.72%), light purple or lilac grey (9.09%), and scarlet red (1.08%). Fruit flesh density is an important characteristic for the determination of fruit volume and weight. A wide range of variation was observed in fruit flesh density, the highest number of genotypes had very loose flesh density (74 genotypes).


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