CHAPTER 1

INTRODUCTION

1.0 General Background

As the world developed, the emerging of new diseases and the number of drug resistant pathogens are increases. The diet habit has also contributed as significant increases of patients that harbouring the non pathogenic born diseases such as cardiovascular disease, diabetes, stroke, and cancer. Atherosclerosis is a major driver of cardiovascular disease in humans and occurs with greater frequency with increasing age, diabetes, smoking, and hypertension (Beckman et al., 2002). It is the main causes of mortality and morbidity in developed countries (Fishbein and Fishbein, 2009). In Malaysia, the atherosclerosis is identified as the main cause of death in 2010 and become the top two causes of death in industrialized countries in China (Zhiyong, 2011). It is an ageing disease with a fastest progress from 40 to 49 years. Atherosclerosis is an inflammatory progressive disease, which begins in the first decade of life and develops silently over decades before clinical manifestations (Lusis, 2000). Atherosclerosis is the build up of plaque on the inside walls of arteries. Plaque is made up of low density lipoprotein (LDL), macrophages, smooth muscle cells, platelets, and other substances. It may narrow the lumen of a blood vessel and restrict blood flow. Plaque rupture can induce the formation of thrombus (blood clot) and block blood flow. This will result in ischemic stroke or heart attack (Frink, 2002).

There are three different stages of atherosclerosis that led to the "clogging" of arteries; firstly, the fatty streak which consists of smooth muscle cells, which are filled with cholesterol and macrophages ; a type of immune system "scavenger" cell that removes harmful substances, such as excess cholesterol particles, from the bloodstream (Thompson, 2010). The fatty streak alone does not cause any symptoms but, over time, can develop into a more advanced form of atherosclerosis called fibrous plaque, secondly, the formation of fibrous plaque in the inner layer of arteries which consist of large numbers of smooth muscle cells, macrophages, and lymphocytes (a type of white blood cell that typically responds to an infection or injury). These cells are all filled with cholesterol. As the fibrous plaque grows, it projects into the space inside the artery where the blood is flowing and lastly the complicated lesion where the fibrous plaque breaks open, exposing the cholesterol and connective tissue underneath. This rupture provokes a strong clotting reaction from your blood, such as when you have a cut. The combination of fibrous plaque and the blood clot is called a complicated lesion (Healthwise, 2010). Although there already have the established drugs used to treat those diseases such lipid lowering therapy and the statin but there still have some problem arise such patient suffered from the side effect (Azevedo et al., 2008).

1.2 Significant of study New drugs discovery from various sources including marine is urge to substitute the current drug in the market such statin that exhibited side effect on the patients which causing liver failure. Expert estimate that the biological diversity in marine ecosystems is higher than the tropical rain forest (Haefner, 2003). Therefore, marine

diversity is indeed in the bioprospecting for natural product as drug candidates. Besides, extremely small number of species have been studied for their pharmaceuticals potential (Jachak and Saklani, 2007). Therefore, this research was also focused on the searching of biopharmaceuticals against atherosclerosis from mangroves Rhizophora apiculata and Acanthus ebracteatus. Mangrove forest is salt tolerant plants confined to the coastal areas and occupies only 5% of the total forest areas of the world. Mangroves producing wide array of natural products classes such as alkaloids, phenol, steroids, terpenoids and tannins that possess bioactivities such as antimicrobial, antiviral, antioxidant,

anticancer, ant proliferative, insecticidal, antimalarial, antifeedant, central nervous system depressant and anti-plasmodial (Jayanta, 2010). As example, the alkaline extract of Rhizophora apiculata leaves was reported to inhibit the HIV replication and HIV-induced cytopathic effects (Premanathan et al., 1999). Pyroligneous acid of this plant possesses antimicrobial, growth promoting agent and antioxidant activity (Loo et al., 2006). Antioxidant has also gained positive feedback as health promoters in cardiovascular problems, atherosclerosis, anticancer and anti-ageing (Packer, 1999). It clearly shows that mangroves have a huge potential source for bioactive compounds for treatment of atherosclerosis. Different mangroves species contains different secondary metabolite. In this study a new potential drugs candidate against atherosclerosis is investigated by using SR-B1 and PPRE promoters. SR-B1 promoter is crucial in order to allow HDL to remain functional to transport the plasma cholesterol away from the blood vessel to the liver to be metabolized (Acton et.al., 1999).

It was demonstrated that SR-B1 promoter contained a binding site for peroxisome proliferator activated receptor gamma (PPAR), a ligand dependent transcription factor that is activated by TZD (ligand). Therefore, in order to determine the assay developed was fully functional, transfected cells were then treated with rosiglitazone, a derivative of TZD as positive control. The chimeric constructs containing PPRE and SR-B1 promoter, respectively, that were ligated to reporter gene (luciferase gene), were used and transfected to human liver, HepG2 cell line. The crude or compounds that increased the PPRE and SR-B1 promoter activities (based on luciferase activity) were identified to have the potential as therapeutic agent against atherosclerosis. Previously, research on PPRE conducted, the peroxisome proliferator activated receptors (PPARs), PPAR,, , and , are ligand activated transcription factors belonging to the nuclear hormone superfamily that also includes the retinoic acid and thyroid hormone receptors. Upon ligand binding, PPARs form heterodimers with one of the three retinoid X receptor proteins which then bind to PPAR response elements (PPRE) within the promoter regions of target genes. They have been shown to regulate diverse cell functions, including adipocyte differentiation, control of inflammation, fatty acid metabolism, cell cycle control, and the development of atherosclerosis (Willson, et al , 2001). A variety of endogenous and exogenous ligands for PPARs have been identified (Guan Y, 2001). PPAR is the target for the hypolipidemic fibrate drugs, and PPAR the target for the antidiabetic thiazolidinediones.

1.3 The scope of Study The Rhizophora apiculata and Acanthus ebracteatus were chosen to for further investigation towards atherosclerosis disease. Both species do not show the cytotoxicity activity against HepG2 cell line. This study also focused on cytotoxicity screening for methanolic crude, fraction and their pure compound. Furthermore, isolated and identified the bioactive compound from Rhizophora apiculata and determined the ability of secondary metabolites towards the transcriptional activity of PPRE and SR-B1 promoter.

2.0 OBJECTIVES: i. To determine the lower cytotoxicity activity of selected mangrove plant extract ii. To determine the biological activities of extract and chemical constituent from Rhizopora apiculata and Acanthus ebracteatus against atherosclerosis using SRBI techniques