ANTIOXIDANT ACTIVITY OF SOUR SOP FRUIT, BARK AND LEAVES RIPE AND UNRIPE
1.1 Background of Study
The use of medicinal plants as major source of formulated drugs in the industrialized society can be traced back to the traditional use in folk medicine. Scientific utilization of medicinal plants is important as they can be toxic, also, can offer a wide range of therapeutic application. Annona muricata (soursop) belongs to the genus Annona of the custard apple tree family and has age-long traditional use as medicinal plant. Soursop is a tropical plant that is also cultivated in Saharan Africa. They are perennial crops with edible fruits and they are commercially underutilized. The plant has been reported to contain nutrients like vitamins B and C; phosphorus, iron and calcium. These nutrients are thought to be complimentary in the traditional use of the plant. For instance, the iron ameliorates effects of massive haemolysis. All parts of soursop are widely applied in alternative medicine. The bark, leaves and seeds possess diverse biological activities and they are traditionally used in the treatment of infectious and chronic non-communicable diseases such as diabetes, hypertension and inflammation. The leave has been demonstrated to be hepatoprotective, antiplasmodic and anti-diabetic. The fruit has been reported to possess antimicrobial, antitumor and antiviral effects. The juice of ripe fruit is applied as diuretic and the powdered immature fruits can be used as remedy for dysentery. Like other medicinal plants, Annona muricata contains phytochemicals that are responsible for their healing properties. The phytochemical screening of this plant revealed the presence of flavonoids (group of polyphenolic antioxidants), saponins, tannins, glycoside and alkaloids. The leaves and seed of Annona muricata contain 50 mono THF acetogenins which is a key intermediate in acetogenins synthesis. Family Annonaceae family, to which soursop belongs, also contain neurotoxic alkaloids named annonacin. This neurotoxin is suspected to cause atypical Parkinsonism and other neurological effects on large or frequent consumption. Reactive oxygen species are formed in minute quantities during physiological cell metabolism. However, at high concentration, excessive free radical production or decreased capacity of endogenous antioxidant leads to state named oxidative stress. This condition has potential of causing damage to cellular macromolecules including as carbohydrate, protein, lipids and nucleic acids thereby disrupting the functional and structural integrity of biological cells]. Oxidative stress plays important roles in the pathogenesis of diseases such as cancer, neurological disorders, atherosclerosis, hypertension, ischaemic disease, diabetes, acute respiratory syndrome, fibrosis, pulmonary disease and asthma. The ability to isolate RNA with good quality and free of contaminants like protein, genomic DNA and secondary metabolites, is crucial for cDNA library construction and molecular analysis, e.g. northern hybridization and reverse transcription-polymerase chain reaction (RT-PCR) (Liu et al., 1998). Several methods have been routinely used for isolation of total RNA (Chirgwin et al., 1979; Chomczynski and Sacchi, 1987; Logemann et al., 1987) and are still being developed due to the fact that plant species from the same genus or related genera may contain variable amounts of diverse substances, like polysaccharides, polyphenolics and secondary metabolites. Therefore, it is not expected to find a unique nucleic acid isolation method adequate for all plants (Loomis, 1974; Weishing et al., 1995; Sharma et al., 2003).
The extraction of RNA from fruit tissue can be affected by several factors, mainly by high levels of RNases and alcohol insoluble substances (AIS) (Romani et al., 1975). Many compounds in the fruit pulp, such as polysaccharides, and phenolic compounds, are known to interfere with nucleic acid extraction (Ikoma et al., 1996; Woodhead et al., 1997; Jaakola et al., 2001). Concerning fruit pulp tissue, several protocols have been described for the isolation of RNA (Podivinsky et al., 1994; Jones et al., 1997; Liu et al., 1998; Woodhead et al., 1997; Asif et al., 2000; Jaakola et al., 2001; Valderrama-Chairez et al., 2002). However, when protocols are applied to new material without further adaptation, RNA quality and yield can be poor or, in some cases, no RNA could be reco- vered. In a current study on soursop fruit ripening, we characterized the expression profile of alternative oxidase (AOX) at the protein level (Brasil, 2002). Further inve-stigations require a reliable protocol that provides the same quality and quantity of RNA from different stages of ripening. Here, we report a simple and efficient method for isolating total RNA from ripe and unripe soursop fruit tissues. This protocol is based partially on a rapid nucleic acid extraction method (Dorokhov and Klocke, 1997), using SDS and potassium acetate for precipitation of contaminants (polysaccharides and proteins) in combination with steps of the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany).