Aritmética Racional

The results above (Tables 4.4-4.6) revealed that there were six main phenolic compounds in the olive leaf extracts. They were oleuropein, hydroxytyrosol, luteolin-7-glucoside, luteolin-4-glucoside, apigenin-7-O-glucoside, verbascoside. These compounds were also found by others in the olive leaf extracts (Benavent et al., 2000; Briante et al., 2002;

Bouzaiz & Sayadi, 2005; Japon-Lujan et al., 2006; Pereria et al., 2007; Altiok et al., 2008;). Benavent et al. (2000) reported that oleuropein was the largest fraction present (24.5%) while other phenolics compounds such as hydroxytyrosol (1.5%), luteolin-7-glucoside (1.4%), verbascoside (1.1%), tyrosol (0.7%), and apigenin-7-O-glucoside (1.4%) were also isolated from the leaves. Atiok et al. (2008) reported the abundance of oleuropein (29%) in an olive leaf crude extract (Atiok et al., 2008). Bouzaiz & Sayadi (2005) reported that six flavonoid (luteolin 7-O-glucoside, luteolin 7-O-rutinoside, apigenin 7-O-glucoside, rutin, luteolin and apigenin) present in Chemlai olive leaf extract (Bouzaiz & Sayadi, 2005). Pereria et al. (2007) quantified oleuropein and lueolin-7-O-glucoside as the most abundant phenolic compounds present in a lyophilised olive leaf extract (Pereria et al., 2007). Meirinhos et al. (2005) quantified eight flavonoid compounds in olive leaf samples and reported luteolin-4-O-glucoside was the major flavonoid compound for Portuguese olive leaf cultivars (Meirinhos et al., 2005).

There were four groups of phenolic compounds identified in the leaf extracts in this study, namely phenolic alcohols (e.g. hydroxytyrosol, tyrsol), phenolic acid (e.g. caffeic acid,P- coumaric acid), flavonoids (e.g. luteolin-7-O-glucoside, luteolin-4-O-glucoside, and apigenin-7-O-glucoside) and secoiridoids (e.g. oleuropein, verbascoside). These

compounds are either secondary metabolites or compounds formed during leaf storage and extraction process (Salvador & Fregapane, 2010). The structures of all phenolic

compounds identified in the three olive leaf extracts are shown in Table 4.7.

Oleuropein, the most abundant secoiridoids present in O. europaea fruits and leaves, is a hydroxytyrosol ester with a β-glycosylated elenolic acid (Briante et al., 2001). Secoiridoids are present exclusively in plants that belongs to the Oleaceae family (Silva et al.,

2009).The formation of oleuropein is related to the iridoids which are produced via

secondary metabolism of monoterpenes as precursors of various indole alkaloids. The leaf has been regarded as the primary site of plant metabolism at the level of both primary and secondary plant products (Ryan et al., 2002). Verbascoside is another secoiridoid derived from hydrotyrosol backbone (Laguerre et al., 2009).

81 | P a g e

Table 4. 7: Phenolic Compounds Present In Crude Olive Leaf Extracts and Their Chemical Structures, Reproduced From (Macdonald-Wicks et al., 2006)

Class Compound Chemical Structure

Phenyl alcohol Tyrosol

Hydroxytyrosol

Phenyl acid P-coumaric acid

Caffeic acid Flavonoids Rutin Apigenin Luteolin Apigenin-7-O-gluocoside Luteolin-7-O-glucoside Luteolin-4-O-glucoside Secoiridoids Oleuropein Verbascoside

82 | P a g e Hydroxytyrosol and tyrosol are the most abundant phenolic alcohol in olives.

Hydroxytyrosol is rarely in the free form in nature with the exception of ripened olives where it occurs through the hydrolysis of oleuropein (De Leonardis et al., 2008).

The dominant flavonoids present in the olive leaf extracts were flavonoid glycosides such as luteolin-7-O-glucoside, luteolin-4-O-glucoside, apigenin-7-O-glucoside and rutin. Other flavonoids present in olive leaves are in aglycone forms which include apigenin and luteolin (Laguerre et al., 2009).

There are three enzymes involved in metabolism of major phenolic compounds in olive fruits and leaves. The enzyme involved in accumulation and degradation of oleuropein is β-glucosidase, which plays an important role in the anabolic and catabolic routes of oleuropein biosynthesis (Gutierrez-Rosales, 1998). Phenylalanine ammonialyase (PAL) is a regulatory enzyme involved in the biosynthesis of a large group of phenylpropanoid- derived secondary products such as flavonoids, isoflavonoids, coumarins, lignins and other phenolic compounds such as tyrosol (Gutierrez-Rosales, 1998). Polyphenol oxidase (PPO) is an enzyme involved in olive fruit and leaf browning. The browning occurs when phenols are oxidized to highly reactive quinones, which then polymerize. This oxidation reaction is catalysed by PPO in presence of oxygen. The substrates for the reaction are simple phenols derived from oleuropein. Polyphenol oxidase activity was significantly higher in fruits than in leaves and it increased during fruit ripening (Ortega-Garcia., 2008).

A wide range of phenolic compounds were identified in the olive leaf extracts. Variability in the type and amount of phenolic compounds in olive leaf extract may be due to

geographical, varietal, seasonal factors and different extraction methods applied (Hayes et al., 2010).The phenolic compounds in the olive leaf extracts may be formed during sample preparation and extraction. The chemical and enzymatic reactions may take place during this process including hydrolysis of glycosides by β-glucosidase, oxidation of phenolic compounds polyphenol oxidase and polymerization of free phenols.

Laguerre et al. (2009) discovered that mature olive leaf extract contained higher levels of verbascoside isomers and glycosylated forms of luteolin, while young ones presented higher contents of oleuropein , ligstroside and flavonoid aglycones. They suggested that there were two main bioconversion scenarios which may occur during maturation of olive leaves (1) a bioconversion of oleuropein and ligstroside into verbascoside isomers and oleuroside, and (2) a bioconversion of flavonoid aglycones into glycosylated forms of

83 | P a g e luteolin (Laguerre et al ., 2009). In this work, oleuropein was not the only predominant form of phenolic extracted but was accompanied with high amounts of luteolin-7-O- glucose (12%-26%), luteolin-4-O-glucoside (11%-12%) and apigenin-7-O-glucoside (4% to 8%). The olive leaves used for this study were collected from the olive oil processing plant; the leaves were green yellowish colour. They could be mature leaves. It may explain the higher amount of glycosylated forms of luteolin (luteolin-7-O-glucose, luteolin-4-O- glucoside and apigenin-7-O-glucose) present in the olive leaf extracts in this study.

4.6.5 Comparison of Phenolic Profiles between Three Different Olive Leaf