*2.3. Isolation of Oleacein (2)*

The above-described property of the secoiridoid phenolic aldehydes of reacting reversibly with water led us to investigate the possibility of extracting secoiridoid phenolic aldehydes from olive leaves using cold water at temperatures ranging between 15 and 25

◦C. In contrast to the common extraction using hot water at boiling temperature that leads to extracts with oleuropein (**9**) as the main phenolic ingredient [69,70], the cold-water extraction led to pure oleacein (**2**). In fact, when the intact leaves are shredded in the presence of cold water, the enzymes of oleuropein glucosidase [71] and demethylase [72] come into contact with their substrate oleuropein (**9**) and transform it to oleacein (**2**). When hot water is used, the enzymes are deactivated and oleuropein (**9**) remains intact. The cold-water extract contains oleaceinediol (**10**) together with several other hydrophilic ingredients such as sugars (i.e., mannitol, glucose, etc.). However, among them, only oleaceinediol (**10**) can be reversibly transformed back to the dialdehyde form of oleacein (**2**) when it comes in contact with solvents such as ethyl acetate (EtOAc) or dichloromethane (CH2Cl2). A simple re-extraction step of the cold-water extract led directly to pure oleacein (**2**) (purity > 95%).

#### *2.4. Isolation of Oleomissional (6a,b,c)*

Surprisingly, when intact olive leaves or olive fruits came in contact with organic solvents such as CH2Cl2, the obtained extract contained only oleomissional (**6a,b,c**) together with lipophilic triterpenoids. Oleomissional (**6a,b,c**) is the enolic form of the open-ring type of oleuropein aglycone (**6a**), which is in equilibrium with two isomeric oleuropeindials (**6b,c**) and is the first product arising from the action of oleuropein glucosidase on oleuropein. Solvents such as CH2Cl2 disrupt the cell membranes permitting the partial activity of glucosidase, which is a very resistant enzymatic system, but do not permit the action of the demethylase. The obtained oleomissional (**6a,b,c**) can be easily separated from the rest of the lipophilic compounds based on its ability to react with water and be reversibly transformed into a hydrophilic diol (oleomissionadiol). Re-extraction of the water solution using EtOAc or CH2Cl2 led to pure oleomissional (**6a,b,c**).

#### *2.5. Conversion of Oleomissional (6a,b,c) to Closed-Type Oleuropein Aglycone (3a,b)*

Oleomissionadiol (**11a,b**) in aqueous solutions is stable only when the pH is slightly acidic. When the pH becomes slightly alkaline then the molecule rearranges from the open form to the closed-ring form of the oleuropein aglycone (**3a,b)**. This molecule exists as a mixture of two isomers (R, S) which can be easily obtained by a simple extraction of the alkaline aqueous solution using organic solvents.
