**2.3 Coconut phosphatidyletanolamines (CocoPEs) separation using vacuum liquid chromatography**

About 5 g of CocoPLs was mixed with 5 g of silica gel in a small amount of chloroform: methanol (9:1, v/v) solution to form a silica slurry. The slurry was then stirred until the mixture was dried and formed fine powder of CocoPLs-SG.

A total of 80 mg of silica gel was poured into a chromatography column and compressed by vacuum. The column was rinsed using chloroform:methanol (9:1, v/v) eluent and vacuumed until all the eluent was eluted. The CocoPLs-SG powder was poured onto the column. Then the column was subjected to compression. Elution was performed using 10 ml of chloroform:methanol (9:1, v/v) solution. Fraction eluted from the column was collected into clean vials. The fraction was analyzed using TLC plate. The spot on the TLC plate was identified with 10% H2SO4 and ninhydrin. Elution was repeated every 10 ml of the eluent until the TLC plate did not show any spot when subjected to identification. The CocoPLs fractions contained ethanolamine species were gathered into an evaporating flask and evaporated at 40°C to obtain dark brownish gel of CocoPEs.

#### **2.4 Characterization of CocoPLs and CocoPEs**

Both CocoPLs and CocoPEs obtained were characterized using FT-IR (Prestige 21 Shimadzu), GC-MS (Shimadzu QP2010S), and LCMSMS (Waters Xevo TQD) and DSC (Shimadzu DSC-60A). The FTIR was employed to probe the phospholipids functional groups. The GC-MS was used to determine the phospholipids fatty acyl chains. The LC-MS/MS was for identifying the chemical component of CocoPEs and the DSC analysis was carried out to explore the CocoPEs phase behavior.

#### **2.5 Vitamin C encapsulation in coconut liposomes**

In this research, vitamin C (VC) was used as a model for hydrophilic drug to be encapsulated in coconut liposome [13, 16, 17, 22]. Stock solution of 500 ppm CocoPEs with cholesterol concentration (0%, 10%, 20%, 30%, 40% w/w) were made. A total of 2 mL of each stock solution was diluted with chloroform to 10 mL and poured into a test tube. The liquid solution was evaporated using N2 gas flow to form a thin layer. After that hydration process was carried out. Around 10 mL of phosphate buffer solution was added to the thin film. The mixture was subjected to freeze-thawing process until the thin film was dispersed completely. The dispersions contained empty coconut liposome and was used as control. Other set of dispersions were prepared by adding 8 ppm (*C*0) VC solution in phosphate buffer pH 7.4 to each 2 mL stock solution and followed by similar process to obtained encapsulated VC in coconut liposome dispersion. The VC concentration in the filtrates obtained after all coconut liposome dispersions were centrifuged were analyzed using UV-Vis spectrophotometer at 265 nm. The concentration of VC was calculated from the filtrate absorbance and represented as Cliposome+VC and Cempty liposome in equation 2.

In addition we used CocoPLs as comparison. The encapsulation efficiency of VC in coconut liposome was determined based on Eqs. (1) and (2): *EE* = \_

$$EE = \frac{C\_0 - C\_r}{C\_0} \times 100\,\% \tag{1}$$

$$\mathbf{C}\_t = \mathbf{C}\_{liposome\text{\textquotedblleft VC}} - \mathbf{C}\_{empty\text{\textquotedblright}} \tag{2}$$

where *EE* is the encapsulation efficiency*; C0* is the initial concentration of VC; and *Ct* is the unencapsulated VC concentration.
