**1. Introduction**

Phospholipids are major constituent of cellular membrane hence they have excellent biocompability. They are amphiphilic molecules which usually built by glycerol backbone with two different polarity groups attached to it. On the one hand is the hydrophilic group renowned as the head group which then becomes the basis of species classification of phospholipids, such as phosphatidylcholine (PC), phosphatidyletanolamine (PE), and phosphatidylserine (PS). On the other hand is the hydrophobic fatty acyl chains distinguished as the tails. The variation of the length and the saturation, the bonding position of fatty acyl chains to glycerol

backbone as well as the head group type become a crucial part of their application, for instance in drug delivery systems.

The development of phospholipids based drug delivery systems have been proven prominent by the emergence of many phospholipid-related drug formulation. Among of them are doxorubicin in stealth liposomes for cancer treatment, which has been on the market since 1995 [1, 2]; Verteporfin in cationic liposomes for molecular degeneration [3] and vincristine in conventional liposome for Non-Hodgkin lymphoma [2]. They have been used in clinic, and achieve good results. Many more phospholipids based liposomal preparation have been developed to find better therapeutic results [4–6]. Furthermore various sources, synthetic and natural, have been explored [2, 7].

The isolation of phospholipids from natural sources cost lower than synthesizing them hence the preference is the isolation of natural phospholipids. For natural origin, the more pure they are, the greater the value is [8]. Phospholipids from natural origin can be refined into diverse levels, comprising food and pharmaceutical grade [2, 9]. In term of natural phospholipids, different source enhance the species variety of phospholipids [7]. Egg yolk and soybean phospholipids mainly consist of phosphatidylcholine species but they have differences in the tail portions which influence their physical, chemical properties and their applications. Other natural phospholipids that currently are being explored extensively are sunflower [10–12], candlenut [13], jack bean [14], sesame [13, 15–17] and coconut [13, 15, 16, 18–22].

Coconut is one of the native plantations in tropical countries and produces mainly copra and coconut oil. Exploration of coconut by-products such as coconut phospholipids needs to be done to increase the added value of these coconut plantations. Previous studies have found that dried coconut contain phospholipids from cephaline species with their fatty acyl chains are dodecanoic and octanoic acyl chains [15]. Purification with eluent chloroform: methanol (9:1) follows by identification using thin layer chromatography (TLC) also detects the presence of phosphatidylcholine (PC), phosphatidyletanolamine (PE), and phosphatidylserine (PS) species in coconut phospholipids (CocoPLs) [20, 21].

In the matter of its application, coconut liposomes (CocoPLs liposomes) have been used in the encapsulation of hydrophilic agent namely carboxyfluoresence and vitamin C and resulted in that CocoPLs liposomes has high efficiency of encapsulation [16, 19, 22]. The addition of cholesterol improves the encapsulation efficiency and low storage temperature reduces CocoPLs liposomes leakage. The results advocated the CocoPLs potency as drug delivery material. Moreover since we have established that CocoPLs consist of many phospholipid species therefore it would be valuable to study the component of the species and their capability as drug delivery system. In this study we explore the isolation and purification of coconut phospholipid species specifically coconut phosphatidylethanolamine (CocoPEs) and utilization of their liposomes (CocoPEs liposomes) for vitamin C encapsulation with various cholesterol concentrations. To our knowledge this is the first study of such.

#### **2. Materials and methods**

#### **2.1 Materials**

Materials used in this study were ripe coconut meat purchased from local market, TLC silica gel 60 F254 plate, silica gel powder 60 G for thin layer chromatography, various solvents and regents for analytical grade.

**183**

*Coconut Phospholipid Species: Isolation, Characterization and Application as Drug Delivery…*

Briefly coconut meat powder was macerated in a chloroform: methanol (2:1, v/v) mixture. The filtrate obtained was washed using 0.9% NaCl. The lipid was evaporated until thick coconut lipid extract were obtained. The extract was then subjected to solvent partition using n-hexane and ethanol 87%. The lower phase

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

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

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

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.

the DSC analysis was carried out to explore the CocoPEs phase behavior.

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

Isolation technique was carried out based on the previous method used [20, 21].

*DOI: http://dx.doi.org/10.5772/intechopen.88176*

**liquid chromatography**

**2.2 Coconut phospholipid (CocoPLs) isolation**

was evaporated to yield brownish yellow extract of CocoPLs.

evaporated at 40°C to obtain dark brownish gel of CocoPEs.

**2.4 Characterization of CocoPLs and CocoPEs**

**2.5 Vitamin C encapsulation in coconut liposomes**

*Coconut Phospholipid Species: Isolation, Characterization and Application as Drug Delivery… DOI: http://dx.doi.org/10.5772/intechopen.88176*

#### **2.2 Coconut phospholipid (CocoPLs) isolation**

*Nano- and Microencapsulation - Techniques and Applications*

for instance in drug delivery systems.

natural, have been explored [2, 7].

coconut [13, 15, 16, 18–22].

backbone as well as the head group type become a crucial part of their application,

The development of phospholipids based drug delivery systems have been proven prominent by the emergence of many phospholipid-related drug formulation. Among of them are doxorubicin in stealth liposomes for cancer treatment, which has been on the market since 1995 [1, 2]; Verteporfin in cationic liposomes for molecular degeneration [3] and vincristine in conventional liposome for Non-Hodgkin lymphoma [2]. They have been used in clinic, and achieve good results. Many more phospholipids based liposomal preparation have been developed to find better therapeutic results [4–6]. Furthermore various sources, synthetic and

The isolation of phospholipids from natural sources cost lower than synthesizing them hence the preference is the isolation of natural phospholipids. For natural origin, the more pure they are, the greater the value is [8]. Phospholipids from natural origin can be refined into diverse levels, comprising food and pharmaceutical grade [2, 9]. In term of natural phospholipids, different source enhance the species variety of phospholipids [7]. Egg yolk and soybean phospholipids mainly consist of phosphatidylcholine species but they have differences in the tail portions which influence their physical, chemical properties and their applications. Other natural phospholipids that currently are being explored extensively are sunflower [10–12], candlenut [13], jack bean [14], sesame [13, 15–17] and

Coconut is one of the native plantations in tropical countries and produces mainly copra and coconut oil. Exploration of coconut by-products such as coconut phospholipids needs to be done to increase the added value of these coconut plantations. Previous studies have found that dried coconut contain phospholipids from cephaline species with their fatty acyl chains are dodecanoic and octanoic acyl chains [15]. Purification with eluent chloroform: methanol (9:1) follows by identification using thin layer chromatography (TLC) also detects the presence of phosphatidylcholine (PC), phosphatidyletanolamine (PE), and phosphatidylserine

In the matter of its application, coconut liposomes (CocoPLs liposomes) have been used in the encapsulation of hydrophilic agent namely carboxyfluoresence and vitamin C and resulted in that CocoPLs liposomes has high efficiency of encapsulation [16, 19, 22]. The addition of cholesterol improves the encapsulation efficiency and low storage temperature reduces CocoPLs liposomes leakage. The results advocated the CocoPLs potency as drug delivery material. Moreover since we have established that CocoPLs consist of many phospholipid species therefore it would be valuable to study the component of the species and their capability as drug delivery system. In this study we explore the isolation and purification of coconut phospholipid species specifically coconut phosphatidylethanolamine (CocoPEs) and utilization of their liposomes (CocoPEs liposomes) for vitamin C encapsulation with various cholesterol concentrations. To our knowledge this is the

Materials used in this study were ripe coconut meat purchased from local market, TLC silica gel 60 F254 plate, silica gel powder 60 G for thin layer chromatog-

raphy, various solvents and regents for analytical grade.

(PS) species in coconut phospholipids (CocoPLs) [20, 21].

**182**

first study of such.

**2.1 Materials**

**2. Materials and methods**

Isolation technique was carried out based on the previous method used [20, 21]. Briefly coconut meat powder was macerated in a chloroform: methanol (2:1, v/v) mixture. The filtrate obtained was washed using 0.9% NaCl. The lipid was evaporated until thick coconut lipid extract were obtained. The extract was then subjected to solvent partition using n-hexane and ethanol 87%. The lower phase was evaporated to yield brownish yellow extract of CocoPLs.
