EXTRACTION, CHARACTERIZATION AND INDUSTRIAL USES OF LECITHIN FROM THREE VARIETIES OF Cucumis melo (MELON SEED) OIL.
Melon seed, Cucumis melo oil and lecithin were evaluated for their physicochemical and possible biopharmaceutical uses as an adjunct in self-emulsifying drug delivery systems (SEDDSs) for future use as safe drug vehicle for poorly aqueous soluble drugs. Melon seed oil was extracted using standard procedure, while the Lecithin was extracted from the seed oil. The oil was subjected to some physicochemical characterization and acute toxicity test. The lecithin also extracted was subjected to physicochemical test as well as solubility and antioxidant evaluations. From the physicochemical studies, the result of the physical properties showed that the colour of the oil is yellow, the mean refractive index of the three oils is 0.0091±0.1specific gravity, 0.9323±0.2 and viscost of 338.89±0.1.The chemical studies showed an acid value of 0.9327± 0.1 mg KOH/g, saponification value 166.13±0.2 mg KOH/g iodine value, 121.8±0.1Wijs, proxide value, 10.67± 0.1 and Ester value of 165.13±0.2 mg KOH/g. The lecithin extracted has mean percentage yield of 0.58±0.1%, and has solubility in acetone, chloroform, petroleum ether but slightly soluble in methanol and water.
The acute toxicity test showed that the oil is not toxic, and has no significant behavioural modification of the animals it was administered up to a dosage of
5000mg/kg body weight. The result in this present study shows that the oil and lecithin extracted from Cucumis melo have a lot of nutritional and biopharmaceutical applications. The developed vitamin E SEDDSs formulations containing melon seed oil showed promise as a possible clinical arsenal for the delivery of poorly water-soluble drugs. The result obtained demonstrated notable usefulness of both the oil and lecithin in health, industry and agriculture. The developed vitamin E SEDDSs formulations containing melon seed oil were found to facilitate maximal delivery, absorption and bioavailability of lipophilic drugs.
1.0 Introduction and Literature Review
Lecithin is an important by-product of vegetable oil processing industries that have important functions in health, agriculture and in the industries (Dreon et al, 1990). Lecithin is a mixture of glycerol-phospholipids obtained from animal, vegetable and microbial sources, containing varying amounts of substances such as triacylglycerols, fatty acids, glycolipids, sterols and sphingolipids (Meek, 1997). The major source of commercial lecithin is soybean oil, and is called 1,2-diacylglycero-3-phosphorylcholine (Dashiell, 2003). The production of lecithin from oil seed is by hydration of the phosphatides using
water or steam (Shanhani, (1980). Lecithin has diverse roles in human metabolism (Orthoefer, 1998), especially in the control of nerve activities and breathing (Gordon, 2000), production and quality could be affected by crude oil storage, soil type, nutrient availability, climatic changes, drying process, and handling manner (Renfree, 2005). Lecithin also has multifunctional uses in agriculture, food confectioneries, pharmaceuticals, paints, plastics, and in the textile industries (Lucas, 1996). Lecithin is an emulsifying, wetting, and dispersing agent. It has antioxidant, surfactant and lipotropic functions, as well as anti-corrosive and anti-spattering roles (Eyster, 2007. In the pharmaceutical industries, lecithin is also important in lowering blood cholesterol levels facilitating optimum absorption of fat-soluble vitamins, maintaining cell membrane integrity, as well as increasing serum choline levels and it also gives relief and cure in the severity of neurological diseases (Kidd, 1997).
The important uses of lecithin in health, industries, and agriculture is increasing; therefore, there is need to explore other sources of lecithin in order to reduce over-dependence on soybean source (Spiller, 2006). Melon seeds are produced in the eastern, middle belt, and northern states of Nigeria, and Nigeria is one of the largest producers in the world (Ofune, 1988). Melon seeds are used for edible purposes in food, cake, seasoning agent, unlike in the western world where its oil is used for soap, cream production, as well as in other pharmaceuticals (Van der Vossen et al, 1992). The deterioration of melon seed and fungal infestation during storage has made farmers to abandon melon production in many parts of Nigeria. Curits, 1964 recognized the possible economic value of the seed oil, their crude protein and by-products of cucurbitaceous plants, the physicochemical characteristics of their oils and by-products attracted the attention of Wentz et al (1983). Bolley et al (1983) characterized their oils as soft drying oils with similarities to soybean oil. Shanhani et al (1980) indicated the possibility of processing the crude oil obtained from such seeds for yielding edible oil and other products. Vasconcellos et al (1982) reported that the oil contents ranged between 35-41 percent. It is believed that if the oil is extracted, and lecithin is produced from it, this will give added value to the melon seeds produced in Nigeria, hence the objective of this research.
EXTRACTION, CHARACTERIZATION AND INDUSTRIAL USES OF LECITHIN FROM THREE VARIETIES OF Cucumis melo (MELON SEED) OIL.
1.1 Lipids, Classification and Uses
Lipids are broadly defined as any fat-soluble (Lipophilic), naturally-occurring molecule, such as fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins (such as vitamins A,D, E and K), monoglycerol diglycerides, phospholipids and others. The main biological functions of lipids include energy storage, acting as structural components of cell membranes, and participating as important signaling molecules (Berg et al 2006). Although the term lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerol and should not be confused with the term fatty acid. Lipids also encompass molecules such as fatty acids and their derivatives (Including tri-, di-, and monoacylglyerol and phospholipids), as well as other sterol-containing metabolites such as cholesterol, (Spiller, 2006). Lipids are classified in to three groups which are simple, compound and complex lipids. These three groups are further divided in to eight sub-groups which are:- Fatty acyls (including fatty acids) are a diverse group of molecules synthesized by chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups.
The fatty acyl structure represents the major lipid building block of complex lipids and therefore is one of the most fundamental categories of biological lipids. The carbon chain may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen and sulphur. Examples of biologically- interesting fatty acyls are the eicosanoids which are in turn derived from arachidonic acid which include prostaglandins, leukotrienes, and thromboxanes. Other major lipid classes in the fatty acyl category are the fatty esters and fatty amides. Fatty esters include important
biochemical intermediates such as wax, esters, fatty , coenzyme A derivatives, fatty acyl thioester, ACP derivatives and fatty acyl carnitines. The fatty amides include N-acyl ethanolamines such as anandamide. (Berg, 2006). Glycerolipids are composed mainly of mono-, di-and tri-substituted glycerols, the most well known being the fatty acid esters of glycerol (triacylglycerols), also known as triacylglycerol. These comprise the bulk of storage fat in animal tissues. Additional subclasses are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
Examples of structures in this category are the digalactosyldiacylglycerols found in plant membranes and seminolipid from mammalian spermatozoa (Holzl: and Doramann 2007). Glycerophospholipids, also referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and signaling. Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar head group at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria or the sn-1 position in the case of archaebacteria. Example of glycerophospholipids found in biological membranes are phosphatidylcholine (also known as PC or GPCho,and lecithin), phosphatidylethanolamine PE or gPEtn) and phosphatidylserine GPSer). In addition to serving as a primary component of cellular membranes and binding sites for intra-and inter-cellular proteins, some glycerophospholipids in eukaryotic cells, such as phosphatidylinositol and phosphatidic acids are either precursors of, or are themselves, membrane-derived second messengers.
Typically, one or both of these hydroxyl group are acylated with long-chain fatty acids, but there are also alkyl-linked and alkenyl-linked (plasmalogen) glycerolphospholipids, as well as diakylether variants in prokaryotes. (Spiller 2006). Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone that is synthesized de novo from serine and a longchain fatty acyl CoA, then converted into ceramides, phosphosphingolipids glycosphingolipids and other species. The major sphingoid base of mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) as a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 14 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramidephosphoinositols and mannose containing head groups. The Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolidpids such as cerebrosides (Bach and Watchtel 2003).
Sterol lipids, such as cholesterol and its derivatives are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins. The steroids, which also contain the same fused four-ring core structure, have different biological roles as hormones and singaling molecules. The C18 steroids include the eostrogen family whereas the C19 steroids comprise the androgens such as testosterone and androsterone.
The C21 subclass includes the progestogens as well as the glucocorticoids and mineralocorticoids. The secosteroids, comprising various forms of Vitamin D, are characterized by cleavage of the B ring of the core structure. Other examples of sterols are the bile acids and their conjugates, which in mammals are oxidized derivatives of cholesterol and are synthesized in the liver (Wang. 2004). Prenol lipids are synthesized from the 5-carbon precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid pathway. The simple isoprenoids (linear alcohols, diphosphates, etc) are formed by the successive addition of C5 units, and are classified according to number of these terpene units.
Structures containing greater than 40 carbons are known as polyterpenes. Carotenoids are important simple isoprenoids that function as antioxidant and as precursors of vitamin A. Another biologically important class of molecules is exemplified by the quinones and hydroquinones, which contain an isoprenoid tail attached to a quinonoid core of nonisoprenoid origin. Vitamin E and Vitamin K, as well as the ubiquinones, are examples of this class. Bacteria synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols
(dolichols) the terminal isoprenoid is reduced. (Kuzuyama and Seto. 2003). Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. in the saccharolipids, a sugar substitutes for the glycerol backbone that is present in glycerolipids and glycerophosphospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the lipid A component of the lipopolysaccharides in gram-negative bacteria.
Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is KdO2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic
acid (KdO2) residues (Heinz, 1996). Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a very large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity, many polyketides are cyclic molecules whose
backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes. Many commonly used anti-microbial, anti-parasitic, and anti-cancer agents are polyketides or polyketide derivatives, such as erythromycins, tetracyclines, ivermectins, and anti-tumor epothilones (Walsh, 2004).
1.1.1 Uses of lipids
Lipids of fats and oils, steroids, waxes and related compounds have their functions divided into to three areas which include; health, industry and griculture.
However, other lipids which are present in their sources in trace quantities function as enzymes, cofactors; electron carriers, light-absorbing pigments, hydrophobic anchors; emulsifying agents, hormones and intracellular messengers (Nelson and Cox 2001).
Table I.I. Some oils used in Industry and Automobiles Paints and varnishes Vernonia oil, safflower oil walnut oil, Tung oil, stillingia oil (Chinese vegetable tallow oil) Chemicals Castor oil, cuphea oil, snow ball seed oil, bladder pod oil crambe oil, Vernonia oil Candle and lighting Neem oil, orange oil, Tonka bean oil, Amur cork tree fruit oil. Insectcides Balanos oil Biofuel and Biodiesel Melon seed oil, Jojoba oil plam kernel oil, Palm oil and Jatropha oil. Lubricants Castor oil, olive oil, Ramtil oil, Dammar oil, Jojoba oil, Tall oil. (Source: Nelson, 1981)
Table 1.2 Some Uses of Oils in Health Care Delivery Medicinal and Antisepitcs Lemon oil, wheat germ oil cashew oil, Almond oil, Borneo tallow nut oil, shea
butter, Snowball seed oil, Corriander seed oil, Perilla seed oil, Amur Cork tree fruit oil, chaulmoogra oil, Brucia havanica oil burdock oil Pharmaceuticals Soybean oil, melon seed oil, cashew nut oil, cocoa butter; Almond oil. Cosmetic and Skin Care Hazelnut oil, coconut oil, cotton seed oil Acai oil, Amaranth oil Borneo tallow oil, Avocado oil Cohune oil, Rape seed oil, Perilta seed oil, Olive oil, Carrot seed oil, lemon oil, Neem oil, Poppy seed oil, Candle nut oil carrot seed oil shea better. Soap and Cleaning products Palm kernel oil, Palm oil, Borneo tallow nut oil, kapok seed oil. Linseed oil, Poppy seed oil, Daminar oil.
Perfumes and Fragrances Palm oil, Castor oil, Copaiba oil, Honge oil, Jojoba oil sunflower oil, (Source: Nelson, 1981)
Table 1.3 Some Uses of oils in Agriculture Animal feed Soybean oil, melon seed oil, Aglae oil, Evening prime rose oil Pesticides Balanos oil
Fertilizers Soybean oil, melon seed oil, palm oil,safflower oil (Source: Nelson, 1981)
18.104.22.168 Oil Extraction
The conventional methods for oil extraction involves three basic approaches namely-Physical, chemical and a combination of both (Owusu-Ansah, 1994). The physical method employed for oil seeds of high oil content example, groundnut, palm fruit and kernel etc, while chemical method is primarily used for oil seeds of low oil content, example soybeans, rice bran, etc (Owusu-Ansah, 1994). The method used could affect the physical and chemical properties of the oil or fat to a considerable extent. In selecting solvent for extraction, the solubility of the oil or fat in the solvent, toxicity and the intended use of the oil are of utmost importance. Petroleum ether, n-hexane, methanol and chloroform are frequently used (Christie, 1982). Enzymes have found use in oil extraction. The application of enzymes in oil extraction can be categorized into: enzyme-assisted processing, enzyme-enhanced solvent extraction, and enzyme-assisted aqueous extraction (Owusu-Ansah, 1994). In all these approaches, the enzymes are used to break the cell walls of the oil bearing material to release the oil.
22.214.171.124 Refining of Crude vegetable Oil
The further processing of edible oils after extraction from the raw materials is concerned with refining and modification (Young et al., 1994). Refining treatment is needed to remove or reduce as far as possible, those contaminants of the crude vegetable oil which will adversely affect the quality of the end-product and the efficient operation of the modification process. Two methods are in use for the refining of oils and fats. These are termed physical and chemical from the means by which free fatty acids are removed from the oil (Young et al., 1994). The fatty acids are distilled off in the physical
process and in the chemical process are neutralized using an alkaline reagent thus forming soap, which are removed from the oil by phase separation.
Pigments such as carotenoids, chlorophyll, Gossypol, and related compounds and the products of degradation and condensation reactions that occur during the handling, storage and treatment of the extracted oils is removed by bleaching. It was later realized that activated absorbents, in particular are responsible for removing at least partially, other impurities such as soaps, trace metals, phosphatides and hydroperoxide compounds containing sulphur. Primary oxidation levels are also reduced by the breakdown of the oxidation product on the absorbent surface followed by absorption of the carbonyl
compounds that are the secondary oxidation products. The process is usually carried out by treating the oil with absorbents such as special clays, and charcoals at high temperature (100 -1100C) and under reduced pressure (Haraldsson, 1983). The operation however provides lighter coloured oil and prepares it for subsequent processing.