The nutritive and antinutritive compositions of S. dulcificum pulp were analysed to augment the available information on the anti-diabetic effect of the plant. Biochemical parameters like liver function enzymes (ALT, AST, ALP) and bilirubin concentrations,serum total protein, serum albumin and globulin, kidney function parameters (creatinine and urea concentrations), blood glucose, serum lipid profile and lipid peroxidation were determined in rats that were administered different concentrations of the methanolic extract to ascertain their effects. The internal organs (liver and kidney) were also removed and used for histopathological studies. From the result of the study, the proximate composition shows that S. dulcificum contains 7.75% protein, 59.55% moisture content, 4.36% ash, 6.24% crude fibre, 3.26% fat and 18.84% carbohydrate.The result of the mineral analysis shows that S.dulcificum pulp contains 100 mg/g calcium, 24.20 mg/g iron, 9.49 mg/g zinc, 6.22 mg/g copper, 0.01 mg/g chromium and 0.01 mg/g cobalt. Vitamin analyses shows that the S. dulcificum pulp contains 0.04% vitamin A, 22.69% vitamin C, 0.01% vitamin D and 0.02% vitamin K. Antinutrient analyses of the pulp show 5.67% oxalate, 0.03% phytates and 0.02% hemagglutanin. Amino acid profile shows that S.dulcificum pulp contains 8.055% tryptophan, 1.35% phenylalanine, 0.7% isoleucine, 0.5% tyrosine, 1.05% methionine, 0.4% proline, 0.69% valine, 1.1% threonine, 0.4% histidine, 0.5% alanine, 1.02% glutamine, 1.6% glutamic acid, 0.7% glycine, 0.3% serine, 1% arginine, 0.1% aspartic acid, 1.23% asparagine, 0.6% lysine and 0.6% leucine. Quantitative phytochemical analysis shows that the pulp contains 3.45% saponins, 57.01%`flavonoids, 7.12% tannins, 0.0001% alkaloids, 0.0001% glycosides, 0.0003% resins, 0.0002% terpenoids, 0.0001% steroids and 0.0003% cyanogenic glycosides.The results of the acute toxicity show that the methanol extract is not toxic to the mice at concentrations up to 5000mg/kg body weight. From the results obtained, the animals receiving 100mg/kg b.w of the methanolic extract showed significantly reduced (p<0.05) serum levels of glucose, bilirubin, low density lipoprotein cholesterol and ALT after the 14 day study compared to the 28 day study. However, no significant difference (p>0.05) was also observed across the groups in their serum ALP, AST, creatinine, urea, cholesterol, TAG, albumin and globulin levels on the 14th day compared with the 28th day. A significant difference (p<0.05) was observed in the malondaldehyde and serum protein concentrations in the 500mg/kg b.w test group while glucose concentration decreased significantly (p<0.05) in the 100mg/kg b.w and 500mg/kg b.w test group after the 14 day study compared with the 28 day study. High density lipoprotein cholesterol level significantly increased (p<0.05) in the 200mg/kg b.w test group. Histopathological examination shows normal liver architecture across the groups at 100mg/kg b.w, 200mg/kg b.w and 500mg/kg b.w. Kidney sections of rats showing normal glomerulus (G) and renal tubules (arrow) at same concentrations.





Title Page





Table of Contents

List of Figures

List of Tables

List of Abbreviations



1.1                   Sweeteners

1.1.1                Common Sweeteners and Their Production             Natural Sweeteners          Honey          Maple Syrup          Molasses          Stevia          Sucrose             Artificial Sweeteners

1.2                   Synsepalum dulcificum

1.3                   Nutrients

1.3.1                Carbohydrates

1.3.2                Proteins

1.3.3                Fats

1.4                   Phytochemicals

1.5                   Antinutrients

1.6                   Vitamins

1.6.1                VitaminA

1.6.2                Vitamin C

1.6.3                Vitamin D

1.6.4                Vitamin E

1.6.5                Vitamin K

1.7                   Antioxidant

1.8                   Some Minerals and Their Biological Functions

1.8.1                Calcium (Ca)             Metabolic Functions and Deficiency Symptoms of Calcium

1.8.2                Magnesium (Mg)             Metabolic Functions and Deficiency Symptoms of Magnesium

1.8.3                Zinc (Zn)             Metabolic Functions and Deficiency Symptoms of Zinc

1.8.4                Iron (Fe)             Metabolic Functions and Deficiency Symptoms of Iron

1.8.5                Copper (Cu)             Metabolic Functions and Deficiency Symptoms of Copper

1.9                   Blood Glucose

1.9.1                Blood Glucose Regulation

1.10                 Lipids

1.10.1              Lipoproteins: Types and Functions           Chylomicrons           Very Low Density Lipoprotein (VLDL)           Low Density Lipoprotein (LDL)        Metabolism of Low Density Lipoprotein via LDL Receptor        Regulation of LDL Receptor           High Density Lipoprotein (HDL)

1.11                 Total Cholesterol andCholesterol Balance in Tissues

1.11.1              Diet and Cholesterol Regulation

1.12                 Liver Function Biomarkers

1.12.1              Alanine Aminotransferase

1.12.2              Aspartate Aminotransferase

1.12.3              Alkaline Phosphatase

1.12.4              Clinical and Diagnostic Significance of Liver Function Enzymes

1.12.5              Bilirubin

1.12.6              Serum Protein

1.12.7              Serum Albumin

1.13                 Renal Function Biomarkers

1.13.1              Blood Urea Nitrogen (BUN)

1.13.2              Creatinine

1.14                 Lipid Peroxidation

1.14.1              Initiation

1.14.2              Propagation

1.14.3              Termination

1.14.4              Types of Lipid Peroxidation           Non- Enzymatic Lipid Peroxidation           Enzymatic Lipid Peroxidation

1.15                 Research Objectives

1.15.1              General Objectives

1.15.2              Specific Objectives



2.1                   Materials

2.1.1                Plant materials

2.1.2                Animals

2.1.3                Chemicals and Reagents

2.1.4                Equipment /Instruments

2.2                   Methods

2.2.1                Experimental Design

2.2.2                Extraction of Plant Material

2.2.3                Determination of the Extract Yield

2.2.4                Toxicological studies             Acute Toxicity Studies and Lethal Dose (LD50) Test

2.2.5                Proximate Analysis             Moisture             Crude Protein             Crude Fat             Crude Fibre             Ash/Mineral Matter             Carbohydrate or Nitrogen Free Extract (NFE)

2.2.6                Estimation of Vitamins             Determination of Vitamin A             Determination of Vitamin C             Determination of Vitamin D             Determination of Vitamin E             Determination of Vitamin K

2.2.7                Determination of Mineral Content of S. dulcificum Pulp             Determination of Phosphorus

2.2.8                Determination of Amino Acid Profile             Defatting of the Pulp             Hydrolysis of the Pulp             Nitrogen Determination             Loading of the Hydrolysate into TSM Analyzer             Method of Calculating Amino Acid values using Chromatogram Peaks

2.2.9                Qualitative Phytochemical Studies on Synsepalum dulcificum Pulp             Test for Alkaloids             Test for Glycosides             Test for Cyanogenic Glycosides             Test for Tannins             Test for Saponins             Test for Flavonoids             Test for Resins             Test for Terpenoids and Steroids

2.2.10              Quantitative Phytochemical Analysis of S.dulcificum Pulp           Determination of Alkaloids           Determination of Cyanogenic Glycosides           Determination of Saponins           Determination of Flavonoids           Determination of Tannins           Determination of Steroids           Determination of Terpenoids

2.2.11              Antinutrient Analysis of S. dulcificum Pulp           Determination of Oxalates           Determination of Phytates           Determination of Haemagglutanins

2.2.12              Blood Sample Collection for Biochemical Analysis

2.2.13              Biochemical Assays           Assay of Alanine Aminotransferase (ALT) Activity           Assay of Aspartate Aminotransferase Activity           Assay of Alkaline Phosphatase (ALP) Activity           Determination of Bilirubin Concentration Using Colorimetric Method        Determination of Total Bilirubin (TB) Concentration           Total Serum Protein Assay           Serum Albumin Concentration           Creatinine           Urea           Blood glucose Assay         Estimation of Serum Lipid Concentrations      Estimation of Total Cholesterol Concentration      Estimation of Low Density Lipoprotein-Cholesterol Concentration      Estimation of High Density Lipoproteins (HDL)–Cholesterol Concentration      Estimation of Triacylglycerol         Estimation of Lipid Peroxidation

2.2.14              Histopathological Examination

2.2.15              Statistical Analysis




3.1       Proximate Composition of S. dulcificum Pulp

3.2       Mineral Composition of S. dulcificum Pulp

3.3       Vitamin Content of S.dulcificum Pulp

3.4       Amino Acid Profile of S. dulcificum Pulp

3.5       Phytochemical Composition of S. dulcificum Pulp

3.6       Antinutritional Composition of S.dulcificum Pulp

3.7       Acute toxicity (LD50) Studies

3.8       Mean Body Weights of Animals

3.9       Effect of S. dulcificumMethanolic Extract Administration on Alkaline Phosphatase (ALP) Activity in Rats

3.10     Effect of S. dulcificumMethanolic Extract Administration on Alanine Aminotransferase (ALT) Activity in Rats

3.11     Effect of S. dulcificumMethanolic Extract Administration on Aspartate Aminotransferase (AST) Activity in Rats

3.12     Effect of S. dulcificumMethanolic Extract Administration on Bilirubin levels in Rats

3.13     Effect of S. dulcificumMethanolic Extract Administration on Total Serum Protein concentration in rats

3.14     Effect of S. dulcificumMethanolic Extract Administration on Serum Albumin Concentration in Rats

3.15     Effect of S. dulcificumMethanolic Extract Administration on Serum Globulinin Rats

3.16     Effect of S. dulcificumMethanolic Extract Administration on Creatinine Level in Rats

3.17     Effect of S. dulcificumMethanolic Extract Administration on Urea Level in Rats

3.18     Effect of S. dulcificumMethanolic Extract Administration on Blood Glucose Concentration in Rats

3.19     Effect of S. dulcificumMethanolic Extract Administration on Cholesterol Concentration in Rats

3.20     Effect of S. dulcificumMethanolic Extract Administration on High Density Lipoprotein Cholesterol Concentration in Rats

3.21     Effect of S. dulcificumMethanolic Extract Administration on Low Density Lipoprotein Cholesterol Concentration in Rats

3.22     Effect of S. dulcificumMethanolic Extract Administration on Triacylglycerol Concentration in Rats

3.23     Effect of S. dulcificumMethanolic Extract Administration on Malondialdehyde Concentration in Rats

3.24     Effect of S. dulcificumMethanol Extract Administration on the Histopathology of Rat Liver [14 days duration]

3.25     Effect of S. dulcificumMethanol Extract Administration on the Histopathology of Rat Liver [28 days duration]

3.26     Effect of S. dulcificumMethanol Extract Administration on the Histopathology of Rat Kidney [14 days duration]

3.27     Effect of S. dulcificumMethanol Extract Administration on the Histopathology of Rat Kidney [28 days duration]





4.1       Discussion

4.2       Conclusion

4.3       Suggestions For Further Studies







Figure 1           Structure of Sucrose

Figure 2           Syvsepalum dulcificum Fruit

Figure 3           Synsepalum dulcificum Tree

Figure 4           Structure of Cholesterol

Figure 5           Mechanism of Non-Enzymatic Lipid Peroxidation

Figure 6           Proximate Composition of S. dulcificum Pulp

Figure 7           Amino Acid Analyses of S. dulcificum Pulp

Figure 8:          Effect of S.dulcificum Methanolic Extract Administration on Alkaline phosphatase Activity in Rat

Figure 9           Effect of S.dulcificum Methanolic Extract Administration on Alanine Aminotransferase Activity in Rat

Figure 10         Effect of S.dulcificum Methanolic Extract Administration on Aspartate Aminotransferase Activity in Rat

Figure 11         Effect of S.dulcificum Methanolic Extract Administration on Bilirubin Concentration in Rat

Figure 12         Effect of S.dulcificum Methanolic Extract Administration on Total Serum Protein in Rat

Figure 13         Effect of S.dulcificum Methanolic Extract Administration on Serum Albumin in Rat

Figure 14         Effect of S.dulcificum Methanolic Extract Administration on Serum Globulin in Rat

Figure 15         Effect of S.dulcificum Methanolic Extract Administration on Creatinine Level in rat

Figure 16         Effect of S.dulcificum Methanolic Extract Administration on Urea Level in Rat

Figure 17         Effect of S.dulcificum Methanolic Extract Administration on Blood Glucose Concentration in Rat

Figure 18         Effect of S.dulcificum Methanolic Extract Administration on Total Cholesterol in Rat

Figure 19         Effect of S.dulcificum Methanolic Extract Administration on High-Density Lipoprotein Cholesterol Concentration in Rat

Figure 20         Effect of S.dulcificum Methanolic Extract Administration on Low-Density Lipoprotein Cholesterol Concentration in Rat

Figure 21         Effect of S.dulcificum Methanolic Extract Administration on Triacylglycerol Concentration in Rat

Figure 22         Effect of S.dulcificum Methanolic Extract Administration on Malondialdehyde Concentration in Rat

Figure 23         Photomicrograph of Liver Sections of Rats 14 days Post Administration With S.dulcificum Methanolic Extract

Figure 24         Photomicrograph of Liver Sections of Rats 28 days Post Administration With S.dulcificum Methanolic Extract

Figure 25         Photomicrograph of Kidney Sections of Rats 14 days Post Administration With S.dulcificum Methanolic Extract

Figure 26         Photomicrograph of Kidney Sections of Rats 28 days Post Administration With S.dulcificum Methanolic Extract




Table 1            Uses for Common Artificial Sweeteners

Table 2            The Levels of Some Minerals in S. dulcificum Pulp

Table 3            Vitamin Content of S.dulcificum Pulp

Table 4            Phytochemical Composition of S.dulcificum Pulp

Table 5            Antinutrient Composition of S. dulcificum Pulp

Table 6                        Result of the Acute Toxicity (LD50) Test of the Methanolic Pulp Extract of S. dulcificum

Table 7:                       The Mean Body Weight of Rats Administered Doses of S. dulcificum Methanolic Pulp Extract





The worsening food crisis and the consequent widespread prevalence of malnutrition in developing and under-developed countries have resulted in high mortality and morbidity rates, especially among infants and children in low-income groups (Enujiugba and Akanbi, 2005). Food has been defined as any substance containing primarily carbohydrates, fats, water, protein, vitamins and minerals that can be taken by an animal or human to meet its nutritional needs and sometimes for pleasure. Items considered as food may be sourced from plants, animals or fungus as well as fermented products like alcohol. Food is also anything solid or liquid that has a chemical composition which enables it provide the body with the material from which it can produce heat or any form of energy, provide material to allow for growth, maintenance, repair or reproduction to proceed and supply substances, which normally regulate the production of energy or the process of growth, repair or reproduction. Food is therefore, the most basic necessity of life (Turner, 2006).


Nutrition is the science that deals with all the various factors of which food is composed and the way in which proper nourishment is brought about. The average nutritional requirements of groups of people are fixed and depend on such measurable characteristics as age, sex, height, weight, degree of activity and rate of growth. Good nutrition requires a satisfactory diet which is capable of supporting the individual consuming it, in a state of good health by providing the desired nutrients in required amounts. It must provide the right amount of nutrients and fuel to execute normal physical activity. If the total amount of nutrients provided in the diet is insufficient, a state of under- nutrition develops.


Plants are primary sources of medicines, food, shelters and other items used by humans everyday. Their roots, stems, leaves, flowers, fruits and seeds provide for humans (Amaechi, 2009; Hemingsway, 2004). Fruits are sources of minerals, fibre and vitamins which also provide essential nutrients for the human health (Anaka et al., 2009). Some fruits are also known to have antinutritional factors such as phytate and tannins,that can diminish the nutrient bioavailability if they are present at high concentrations (Baum, 2007). It has been reported that these anti-nutritional factors could also help in the treatment and prevention of certain important diseases like the anti-carcinogenic activities reported for phytic acid which has been demonstrated both invivo and invitro (Anaka et al., 2009).


The reliance on starchy roots and tubers and certain cereals as main staples result in consumption of non-nutritious foods. The insufficient availability of nutrient rich diets and the high cost of available ones have prompted an intense research into harnessing the potentials of the lesser known and underutilized crops, which are potentially valuable for human and animal foods to maintain a balance between population and agricultural productivity, particularly in the tropical and sub-tropical areas of the world. The challenge of improper nutrition especially in developing countries which include Nigeria, is indeed alarming. The World Health Organization (WHO, 2007) reported that poor nutrition contributes to one out of two deaths associated with infectious diseases among children within five yearsand the aged. Poor diet can have an injurious impact on health, causing deficiency diseases such as scurvy, beriberi and kwashiokor, health-threatening conditions such as obesity, metabolic syndrome, and such common other diseases as cardiovascular diseases, diabetes and osteoporosis. Under-nutrition among pregnant women in developing countries leads to one out of six infants being born with low birth weight, which is a risk factor for neonatal deaths, learning disabilities, mental retardation, poor health and premature death. One out of three people in developing countries is affected by vitamin and mineral deficiencies making them prone to infectious diseases and impaired psycho intellectual development. Under and chronic nutrition problems and diet related chronic diseases account for more than half of the world’s diseases (WHO, 2007). In most of these side effects or diseases, the biochemical and haematological parameters are usually altered. For a food to be considered safe for human and animal consumption, its effect on these parameters need to be investigated to understand the nutritional potentials and safety of such foods with a view to determining their acceptability.



1.1              Sweeteners


Sweeteners are food additives that are used to improve the taste of everyday foods. Natural sweeteners are sweet-tasting compounds with some nutritional value; the major ingredient of natural sweeteners is either mono- or disaccharides. Artificial sweeteners, on the other hand, are compounds that have very little or no nutritional value. This is possible because artificial sweeteners are synthesized compounds that have high-intensities of sweetness, meaning less of the compound is necessary to achieve the same amount of sweetness. Artificial sweeteners are used in products intended to limit caloric intake or prevent dental cavities. Sugar alcohols are natural compounds with varying degrees of sweetness which are often added to boost or fine tune flavours of products while increasing their sweetness. They are often used in conjuncture with natural or artificial sweeteners in order to achieve a desired degree of sweetness, taste or texture. Sugar alcohols typically provide some amount of nutrition but have other benefits such as not affecting insulin response or promoting tooth decay which makes them a popular sweetening choice.



1.1.1          Common Sweeteners and Their Production


A sugar substitute is a food additive that replicates the effect of sugar in taste, but usually has less food energy. Some sugar substitutes are natural while others are synthetic, those that are not natural are referred to as artificial sweeteners (Mattes and Popkin, 2009). An important class of sugar substitutes is known as high-intensity sweeteners. These are compounds with sweetness that is many times that of sucrose, a common table sugar. As a result, much less sweetener is required, and energy contribution often negligible. The sensation of sweetness caused by these compounds is sometimes notably different from sucrose, so they are often used in complex mixtures that achieve the most natural sweet sensation. This may be seen in soft drinks labelled as "diet" or "light"; they contain artificial sweeteners and often have notably different mouth feel. In the United States, six intensely-sweet sugar substitutes have been approved for use (Mattes and Popkin, 2009). They are saccharin, aspartame, sucralose, neotame, acesulfame potassium, and stevia. The US Food and Drug Administration regulates artificial sweeteners as food additives. The majority of sugar substitutes approved for food use are artificially-synthesized compounds. However, some bulk natural sugar substitutes are known, including sorbitol and xylitol, which are found in berries, fruit, vegetables and mushrooms (Mattes and Popkin, 2009). Some non-sugar sweeteners are polyols, also known as "sugar alcohols." These are, in general, less sweet than sucrose, but have similar bulk properties and can be used in a wide range of food products. Sometimes the sweetness profile is 'fine-tuned' by mixing high-intensity sweeteners. As with all food products, the development of a formulation to replace sucrose is a complex proprietary process. Natural Sweeteners


Natural sweeteners are extracted from natural products without any chemical modifications during the production or extraction process. Some of these sweeteners have been in use for decades while other for centuries. Natural sweeteners are well known and their production processes have been perfected over time making their cost low and leaving their demand high. Honey


Honey is a sweet food made by certain insects using nectar from flowers. The variety produced by honey bees is the one most commonly referred to and is the type of honey collected by beekeepers and consumed by humans. Honey produced by other bees and insects has distinctly different properties. Honey bees transform nectar into honey by a process of regurgitation and evaporation. They store it as a food source in wax honeycombs inside the beehive (National Honey Board, 2012). Beekeeping practices encourage overproduction of honey so that the excess can be taken without endangering the bee colony. Honey gets its sweetness from the monosaccharides fructose and glucose and has approximately the same relative sweetness as that of granulated sugar (74% of the sweetness of sucrose, a disaccharide) (NHB, 2012). It has attractive chemical properties for baking, and a distinctive flavour which leads some people to prefer it over sugar and other sweeteners. Most micro-organisms do not grow in honey because of its low water activity (Arcot and Brand-Miller, 2005). The main uses of honey are in cooking, baking, as a spread on breads, and as an addition to various beverages such as tea and as a sweetener in some commercial beverages. Honey is also used as an adjunct in beer. Its glycaemic index ranges from 31 to 78, depending on the variety (Arcot and Brand-Miller, 2005).


Honey is a mixture of sugars and other compounds. With respect to carbohydrates, honey is mainly fructose (about 38.2%) and glucose (about 31.0%).The remaining carbohydrates in honey include maltose, sucrose, and other complex carbohydrates (Martos et al., 2000). Honey contains trace amounts of several vitamins and minerals (Gheldof et al., 2002). As with all nutritive sweeteners, honey is mostly sugars and is not a significant source of vitamins or minerals. Honey also contains tiny amounts of several compounds thought to function as antioxidants, including chrysin, pinobanksin, vitamin C, catalase, and pinocembrin (Gheldof et al., 2002). The specific composition of any batch of honey depends on the flowers available to the bees that produce the honey. A typical honey analysis shows the following: fructose: 38.2%, glucose: 31.0%, sucrose: 1.5%, maltose: 7.2%, water: 17.1%, higher sugars: 1.5%, ash: 0.2%. Honey has a density of about 1.36 kg/L (36% denser than water) (NHB, 2012). The pH of honey is between 3.2 and 4.5. This relatively acidic pH level prevents the growth of many bacteria (Arcot and Brand-Miller, 2005).                    Maple Syrup


Maple syrup is a sweetener made from the sap of some maple trees. In cold climate areas, these trees store sugar in their roots before the winter and the sap which rises in the spring can be tapped and concentrated (Ball, 2007). The sap has only 3 to 5% total solids, consisting mainly of sucrose. Other components of the maple syrup include organic acids (primarily malic acid) and minerals (potassium and calcium), amino compounds (trace) and vitamins (trace). Maple Syrup has about the same 50 cal/tbsp as white cane sugar. However, it also contains significant amounts of potassium (35 mg/tbsp), calcium (21 mg/tbsp), small amounts of iron and phosphorus, and trace amounts of β- complex vitamins. Its sodium content is as low as 2 mg/tbsp. The sugar content of sap averages 2.5% and the sugar content of syrup averages 66.5% (Ball, 2007).                      Molasses


Molasses is a viscous byproduct of sugar cane or sugar beets processing into sugar. The quality of molasses depends on the maturity of the sugar cane or sugar beet, the amount of sugar extracted, and the method of extraction exployed (Taubes, 2011). Molasses has the molecular formula C6H12NNaO3S, molecular weight of 201.22 g/mol, and a density of 1.41 g/cm3 (Taubes, 2011). A typical composition of molasses shows the following substances: sucrose 35.9 %, fructose 5.6 %, nitrogen 1.01 %, reducing substances 11.5 %, glucose 2.6 %, and sulfur 0.78 % (Taubes, 2011).                      Stevia


Stevia is one of the newest sweeteners available in the market. It has been known since 1899 for its sweet taste and has been cultivated in Japan since 1970. It was not until recently that a safe and successful extraction of glycosides (the chemical in the Stevia plant which gives it a sweet taste) allowed for the Food and Drug Administration (FDA) to approve Stevia as a general sweetener (Raji and Mohamed, 2012). Stevia is also known under different trade names as TruViaand PureVia patents by Coca Cola and Pepsi(Raji and Mohamed, 2012). Many different forms of Stevia as sweeteners exist such as: Reb A, B, C, D, Rebiana, Stevioside, SunCrystalsand Enliten. Each has a small variation in the manufacturing process or how it is used.


Stevia is an all natural sweetener because it is extracted from the Stevia plant and undergoes no chemical changes in the manufacturing process. This makes it very desirable to many consumers looking for healthy alternatives to sucrose sugar. Stevia is a general term referring to a plant, Steviarebaudiana (Bertoni), native to Paraguay. The plant contains a number of diterpene glycosides that taste sweet; the main ones are stevioside and rebaudioside A. These glycosides are 200 and 300 times sweeter than sucrose respectively (Mattes and Popkin, 2009).                      Sucrose


Sucrose is a disaccharide, formed from the monosaccharides glucose and fructose. It is the organic compound commonly known as table sugar and sometimes called saccharose.It has the molecular formula C12H22O11 and a molecular weight of 342.30 g/mol. In sucrose, the component sugars glucose and fructose are linked via an α (alpha) 1 on the glucose, to a β (beta) 2 on the fructose glycosidic linkage.


Sucrose forms a major element in confectionery and desserts. Cooks use it for sweetening, its fructose component which has almost double the sweetness of glucose makes sucrose distinctively sweet in comparison to other carbohydrate foods (Taubes, 2011). It can also act as a food preservative when used in sufficient concentrations. It is a common ingredient in many processed and junk foods.

Fig 1: Structure of sucrose (Stryer, 1995) Artificial Sweeteners


Table 1: Uses for common artificial sweeteners


Trade Names








Sweet One®

200 times

Found in more than 4,000 productsincluding



sweeter than

candies, tabletop sweeteners, chewing gums,




beverages, dessert and dairyproduct mixes,




baked goods,alcoholic beverages, syrups,




refrigerated and frozen desserts,and sweet




sauces and toppings.







180 times

Found in more than 6,000 productsincluding



sweeter than

carbonated powderedsoft drinks, chewing gum,




confections, gelatins, dessertmixes, puddings




and fillings, frozendesserts, yoghurt, tabletop




sweeteners, and somepharmaceuticals.






None yet


Approved for use in beveragesdairy products,



times sweeter

frozen desserts,baked goods, and gums.



than sugar







Sweet N Low®


Fountain Diet Coke® and pepsi®,Tab®, and



sweeter than

often mixed withaspartame.











600 times

Found in everything from frozendesserts,



sweeter than

cookies, gum, sodas,candies. Can also be used









Source:( 5/14/2013 5:03pm


Artificial sweeteners are derived from chemical synthesis of organic compounds which may or may not be found in nature. They are relatively new and their uses are being researched and extended every day. Much controversy surrounds artificial sweeteners and their health effects as they may break down into harmful chemical sub-compounds. New artificial sweeteners are always being researched and due to their low cost and ease of production, they will likely become the primary sweetening compounds in the future (Mattes and Popkin, 2009).



1.2              Synsepalum dulcificum


Synsepalum dulcificumis a shrub that grows up to 6.1m high in its native habitat but does not usually grow higher than 10ft (3.048m) in cultivation (Wiersema and Leon, 1999).Its leaves are 5-10cm long, 2-3.7cm wide and glabrous below. They are clustered at the end of the branchlets. It is an evergreen plant that produces small orange fruits (Duke and Ducellier, 1993). The seeds are about the same size as coffee beans (fig. 2). The plant is also known as Richardelladulcificum (old name), miracle fruit, magic fruit, miraculous or flavor fruit (Duke and Ducellier,1993). The miracle fruit plant (Synsepalum dulcificum) produces fruits or berries that, when eaten, causes sour foods (including lime and lemon) consumed later to taste sweet (fig. 3) (Joseph et al., 2009). The fruit was first documented by explorer Chevalier des Marchais who searched for many different foods during a 1725 excursion to its native West Africa (Roecklin and Leung, 1987). Marchais noticed that local tribes picked the fruit from shrubs and chewed it before meals.


The berry contains an active glycoprotein molecule, with some trailing carbohydrate chain called miraculin (Forester and Waterhouse, 2009). When the fleshy part of the fruit is eaten, the molecule binds to the tongue’s taste buds, causing sour foods to taste sweet. While the exact cause of this change is unknown, one theory is that the glycoprotein, miraculin works by distorting the shape of sweetness receptors so that they become responsive to acids, instead of sugar and other sweet things (Duke and Ducellier,1993).This effect can last for 10min-2hr (Joseph et al.,2009).


In Africa, S. dulcificum leaves are attacked by lepidopterous larvae and fruits are infested with larvae of fruit flies. A fungus which has been found on this plant is microporous (Duke and Ducellier, 1993). In tropical West Africa where this specie originates, the fruit pulp is used to sweeten palmwine (Joseph et al., 2009). Attempts have been made to make a commercial sweetener from this fruit with an idea of developing this for patients with diabetes (Joseph et al., 2009). Fruit cultivators also report a small demand from cancer patients, because the fruit allegedly counteracts a metallic taste in the mouth that may be one of the many side effects of chemotherapy. This claim has not been researched scientifically. In Japan, miracle fruit is popular among patients with diabetes and dieters (Duke and Ducellier, 1993).



The detailed scientific classification of the plant is as follows:

 Kingdom: Plantae

 Superdivision: Angiosperms

 Division: Eudicots

 Class: Asterids

 Order: Ericales

 Family: Sapotaceae

 Genus: Synsepalum

 Species: S.dulcificum

 Binomial name: Synsepalumdulcificum

 (Source: Wiersema and Leon, 1999)

Fig. 2: Synsepalum dulcificum fruit (taken at source)



Fig. 3: Synsepalum dulcificum tree (taken at source)


1.3              Nutrients


A nutrient is any substance that is assimilated by an organism to promote growth (Harper, 1999). Nutrients consist of various chemical substances in the foods that make up each diet. Many nutrients are essential for life and an adequate amount of the nutrients in the diet is necessary for providing energy, building and maintaining of the body organs and for various metabolic processes (Morrison and Mark, 1999). There are six major classes of nutrients found in the food: carbohydrate, protein, fats, vitamins (both fat soluble and water soluble), mineral and water.



1.3.1        Carbohydrates


Carbohydrates are one of the main dietary components of food. This category of foods includes sugars, starches and fibres. Carbohydrates are important in the body as sources of energy. They can be found in a wide range of plant and animal food sources. In plants, they are generally end products of photosynthesis- the process in which plants convert carbondioxide and water into simple sugars such as glucose. In foods, carbohydrates are important for adding flavour, texture and colour (Harper, 1999).



1.3.2        Proteins


Dietary proteins are powerful compounds that build and repair body tissues from hair and fingernails to muscles. In addition to maintaining the body’s structure, proteins as enzymes speed up chemical reactions in the body, as well as serve as chemical messengers in the body, fight infection and transport oxygen from the lungs to the body’s tissues. Proteins play an important role in biochemical, biophysical and physiological processes. The deficiency of proteins lead to weakness, anaemia, protein-energy malnutrition (kwashiorkor and marasmus), delayed wound and fracture healing, decreased resistance to infection because antibody formation is decreased and sprue syndrome (Wardlaw,1999).



1.3.3        Fats


Fats  in  the  body  serve  as  energy  sources  and  as  protective  cushion  around  organs.


Saturated fats are usually solid at room temperature while unsaturated fats remain liquid at room temperature. They provide insulation for the body, protect vital organs, and aid in the absorption and transportation of the fat soluble vitamins A, D, E and K. A lot of health disorders arise when proper amount of essential fats are not absorbed. This leads to autoimmune, inflammatory and cardiovascular diseases (Wardlaw, 1999). Those suffering from degenerative diseases such as obesity, cancer, cardiovascular disease, diabetes and liver disorders usually have low levels of essential fatty acids in their tissues. A deficiency of some essential fats will retard growth and produce eczema, acne, dry skin and dandruff, dull, brittle and sparse hair, soft brittle and flaking nails, dry eyes and mouth, diarrhoea, allergies, varicose vein, decreased or increased weight,gallstone, decreased radiation resistance, heart disease ,cancers, deterioration of skin, sterility, swollen joints, liver deterioration, fatigue, emotional agitation, decreased immunity, e.t.c. Excess fat has been shown to produce an abnormal weight gain and diminishing metabolism (Wardlaw, 1999).



1.4              Phytochemicals


Phytochemicals  are  naturally  occurring,  biologically  active  chemical  compounds  in plants. They act as a natural defence system for host plants and provide colour, aroma and flavor. Phytochemicals are protective and disease-preventing particularly for some form of cancer and heart disease. The most important action of these chemicals with respect to human beings is somewhat similar in that they function as antioxidants that react with the free oxygen molecules or free radicals in our bodies (Sofowora, 1993). Phytochemicals that have been discovered are grouped based on function and sometimes sources. These groupings include the flavonoids, phyto-estrogens, phytosterols and carotenoids. These classes and others can be further divided into subclasses (Frantisek, 1991). The flavonoids include more than 1500 separate compounds with varied functions. Flavonoids enhance the effect of vitamin C and function as antioxidants. They are also known to be biologically active against liver toxins, tumours, viruses and other microbes, allergies and inflammation (Sofowora, 1993). Some of the important flavonoids include hesperidin, quercitin, tangeretin, resveratrol and anthocyanins. Phyto-oestrogens are naturally occurring plant compounds that structurally resemble mammalian oestrogen. They copy or counteract the effect of oestrogen in the body. Consumption of isoflavone, a phytoestrogen, is associated with cancer prevention, improved cardiovascular health and bone health (Evans, 2005). Phytosterols are plant sterols that occur in many plant species but appear to be more abundant in the seed of green and yellow vegetables. They are important in the human diet because they help to reduce the amount of dietary cholesterol absorbed by the body by blocking uptake in the intestine. They also facilitate cholesterol excretion from the body. Carotenoids are plant pigments found in bright yellow, orange and red fruits and vegetables. Carotenoids are generally well known as vitamin A precursors (Frantisek, 1991). Phytochemicals are found in all plant products. Some good sources include vegetables, spinach, tomatoes, peppers, carrots, watermelon, citrus fruits, mangoes, papaya, grapes, apples, red grape, pears, oats, barley, sweet potatoes, corn, ginger, thyme, onions, green tea (Okaka etal., 1992).



1.5              Antinutrients


Antinutrients are chemical substances found in food that usually interfere with digestion, absorption or utilization of proteins (Price etal., 1987). The three broad classes of antinutrients are antiproteins, antivitamins and antiminerals.


Antiproteins are substances that interfere with the digestion, absorption or utilization of proteins. They occur in many plants and some animals (Ayyagari etal., 1989). Various protease inhibitors affect proteolytic enzymes of the gut usually by binding to the enzyme’s active site. Lectins are antiproteins that have binding site for cell receptors similar to what antibodies have. Haemaglutinins cause red blood cell to agglutinate. Trypsin and chymotrypsin inhibitors can be found in legumes, vegetables, milk, wheat and potatoes (Ayyagari etal., 1989).


Antivitamins are substances that inactivate or destroy vitamins or inhibit the activity of a vitamin in a metabolic reaction and increase an individual’s need for the vitamins. They destroy or inhibit the metabolic effect of vitamins. Examples of antivitamins in foods include thiaminase (an antivitamin B present in raw fish and other animal foods), caramel colourants (antivitamin B6) and dicoumarol (antivitamin K). Antinutrients are sometimes consumed as natural component of food or medication (Liener, 1980). These vitamins can cause deficiency symptoms similar to those observed when the corresponding vitamins are not present. The administration of the specific vitamins reverses the deficiency symptoms. Isotonic acid hydrazide, also called isoniazid used to treat tuberculosis, can cause deficiency of niacin and vitamin B6. The deficiency symptoms are reversed after giving supplement of these two vitamins.


Antiminerals are substances that interefere with absorption and metabolic utilization of minerals. Some examples are phytates, oxalates, glucosinolates, dietary fibre and gossypol. Phytic acid is found in bran and germ of many seeds and grains, legumes and nuts. In addition, phytic acid can compromise the absorption of magnesium, zinc, copper and manganese, usually forming precipitates. Formation of soybean-phytate complexes during processing has been associated with a reduction in bioavailability of minerals such as Ca, Zn, Fe and Mg. On the other hand, fermentation and other processing techniques are useful in reducing phytate levels (Liener, 1980). Oxalic acid, like phytic acid reduces the availability of bivalent cations. Sources of oxalic acid include rhubarb, spinach, beets, potatoes, teas, coffee and cocoa. Glucosinolates reduce an enlargement of the thyroid gland and inhibit iodine uptake into the thyroid. Rutabaga, turnips, cabbage, peaches and strawberries are good sources of glucosinolates (Liener, 1980).



1.6              VITAMINS


Vitamins are essential organic substances needed in small amounts in the diet for the normal function, growth and maintenance of body tissues. Although vitamins themselves provide no energy to the body, some can facilitate energy–y ielding chemical reactions. Vitamins A, D, E and K dissolve in organic solvents such as ether and benzene and are referred to as fats – soluble vitamins. The B-vitamins and vitamins C, in contrast, dissolve in water and are the water soluble vitamins.


Vitamins are generally indispensable in human diets because they can’t be synthesized in sufficient quantities to meet individual needs. Again synthesis is curtailed by environmental factors or they also can’t be synthesized at all (Hampl and Gordon, 2007).


To be classified as a vitamin, the compound must be organic and must meet the criteria to be an essential nutrient – the body is unable to sy nthesize enough of the compound to maintain health and the absence of the compound from the diet for a defined period of time produces deficiency symptoms that, if caught in time, are quickly cured when the substance is resupplied. A substance does not qualify as a vitamin merely because the body can’t make it. Evidence must suggest that health declines when the substance is not consumed (Hampl and Gordon, 2007).



1.6.1        VitaminA (Beta-carotene)


Beta-carotene  is  an  unstable  fat-soluble  primary  alcohol.  It  is  necessary  for  the production and resynthesis of rhodopsin (visual purple) and may protect against (or reverse) radiation damage (Watty, 2000). Beta-carotene acts as an antioxidant to scavenge radiation induced oxygen radicals and reduce lipofuscin (a component of drusen).

Consuming foods rich in beta-carotene appears to protect the body from damaging molecules called free radicals (Gaziano et al., 2007). The antioxidant action of beta-carotene makes it valuable in protecting against and in some cases even reversing precancerous conditions affecting the breast, mucous membranes, throat, mouth, stomach, prostate, colon, cervix and bladder (Gaziano et al., 2007). Individuals with high levels of β-carotene intake have lower risks of lung cancer, coronary artery heart disease, stroke and age-related eye diseases than individuals with low levels of β-carotene intake. Too much intake of β-carotene may cause or and may be mistaken for jaundice (Gaziano et al., 2007). Beta-carotene is richly found in yellow, orange and green leafy fruits and vegetables such as carrots, spinach, lettuce, tomatoes, sweet potatoes, broccoli, cantaloupe and winter squash (Bjelakovic, 2007). Deficiency of vitamin A causes night blindness, xerophthalmia (an extreme dryness of the conjunctiva), keratosis (an epidermal lesion of tissue overgrowths) and infections (Watty, 2000).



1.6.2        Vitamin C (Ascorbic acid)


Ascorbic acid is a sugar acid with antioxidant properties. Its appearance is white to light-yellow crystals or powder, and it is water-soluble. One form of ascorbic acid is commonly known as vitamin C (Shigeoka et al., 2002). Most animals are able to produce this compound in their bodies and do not require it in their diet. In cells, it is maintained in its reduced form by reaction with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxins (Jacob, 1996). Ascorbic acid is a reducing agent and can reduce and neutralize reactive oxygen species generated by molecules such as H2O2 (Shigeoka et al., 2002). Vitamin C neutralizes potentially harmful reactions in the aqueous parts of the body, such as the blood and the fluid inside and surrounding cells (Khaw and Woodhouse, 1995). Vitamin C may help decrease total LDL cholesterol and triacylglycerol, as well as increase HDL levels. Vitamin C antioxidant activity may be helpful in the prevention of some cancers and cardiovascular diseases (Padayatty, 2003). It is found in high concentrations in ocular tissue. It is a potent antioxidant and prevents scurvy, a condition that causes ulceration of the gums, skin and mucous membranes. The antioxidants properties of vitamin C are thought to protect smokers, as well as people exposed to secondary smoking (passive smokers), from the harmful effects of free radicals (i.e. prevents the conversion of nitrates from tobacco smoke). As a powerful antioxidant, vitamin C may help to fight against cancer by protecting healthy cells from free-radical damage  and inhibiting the proliferation of cancerous cells (Bjelakovic, 2007). In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the antioxidant enzyme ascorbate peroxidase, a function that is particularly important in stress resistance in plant (Shigeokaet al., 2002). Foods containing the highest sources of vitamin C include green peppers, citrus fruit and juices, strawberries, tomatoes, pineapple, pawpaw, sweet and white potatoes, and cantaloupe (Jacob, 1996).



1.6.3        Vitamin D


Vitamin D is a fat soluble vitamin that is used by the body in the absorption of calcium which is essential for normal development and maintenance of healthy teeth and bones. It helps in maintaining adequate blood levels of calcium and phosphorus. It is also called the ‘sunshine vitamin’ because the body manufactures the vitamin after being exposed to sunshine. Vitamin D is found in the following foods: dairy products like cheese, butter, margarine, cream, fortified milk, fish, oysters and fortified cereals. Deficiency of vitamin D leads to osteoporosis in adults or rickets in children. Excessive doses of vitamin D can result in increased calcium absorption from the intestinal tract. This may cause increased calcium resorption from the bones, leading to elevated levels of calcium in the blood. Kidney stones, vomiting and muscle weakness may also occur due to the ingestion of too much vitamin D.



1.6.4 Vitamin E


Vitamin E is a fat-soluble antioxidant vitamin known to occur in the human body and it prevents free radical damage of biological membranes (Traber and Atkinson, 2007). Vitamin E is actually a generic term that refers to all entities that exhibit biological activity of the isomer α - tocopherol. The alpha-tocopherols are the most widely available isomer that have the highest bio-potency effect in the body (Schneider, 2005).


Vitamin E appears to be the first line of defence against peroxidation of polyunsaturated fatty acids contained in cellular and subcellular membrane phospholipids (Murray et al., 2003). The phospholipids of the mitochondria, endoplasmic reticulum and plasma membranes possess affinities for α–tocopherol, and the vitamin appears to concentrate at these sites. The tocopherol acts as antioxidants, breaking free-radical chain reactions as a result of their ability to transfer phenolic hydrogen to a peroxyl free radical of a per-oxidized polyunsaturated fatty acid. The phenoxy free radical formed may react with vitamin C to regenerate tocopherol or it reacts with a further peroxyl free radical so that the chromane ring and the side chain are oxidized to the non-free radical product (Murray et al., 2003).


Vitamin E is an antioxidant that helps to stabilize cell membranes and protect the tissues of the skin, eyes, liver, breast and testis, which are more sensitive to oxidation (Watty, 2000). It retards cellular aging of the eyes due to oxidation, it strengthens the capillary walls and supplies oxygen to the blood, which is then carried to the eyes (Watty, 2000). Vitamin E is a blood thinner, which should be used with caution in cases of exudative (wet) muscular degradation. Vitamin E is found in many common foods, including vegetable oils (such as soybean, corn, cotton seed and safflower) and products made from these oils (margarine),avocado, milk, egg, wheat germ, nuts and green leafy vegetable (Schneider, 2005).  Vitamin K


Vitamin K is a fat soluble vitamin that helps blood to clot and stop bleeding. Food sources of vitamin K include cabbage, cauliflower, spinach and other green leafy vegetables as well as cereals. Vitamin K is also made in the body by normal beneficial gastrointestinal bacteria. Deficiency problems of vitamin K are thin blood that does not adequately coagulate.



1.7              Antioxidant


Antioxidants are radical scavengers which protect the human body against free radicals (Poteract, 1997). A free radical is an atom or molecule that has one or more unpaired electron(s) and is capable of independent existence (Halliwell et al., 1995). The most biological significant free radicals are the reactive oxygen species (ROS) (Murray etal., 2000), which include hydroxyl radical (OH˚) and superoxide radical (O 2˚). ROS are formed due to various exogenous and endogenous factors such as exposure to radiation from the environment and the utilization of oxygen during aerobic respiration (Krishnaiah et al., 2007).


Imbalance in favour of the generation of reactive oxygen species against the activity of the antioxidant defences leads to a pathophysiological condition known as oxidative stress. Oxidative stress is defined, in general, as excess formation and/or insufficient removal of highly reactive molecules such as ROS (Johansen et al., 2005). Oxidative stress is associated with a lot  of diseases such as cancer, atherosclerosis, diabetes, rheumatoid arthritis, Parkinson’s disease, malaria and HIV/AIDS (Aruoma, 1993).





Minerals of biological importance are classified into macro and micro (trace) elements.


Macro minerals are those that are required by the system in large amounts while micro (trace) minerals are required in minute quantities. Macro minerals include calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K) while micro minerals include iron (Fe), copper (Cu), zinc (Zn), iodine (I), chromium (Cr), selenium (Se) and manganese (Mn) (Chaney, 2002).


These minerals play very important roles in physiological activities.



1.8.1 Calcium (Ca)


Calcium is essential for living organisms in particular in cell physiology. A 70kg normal adult human body has about 1200g of calcium which amounts to about 1–2% of body weight. About 99% of it is found in mineralized tissues such as bones and teeth. The remaining 1% is found in the blood extra- cellular fluid, muscles and other tissues. In food, calcium occurs as salt or it gets associated with other dietary constituents in the form of complexes of calcium ions. Calcium must be released in a soluble and ionized form before it can be absorbed. Absorption occurs basically in the intestine (Girventet al., 2005).  Metabolic functions and deficiency symptoms of calcium


Calcium  is  required  for  normal  growth  and  development  of  the  skeleton.  Adequate


calcium intake is critical to achieving optimal peak bone mass (PBM) and modifies the rate of bone loss associated with aging (Girventet al., 2005). Calcium mediates some hormonal responses and is required by many enzymes as co-factor. Muscle contractility and normal neuromuscular activity and irritability require the presence of calcium (Chaney, 2002).


Calcium deficiency results in muscle cramp and osteoporosis. Chronic inadequate intake or poor intestinal absorption of calcium is suspected to play some role in the aetiologies of hypertension and colon cancer (Girventet al., 2005).


1.8.2 Magnesium (Mg)


Magnesium, another abundant mineral in the body is essential for healthy functions of the system. Total magnesium (50-60%) is found in bone while the other half, is found within body tissues and organs. About 1% is found in the blood (Rude, 1998; Girventet al., 2005).  Metabolic functions and deficiency symptoms of magnesium


Magnesium is required for several enzyme activities particularly those involving ATP synthesizing as ATP–Mg 2+ complex; and for neuromuscular transmission (Chaney, 2002). It also enhances the condensation of chromatin.


Magnesium deficiency does not appear to be a problem in healthy individuals since its homeostasis can be maintained by a wide range of intakes. Its deficiency is only seen as a secondary complication of a primary disease state as in cardiovascular and neuromuscular mal-functions, endocrine disorders and muscle wasting (Girventet al., 2005).



1.8.3 Zinc (Zn)


Zinc is a ubiquitous mineral in the body. It is the most abundant intracellular trace element. About 2g of zinc is found in adults with 60% and 30% are present in muscles and bones respectively. It is absorbed from the small intestine and transported in the plasma by albumin and a 2–macroglobulin (Girvent et al., 2005).  Metabolic functions and deficiency symptoms of zinc


Zinc functions as a co-factor. Over 300 zinc metalloenzymes that have been described to date include a number of regulatory proteins and both RNA and DNA polymerases (Chaney, 2002). The structural functions are found in the zinc finger motif in proteins. Zinc is required by protein kinases that participate in signal transduction processes (Girventet al., 2005).


Zinc deficiency in children is usually marked by poor growth and impairment of sexual development (Chaney, 2002). Poor wound healing results from zinc deficiency in both adults and children. Other malfunctions resulting from zinc deficiency include decreased taste sense and impaired immune function (Girvent et al., 2005).

1.8.4        Iron (Fe)


The iron content of a typical 70kg adult man is approximately 4–5g. About two–thirds of this is utilized as functional iron such as haemoglobin, myoglobin and other haem (cytochromes and catalase) and non-haem (NADH dehydrogenase) enzymes. Others are stored as ferritin and hemosiderin (Girvent et al., 2005).


Iron from food is absorbed mainly in the duodenum by an active process that transports iron from the gut lumen into the mucosal cell. When required by the body for metabolic processes, iron passes directly through the mucosal cell into the blood stream where it is transported by transferrin, together with the iron released from old blood cells to the bone marrow and other tissues. Iron absorbed in excess is stored in the liver, spleen or bone marrow. It is usually released from these stores for utilization in times of high need, such as during pregnancy (Girventet al., 2005).  Metabolic functions and deficiency symptoms of iron


Iron present in haemoglobin and myoglobin is required for transport of oxygen during cellular respiration and storage in muscles. Being part of the tissue enzymes makes it critical for energy production. It also plays a role in the functioning of the immune system (Girvent et al., 2005).


A major deficiency symptom of iron is anaemia. This results from insufficient haemoglobin for the production of new erythrocytes. This is most common in infants, preschool children, adolescents and women of child–bearing ag e particularly in developing countries (Chaney, 2002).



1.8.5 Copper (Cu)


Copper is a micronutrient present in a number of important metallo enzymes including cytochrome C oxidase, dopamine-β-hydroxylase and superoxide dismutase (Chaney, 2002).


About 50–75% dietary copper is absorbed mostly thr ough the intestinal mucosa from a typical diet. The absorption of copper is primarily influenced by the amount ingested; increased ingestion leads to decreased absorption (Chaney, 2002). Other factors that influence the absorption of copper or that affect its bioavailability include the antagonistic effects of zinc, iron, ascorbic acid, sucrose and fructose (Girvent et al., 2005).  Metabolic functions and deficiency symptoms of copper


As a component of several enzymes, co-factors and proteins, it is essential for important bioactivities. It is required for proper functioning of the immune, nervous and cardiovascular systems. It plays a role in iron metabolism and formation of erythrocytes. It also functions as an electron transfer intermediate in redox reactions (Girventet al., 2005).


This is relatively rare in humans and animals on typical, varied diets. Most features of severe copper deficiency can be explained by a failure of one or more of the copper-dependent enzymes like superoxide dismutase, lysyl oxidase, tyrosinase, e.t.c. For instance, lysyl oxidase plays one of the most important and best understood roles of copper in the body (Girvent et al., 2005). This is the main enzyme involved in cross- linking of connective tissues. Optimal functioning of lysyl oxidase ensures the proper cross-linking of collagen and elastin, vital for the strength and flexibility of our connective tissue. A reduction in lysyl oxidase activity affects the integrity of numerous tissue including the skin, bones and blood vessels. Not surprising, some of the hallmarks of copper deficiency are connective tissue disorders, osteoporosis and blood vessel damage (Chaney, 2002).



1.9                Blood glucose


Glucose transported through the blood stream from the intestines to other tissues and organs is the primary source of energy for the body’s cells (Spiller, 1992). Blood sugar concentration or glucose level is tightly regulated in the human body. Normal blood glucose level is maintained between 4 and 6mM. Normal blood glucose concentration (homeostasis) is about 90mg/100ml; which works out to 5mM/L as the molecular weight of glucose. The normal total amount of glucose in circulating blood is therefore about 3.3 to 7.0g (Henry, 2001). Glucose concentration rises after meal for an hour or two and is usually lowest in the morning, before the first meal of the day. Failure to maintain blood glucose in the normal range leads to conditions of persistently high (hyperglycaemia) or low (hypoglycaemia) blood sugar. Although it is called ‘blood sugar’, other simple sugars such as fructose and galactose aside from glucose are found in the blood. Only glucose concentrations are used as metabolic regulation signals (Sacher and Mcpherson, 2001). Despite the long intervals between meals and the occasional consumption of meals with a substantial carbohydrate load, human blood glucose concentrations normally remain within a remarkable narrow range. In most humans, this varies from about 80mg/dl to perhaps 120mg/dl (3.9 to 6.0mml/litre) except shortly after eating when the blood glucose concentration rises temporarily. In a healthy adult male of 75kg body weight with a blood volume of 5litres, a blood glucose level of 100mg/dl or 5.5mmol/litre corresponds to about 5g in the total body water (Henry, 2001).



1.9.1 Blood glucose regulation


The homeostatic mechanism which keeps the blood value of glucose in a remarkably narrow range is composed of several interacting systems, of which hormone regulations is the most important. There are two types of mutually antagonistic metabolic hormones affecting blood glucose levels: catabolic hormones such as glucagon, growth hormone (e.g. pituitary hormone), glucocorticoids(e.g. cortisol) and catecholamines (e.g. norepinephrine, epinephrine,dopamine) which increase blood glucose; anabolic hormone (insulin), which decreases blood glucose.


The human body maintains blood glucose in a very narrow range. Insulin and glucagon are the hormones which make this possible(John and Harry, 2001). Both insulin and glucagon are secreted from the pancreas, and thus are referred to as pancreatic endocrine hormones. It is the production of insulin and glucagon by the pancreas which ultimately determines if a patient has diabetes, hypoglycemia, or some other forms of sugar problems (John and Harry, 2001).


Insulin is normally secreted by the beta cells (a type of islet cells) of the pancreas. The stimulus for insulin secretion is high blood glucose. Although there is always a low level of insulin secreted by the pancreas, the amount secreted into the blood increases as the blood glucose rises. Similarly, as blood glucose falls, the amount of insulin secreted by the pancreatic islets goes down. Insulin has an effect on a number of cells, including muscle, red blood cells, and fat cells. In response to insulin, these cells absorb glucose out of the blood, having the net effect of lowering the high blood glucose levels the normal range (John and Harry, 2001).


Glucagon is secreted by the alpha cells of the pancreatic islets in much the same manner as insulin except in the opposite fashion. If blood glucose is high, then no glucagon is secreted. When blood glucose goes low, however, (such as between meals and during exercise), more and more glucagon is secreted. The effect of glucagon is to make the liver release the glucose it has stored in its cells into the blood stream, with the net effect of increasing blood glucose.


1.10          Lipids


Lipids  constitute  a  group  of  naturally  occurring  molecules  that  include  fats,  waxes, sterols, fat soluble vitamins (such as vitamins A, D, E and K), monoacylglycerol, diacylglycerol, triacylglycerol, phospholipids and others (Fahy et al., 2009). The main biological function of lipids includes energy storage, signaling and acting as structural components of cell membranes (Fahy et al., 2009). Lipids have found application in cosmetic and food industries as well as in nanotechnology (Mashaghi et al., 2013).


Lipids may be broadly defined as hydrophobic or amphiphilic small molecules, the amphiphilic nature of some lipids allow them to form structures such as vesicles, liposomes or membranes in an aqueous environment. Biological lipids originate entirely or in part from two distinct types of biochemical subunits or “building blocks”: ketoacyl and isoprene groups (Fahy et al., 2009). Although the term lipids is sometimes used as alternative for fats, fats are a group of lipids called triacylglycerol. Lipids also encompass molecules such as fatty acids and their derivatives as well as other sterol containing metabolites such as cholesterol. Although humans and other mammals use various biosynthetic pathways to breakdown and synthesize lipids, some essential lipids cannot be made this way and must be obtained from the diet (Fahy et al., 2009).



1.10.1    Lipoproteins: Types and Functions


Lipoproteins consist of a non polar core and a single surface layer of amphipathic lipids. The non polar core consists of mainly triacylglycerol and cholesteryl ester and is surrounded by a single surface layer of amphipathic phospholipid and cholesterol molecules. These are oriented so that their polar groups face outwards to the aqueous medium, as in the cell membrane. The protein moiety of a lipoprotein is known as apolipoprotein or apoprotein, constituting nearly 70% of some HDL as little as 1% of chylomicrons (Murray etal., 2008).


Because fat is less dense than water, the density of a lipoprotein decreases as the proportion of lipid to protein increases. In addition to FFA, four major groups of lipoproteins have been identified that are important physiologically and in clinical diagnosis. These include:


Chylomicrons, derived from intestinal absorption of triacylglycerol and other lipids;


Very low density lipoproteins (VLDL, or pre- β - lipoproteins), derived from the liver for the export of triacylglycerol;

 Low-density lipoproteins (LDL, or β -lipoproteins), representing a final stage in the catabolism of VLDL; and


High- density lipoproteins (HDL, or α- lipoprotein), involved in VLDL and chylomicron metabolism and also in cholesterol transport.


Triacylglycerol is the predominant lipid in chylomicrons and VLDL, whereas cholesterol and phospholipids are the predominant lipids in LDL and HDL, respectively. Lipoproteins may be separated according to their electrophoretic properties into α-,β-, and pre- β- lipoproteins.                      Chylomicrons


Chylomicrons in connection with the movement of dietary triacylglycerols from the intestine to other tissues are the largest of the lipoproteins and the least dense, containing a high proportion of triacylglycerol. Chylomicrons are synthesized in the endoplasmic recticulum of epithelial cells that line the small intestine, then move through the lymphatic system and enter the bloodstream via the left subclavian vein (Nelson and Cox, 2005).


Larger particles are catabolized more quickly than smaller ones. Fatty acids originating from chylomicron triacylglycerol are delivered mainly to the adipose tissue, heart and muscle (80%), while about 20% goes to the liver (Murray etal., 2008). However, the liver does not metabolize native chylomicrons or VLDL significantly; thus, the fatty acid in the liver must be secondary to their metabolism in extrahepatic tissues (Murray etal., 2008).


The apoproteins of chylomicrons include apo B-48(unique to this class of lipoproteins), apoE, and apoC-II. ApoC-II activates lipoprotein lipase in the capillaries of adipose, heart, skeletal muscle, and lactating mammary tissues, allowing the release of free fatty acids to these tissues. Chylomicrons thus carry dietary fatty acids to tissues where they will be consumed or stored as fuel. The remnant of chylomicrons (depleted of most of their triacylglycerols but still containing cholesterol, apoE, and apoB-48) move through the bloodstream to the liver. Receptors in the liver bind to the apoE in the chylomicron remnants and mediate their uptake by endocytosis. In the liver, the remnants release their cholesterol and are degraded in lysosomes (Murray etal., 2008).                      Very Low Density Lipoprotein (VLDL)


When diets contain more fatty acids than are needed immediately as fuel, they are converted to triacylglycerol in the liver and packaged with specific apolipoproteins into very-low-density-lipoprotein (VLDL). Excess carbohydrates in the diet can also be converted to triacylglycerols in the liver and exported as VLDLs (Nelson and Cox, 2005).


In addition to triacylglycerols, VLDLs contain some cholesterol and cholesteryl esters, as well as apoB-100, apoC-I, apoC-II, apoC-III and apo-E.These lipoproteins are transported in the blood from the liver to muscle and adipose tissue, where activation of lipoprotein lipase by apoC-II causes the release of free fatty acids from the VLDL triacylglycerols. Adipocytes take up these fatty acids, reconvert them to triacylglycerols and store the products in intracellular lipid droplets; mycocytes in contrast, primarily oxidize the fatty acids to supply energy. Most VLDL remnants are removed from the circulation by hepatocytes. The uptake, like that for chylomicrons, is receptor-mediated and depends on the presence of apoE in the VLDL remnants. The loss of triacylglycerol converts some VLDL to VLDL remnants (also called intermediate density lipoprotein, IDL) (Nelson and Cox, 2005).                      Low Density Lipoprotein (LDL)                Metabolism of low density lipoprotein via LDL receptor


The liver and many extrahepatic tissues express the LDL (B-100, E) receptor. It is so designated because it is specific for apoB-100 but not B-48, which lacks the carboxyl terminal domain of B-100 containing the LDL receptor ligand, and it also takes up lipoproteins rich in apoE. This receptor is defective in familial hypocholesterolemia. Approximately 30% of LDL is degraded in extrahepatic tissues and 70% in the liver. A positive correlation exists between the incidence of coronary atherosclerosis and the plasma concentration of LDL cholesterol (Murray et al., 2008).                Regulation of LDL receptor


Low density lipoprotein (LDL) receptor is highly regulated. LDL (apo B-100,E) receptors occur on the cell surface in the pits that are coated on the cytosolic side of the cell membrane with a protein called clathrin. The glycoprotein receptor spans the membrane the B-100 binding region being at the exposed amino terminal end. After binding, LDL is taken up intact by endocytosis. The apoprotein and cholesteryl esters are then hydrolysed in the lysosome and cholesterol is translocated into the cell. The receptors are recycled to the cell surface. This influx of cholesterol inhibits in a co-ordinated manner HMG-CoA synthase, HMG CoA reductase and therefore cholesterol synthesis; stimulates ACAT activity and down-regulates synthesis of LDL receptor. Thus, the number of LDL receptors on the cell surface is regulated by the cholesterol requirement for membranes, steroid hormones, or bile acid synthesis. The apo B-100, E receptor is a ‘high affinity’ LDL receptor, which may be saturated under most circumstances. Other ‘low- affinity’ LDL receptors also appear to be present in addition to a scavenger pathway, which is not regulated (Murray etal., 2008).


In Western countries, the total plasma cholesterol in humans is about 5.2mmol/L, rising with age, though there are wide variations between individuals. The greater part is found in the esterified form. It is transported in lipoprotein of the plasma and the highest proportion of cholesterol is found in the LDL. Dietary cholesterol equilibrates with the plasma cholesterol in days and with tissue cholesterol in weeks. Cholesteryl esters in the diet are hydrolysed to cholesterol, which is then absorbed by the intestine together with dietary unesterified cholesterol and other lipids. With cholesterol synthesized in the intestines,it is then incorporated into chylomicrons. Of the cholesterol absorbed, 80-90% is esterified with long-chain fatty acids in the intestinal mucosa. Ninety-five percent of the chylomicron cholesterol is delivered to the liver in chylomicron remnants, and most of the cholesterol secreted by the liver in VLDL is retained during the formation of LDL and ultimately LDL, which is taken up by the LDL receptor in liver and extrahepatic tissues (Murray etal., 2008).


Further removal of triacylglycerol from VLDL produces low density lipoprotein (LDL). Very rich in cholesterol and cholesteryl esters and containing apoB-100 as their major apolipoprotein, LDLs carry cholesterol to extrahepatic tissues that have specific plasma membrane receptors that recognize apoB-100. These receptors mediate the uptake of cholesterol and cholesteryl esters (Nelson and Cox, 2005).          High Density Lipoprotein (HDL)


The fourth major lipoprotein type, high-density lipoprotein (HDL), originates in the liver and small intestine as small, protein-rich particles that contain relatively little cholesterol and no cholesteryl esters. HDLs contain apoA-I, apoC-I, apoC-II, and other apolipoproteins, as well as the enzyme lecithin-cholesterol acyl transferase (LCAT), which catalyses the formation of cholesteryl esters from lecithin (phosphatidyl choline) and cholesterol. LCAT on the surface of nascent (newly forming) HDL particles converts the cholesterol and phosphatidyl choline of chylomicron and VLDL remnants to cholesteryl esters, which begin to form a core, transforming the disk-shaped nascent HDL to a mature, spherical HDL particle. This cholesterol- rich lipoprotein then returns to the liver, where the cholesterol is unloaded, some of this cholesterol is converted to bile salts (Nelson and Cox, 2005).


HDL may be taken up in the liver by receptor mediated endocytosis, but at least some of the cholesterol in HDL is delivered to other tissues by a novel mechanism.HDL can bind to plasma membrane receptor proteins called SR-B1 in hepatic and steroidogenic tissues such as the adrenal gland. This receptor mediates not only endocytosis but also partial and selective transfer of cholesterol and other lipids in HDL into the cell (Nelson and Cox, 2005).


Depleted HDL then dissociates to recirculate in the bloodstream and extract more lipids from chylomicron and VLDL remnant. Depleted HDL can also pick up cholesterol stored in extrahepatic tissues and carry it to the liver, in reverse cholesterol transport pathways. In one reverse transport path, interaction of nascent HDL with SR-B1 receptors in cholesterol-rich cells triggers passive movement of cholesterol from the cell surface into HDL, which then carries it back to the liver. In a second pathway, apoA-I in depleted HDL interacts with an active transporter, the ABC1 protein, in a cholesterol- rich cell. The apoA-1(and presumably the HDL) is taken up by endocytosis, then resecreted with a load of cholesterol, which it transports to the liver (Nelson and Cox, 2005).


The ABC1 protein is a member of a large family of multidrug transporters, sometimes called ABC transporters, because they all have ATP- binding cassettes; they also have two transmembrane domains with six transmembrane helices. These proteins actively transport a variety of ions, amino acids, vitamins, steroid hormones and bile salt across plasma membranes. The CFTR protein that is defective in cystic fibrosis is another member of this ABC family of multidrug transporters (Nelson and Cox, 2005).



1.11          Total cholesterol and cholesterol balance in tissues


Cholesterol is a lipid that is made in the liver from fatty foods.  It  is found in cell membranes of all tissues and is transported in blood plasma of all animals. Cholesterol is also considered a sterol (Stryer, 1995). Most of the cholesterol in the body is synthesized by the body and some have dietary origin. Cholesterol is more abundant in tissues which either synthesize more or have more abundant densely packed membranes, for example, the liver, spinal cord and brain. It plays a central role in many biochemical processes such as the composition of cell membranes and the synthesis of steroid hormones (Smith, 1991). Since cholesterol is insoluble in blood, it is transported in the circulatory system within lipoproteins, complex spherical particles which have an exterior composed mainly of water, soluble proteins; fats and cholesterol are carried internally (Stryer, 1995). Cholesterol is required to build and maintain cell membranes; it regulates membrane fluidity over a wide range of temperature. Some research indicates that cholesterol also aid in the manufacture of bile and is also important for the metabolism of fat soluble vitamins and of the various steroid hormones (Haines, 2001). Conditions with elevated concentrations of oxidized LDL particles are associated with atheroma formation in the walls of arteries, a condition known as atherosclerosis, which is the principle cause of coronary heart disease and other forms of cardiovascular diseases. Abnormally low levels of cholesterol are termed hypocholesterolemia. Research into the cause of this state is relatively limited but some studies suggest a link with depression, cancer and cerebral haemorrhage. It is unclear whether the low cholesterol concentrations causes for these conditions or something which occurs along side them (Shepherd et al., 1995). Normal values for serum cholesterol are 3.6 or 5.0 – 6.5mmol/l or 120 or 140 – 200 or 250mg/dl (Deepak et al., 2007).


In tissues, cholesterol balance is regulated as follows: cell cholesterol increase is due to uptake cholesterol- containing lipoproteins by receptors e.g. the LDL receptor or the scavenger receptor; uptake of free cholesterol from cholesterol-rich lipoproteins to the cell membrane; cholesterol synthesis, and the hydrolysis of cholesteryl esters by the enzyme cholesteryl ester hydrolase. Decrease is due to the efflux of cholesterol from the membrane to HDL, promoted by LCAT (lecithin cholesterol acyltransferase); esterification of cholesterol by ACAT (acyl coA: cholesterol acyltransferase); and utilization of cholesterol for synthesis of other steroids, such as hormones or bile acids in the liver (Illingworth, 2000).


Fig. 4: Structure of cholesterol (Murray et al., 2008)



1.11.1 Diet and cholesterol regulation


Hereditary factors play important roles in determining individual serum cholesterol concentrations; however, dietary and environmental factors may also play some parts, and the most beneficial of these is the substitution in the diet of polyunsaturated and monounsaturated fatty acids for saturated fatty acids. Plant oil such as corn oil and sunflower seed oil contain a high proportion of polyunsaturated fatty acids, while olive oil contains a high concentration of monounsaturated fatty acids. On the other hand, butter fat and beef fat contain a high proportion of saturated fatty acids. Sucrose and fructose have a greater effect in raising blood lipids, particularly triacylglycerol, than do other carbohydrates (Murray etal., 2008).


The reason for the cholesterol-lowering effect of polyunsaturated fatty acids is still not fully understood. It is clear, however, that one of the mechanisms involved in the up-regulation of LDL receptors by poly and monounsaturated as compared with saturated fatty acids, causing an increase in the catabolic rate of LDL, the main atherogenic lipoprotein. In addition, saturated fatty acids cause the formation of smaller VLDL particles that contain relatively more cholesterol, and they are utilized by extra hepatic tissues at a slower rate than are larger particles-tendencies that may be regarded as atherogenic (Ness and Chambers, 2000).





Liver function tests are groups of clinical blood assays designed to give information about the state of a patient’s liver. The tests specifically detect the levels of some liver enzymes which leak into the blood stream in the event of a damage. Some of these enzymes include – alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP).



1.12.1 Alanine aminotransferase


Alanine aminotransferase (ALT), formerly called serum glutamate-pyruvate transminase (SGPT), catalyses the transfer of α-amino group from alanine to α-keto-glutarate with the release of pyruvate and glutamate.


Alanine aminotransferase can also be found in several tissues throughout the body, but the concentrations in the liver are considerably higher than elsewhere (Murray et al., 2003). At physiologic pH, the reaction is energetically favoured towards the formation of L– alanine and a -oxoglutarate.In vivo, the reaction goes to the right to provide a source of nitrogen for the urea cycle. The glutamate thus produced is deaminated by glutamate deydrogenase resulting in ammonia and regeneration of a -oxoglutarate (a -ketoglutarate) whereas, the pyruvate thus generated is available for entry into the citric acid cycle. The reaction is reversible; the chemical equilibrium favours the formation of alanine and a -oxoglutarate (Murray et al., 2003).

Alanine aminotransferase is found in high concentrations in the hepatocytes, and in much smaller concentrations in other tissues such as kidney, heart, skeletal muscle, spleen and serum. There is more than one form of alanine aminotransferase in the body. The mitochondrial form is low in concentration and very unstable as compared to the cytosolic form. The different electrophoretic components have been identified. They include alanine glutamate transaminase and alanine pyruvate transaminase. The former is very specific for alanine and glutamic acid, whereas the latter is non-specific and has alanine and pyruvate as principal substrates and can also act on other amino acids though at a very low rate (Murray et al., 2003).



1.12.2    Aspartate aminotransferase


Aspartate   aminotransferase   (AST),   formerly   known   as   glutamate-oxaloacetate transaminase (GOT) or serum glutamate–oxaloacetate transaminase (SGOT), catalyses the transfer of the α-amino group from aspartate to α-ketoglutarate with the release of oxaloacetate and glutamate.


Aspartate aminotransferase is located in the cytosol and mitochondria of the liver cells. There are individual iso–enzymes, and the main seru m component is from the cytosolic fraction. This enzyme is also located in the cardiac muscle, skeletal muscle, brain, kidney, pancreas, erythrocytes and serum. The hepatic mitochondrial cytosolic AST isoenzymes are genetically distinct and different in their amino acid composition, kinetic behaviour, electrophoretic mobility and immunochemical properties. Isoelectric focusing shows that mitochondrial isoenzymes from human liver exist in a single form whereas the cytoplasmic isoenzymes have at least three sub-forms with similar immunochemical behaviour (Nelson and Cox, 2000).


1.12.3    Alkaline Phosphatase


Alkaline  phosphatase  is  the  name  given  to  a  group  of  enzymes  that  catalyse  the hydrolysis of phosphate esters in alkaline pH. This enzyme is widely distributed in human tissues, including liver, bone, placenta, intestine, kidney and leukocytes. In the liver, the enzyme is mainly bound to canalicular membranes (Nelson and Cox, 2000).


Liver and bone isoenzymes are the major fractions of the serum alkaline phosphatase in healthy adults. In children and adolescents, where bone growth is active, the serum alkaline phosphatase may increase up to three fold and the boneisoenzymes become the major fraction. The placenta isoenzyme is prominent in pregnant women, particularly during the third trimester. An intestinal component is often present in Lewis antigen secretors of blood groups O and B, particularly after ingesting a fatty meal.


Although the prime metabolic function of the enzymes is not yet understood, the enzyme is closely associated with the calcification process in bones. Alkaline phosphatase displays considerable inter ad intra–tissue heterogeneity, b ut there are rarely more than two or three forms in any one serum specimen.


The isoenzymes of alkaline phosphatase exhibit optimal activity invitro at a pH of about 10, although the optimal pH varies with the nature and concentration of the substrate acted upon, the type of buffer or phosphate acceptor present, and to some extent, the nature of the isoenzymes. Alkaline phosphatase acts on a large variety of naturally occurring synthetic substrates but the natural substrates on which they act in the body are not known.


Some divalent ions such as Mg(II), Co(II) and Mn(II) are activators of the enzyme and Zn(II) is a constituent metal ion. The correct ratio of Mg(II)/Zn(II) ion is necessary to obtain optimal activity (Nelson and Cox, 2000).



1.12.4    Clinical and Diagnostic Significance of Liver Function Enzymes


Analysis of some enzyme activities in blood serum gives valuable diagnostic informationfor a number of disease conditions. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important in the diagnosis of heart and liver damage caused by heart attack, drug toxicity or infection. After a heart attack, a variety of enzymes, including these aminotransferases, leak from the injured heart cells into the blood stream. Measurements of the blood serum concentration of the two aminotransferases and alkaline phosphatase by SGPT, SGOT and alkaline phosphatase tests and of another enzyme, creatine kinase and is the first heart enzyme to appear in the blood after a heart attack; it also disappears quickly from blood. AST is the next to appear and ALT follows later.


The AST and ALT tests are also important in industrial medicine, to determine whether people exposed to carbon tetrachloride, chloroform, or other industrial solvents have suffered liver damage. Aminotransferases are most useful in the monitoring of people exposed to these chemicals because they are very active in liver and their activity can be detected in very small amounts (Nelson and Cox, 2000).



1.12.5    Bilirubin


Bilirubin is a product of red cell breakdown in the liver, spleen, and bone marrow. A small amount is produced form the breakdown of haem-containing proteins such as myoglobin (oxygen-transporting muscle protein), and the enzymes catalase, cytochromes, and peroxidases.


The haem (iron porphyrin part is converted to biliverdin which is then reduced to bilirubin. This bilirubin is referred to as unconjugated (indirect) bilirubin. It is not soluble in the blood to the liver. In the liver cells, the enzyme glucuronosyl-transferase joins (conjugates) glucuronic acid to bilirubin forming bilirubin glucuronides (mainly diglucuronide). This bilirubin is known as conjugated (direct) bilirubin. It is water-soluble and non-toxic. Conjugated or direct bilirubin refers to bilirubin which has been conjugated in the liver to form water-soluble mono-and diglucuronides of bilirubin, in certain forms of jaundice (not haemolytic) it can be found in urine. Conjugated bilirubin passes into the bile canaliculi, through the bile duct, and into the intestine. In the terminal ileum and colon, the conjugated bilirubin is reduced by bacteria to various pigments and colourlesschromogens (urobilinogen), most of which are excreted in the faeces. One of the urobilinogenchromogens excreted in the faeces is stercobilinogen (Cheesborough, 1987).


Some of the urobilinogen from the intestine is absorbed into the portal circulation and reaches the liver. Where it re- enters the intestine in the bile and is excreted in the faeces. A small amount of this reabsorbed urobilinogen is carried in the blood through the liver and transported to the kidneys where it is excreted in the urine. Urobilinogen is rapidly oxidized to the coloured pigment urobilin (stercobilinogen to stercobilin).


The normal concentration of total bilirubin (unconjugate and conjugated) in the blood of an adult is usually 3-17 µmol/L (0.2-0.9mg%). When the plasma bilirubin reaches around 34 µmol/ L (2 mg%) a person will become jaundiced, wit h the skin and particularly the white part of eyes appearing yellow-coloured. (Cheesborough, 1987).


In haemolytic (prehepatic) jaundice, more bilirubin is produced than the liver can metabolise e.g. in severe haemolysis. The excess bilirubin which builds up in the plasma is mostly of the unconjugated types and is therefore not found in the urine.


In hepatocellular (hepatic) jaundice, there is a build up of bilirubin in the plasma because it is not transported, conjugated or excreted by the liver cells since they are damaged e.g. in viral hepatitis. The excess bilirubin is usually of both the unconjugated and conjugated types with bilirubin being found in the urine.


In obstructive (posthepatic) jaundice, bilirubin builds up in the plasma because it is obstructed in the small bile channels or in the main bile duct. This can be caused by gall stones or a tumour obstructing or closing the biliary tract. The excess bilirubin is mostly of the conjugated type and is therefore found in the urine.



1.12.6    Serum protein


Blood proteins also called serum protein, are found in blood plasma. Serum total protein in blood is 7 g/dl, which makes 7% of total body weight (Anderson and Anderson, 1977). They serve many different functions including circulating transport molecules for lipids, hormones, vitamins and minerals, enzymes, complement components and protease inhibitors, and in regulation of cellular activity and functioning and in the immune system. About 60% of plasma proteins are made up of the protein, albumin which is a major constituent to osmotic pressure of plasma assists in the transport of lipids and steroid hormones. Globulins make up 35% of plasma proteins and are used in the transport of ions, hormones and lipids assisting in immune function; 40% is fibrinogen and this is essential in the clotting of blood and can be converted to insoluble fibrins (Adkins etal., 2002). A total serum protein test measures the total amount of protein in the blood. It also measures the amounts of the two major groups of proteins in the blood; albumin and globulin (Fischbach and Dunning, 2004). Normally, there is little more albumin than globulin and the ratio is greater than 1.A ratio less than 1or much greater than 1 can give clue about problems in the body (Pagana and Pagana, 2002).



1.12.7    Serum albumin


Human serum albumin is the most abundant protein in human blood plasma. It is produced in the liver. Albumin comprises about half of the blood protein. The reference range for albumin concentration in blood is 3.0-5.5 g/dl(Pagana and Pagana, 2002). It has a serum half life of approximately twenty days. It has a molecular mass of 67 KDa (Mohamadi-Nejadet al., 2002). Albumin transports thyroids hormones and other hormones particularly fat soluble hormones, unconjugated bilirubin and many drugs to the liver and other important organs. Low blood albumin concentrations (hypoalbuminaemia) can be caused by liver disease/cirrhosis of the liver, decreased production (as in starvation/malnutrition/malabsorbtion), excess excretion by the kidney, excess loss in bowel, burns, redistribution, acute disease states, and mutation causing analbuminaemia. Hyper albuminaemia typically is a sign of severe dehydration.





1.13.1 Blood urea nitrogen (BUN)


Urea is a waste product of the liver and part of the urea cycle. Urea is removed from the blood by the kidneys. Urea clearance is similar to creatinine clearance but urea is both filtered and reabsorbed and urea levels vary with the state of hydration and diet. Urea clearance is therefore less than glomerular filtration rate (GFR), if protein intake and metabolism are constant. However, plasma levels increase as the GFR declines. If there is no tubular adaptation, urea levels change because urea is primarily excreted by glomerular filtration. BUN levels are measured by chemical colorimetric method.


The concentration of urea nitrogen in the blood reflects glomerular filtration and urine-concentrating capacity. Urea is filtered at the glomerulus and as a result, BUN levels increase as glomerular filtration drops. BUN rises in states of dehydration and acute and chronic renal failure when passage of fluids through the tubules is slowed, because urea is reabsorbed by the blood through the permeable tubules. BUN also varies as a result of changed protein intake and protein catabolism and therefore is a poor measure of GFR. BUN is used for the detection of chronic kidney injury, as BUN levels do not change until there is extensive renal damage.


Increases are usually caused by excessive protein intake, kidney damage, certain drugs, low fluid intake, intestinal bleeding. Decreased levels may be due to a poor diet, malabsorption, liver damage or low nitrogen intake (Girventet al., 2005).



1.13.2    Creatinine


This is basically the waste product of muscle metabolism. Its level is a reflection of body muscle mass. Low levels are sometimes seen in kidney damage, protein starvation, liver diseases or pregnancy. Elevated levels are seen in kidney diseases (since kidney is involved in its excretion), muscle degeneration and some drugs involved in impairment of kidney function (Jaeger and Hedegaard, 2002).


Creatinine is a break-down product of creatine phosphate, which is used as an energy resource in the muscles. Creatinine is produced by the muscles and excreted into the blood at a relatively constant rate. Creatinine is commonly used in the clinic to determine glomerular filtration rate (GFR) in a patient.


GFR is a measurement of the functioning of the glomerulus. Creatinine is freely filtered at the glomerulus. Small amounts are secreted by the tubules, which leads to a small but acceptable overestimation of GFR. These qualities make blood creatinine levels a good measure of GFR. When the body is in steady state, the amount produced by the body approximates the amount filtered and excreted in the kidneys. The plasma concentration of creatinine changes until the amount excreted again equals the production if either the rate of production or the GFR changes. Therefore, if GFR levels decline (e.g. chronic renal failure), the plasma creatinine level increases by a reciprocal amount. The plasma levels continue to increase as the GFR decreases, because no significant tubular adjustment occurs for creatinine. This relationship between creatinine blood concentration and renal excretion of creatinine allows plasma creatinine concentration to serve as an estimate of changing glomerular function.



1.14            Lipid Peroxidation


Lipid peroxidation is a major form of oxidative stress. It is the oxidative deterioration of unsaturated lipids containing methylene-interrupted double bonds. Lipid peroxidation is a source of free radicals. In the presence of the free radical like the hydroxyl radicals, lipids undergo peroxidation. Hydroxyl radicals are capable of initiating lipid peroxidation by abstracting hydrogen atom from fatty acid side chain (Kanner et al., 1997). Lipid peroxidation involves the direct reaction of lipids with free radical intermediates and semi stable peroxides. Peroxidation (auto-oxidation) of lipids exposed to oxygen is responsible not only for deterioration of food (rancidity) but also for damage to tissues in vivo, where it may be a cause of cancern inflammatory diseases,atherosclerosis and ageing (Murray et al., 2003). The deleterious effects are considered to be caused by free radicals (ROO; RO;OH) produced during peroxide formation from fatty acids containing methylene interrupted double bonds i.e. those found in the naturally occurring polyunsaturated fatty acids. Lipid peroxidation can be said to be the oxidative degradation of lipids. It is the process whereby free radicals “steal”’ electrons from the lipids in cell membranes (Halliwell etal., 1999), resulting in cell damage. This proceeds by a free radical chain reaction mechanism. Most often it affects polyunsaturated fatty acids, because they contain multiple double bonds which lie between methylenes (CH2-) groups they possess especially, reactive hydrogen. As with any radical reaction, lipid peroxidation is a chain reaction providing a continuous supply of free radicals that initiate further peroxidation (Kanner etal., 1997). The reaction consists of three major steps: initiation, propagation and termination.


1.14.1    Initiation


Initiation is the step whereby a fatty acid radical is produced. The initiators in living cells are most notable ROS, such as OH, which combines with a Hydrogen atom to make water and a fatty acid radical (Halliwell, 1994).

(Reaction 3)


The products of the initiation phase could undergo molecular rearrangement to form conjugated dienes.


1.14.2    Propagation


The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen, thereby creating a peroxyl fatty acid radical. This too is an unstable specie that reacts with another free fatty acid producing a different fatty acid radical and a hydrogen peroxide or cyclic peroxide if it had reacted with itself. This cycle continues as the new fatty acid radical reacts in the same way. (Aruoma et al., 1989)

The hydrogen peroxide is unstable and in the presence of a metal catalyst such as iron forms a reactive alkoxy radical (Braughler etal., 1996).



1.14.3    Termination


When a radical reacts it always produces another radical, which is why the process is called a “chain” reaction mechanism”. The radical r eaction stops when two radicals react and produce a non-radical species. This happens only when the concentration of radical species is high for the probability of two radicals colliding. Living organisms have different molecules that speed up termination by catching free radical and therefore protect the cell membrane. One important of such antioxidants is alpha-tocopherol, also known as vitamin E. Other antioxidants made within the body include the enzymes: superoxide dismutase, catalase and peroxidase (Gutteridge 1997).


In addition, end-products of lipid peroxidation may be mutagenic and carcinogenic. For instance, the end-product; malondialdehyde reacts with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts (Gutteridge, 1996).


Since the molecular precursor for the initiation process is generally the hydrogen peroxide product-ROOH, lipid peroxidation is a chain reaction with potentials of devastating effects. To control and reduce lipid peroxidation both humans in their activities and nature invoke the use of antioxidants. Propyl gallate, butylated hydroxyl toluene(BHT) are antioxidants used as food additives (Murray etal, 2003).



1.14.4                            Types of Lipid Peroxidation                      Non- Enzymatic Lipid Peroxidation


Lipid peroxidation is probably the most extensively investigated free radical-induced process (Gutteridge and Halliwell,1990). Polyunsaturated fatty acids (PUFAs) are particularly susceptible to peroxidation and once the process is initiated, it proceeds as a free radical-mediated chain reaction involving initiation, propagation and termination. Initiation of lipid peroxidation is caused by attack of any specie that has sufficient reactivity to abstract a hydrogen atom from a methylene group upon a PUFA. Since hydrogen atom in principle is a radical with a single unpaired electron on the carbon to which it was originally attached. The carbon-centred radical is stabilized by a molecular rearrangement to form a conjugated diene, followed by reaction with oxygen to give a peroxyl radical. Peroxyl radicals are capable of abstracting a hydrogen atom from another adjacent fatty acid side chain to form a lipid hydrogenperoxide, but can also combine with each other or attack membrane proteins, when the peroxyl radical abstracts a hydrogen atom from fatty acid, the new carbon-centered radical can react with oxygen to form another peroxyl radical, and so the propagation of the chain reaction of lipid peroxidation continues (Gutteridge, 1996).


Fig 5 Mechanism of non-enzymatic lipid peroxidation (Source: Gutteridge, 1996).                      Enzymatic lipid peroxidation


Cyclooxygenase and lipoxgenase catalyse lipid peroxidation (Vane and Botting, 1995). The peroxidation of PUFAs can proceed not only through non-enzymaticfree radical induced pathways, but also through processes that are enzymatically catalysed. Enzymatic lipid peroxidation may be referred only to the generation of lipid hydroperoxides achieved by insertion of an oxygen molecule at the active center of an enzyme (Halliwell etal., 1999). Free radicals are probably important intermediates in the enzymatically catalysed reaction, but are localized to the active site of the enzyme. Cyclooxygenase (COX) and lipoxygenase carry out enzymatic lipid peroxidation when they catalyse the controlled peroxidation of various fatty acid substrates. The hydroperoxides and endoperoxides produced form enzymatic lipid peroxidation become stereospecific and have important biological functions upon conversion to stable active compounds. Both enzymes are involved in the formation of eicosanoids, which comprise a large and complex family of biologically active lipids derived from PUFAs with 20 carbon atoms. Prostaglandins are formed by cyclooxygenase-catalysed peroxidation of arachidonic acid (Samuelson etal., 1975). Cyclooxygenase exist in at least two isoforms (Vane and Botting,1995). Cylooxygenase-1 is present in cells under physiological conditions, whereas cyclooxygenase-2 is induced in macrophages, epithelial cells and fibroblasts by several inflammatory stimuli leading to release of prostaglandins (Halliwell etal., 1999).




 1.15.1    General Objective


The major objective of this work is to determine the nutritive composition of the pulp of S.dulcificum and to ascertain whether or not the methanolic pulp could have beneficial effects on some biochemical parameters such as liver function status, kidney function parameters, blood glucose, serum lipid profile and lipid peroxidation/antioxidant activity of rats as the animal model for the research.



1.15.2    Specific Objectives


Thespecificobjectives of this research work are:


To determine the nutritive and antinutritive composition of pulp of S. dulcificum.

To determine the LD50 (lethal dose) of the methanolic pulp extract of S. dulcificum in rats.


To determine the effect of the methanolic pulp extract of S.dulcificum on some liver function enzymes (ALT, AST and ALP) and liver function status (serum total protein, serum albumin, serum globulin and bilirubin) concentration in rats.


To determine the effect of the methanolic pulp extract of S. dulcificumon kidney function parameters (creatinine and urea concentration) in rats.


To establish already known anti-diabetic properties of the plant

To determine the effect of the methanolic pulp extract of S. dulcificum on serum lipid profile (total cholesterol, LDL cholesterol, HDL cholesterol, TAG concentrations) in rats.


To determine lipid peroxidation/antioxidant activity.

To study the effect of the methanolic pulp extract of S.dulcificum on the histology of liver and kidney of the rats so as to confirm the toxicity studies.


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