EVALUATION OF PROXIMATE CONSTITUENTS IN LEAFS AND ROOT OF SIMAROUBA GLAUCA D.C. (PARADISE TREE)

ABSTRACT

The proximate content; moisture, crude protein, crude ash, crude fat, crude fibre and carbohydrate, of Simarouba glauca were determined, along with other properties to include the glucose and amino acid contents, and the qualitative macromolecular content of the plant. The proximate properties of the plant were determined based on the A.O.A.C (1990) methods for moisture, crude protein and crude ash; A.O.A.C (1984) for crude fibre and carbohydrates; and the acid base extraction technique described by Phillips et.al. (2001). The concentration of different amino acids were determined using the ninhydrin, while the glucose content was determined Nelson-Simogy’s method. The moisture contents of the

leaves and the roots were quite high, over 60% in both. The root of the plant was also found to be rich in fibre and low in fat, meanwhile the leaves recorded a higher fat concentration than fibre. With the moisture and fibre content of the roots taking a large percentage, it was found to be pretty low in crude ash, carbohydrates, crude protein and crude fat. The leafs however was much higher in ash, carbohydrates and protein, and a little higher in fat. Among the essestial amino acids, methionine was the highest while phenyl alanine was negative. The highest amino acid was cysteine, which is a conditionally essential amino acid. From these results, it is obvious that Simarouba glauca roots and leaf extract might not enough nutritional supplements while being used medicinally. They can serve as a good source of animal feed, however, proteins and fat would need to be supplemented.

TABLE OF CONTENTS

TITLE PAGE       -        -        -        -        -        -        -        -        -        ii

CERTIFICATION         -        -        -        -        -        -        -        -        -        iii

DEDICATION     -        -        -        -        -        -        -        -        -        iv

ACKNOWLEDGEMENT       -        -        -        -        -        -        -        v

TABLE OF CONTENTS  -          -         -         -         -         -         -         -      vi

ABSTRACT        -        -        -        -        -        -        -        -        -        viii

CHAPTER ONE

1.1 INTRODUCTION             -        -        -        -        -        -        -        1

1.2 MOISTURE CONTENT             -        -        -        -        -        -        2

1.3 CARBOHYDRATES        -        -        -        -        -        -        -        3

1.4 PROTEINS    -        -        -        -        -        -        -        -        -        7

1.5 CRUDE ASH -        -        -        -        -        -        -        -        -        8

1.6 FAT AND FIBRE    -        -        -        -        -        -        -        -        9

1.7 AIM OF THE RESEARCH WORK     -        -        -        -        -        9

CHAPTER TWO

2.1 LITERATURE REVIEW  -        -        -        -        -        -        -        10

2.2 PHYSICAL QUALITIES OF SIMAROUBA GLAUCA:    -        -        11

2.3 MEDICINAL IMPORTANCE OF SIMAROUBA GLAUCA:      -        -        12

2.4 BIOFUEL PRODUCTION:        -        -        -        -        -        -        -        15

2.5 CONSTITUENTS OF SIMAROUBA SEEDS:       -        -        -        -        16

CHAPTER THREE

MATERIALS AND METHODOLOGY     -        -        -        -        -        20

3.1 MATERIALS -        -        -        -        -        -        -        -        -        20

3.1.1 PLANT -       -         -            -           -         -          -          -         -            -         20

3.2 CHEMICALS/REAGENTS        -        -        -        -        -        -        -        20

3.3 APPARATUS          -        -        -        -        -        -        -        -        -        21

3.4 EQUIPMENTS -      -        -        -        -        -        -        -        -        22

3.5 METHODS    -        -        -        -        -        -        -        -        -         22

3.5.1 DETERMINATION OF MOISTURE  CONTENT -      -        -         23

3.5.2 QUANTITATIVE DETERMINATION OF PROTEIN CONTENT -     24

3.5.3 QUANTITATTIVE DETERMINATION OF ASH CONTENT   -          27

3.5.4 DETERMINATION OF CRUDE FIBRE CONTENT -      -       -            28

3.5.5 DETERMINATION OF CRUDE FAT CONTENT -          -       -            28

3.5.6 QUANTITATTIVE DETERMINATION OF CARBOHYDRATE CONTENT- -          -        -        -        -        -        -        -          -          29

3.5.7 QUANTITATTIVE DETERMINATION SOME MACROMOLECULES      -    -          -        -        -        -        -        -        -          -         -          -          30

3.5.8 QUALITATIVE DETERMINATION OF PROTEIN CONTENT -           32

3.5.9 QUANTITATIVE DETERMINATION OF GLUCOSE CONTENT -       33

3.5.10 QUALITATIVE DETERMINATION OF SOME AMINO ACIDS (NINHYDRIN METHOD) -        -        -         -        -         -         -         -            24

CHAPTER FOUR

4.1     PROXIMATE ANALYSIS ON SIMAROUBA LEAF

AND ROOT EXTRACT         -        -        -        -        -        -        -        35

4.2     QUALITATIVE TEST FOR MACROMOLECULES IN LEAVES AND                           ROOTS      -        -        -        -        -        -        -          -           -        -       35

4.3      RESULT FOR QUANTITATIVE ANALYSIS OF GLUCOSE IN SIMAROUBA LEAVES AND ROOT   -        -        -        -         -          37   

CHAPTER FIVE

5.1     DISCUSSION      -        -        -        -        -        -        -        -        38

5.1.1   MOISTURE CONTENT        -        -        -        -        -        -         38

5.1.2   CRUDE PROTEIN AND AMINO ACIDS         -        -        -        -        38

5.1.3   CRUDE ASH     -        -        -        -        -        -        -         -         39

5.1.4   CARBOHYDRATE, GLUCOSE AND CRUDE FIBRE       -        -        40

5.2     CONCLUSSION  -        -        -        -        -        -        -        -        41

REFERENCES    -        -        -        -        -        -        -        -        42

APPENDIX 1       -        -        -        -        -        -        -        -        45

CHAPTER ONE

1.1 INTRODUCTION

Plants undergo photosynthesis and they constitute a primary resource of carbon, vitamins, minerals, protein, essential fatty acids, and utilizable energy for food production (Young and Pelett, 1994). Plants have played a significant role in maintaining the health and improving the quality of human life for thousands of years. (Mishra, 2010).  They provide a major source of food and nourishment for man and animal.

 Nutrition is a science of food and its relationship to health. Nutrition refers to nourishment that sustains life. The study of nutrient requirements and the diet providing these requirements is also known as ‘nutrition’ (Chutani, 2008). Pike and Brown, 1984 defined it as “the science that interprets the relationship of food to the functioning of living organism. It includes the uptake of food, liberation of energy, elimination of wastes and all the processes of synthesis essential for maintenance, growth and reproduction (Chutani, 2008).  Apart from maintaining normal body functioning, nutrition is important in fighting infections and in the recuperation of an ill person. Nutrition interacts with infections in a synergistic manner, such that recurrent infections lead to a loss of body nitrogen and worsen nutritional status; the resulting malnutrition, in its turn, produces a greater susceptibility to infection (Kurpad, 2005). Aristotle (384-322 B.C.) was the first to suggest that the composition of foods in the normal diet might contribute to health.

In an 1897 literature on metabolic investigations, Atwater divided food composition into five classes; protein, fat carbohydrate, energy and water. However, today, proximate composition is the term usually used to describe six components of food namely; moisture, crude protein, crude ash, crude fibre, crude fat and carbohydrate (nitrogen free extract) which are all expressed in percentage (%) or gram per 100 grams (g/100g). The study of proximate analysis on foods was devised over a hundred years ago by two German scientists, Henneberg and Stohmann, and even though new techniques have been introduced, their system of proximate still forms the basis for the statutory declaration of the composition of foods. (Dublecz, 2011).

1.2 MOISTURE CONTENT

Water is essential for every living organism. In the human body, water content ranges from 50-70% in different tissues. It is present in different fluid compartments of the human body- Intracellular (fluid inside the cells) and extra cellular. Plasma, interstitial fluid, cerebrospinal fluid, ocular fluid, lymph, peritoneal, pericardial, pleural and synovial fluids are part of the extra cellular fluid (Chutani, 2008).  However, the moisture content of a feed is seldom of interest nutritionally as water is usually taken on its own.

The active ingredients from the view of feed nutrition are present in the part of dry matter (solid matter); therefore the level of moisture content is an important factor in both economy and storage.  At high temperature and humidity  the risk of putrefaction is predicted due to the proliferation of molds, etc., or self-digestion by enzymes in the feed when moisture in the feed is not less than about 15%.  As the assay for moisture in the feed measures loss on drying by heating at normal pressure as moisture, the result includes most of volatile substances other than H₂O.  Therefore, it may be more appropriate to be referred to as volatile matter rather than moisture for accuracy.  Organic acids such as acetic acid and butyric acid in silage as well as ammonia and flavor components in feed materials are also vaporized and thus measured as moisture.  Because the content of these in most feed is extremely low, there has hardly been a need to consider their influence on the measured value. (Chutani, 2008). 

1.3 CARBOHYDRATES

Photosynthesis is a process used by plants and other organisms to convert light energy from the sun, into chemical energy that can be later released to fuel the organisms' activities. The light energy harnessed from the sun drives the reduction of carbon from CO₂ to produce O₂ and fixed carbon in form of carbohydrate.

Early in the twentieth century, it was mistakenly thought that light absorbed by photosynthetic pigments directly reduced CO₂ which then combined with water to form carbohydrate. In fact, photosynthesis in plants is a two stage process in which light energy is harnessed to oxidise H₂O:

                                 2H₂O        →          O₂ + 4 [H⁺]

The electrons thereby obtained subsequently reduce CO₂:

4H⁺ + CO₂ → (CH₂O)_n + H₂O

The two stages of photosynthesis are traditionally referred to as the light reactions and the dark reactions:

1.     In the light reactions, specialised pigment molecules capture light energy and are thereby oxidized. A series of electron- transfer reactions which culminate with the reduction of NADP⁺ to NADPH, generate ATP from ADP + Pi. The oxidised pigment molecules are reduced to H₂O, thereby generating O₂.

2.     The dark reactions use NADPH and ATP to reduce CO₂ and incorporate it into the three-carbon precursors of carbohydrate.

The light reactions takes place in the thylakoid membrane of chloroplasts in leaves and green parts of plants.  The inside of the thylakoid is referred to as the lumen. The light reactions are catalysed by enzymes located in the thylakoid membrane, whereas the dark reactions take place in the stroma. The principal photoreceptor of light is chlorophyll. These chlorophyll molecules do not participate directly in photochemical reactions but function to act as light harvesting antennas. The absorbed photons are transferred from molecule to molecule until it reaches the photosynthetic reaction centre.

In the respiratory chain, electrons flow from NADH + H⁺ to O₂, with the production of water and energy. However in photosynthesis, electrons are taken up from water and transferred to NADP⁺, with an expenditure of energy. Photosynthetic electron transport is therefore energetically “uphill work.” To make this possible, the transport is stimulated at two points by the absorption of light energy. This occurs through two photo systems protein complexes that contain large numbers of chlorophyll molecules and other pigments Another component of the transport chain is the cytochrome b_f complex, an aggregate of integral membrane proteins that includes two cytochromes (b563 and f). Plastoquinone, which is comparable to ubiquinone, and two soluble proteins, the copper containing plastocyanin and ferredoxin, function as mobile electron carriers. At the end of the chain, there is an enzyme that transfers the electrons to NADP⁺. Because photosystem II and the cytochrome b/f complex release protons from reduced plastoquinone into the lumen, photosynthetic electron transport establishes an electrochemical gradient across the thylakoid membrane, which is used for ATP synthesis by an ATP synthase.

ATP and NADPH + H⁺, which are both needed for the dark reactions, are formed in the stroma. (Voet et al., 2013).

1.3aCalvin cycle

The actual CO₂ fixation i.e., the incorporation of CO₂ into an organic compound is catalysed by ribulosebisphosphate carboxylase/oxygenase (“rubisco”). Rubisco, the most abundant enzyme on Earth, converts ribulose 1,5-bis-phosphate, CO₂ and water into two molecules of 3-phosphoglycerate. These are then converted, via 1,3-bisphosphoglycerate and 3-phosphoglycerate, into glyceraldehyde 3-phosphate. In this way, 1,2-glyceraldehyde 3-phosphates are synthesized from six CO₂. Two molecules of this intermediate are used by gluconeogenesis reactions to synthesize glucose 6-phosphate. From the remaining 10 molecules, six molecules of ribulose-1,5-bisphosphate are regenerated, and the cycle then starts over again. In the Calvin cycle, ATP is required for phosphorylation of 3-phosphoglycerate and ribulose-5-phosphate. NADPH + H⁺, the second product of the light reaction, is consumed in the reduction of 1,3-bisphosphoglycerate to glyceraldehyde-3- phosphate.

Carbohydrates are the most abundant biomolecules produced on earth; photosynthetic plants and algae convert over 100 billion metric tons of CO₂ and water into sugars, starches, and cellulose like substance. Carbohydrates supply energy for the human body to function. They are the most abundant bulk nutrients and form the major source of biological energy through their oxidation in the tissues. They also furnish organic precursors for the biosynthesis of many cell components. Carbohydrates are not essential in the human diet, but because carbohydrate rich foods are abundant and cheap, compared with fats and protein, they naturally form a major part of the diet in most of the world. (Voet et al., 2013).

1.4 PROTEINS

The requirements for total protein, at various stages during the life cycle of humans, were reviewed and evaluated by a joint panel of the Food and Agriculture Organization, the World Health Organization, and the United Nations University (FAO/ WHO/UNO).

The requirement for dietary protein consists of two components:

1)     the requirement for the nutritionally indispensable amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) under all conditions and for conditionally indispensable amino acids (cysteine, tyrosine, taurine, glycine, arginine, glutamine, proline) under specific physiological and pathological conditions and

2)    the requirement for nonspecific nitrogen for the synthesis of the nutritionally dispensable amino acids (aspartic acid, asparagine, glutamic acid, alanine, serine) and other physiologically important nitrogen-containing compounds such as nucleic acids, creatine, and porphyrins.

With respect to the first component, it is usually accepted that the nutritive values of various food protein sources are to a large extent determined by the concentration and availability of the individual indispensable amino acids. Hence, the efficiency with which a given source of food protein is utilized in support of an adequate state of nutritional health depends both on the physiological requirements for the indispensable amino acids and total nitrogen and on the concentration of specific amino acids in the source of interest (Young and Pelett, 1994).

Proteins, are synthesized from a complex series of steps which involves the transcription of DNA already present in each cell of an organism, and its consequent transcription into a polypeptide chain. This chain is modified by other inherent mechanisms in the cell to yield protein.

1.5 CRUDE ASH

Ash is the inorganic residue remaining after the water and organic matter have been removed by heating in the presence of oxidizing agents, which provides a measure of the total amount of minerals within a food. The ash content is a measure of the total amount of minerals present within a food. Minerals are required for many purposes like forming the frame and rigid structure of the body, as part of the body/cell fluids and for number of cellular and sub cellular physiological functions (Chutani, 2008).  The mineral content includes specific inorganic components present within a food, such as Ca, Na, K and Cl. Determination of the ash and mineral content of foods is important for a number of reasons. The most important reason in regards a plant like S. glauca is the nutritional importance. Some minerals are essential to a healthy diet (e.g.  calcium, phosphorous, potassium and sodium) whereas others can be toxic (e.g. lead, mercury, cadmium and aluminum).

1.6 FAT AND FIBRE

The importance of fat and fibre in nutrition cannot be underestimated.  Crude fat contains fat, complex lipid, sterols, fatty acids and fat soluble dyes; while crude fibre contains cellulose, hemicellulose, and lignin.

1.7 AIM OF THE RESEARCH WORK

This study is designed to screen the proximate constituents of the leaf extracts of Simarouba glauca which includes moisture, protein, carbohydrates, ash fibre and lipid content of Simarouba glauca. In addition to this, to determine the quantitative carbohydrate and amino acid constituents of Simarouba glauca. Thus, the nutritional value of Simarouba roots and leaves with some emphasis on their possible use both as a medicinal and nutritional food for the sick or convalescent.

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