EXTRACTION OF ALKALOIDS FROM THREE NIGERIAN PLANTS, KOLA ACCUMINATA (OJI IGBO), KOLA VERA (OJI HAUSA), AND GAXCINA KOLA (BITTER KOLA).

ABSTRACT

The   extraction of alkaloid from Gaxcina  kola (bitter Kola), Kola acuminated (Oji Igbo) and Kola Vera (Oji Hausa) were carried out using 10% enthanoic acid and 10% ethanol to separate the alkaloid from the residue. The alkaloid was extracted using 5% ammonium hydroxide. From the sample results obtained on alkaloid for the sample 9.16% oji Igbo, 6.20% oji Hausa and 8.20% Bitter kola. It was observed that the percentage of alkaloids was highest in Oji Igbo and the least for Oji Hausa.

TABLE OF CONTENT

Certification -  -      -      -      -      -      -      -      -      i

Dedication -    -      -      -      -      -      -      -      -      ii

Acknowledgements -      -      -      -      -      -      -   iii-iv

Table of content -   -      -      -      -      -      -      -  v-vii

Abstract -       -      -      -      -      -      -      -      -      - -  viii

CHAPTER ONE

1.1      Introduction - -      -      -      -      -      -      -      1

1.2      Occurrence -   -      -      -      -      -      -      -      1

1.3      Plant Source - -      -      -      -      -      -      -      3

1.4      Alkaloid Classification -   -      -      -      -      -      3

1.5      Physiochemical Properties -    -      -      -      -      6

1.6      Presence of Nitrogen in their molecule- -      -      7

1.7      Presence of Monoacid as a Biosynthetic material - 7

1.8      Alkalinity of Alkaloids -   -      -      -      -      -      8

1.9      Solubility of Alkaloids -   -      -      -      -      -      10

1.10  Kola Nut -       -      -      -      -      -      -      -      -      12

1.11  Pharmacological effects of kola nuts -   -      -      14

1.12  Chemical composition of kola Nuts -     -      -      15

CHAPTER TWO

2.0  Literature Review - -      -      -      -      -      -      16

CHAPTER THREE

3.0      Materials and Methods - -      -      -      -      -      24

3.1  Materials -      -      -      -      -      -      -      -      24

3.2  Reagents -      -      -      -      -      -      -      -      24

3.3  Reagents preparation -  -      -      -      -      -      24

3.4  Sample Collection

3.5  Sample preparation -     -      -      -      -      -      26

3.6  Extraction of Alkaloid -   -      -      -      -      -      27

3.7  Qualitative test of Alkaloid -   -      -      -      -      28

CHAPTER FOUR

4.0      Results and Discussion - -      -      -      -      -      29

4.1  Results -  -      -      -      -      -      -      -      - -   29

4.2  Discussion -    -      -      -      -      -      -      -      32

4.3  conclusion -    -      -      -      -      -      -      -      35

4.4  Recommendation for further study -     -      -      37

References  -  -      -      -      -      -      -      -      38

APPENDIX   -   -      -       -     -       -       -

                                 CHAPTER ONE

1.1   INTRODUCTION

Alkaloids are a class of organic compounds that typically contain nitrogen and have complex ring structures. They occur naturally in seed bearing plants and are found in berries, bark, fruits, roots and leaves. That is, they are derived from plants. Often, they are bases that have some physiological effects.

Many of the earliest isolated pure compounds with biological activity. This was due to the ease of isolation. The nitrogen generally makes the compound basic and the compound exists in the plant as a salt. This, alkaloids are often extracted with water or mild acid and then recovered as crystalline material by treatment with base. Prior to approximately 300 years ago malaria was the scourage of Europe, likely  having been introduced though the middle East. Malaria is caused by protozoa of the genus plasmodium, contained as spores in the gut of the anopheles mosquitoes, which then spread the spores to humans when they bites. As the Spanish and Portuguese explores began to colonize. South America they discovered a cure for Malaria known to the native Indians. This was the bark of the cinchona tress. The use of cinchona bark to treat Malaria was first reported in Europe in 1633, and the first bark reached Rome about 12 years later teas made from the bark cures people suffering from Malaria, one of the major scourges in Europe at the time and the bark became known as Jesuits bark. Because of the philosophical differences between protestants and catholic, many protestants refused to be treated with the bark. One of the most prominent protestants of the time, Olive Isolate originally from cinchona Succirubra, quinine is one of 31 alkaloids with related structure, and the principal ant malaria compound, in the plant. Alkaloids have been defined in various ways, but one definition come fairly close to actuality. An alkaloid is plant – derived compounds that is toxic or physiologically active, contain nitrogen in a heterocyclic ring, and is basic, has a complex structure, and is of limited distribution in the plant kingdom,. Malaria is still a major problem throughout the world, and although synthetic antimalaria drugs largely supplanted quinine as the treatment for malaria during world warII, quinine is often once again the drug of choice as strain of malarial have become resistant to the synthetic drugs. However, the search for other antimalaria drugs from natural sources has also continued.

One of the most promising new drugs is qinghaosu, isolated from Artemisia annua.

Among the most famous of the alkaloids are the slanaceae or tropane alkaloids. Plants containing these alkaloids have been used throughout recorded history as poisons, but many of the alkaloids do have valuable pharmaceutical properties. Atropine, the racimic form of hyoscyamine, comes from Atropa belladonna (deadly nightshade) and is used to dilate the pupils of the eye. Atropine is also a central Nervous system (CNS) stimulant and is used as a treatment for nerve gas poisoning. Scopolamine, another member of this class is used as treatment for motion sickness. Cocaine from Erythroxylum coca, is closely related in structure,  is also a CNS stimulant, and has been used as a topical anesthetic in ophthalmology. It is also drug abuse. Cocaine was found in very small amounts in the original coca – cola formula, but was not the main concern of the USDA at the time. Caffeine was considered to be the major problem with the, drink. Datura stramonium (Jimsonweed), a plant found in Virginia contains similar compounds.

The ergot alkaloids come from a fungus, claviceps purpurea, which is a parasite on rye and wheat. The ergot alkaloids are responsible for ergotism, resulting in loss of limbs, or convulsive erogotism, resulting in hallucinations. In both cases, death usually follows and out breaks of ergotism caused 11,000 deaths in Russia as late as 1926. Today the problem is recognized and controlled. Some of the ergot alkaloids have been used to trial migraine headaches and sexual disorders in clinical applications. The most famous of these alkaloids is lysergic acid diethylamide, LSD, a powerful hallucinogen that is a synthetic derivative of the natural products. Similar alkaloids, particularly ergine, are also found in Mexican morning glories, such as iponeoa tricolore³.

The morphine alkaloids, derived from the opium poppy, papaver somniferum, are powerful pain relievers and narcotics. The narcotic activity of P. somniferum was  noted on Sumerian tablets in 3500 B.C. Making  it one of the oldest drugs known. Opium is the dried latex of the seed head of p.somniferum and has been used as an analgesic (eliminates or  relieves pain) and narcotic (induces sleep or drowsiness) in preparation such as laudanum and paregoric. Morphine is the principal alkaloid and was first isolated between 1803 and 1806. it was widely used for pain relied beginning in the 1830’s but was also recognized as being addictive. In an attempt to make morphine less addictive, Bayer chemist acetylated the hydroxyl groups to produce diacetylmorphine. This was marketed as a non- addictive pain reliever under the trade name heroin for about two years in the early 1900’s, until it was recognized to be more addictive than morphine have been developed and found use as opiate antagonist or as animal tranquilizers³.

Vincristine, one of the most potent antileukemic drugs in use today, was isolated in a search for diabetes treatment from vinca rosea (now Catharanthus roseus) in the 1950’s along with vinblastine, a homologue in which the N- methyl group is oxidized to an aldehyde moiety. This is such a complex structure that is still isolated from the plant (the Madagascan periwinkle) today rather than prepared by synthesis.

Vincristine (leurocristine, VCR) is most effective in treating childhood leukemia’s and non – Hodgkin’s lymphomas, while vinvlasbtine (vincaleukoblastine, VLB) is used to treat Hodgkin’s disease⁴.

Thin layer Chromatography (TLC) techniques are frequently used for evaluating medicinal plant materials and their preparations (world health organisation 1998). The ascending technique was used.

1.2             OCCURRENCE

Alkaloids are found in every habitat in which vascular plants grow. They are not widely distributed in the vegetable kingdom. They are derived mainly from the angiosperms, the seed bearing or flowering plants. They occur mainly in the dicotyledons (two seed leaves). They are rarely found in non-seed bearing plants. Generally, they occur in plant tissues at points of intense cell activity (leaves, roots, barks and seeds) and they are generally found in living cells rather than in dead tissues.

Alkaloids may be found in solution in cell sap, and especially in the parenchymatous tissues in the other bark and may be stored in the solid state usually in the form of salts. Alkaloids from plants of closely related family are similar in structure and are not localized, but appear to be characteristics of the organs.

However, not all organs of any one species must have alkaloids. For example, the seed of tobacco does not contain alkaloids. The periods of maximum accumulation of  alkaloids appear only in early flowering stage. Some alkaloids are virtually absent in a young plant, but increase to insoluble amounts as the plant approaches maturity. As a rule alkaloids are not found free in plants, but are combined with some acids in form of salts. The acids most encountered are malic, citric, tannic, succinic, oxalic, sulphric, phosphoric and hydrochloric acid.

1.3   Plant source;

Alkaloids occur in many different species in numerous genera and families of vascular plants as well as in certain species of fungi. It has been estimated that some fifteen percent or more of all vascular plants contain alkaloids. A number of amines produced by animals possess physical and chemical properties rather similar to those of alkaloids. By traditions and conventions, these animals amines are generally not considered as alkaloids. The occurrence of alkaloids in different plant organs and tissues and their relationship to aspects of the physiology of the plants are interesting part of alkaloids.

1.4   Alkaloid Classification

Alkaloids are usually classified by their common molecular heat, based on the metabolic pathway used to construct the molecule. When not much was known about the biosynthesis of alkaloids, they were grouped under the names of known compounds, even some non – nitrogenous ones (since those molecular structures appear in the finished product, the opium alkaloids are sometimes called phenanthrenes, for example) or by the plant or animal they were isolated from. When more is learnt about a certain alkaloid, the grouping is changed to reflect the new knowledge, usually taking the name of a biologically important amine that stands out in the synthetic process.

Pyridine group includes piperine, conine, trigonelline, arecaidine, guvacine, pilocarpine, cytosine, nicotine, sparteine, pelletierine.

Pyrolidine group is made up of hygrine, cuscohygrine, nicotine.

Tropane group: atropine, cocaine, ecgonine, scopolamine, catuabine.

Quinoline group: quinine, guanidine, dihydroquinine,  strychinine, brucine, veratine, cevadine.

Isoquinoline group: the opium alkaloids (morphine, codeine, thebaine, isopapa – dimethoxy – aniline, papaverine, narcotine, sanguinarrine, narceine, hydrstine berberine), emetine.

Phenethylamine group: mescaline, ephedrine, dopamine, amphetamine.

Indole group: Tryptamines: DMT, N – Methyltryptamine psilocybin, and serotonin. Ergolines, the ergot alkaloids. Ergine, ergitamine, lysergic acid etc).

Beta – carbolines, haemine, harmaline, yolimbine, reserpine.

Rauwolfia alkaloids; Reserpine.

Purine group: Xanthines, Caffeine, the theobromine, theophylline.

Terpenoid group: Aconite alkaloids, aconitine, steroids, solanine, samandaris (quaternary) ammonium compound  muscarine, choline, neurine. Vinca alkaloids: vinblastine, vincristine, they are antineoplastic and bind free tublin dimmers thereby distrupting balance between microdubule polymerization and delpolmerization resulting in arrest of cells in metaphase.

1.5   physiochemical properties

Alkaloids are colourless, crystalline, non – violatile solids that are insoluble in water but soluble in organic solvents such as ether, chloroform e.t.c. They have the molecular weights ranging from 100 to 900.

Some alkaloids contain oxygen atoms while the others do not.

Those without oxygen atoms in the structure are usually liquid at ordinary temperature (e.g) nicotine, sparteine and coniine)

Those with oxygen atoms are crystalline (e.g berberine yellow). Most are optically active (usually leavorotatory) but not those from the group.

1.6   Presence of Nitrogen in their molecule

Alkaloids contain one or more nitrogen atoms in their molecules. A large number of alkaloids contain at least one nitrogen atoms in a heterocyclic ring. But in a number of alkaloidal amines (e.g. ephedrine, mescaline, colchineds) which are generally considered as alkaloids, the nitrogen atom in the molecule is not in the ring.

1.7   Presence Amino acid as a Biosynthetic materials;

Certain alpha amino acids have been experimentally shown to serve as precursor in the nitrogen containing portion of the alkaloid molecules but in some cases also with respect to the non-nitrogenous portion as well. Among the amino acids which have been either experimentally established or postulated on the basis of theoretical chemical considered as common ones are;

phenylnine, tyrosine, lysine, omthine, histidine, tryptophan and anthracilic acid.  However, some alkaloids are, in their biogenesis, more directly derived from isoprene or other precursor compounds of carbohydrate metabolism.

1.8               Alkalinity of Alkaloids

Alkaloids are basic (alkaline) in reaction, due to the presence of nitrogen atom in their molecule.The term alkaloids mean alkali like. Therefore they form salts with various acids. Most of the alkaloid bases are crystalline solids, a few are liquid (eg nicotine, pilocarpine). The alkaloid salts are crystalline and the microscopic examination of their crystalline structure serves as an aid to their identification.The alkaloid salts in solution release the free alkaloid bases when the solution is made alkaline, most commonly with ammonia, sodium carbonate or calcium hydroxide. All alkaloids do not have the same degree of alkalinity. Apart from the influence exerted on the electronic disposition of the nitrogen atom in the molecule by side chains and various substitution, one important factor contributing to the different degree of alkalinity among alkaloids is whether a given alkaloid contains primary, secondary, tertiary or quaternary nitrogen atom or atoms. Such differences in the degree of alkalinity arise from the various structural characters and one reflected in the different alkaline values for the different alkaloids. The weaker bases (those with low alkaline value) would require a more acidic medium to form salts with the acid than would the strongly basic alkaloids, having higher alkaline value.

1.9             Solubility of Alkaloids

Most alkaloid bases are practically water – insoluble. They are fairly soluble in a  number of organic solvents such as Chloroform, Ether and lower alcohols. There are of course, exceptions. For instance, Morphine and Colchicines are practically insoluble in ether. Also many alkaloids which contain phenolic hydroxyl group in their molecules are soluble in aqueous solutions of caustic alkali.

On the other hand, the alkaloid salts are generally soluble in water and in alcohol, and mostly nearly insoluble in the immiscible organic solvents.

A few alkaloid bases are rather soluble in water, but there are exceptions. Some examples of water – soluble alkaloids are:

Ephedine, Colchicines, Ergonovine and the Clavinet – type of ergot alkaloid. It should be noted however that  these water – soluble alkaloids are still relatively more soluble in a number of organic solvents such as chloroform or ether, than they are in water, and their salts are nearly insoluble in the immiscible organic solvents such as chloroform or ether, than they are in water, and their salts are nearly insoluble in the immiscible organic solvents.

Many alkaloids in small quantities (10 – 100:g) in soluble may form precipitation or turbidity with certain reagents. Most alkaloids are also precipitated by tannins. With special techniques some alkaloids smaller in quantities than  :g may be detected in this way, on this basis. These alkaloid precipitating reagents are sometime used in testing the presence or absence of alkaloids in crude extracts of plant materials, and in testing whether a particular step in an extraction procedure has exhausted the alkaloidal contents. A negative response can be taken to mean absence of alkaloids, but a positive test may or may not be due to the presence of alkaloids in the solution or extract  being tested, as some non-alkaloid substances may also give such a positive response to these reagents.

1.10         Kola Nut

Kola nut is a genus of about 125 species of trees native to the tropical rainforest of Africa classified in the family malvaceae, subfamily sterculioideae (or treated in the separate family sterculiaceae) It is related to south American genus Theobroma (Cocao). They are evergreen trees growing to 20m tall, with glossy ovoid leaves up to 30cm long⁵.

The kola nut has a bitter flavour and caffeine content, and is chewed in many African cultures, individually or in a group setting. It is often used ceremonically, presented to tribal chiefs or presented to guests. Chewing kola nut can ease hunger pangs. Frequent chewing of the kola nut can also lead to stained teeth. Among the urban youths of West Africa, Kola nut is becoming less popular⁵. Kola was originally used to make cola soft drinks, though today most of these mass – produced beverages use artificial flavourings. Some exceptions are barr’s Red Kola, Harboe original Taste Cola, foxon Park Kola, Blue sky Organic Cola, Whole food market 365 Cola, sprecher’s puma kola and cricket cola, the latter being made from kola nuts and green tea. In 2007, united kingdom supermarket, Tesco introduced an American premium cola that uses kola nuts, species and vanilla. The merging energy drink mix, blow, contains 50mg of kola nut extract per vial (in addition to 240mg of caffeine per serving).

Outside of Africa, some species are cultivated for their nuts. Examples in Indonesia, Brazil, Jamaica and elsewhere in the humid tropics. Kola nuts are often used to treat whopping cough and asthma. The caffeine present acts as a bronchodilator, expanding the bronchiole air passages.

1.11         Pharmacological Effects Of Kola Nuts:

kola nuts are used mainly for their stimulant and euphoria qualities. They have effects similar to other xanthenes containing herbs like cocoa, tea coffee, quarana and Yerba mate. However, the effects an distinctively different, producing a stronger state of euphoria and well being. They have stimulant effects on the central nervous system and heart. Animal experiments indicate that kola nuts have analeptic and lipolytic (fat - burning) properties, and stimulate the secretion of gastric juices. Human studies show kola nuts have positive chronotropic and weak diuretic effects. In human, it enhances alertness and physical energy, elevates mood, increases tactile sensitivity, suppresses the appetite and is used in Africa as an aphrodisiac. Autonomic changes include. Increased body temperature, increase blood pressure and increased respiratory rate. Effects may last up to 6 hours after ingestion

1.12         Chemical composition of Kola Nuts

Caffeine (2-3.5%), Theobromine (1.0-2.5%) Thophylline, catechine, Epicatechine, D – Catechine, phenolics phlobaphens, kola red, Betaine, protein, starch, fat, thiamine, Riboflavin, Niacin, Ascorbic acids, Betaine, sugar, Gum, cellulose, water calcium, potassium, Iron, Beta – carotene and tannic acid⁶.

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