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ABSTRACT

The aqueous methanolic extracts of 20 selected plant parts were screened for in-vitro antimicrobial activity against clinical strains of Staphylococcus aureusBacillus subtilisKlebsiella pneumoniaSalmonella typhiEscherichia coliPseudomonas aeruginosaAspergillus niger and Candida albicans at a test concentration of 10mg/ml using the agar well diffusion method and penicillin G, chloramphenicol and Nystatin as controls.  The seeds of Parkia biglobosaGlycine maxVoendzeia substerrana,  Arachis hypogeal, Cajanus cajanSamonea samanManihot esculenta (root bark)  Uapaca esculenta (stem bark and root bark), exhibited no meaningful anti-microbial activity against all the tested organisms.  Uapaca esculenta (leaf) exhibited significant activity against kleb. (IZD= 16mm), Samanea saman (stem bark and leaves) showed significant anti-staphylococcus aureus activities (IZD=17mm, 15mm respectively) which compared favourably to the controls penicillin G and chloramphenicol.   Detarium microcarpum (seed), Phyllanthus mullerianus (leaf) and Phyllanthus discoideus (leaf) exhibited a broad spectrum anti-microbial activity against all the test micro-organisms with IZD’s ranging from (10mm – 20mm) an indication that they could be  effective in severe bacterial infections. Interestingly to know is the anti-staphylococcus activity. (IZD=19mm) leaf extract higher than the control penicillin G (IZD=17mm). Crude 95% aqueous methanolic extract of  Phyllanthus discoideus (yield = 29.75%) gave 14.315% of hexane solubles, 13.965% of ethylacetate solubles, 70.194% of absolute MeOH solubles and 1.624% of absolute MeOH insolubles.  These solubility fractions were similarly screened in vitro for anti-microbial activity against the same micro-organisms.  The absolute methanol soluble fraction displayed the widest spectrum of antimicrobial activity (IZD range 9mm-24mm). particularly noteworthy are the anti-E.coli activities of the absolute  MeOH soluble fraction  (IZD = 20mm), and its precipitate  (IZD=18mm) and  the anti-salmonella typhi  activity (IZD=17mm), its precipitate  (IZD=15mm),  against chloramphenicol and penicillin G – resistant strains of E.coli and Salmonella typhi .These are accentuated compared to those of the crude extract. The most active absolute methanol soluble fraction was separated by PTLC in to fifteen bands  on silica gel 60 GF254with solvent system acetone: chloroform:formic acid:water (6:6:1:05 v/v/v/v).  The impure bands were further  purified by using different elutants of increasing polarities  into twelve additional fractions.  The twenty seven fractions were similarly screened in-vitro for anti-microbial activity against the same micro-organism.  Bands 1A, 2A, 3A,4A and 8A displayed significant anti-microbial activity particulary against Staphylococcus aureusBacillus subtilisPseudomonas aureginosa & Klebsiella pneumonia which are comparable with that of controls.

The glycosidic band 8A gave anti-Bacillus subtilis and anti-Staphylococcus aureus (IZD=18mm & 20mm respectively) which are comparable with that of the control penicillin G (IZD=20mm & 22mm respectively).  The flavonoid glycosides 2A gave anti-Bacillus subtilis (IZD = 20mm, MIC 1.00mg/ml) which is the same as the control pencillin G (IZD = 20mm, MIC 1.037mg/ml).  The phenolic fraction 1A and 9 gave anti-Candida albican activities which are comparable with that of Nystatin the control.  Qualitative analysis of the bioactive bands/fractions using a combination of chemical and UV/visible spectrophotometric methods showed band 1A, 1B, 2A, 2B to contain tannins, flavonoids as glycosides while band 3 – 13 showed different form of glycosides and band 14 and 15 are phenolic compounds.  The UV-spectra of the bands confirmed the phytochemical results for the presence of minor flavonoids of the isoflavonoid and flavones classes (λmax 207mm, 276mm) quinine glycosides (270nm – 275nm), flavan (λmax 207nm, 276nm) quinine glycosides (270nm – 275nm)

This study reveals that a flavonoid glycosides, a minor flavonoid, alkaloids, Tannins are likely to be the active principles of Phyllanthus discoideus leaf with the possibility of synergistic action

 

 

 

TABLE OF CONTENTS

 

Title page———————————————————————————————————

Certification——————————————————————————————————i

Dedication——————————————————————————————————–ii

Acknowledgment————————————————————————————————iii

Abstract————————————————————————————————————iv

Table of contents———————————————————————————————–v

List of tables—————————————————————————————————–ix

List of figure—————————————————————————————————–ix

Appendices——————————————————————————————————-ix

 

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW                                                                              Page

  • Introduction ———————————————————————————————-1

1.1 The phyllanthus genus –morphology—————————————————————-1

1.2 Plant Description —————————————————————————————–2

1.3 Chemical constituent of genus phyllanthus and the biological activities——————–2

1.4 The phyllanthus discoideus plant ——————————————————————–6

1.5 Aim of the study——————————————————————————————-7

1.6 The phenolic compounds ——————————————————————————-7

1.6.1.0 Tannins- Distribution and chemistry ————————————————————8

1.6.1.1 Economic and pharmacological relevance of Tannins—————————————9

1.6.2.0  Flavonoids compounds and their pharmacological importance————————–9

1.7.0  Alkaloids classification/pharmacological importance —————————————–11

1.8.0  Terpenes structures/Distribution and Biosynthesis——————————————–15

1.8.1 Type of terpenes —————————————————————————————-16

1.8.2 Pharmacological importance of some known Terpenes ————————————-17

1.9 Steriods chemistry/Distribution ———————————————————————-18

1.9.1 Classification of steroids ——————————————————————————18

1.10 Saponins chemistry and Distribution —————————————————————19

1.10.1 Physicochemical properties of saponins——————————————————–20

1.10.2 Classification and Distribution of saponins —————————————————-20

1.10.3 General Application of Saponins——————————————————————21

1.11 Clinical significance of pathogenic organisms —————————————————-22

1.11.1.0 Clinical significance of Staphylococcus aureus(Micrococcaceae)————————22

1.11.1.1 Pathogenicity of Staphylococcus plants——————————————————–23

1.11.2 Clinical significance of Bacillus species (Bacillaceae) —————————————–23

1.11.3 Clinical significance of pseudomonas aeruginosa (Non_ Fermetative

Bacteria———————————————————————————————24

1.11.3.1 Pathogenicity of pseudomonas aeruginosa—————————————————24

1.11.4 Clinical significance of klebsiella pneumonia (Enterrobacteriaceae) ———————25

1.11.4.1 Pathogenicity of klebsiella pneumonia ———————————————————25

1.11.5 Clinical significance of Salmonella paratyphi (Enterrobacteriaceae) ——————–26

1.11.6 Clinical significance of Escherichia Coli———————————————————–26

1.11.7.0 Clinical significance of Candida albicans (Yeast)———————————————27

1.11.7.1  The Pathogenicity of Candida albicans——————————————————-28

 

CHAPTER TWO

MATERIALS AND METHODS

2.1.0 Materials ————————————————————————————————–29

2.1.1 Instrumentation—————————————————————————————-29

2.1.2 Reagents and solvents———————————————————————————-29

2.1.3 Plants materials Collection, Identification and Preparation———————————-29

2.2.0 Methods —————————————————————————————————30

2.2.1 Extraction of plant materials————————————————————————–30

2.2.2 Fractionation of the crude aqueous methanolic extraction of phyllanthus discoideus leaves—————————————————————————————————-30

2.2.3 Preparation of standard samples of extracts, fractions and standard drugs       (controls) in DMSO——————————————————————————-33

2.3.0 Antimicrobial Screening of test samples and standard drugs ——————————-33

2.3.1 Micro-Organisms—————————————————————————————33

2.3.2 Culture Media——————————————————————————————-33

2.3.3 Determination and sensitivities of pathogenic microorganisms to plant extracts, solvent fraction and PTLC fractions—————————————————————33

2.3.4 Determination of the minimum inhibitory concentration (MIC) —————————-34

2.4.0 Phytochemical Analysis ——————————————————————————–34

2.4.1 Test for Alkaloids—————————————————————————————-34

2.4.2 Test for Tannins——————————————————————————————35

2.4.3 Test for Flavonoids————————————————————————————–35

2.4.4 Test for Saponins—————————————————————————————–35

2.4.5 Test for terpenoids and Triterpenoids/steroids————————————————–36

2.4.6 Test for carbohydrates (Molisch’s test) ————————————————————36

2.4.7 Test for reducing sugar (Fehling’s test) ————————————————————36

2.4.8 Test for Glycosides—————————————————————————————37

2.5.0 Extraction/Purification of alkaloid from Absolute methanol insoluble.——————–37

2.6.0 Chromatographic Separation of the Absolute methanol soluble fractions of aqueous methanol leaf extract of phyllanthus discoideus——————————————37

2.6.1 Preparative TLC of the Absolute methanol soluble fraction ——————————38

2.7 Determination of Ultra-Violet spectra of PTLC fractions————————————–38

CHAPTER THREE

RESULT AND DISCUSSION

3.1 Results and discussion of preliminary Antimicrobial screening of 20 selected Leguminaceous and Euphorbiaceous plants——————————————39

3.2 Result and Discussion on the percentage Yields of Extracts and solvent fractions. ——-40

3.3 Results and Discussion Antimicrobial screening of various morphological                         parts of phyllanthus  discoideus and the various fractions of the                       Aqueous methanolic Leaf extracts.——————————————————-42

3.4 Results and Discussion of the phytochemical screening on the phyllanthus discoideus leaves and solubility fractions. ———————————————————44

3.5 Results and Discussion on Adsorption TLC Trials of the Absolute methanol-soluble fraction —————————————————————————————-45

3.6 Result of the melting point determination of the isolate from phyllanthus discoideus Leaf———————————————————————————————46

3.7 Results and Discussion on the phytochemical screening of PTLC fractions of Absolute methanol soluble fractions of phyllanthus discoideus

methanol Leaf extract. ———————————————————————-47

3.8 Results and Discussion of the Antimicrobial screening/(MICs)of the PTLC  fraction of the Absolute methanol soluble fraction of phyllanthus discoideus methanol Leaf extract.—————————————————————————————-49

3.9 Results and Discussion of the Absorption Maxima and Absorbance ————————–51

 

CHAPTER FOUR

4.1 CONCLUSION,————————————————————————56

4.2  LIMITATION————————————————————————–56

4.3 RECOMMENDATION—————————————————————57

REFFERENCES——————————————————————————58

APPENDICES———————————————————————————-68

 

CHAPTER ONE

                         INTRODUCTION AND LITERATURE REVIEW

1.0 INTRODUCTION

Traditional medicine has contributed immensely to health care in Nigeria. This is due in part to the recognition of the value of traditional medicinal systems, particularly the Asian systems and the identification of medicinal plant from emergence of indigenous pharmacopoeias, which have environment is probably the least explored in terms of its available in diverse vegetation in our diverse vegetation, cheap and has the potentials of introducing new dimensions into modern medicine (Vogel et al, 2002).

The medicinal value of plants lies in some chemical constituents that produce some definite physiological changes in the human body and the pathogens. The most important of these bioactive constituents of plants are alkaloids, phenolic compounds particularly tannins and favonoids (Hill, 1952). Many of these indigenous medicinal plants are used as spices and food plants.

Current advances in drug discovery technology have increase the volume of researches on the validation of their claimed activities but have not led to a commensurate increase in the number of new drugs.

1.1 THE PHYLLANTHUS GENUS – MORPHOLOGY.

The genus phyllanthus is one of the largest genera of the Euphobiaceae family, of about 833 species of diverse morphology and secondary metabolites.

(Ahn et al. 1995). It is subdivided into the following Eleven (11) sub-genera sub-genera, including Isocladus, Kirganella, Cicca, Emblica, Conani, Gamphidium, Phyllanthodendrion, Xylophylla, Botryanthus, and Ericocus. (Govaerts, et al; 2000). They are widely distributed in most tropical and subtropical countries and have long been used in different folk medicines to treat kidney and urinary bladder disturbances, intestinal infections, diabetes and hepatitis B. In recent years. The interest in phyllanthus species has increased considerably, especially regarding their therapeutic potentials and substantial progress on the chemistry and biological activities of some phyllanthus species have been made (Calixto et al; 1998).

 

1.2 PLANT DESCRIPTION

They are trees, shrubs, or herbs; monoecious or (less commonly) dioecious. Their leaves are alternate, spiral or distichous simple and entire, pinnately veined, stipulated. A locule has 1 -2 seeds and the seed – coat is dry and not ventrally invaginated: endosperm present (Dassanayake 1997).

1.3 CHEMICAL CONSTITUENTS OF GENUS PHYLLANTHUS AND THEIR BIOLOGICAL ACTIVITIES

The members of genus Phyllanthus are diverse in their chemical constituents which include alkaloids, flavonoids, lactones lignans, monoterpenes, diterpenes, triterpenes, sesquiterpenes, sterols, phenolic glycosides, tannins, vitamins, carbohydrates, lipids, phytallates and alkamides. ( Table 1)

1.4 PHYLLANTHUS discoideus PLANT

Phyllanthus discoideus, locally called Atara by the Igbo of Nigeria, marike by the Hausa, Ayin by the Yoruba, and Ujaluanwa by the Igala is a tropical West Africa shrub of about 5 – 6m high belonging to the plant family Euphorbiaceae. It can be found in rain forest zones and the drier savannah areas of Africa. Phyllanthus discoideus is a biennial shrub with greenish brown stem and glabrous leaves which exists in Nigeria’s geographical regions.

 

Phyllanthus discoideus has a pharmacognostic profile as:

Kingdom                                                   Plantae

Division                                              Spermatophytae

Phylum                                                Angiospermae

Class                                                    Dicotyledoneae

Family                                                  Euphorbiaceae

Order                                                      Tubiflorae

Genus                                                    Phyllanthus

Species                                                    discoideus

(www.wikipedia.com)

P.discoideus (Euphorbiaceae) is widely used in tropical West Africa in local medicine. Decoction of leaves of P. discodeus has been  reported to treat various nervous system diseases: anxiety, convulsion, epilepsy and madness. (CarriA re; 2000). The invivo models of epilepsy were used to evaluate the anticonvulsant  properties of the plant and also the potentiation of sleep induced by diazepam in mice for the determination of the sedative properties, (E. Ngobum et al; 2009).

In Guinea Conakry, the bark of this plant is used in the treatment of diarrhea and belly worms (Carriare; 2000). The leaves are used as tonic and in various infections diseases. The bark is given mainly as a purgative and antipyretic. In Southwest part of Nigeria, the bark extract is used locally to cure stomach ache and lumbago. It is also used in the treatment of helminthes infections. The leaves is also used as animal feed, on this note, the nutritional value of Pdiscoideus leaves was evaluated. (Osakwe et al 2000).

According to useful plants of West Tropical Africa by H.M Burkill, the leaves are considered to be antiseptic. Preparations are commonly applied to craw-craw in the West Africa and to itch in Southern Asia. The bark in combination with other herbs is used to cure pile in Western part of Nigeria.

The reputed efficacies of these plants have been experienced and passed on from one generation to the other. Apparently, lack of scientific proof of efficacies claimed by traditional medical practitioners in Nigeria called for the present studies on Phyllanthus discoideus.

 

1.5 AIM OF THE STUDY

This study is aimed at;

  • A scientific and comparative evaluation of the anti-microbial properties of crude extacts of different morphological parts of some leguminous crops and few euphorbiaceous plants.
  • Fractionation of the crude extract with the most interesting antimicrobial activity and screening them for anti-microbial activity
  • Seperation of the constituents of the fraction(s) with the best anti-microbial activity by LSAC
  • The identification of the anti-microbial constituent(s) and
  • Semi- characterization of the active constituent(s)

 

1.6 THE PHENOLIC COMPOUNDS

Phenols sometimes called phenolics are a class of chemical compounds consisting of one or more hydroxyl groups  attached to aromatic hydrocarbon ring. The simplest of the class is phenol (C6H5OH). Although similar to alcohols, the phenols have unique properties and are not classified as alcohols where the hydroxyl group(s) is/are bonded to  saturated carbon atom(s). The plant phenolics can be classified as tannins, flavonoids or phytosterols (plants sterols).

1.6.1.0  TANNINS –DISTRIBUTION AND CHEMISTRY

Bate-smith defined tannins as “water soluble compounds having molecular weights between 500 – 3000, giving the usual phenolic reactions and having special properties such as the ability to precipitate alkaloids, gelatin and other proteins.” Haslam has more recently substituted the term  “polyphenol” for “tannin”, in an attempt to emphasize the multiplicity of phenolic groups characteristic of these compounds. He also noted that molecular weights as high as 20,000 have been reported, and that tannins complex not only with protein and alkaloids but also with certain polysacccarides.

Tannins are distributed all over the plant kingdom. They are commonly found in both gymnosperms as well as angiosperms. Tannins are found in leaf, bud, seed, root and stem tissues, where they may help to regulate the growth of these tissues. They are also found in the heartwood of conifers and may play a role in inhibiting microbial activity, thus, resulting in the natural durability of the wood.

Tannins are classified as hydrolysable and condensed tannins. The hydrolysable tannins are hydrolyzed by weak acids and bases to produce carbohydrates and phenolic acids. For example, gallotannins (i) are the gallic acid esters of glucose in tannic acid (C78H52O46). Condensed tannins, also known as proanthocyanidins, are polymers of  2 to 50 flavonoid units or more that are not susceptible to hydrolytic cleavage. While hydrolysable tannins and most condensed tannins are water soluble, some very large condensed tannins are water insoluble.

 

 

O
O
OH
OH
HO
C
OH
OH
HO
C
O
O
OH
OH
HO
C
O
O
OH
OH
HO
C
O
O
O
OH
OH
HO
C
O
O
(i)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                    

 

 

 

1.6.1.1 Economic  and Pharmacological Relevance of Tannins

1) Tannins are important ingredients in the process of tanning of leather. Oak bark has traditionally been the primary source of tannery tannins, though inorganic tanning agents are also in use today.

2) The anti-inflammatory effect of tannins help to control all indications of gastritis, esophagitis, enteritis and irritating bowel disorders.

3) Tannins have shown potential antiviral (Research 55 (2002), antibacterial (Akiyama et al 2001), and anti parasitic effects (phytochemistry 66 (2005).

4) Tannins have been developed into adhesive for particle board jointly by the Tanzania Industrial Research and Development Organization and Forintek Labs Canada.

5) Tannins are astringents. They can draw out irritants e.g poisons from bee strings from the skin by tightening the pores.

6) Tannins do not only heal burns and stop bleeding; they also stop infection by forming a protective layer over the exposed tissue while they continue to heal the wound internally.

7) Tannin can also be effective in protecting the kidneys.

 

 

 

 

1.6.2.0 FlAVONOID COMPOUNDS AND THEIR PHAMACOLOGICAL IMPORTANCE.

 

The term flavonoids (or bioflavonoids) refers to a class of plant secondary      metabolites derived from 2-phenyl-1,4-benzopyrone (flavonoids) or from  3-     phenyl- 1,4-benzopyrone(isoflavonoids) or from 1,2-benzopyrone (neoflavonoids).

Flavonoids are most commonly known for their antioxidants activity. However, it is now known that the health benefits they provide against cancer and heart disease result from other mechanisms (David stauth, Eurekalert; 2007).

Flavonoids are widely distributed in plants where they serve various functions including production of pigments of various colours in flowers and protection from attack by microbes and insects. The wide spread distribution of flavonoids, their diversity and relatively low toxicity compared to other active plant compound (e.g alkaloids) mean that many animals, including humans, ingest significant quantities in their diets. Flavonoids have been referred to as “nature’s biological response modifier” because of strong experimental evidence of their inherent ability to modify the body’s reaction to allergens, viruses and carcinogens. They show anti-allergic anti-inflammatory, anti-microbial and anti-cancer activity. Consumers and food manufacturers have become interested in flavonoids for their medicinal properties, especially their potential role in the prevention of cancers and cardiovascular disease. The beneficial effects of fruit, vegetables and tea or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins.

  1. i) Quercetin: Quercetin(ii) is a flavonoid and to be more specific, a flavonol that constitutes the aglycone of the glycosides rutin and quercitrin. In studies, quercetin is found to be the most active of the flavonoids and many medicinal plants own much of their activity to their high quercetin content. Quercetin has demonstrated significant anti-inflammatory activity because it directly inhibits the production and release of histamine and other allergic/inflammatory mediators. In addition, it exerts potent antioxidant activity and vitamin C- sparing action. It can be found in Hawthorn-based herbal products which are used for acute symptoms of congestive heart failure.
  2. ii) Epicatechin: Epicatechin (iii) improves blood flow and thus seems for cardiac health.
OH
OH
OH
OH
OH
(ii)
(iii)
OH
OH
OH
OH
OH
(iv)
OH
OH
OH
OH
OH

 

 

 

 

 

 

 

 

 

 

 

 

 

1.7.0 ALKALOIDS CLASSIFICATION AND PHARMACOLOGICAL IMPORTANCE

Alkaloids are a heterogeneous group of compounds which defy any adequate definition. They are cyclic, basic, nitrogenous natural plant compounds containing one or more nitrogen atom. They are usually colorless, crystalline non-volatile solids which are insoluble in water but soluble in aqueous HCl, ethanol, ether, chloroform and other organic solvents. Only very few are liquids which are soluble in water i.e coniine (iv) and nicotine (v) and a few are coloured e.g berberine is yellow. Most alkaloids have a bitter taste and are optically active. Some alkaloids have been isolated from animals like frogs and salamanders. Most alkaloids are physiologically active while some are extremely poisonous.

Alkaloids have been classified through so many rational. Some have classified alkaloids according to their natural sources like the cinchona, ergot-alkaloids etc. Some have classified alkaloids based on the nature of their biosynthetic precursor, as lysine, phenylalanine, tyrosine and tryptophan alkaloids. In chemistry, alkaloids are classified according to the nature of the nucleus present in the molecule. Alkaloids are classified into: Phenylethylamine, Quinoline, Isoquinoline, Phenanthrene, (opium), Pyrrolidine, Pyridine, Indole, Pyrolidine – pyridine and Pyrolidine – piperidine types.

  1. a) Phenylethylamine alkaloids: The basic structure is phenylethylamine(vi). Their outstanding physiological action is to increase the blood pressure. Examples of phenylethylamine alkaloids are mescaline(vii) and ephedrine(viii):
CH3
N
N
(iv)
N
H
CH2CH2CH3
(v)

 

 

 

 

 

                                                                   

 

CH2-CH2-CH3
(vi)
O CH3
O CH3
CH3  
CH2CH2NH2 

 

(vii)

 

 

 

 

 

 

 

 

 

  1. b) Quinoline alkaloids: Their basic structure is quinoline(i). Most alkaloids in this group have anti – malarial activity and antriprotozoal activities.Examples of antimalarial quinoline alkaloids are chloroquine(ii),
Compound R RI
Chloroquine CH(CH2) N(C2H5)2  H
CH3

Sontoquine

CH(CH2)3 N(C2H5)2  CH3 
Amodiaquine
CH2 N(C2H5)2
H

 

 

 

N
N

 

 

 

 

 

 

 

 

 

  1. c) Isoquinoline Group: The basic structure is isoquinoline(iii). The therapeutic values of the isoquinoline alkaloids differ according to the sub-class, which include papaveraceae, protopine, protoberberines and ipecac isoquinolines. The protoberberines which includes berberine(iv) and canadine(v) are anti-bacterial, anti –protozoal, astringent, tonic, and circulatory stimulants
O
O
O
O
N
(iv)
(v)
N
O
O
N
(iii)ilucocorticoids(ivn(istrogen(ii

 

 

 

 

 

 

 

 

  1. d) Phenanthrene Group: They contain phenanthrene(i) nucleus. Most opium alkaloids are in this group. Examples are Morphine(ii), codeine(iii), heroin and ethylmorphine etc. They are very effective pain killer and are used in medicine when pain is absolutely intolerable. Codeine is a fairly good analgesic but used as a cough suppressant and as an ant diarrheal drug.
  2. e) Pyridine Group: The basic nucleus in these alkaloids in pyridine(v). These are mainly lysine derived alkaloids. Examples are coniine, halosalline etc.
(i)
(iv)ilucocorticoids(ivn(istrogen(ii
N
H
H
N
(v)
OH
  OCH3
CH3
N
(iii)
N
(vi)
OH
OH
CH3
N
(ii)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. f) Indole Alkaloids: These alkaloids contain indole(iv) nucleus. Indole alkaloids such as serotonin harmine and reserpine have a sedative effect on central nervous system while a few are hallucinogenic. The Rauwolfia alkaloids (reserpine type) are used as anti-hypertensive agents.
  2. g) Pyrolidine – pyridine alkaloids: The basic structural unit in this class is pyrrolidine (v) linked or fused with pyridine (vi). An example is nicotine. Nicotine stimulates the sympathetic nervous system resulting in an increase in heart rate, dilation pupils and increased blood sugar level. It is also an effective insecticides.
  3. h) Pyrolidine alkaloids: The basic structural unit is the pyrrolidine nucleus(v). The members of the group are hygrine, stachydrine, cuscohygrine.
  4. i) Pyrrolidine- Piperidine Group: The basic structural unit in these alkaloids are the pyrrolidine(i) ring linked or fused with piperidine(ii). Most alkaloids in this groups affect the peripheral nervous system, inhibiting the asympathetic nervous system and stimulating the sympathetic nervous system and the vagus nerve is inhibited. These cause vasodilatation, bronchial dilation and reduced peristalsis. Examples are atropine(iii), hyoscyamine(iv), cocaine(vi) and scopolamine.
(iv)
O 

 

O
Ph 

 

N-CH3
C
CH 

 

OH 

 

(+) iii 

 

O 

 

O
Ph 

 

N-CH3
C
CH 

 

OH 

 

 

 

 

 

 

 

O
C
O
CH3 

 

C
O
O 

 

N 

 

CH3 

 

(vi)

 

 

 

 

 

 

 

 

1.8.0  TERPENES  – STRUCTURE/DISTRIBUTION AND BIOSYNTHESIS

Terpenes is the generic name derived from the word “turpentine” of a group of hydrocarbons with the general formular (C5H8)n  (­­ where n is the number of linked isoprene units) and their oxygen derivatives mainly alcohols, aldehyde and ketone. Terpenes are primarily produced by a wide variety of fragrant plants, particularly conifers, and by some insects such as swallow tail butterflies, which emit terpenes from their osmeterium (www.chemie.fuberlin;2008).

Terpenes are the major components of resins, and of turpentine produced from resins. In addition to their roles as end-products in many organisms, terpenes are major biosynthetic building blocks in nearly every living creature. Steroids, for example, are derivatives of the triterpene squalene.

Terpenes and their derivatives (terpenoids or isoprenoids) are the primary constituents of the essential oils of many plants and flowers.

     The isoprene units are linked together head to tail to form linear chains or to form rings. Isoprene itself exists in the activated forms namely isopentenyl pyrophosphate(IPP)(i) and dimethylallyl pyrophosphate (DMAPP)(ii). IPP is formed from acetyl CoA with mevalonic acid as an intermediate..

 

 

CH3 

 

CH2OH 

 

(v)
CH3 

 

CH2 

 

(vi)

 

 

 

 

 

 

 

 

 

1.8.1 TYPES OF TERPENES AND THEIR PHARMACOLOGICAL IMPORTANCE

Terpenes are classified into the following based on the size of the isoprene chain length (number of isoprene units):

  1. Hermiterpenes: they consist of a single isoprene unit. Isoprene itself is considered the only hermiterpene, but oxygen – containing derivatives such as prenol and isovaleric acid are hermiterpenoids.
  2. Monoterpenoids consist of two isoprene units and have the molecular formular C10 16. Examples are geraniol(v), limonene(vi) and terpineol.
  3. Sesquiterpenes consist of three isoprene units and have the molecular formular C15H24­. Example of sesquiterpenes is farnesol.
  4. Diterpenes are composed of four isoprene units and have the molecular formular C20H They are derived from geranyl geranyl pyrophosphate. Examples of diterpenes are cafestol, kahweol, cambrene and taxadiene. Diterpenes also form the basis of biologically important compounds such as retinol, retinal and phytol. Diterpenes are known to be antimicrobial and anti-inflammatory.
  5. Sesterterpenes, are terpenes having 25 carbons i.e 5 isoprene units are relative to the other sizes. The sester – prefix means half to three, i.e two and a half.
  6. Triterpenes consist of six isoprene units and have the molecular formular C30H48. The linear triterpene squalene is derived from the reductive coupling of two molecules of farnesyl pyrophosphate. Squalene is the natural precursor of lanosterol or cycloartenol which are  the structural precursors to all the steroids.
  7. Tetraterpenes contain eight isoprene units and have the molecular formular C40H64. Biologically important tetraterpenes include the acyclic lycopene the monocyclic gamma-carotene, and the bicyclic alpha and beta-carotenes.
  8. Polyterpenes consist of long chains of many isoprene units. Natural rubber consist of polyisoprene in which the double bond are cis. Some plants produce a polyisoprene with trans double bonds, known as gutta-percha. (http://www.wikipedia.org).

1.8.2 Pharmacological importance of some known Terpenes

  1. i) Menthol is a topical pain reliver and antipyretic
  2. ii) Borneol (monoterpene) is used as a disinfectant and deodorant

iii) Camphor (monoterpenes) is used as counterirritant an aesthetic, expectorant and antipruritic.

  1. iv) Eugenol is a local anaesthetic, which is utilized in dentistry.
  2. V) Periplanone B is the female sex attractant a species of cockroach. Santonin (sequiterpence) is a photosensitizer.

1.9 STEROID CHEMISTRY/DISTRIBUTION

A steroid is a terpenoid lipid characterized by a carbon skeleton with four fused rings, generally arranged in a 6-6-6-5 fashion.

Steroids vary by the functional group attached to these rings and the oxidation state of the rings. Hundreds of distinct steroids are found in plants, animals and fungi. All steroids are made in cells either from the sterol lanosterol(i) (animal and fungi) or the sterol cycloartenol (plants). Both sterols are derived from the cyclization of the triterpene squalene.

HO

 

 

 

 

 

 

 

 

(i)

 

1.9.1 CLASSIFICATION OF STEROIDS

Some of the common categories of steroids are:

  1. a) Animal Steroids: These includes insect steroids e.g ecdysterone and vertebrate steroids. Numerous vertebrate steroids are known. Examples are:
  2. i) Steroid Hormones: These are categorizes into;

– Sex steroids (androgens(i), progesterone(ii) estrogen etc) that produces sex differences or support reproduction.

– Corticosteroids (Glucocorticoids(iii), mineralocorticoids) which regulate many aspects of metabolism, immune function, maintenance of blood volume and renal excretion of electrolytes.

– Anabolic Steroids: Are class of steroids hormones that interact with androgen receptors to increase muscles bone synthesis. There are natural and synthetic anabolic steroids.

  1. ii) Cholesterol: Which modulates the fluidity of cell membrane and is the principle constituent of the plagues implicated in atherosclerosis.
O
O
(ii)
  1. b) Plant Steroids: examples are phytosterols and Brassinosteroids
OH
O
(i)
  1. c) Fungus steroids: Example is Ergosterols(iv)

                    

 

 

 

(iii)
C
O
O
CH2OH
R2 

 

(iv)
 CH3 

 

 

 

 

 

 

 

 

 

 

 

1.10 SAPONIN CHEMISTRY AND DISTRIBUTION

Saponins are glycosides with distinctive foaming characteristics. They are natural detergents found in certain plants. They got their name from the soapwort plant (Saponaria), the root of which was used historically as a soap. Saponins have detergent or surfactant properties because they contain both water soluble and fat soluble components. They consist of a polycyclic aglycone that is either a choline steroid or triterpenoid attached via C3­ and an ether bond to a sugar side chain. The aglycone is referred to as the sapogenin and steroid saponins are called saraponins.

The two major commercial sources of saponins are yucca schidigera, which grows in the arid Mexican desert country of Baja California and  Quillaja Saponaria (soap bark tree), found in arid areas of Chile. The Yucca saponins have a steroid nucleus while the Quillaja saponins have a triterpenoid nucleus.

1.10.1 Physicochemical Properties of Saponins

Saponins are amphipathic compounds, possessing both hydrophilic and lipophilic portions. They are therefore surface active and can be used as emulsifiers. Molecular weight is of the order 180 – 2000 Daltons. Some saponins are bitter while others are sweet. Saponins can undergo enzyme and acid hydrolysis to generate free sugars and the aglycones.

 

1.10.2 Classification and Distribution of Saponins

Saponins are classified on the basis of the sapogenin skeleton present. Two broad classes are identified: the steroid saponins and the triterpenoid saponins. The steroid saponins are also known as the saraponins.

  1. a) Steroidal Saponins: The aglycones (sapogenins) are based on the steroid nulcleus (commonly tetracyclic triterpenoids (i) an example is ginsenoside.

 

 

 

 

RIO
OH
OR2

 

 

RI   =  D – GLUC  (1 – 2 )   D –gluc

R2 = Ara (pyr)  (1 – 6) D – gluc

 

 

These saponins are less widely distributed in nature than the pentacyclic triterpenoid type. Phytochemical surveys have shown their presence in many monocotyledonous families particularly Dioscoreaceae, Amaryllidaceae. In dicotyledons they are found in Solanaceae, Leguminoseae though to lesser extent. (G.E Trease and W.C Evans; 1985).

  1. b) Pentacyclic Triterpenoid Saponins: The aglycones are based on the pentacyclic triterpenoid nucleus and an example is quillaja saponins.

1.10.3    GENERAL APPLICATIONS OF SAPONINS

1) Use in Agriculture: Saponins mainly from quillaja are used in agriculture as wetting agent, nematicide, insect repellant, for increasing crop production and for fungal control.

  1. a) Natural fungi control: A saponins extract from quillaja is used in integrated fungi management system. The saponins form complexes with cell wall sterols of fungi producing a “microporation” which induces cell lyses (Turner; 1960).
  2. b) Natural Nematode Control: A saponin extract from quillaja is used to control nematodes such as meloidogine. It appears nematicide effect is as a result of saponins binding to plant sterol components, inhibiting nematode cholesterol synthesis, essentially for nematode growth and survival.
  3. c) Natural bio-repellant: The addition of saponins with other natural products like cinnamic aldehyde has been proven to be an effective repellant for pest such as insects, arachnids, aphids etc. (A. Osbornne; 1996).
  4. d) Plant Growth Stimulants: Plants growth stimulation research showed that the germination rates of certain seeds increases with the use of quillaja extracts. These effects seem to be related to a better transfer of nutrients of the plant due to changes in the cell wall permeability induced by saponins.

2) Use in Mining: Quillaja saponins reduce acid mist by decreasing the surface tension.

3) Use in Food and Beverage: Natural foaming agents from quillaja extracts are used in the production of slush type drinks, root beer etc. Saponins are generally used as emulsifiers and to achieve low cholesterol in foods.

4) Pharmaceuticals Applications: The excellent surfactant properties of saponins are made use of in pharmaceutical formulations as wetting agents, emulsifying agents and solubilizing agents. It is also used to wash hair due to its good antidandruff properties and reduce hair loss.

 

1.11 CLINICAL SIGNIFICANCE OF PATHOGENIC ORGANISMS

1.11.1.0 CLINICAL SIGNIFICANCE OF STAPHYLOCOCCUS aureus

(MICROCOCCACEAE)

Staphylococcus aureus is a species of the Staphylococcus genus of the Gram-positive Micrococcaceae family of the cocci bacteria. Species of this genus grow characteristically in bunches or clustered colonies which are opaque, round, smooth, and either golden yellow like staphylococcus aureus, or white for other species. Staphylococcus aureus, like other species, is a facultative anaerobic bacterium capable of lactic acid fermentation. Other species include: Staphylococcus epidermidis, Staphylococcus saprophyloccocus saprophticus,  Staphylococcus equroum, etc.

1.11.1.1 Pathogenicity of Staphylococcus aureus

Staphylococcus aureus, is the predominant pathogenic Staphylococcus in man (Pen, 1961). It is associated with bites, burns, wounds, car, eye, skin, gastrointestinal tract, urinary tract, and vagina infections linked to poor blood supply to the affected tissues or organs. (Baron andFine gold, 1990 ;and Pen, 1961). Opportunistic infections in cystic fibrosis are also connected with Staphylococcus aureus (Baron, and Finegold, 1990).

Staphylococcus aureus produces a number of extra-cellular proteinous toxins like haemolysins, leukocidin enterotoxin and exfoliatin. Typical example is the a-toxin, the dermonecrotic toxin. Enterotoxins A-E, with powerful emetic effect in man and certain animals are produced by strains of Staphylococcus aureus. Enterotoxin “B” has, for example, been associated with the Toxic Shock Syndrome” (Schlievert, 1986). Generally Staphylococcus infections occur in individuals with weakened host defense mechanism.

Typical clinical conditions associated vith Staphylococcus aureus infections include:

  • Folliculitis (a superficial   skin   infection   symptomized   as   minute

erythematous nodules around hair follicles), • Sinustitis ( Nasal infection),• Food poisoning, • Bacterial conjuctivitis,•Mastitis, etc.

Many penicillin G resistant strains of  Staphylococcus aureus have been reported. Presently, about 85% of clinical isolates of S. aureus are penicillin G. resistant (Sabath, 1982).   This is because they produce the enzyme penicillinase, a beta lactamase that inactivate the beta lactam ring of penicillins.

 

 

1.11.2 CLINICAL SIGNIFICANCE OF BACILLUS SPECIES (BACILLACEAE)

The Bacillus genus comprises of the spore forming Gram- positive aerobic bacilli. Over 40 species have been isolated. Their natural habitat is usually the soil. B. anthracis is the most virulent specks. It is the etiological agent of anthrax. B, subtilis is highly ubiquitous in the environment. It forms spores which withstand prolonged boiling. It is a common contaminant during bacteria typing as it is able to grow optimally at temperature of 37oC. The colonies of B. subtilis are usually large, flat, and dull with a ground glass appearance (Baron, and Finegold, 1990). Clinically, B.subtilis has been associated with allergic reactions and hypersensitivity pneumonitis due to exposure to contaminated wood dust. (Baron and Finegold, 1990). B. cereus, a closely related species to B. subtilis is associated with food poisoning, septicemia, endocarditis, wounds and pulmonary infections.

(Burdon, et al, 1967).

Bacillus spp. generally produces dermonecrotic and lethal toxins. They also produce lecithinase and emetic toxin. Death in Bacillus spp infection is not necessarily due to their presence in the host but due to the effect of these toxins. B.anihracis produces the three anthrax toxins known as the Lethal Factor (LF), Protective Antigen (PA), and Edema Factor (EF). The LF and PA have caused edema in rodents. The LF has adenylate cyclase activity {i.e increasing concentration of cyclic AMP in the cell (Leppla, 1982). Certain Bacillus spp, have been reported to be resistant to pencillin G, and the cephalosporins (Coonrod, et al, 1971)

1.11.3 CLINICAL SIGNIFICANCE OF PSEUDOMONAS aeruginosa (FAMILY NON-FERMENTATIVE BACTERIA NFB)

The Pseudomonas genus is a very large and heterogeneous genus of Gram.-negative non-fermentative (NFB) aerobic motile rod-Like bacteria with polar flagella. Species include P. aeruginosa and P fluorescence. Pseudomonas aeruginosa is the predominant Pseudomonas in clinical isolates (Howard, et al, 1987). It is known to pose serious problems in the hospitals, pharmaceuticals and toiletries. This is because:

(1) They have a highly impermeable and protective outer

lipopolysaccharide membrane which makes it difficult for antibiotics and disinfectants to penetrate the cell.

(2) They possess a resistance gene known as the P-factor multiple, resistance plasmid” that encodes multiple resistance mechanisms active against antibiotic agents (Pen, 1991)

Pseudomonas aeruginosa causes clinical conditions like nosocomial infections; respiratory tracts infections, and opportunistic infections of wounds and burns. Others include; ecthyma, gangrenosum –a skin lesion in infants and immuno-compromised patients, otitis external [swimmer’s ear], follicolitis, and corneal ulcers an eye infection which progress if not treated, to panophthalmitis. Panophthalmitis is an inflammation involving all the tissues of the eyeball that leads finally to blindness. Meningitis and urinary treat infections are also associated with Pseudomonas aeruginosa.

1.11.3.1 Pathogenicity of Pseudomonas aeruginosa

The pathogenicity of Pseudomonas aeruginosa has been associated with its ability to produce virulent factors like proteolytic enzymes, Exotoxin A2, and Phospholipases, The proteolytic enzymes e.g Elastin are thought to be responsible for the haemorrhagic lessions seen in skin, internal organs and cornea due to invasion by Pseudomonas aeruginosa. Exotoxin A interferes with protein synthesis by inhibiting the action of the elongation factor just like the diphtheria toxin (Howard et al, 1987,), Pseudomonas aeruginosa is resistant to kanamycin and penicillin. G.

1.11.4.0 CLINICAL SIGNIFICANCE OF KLEBSIELLA pneumonia (ENTEROBACTERIACEAE)

The enterobacteriaceae is a large family of non-motile Gram-negative non-spore forming bacilli. They are oxidase negative and also ferment glucose with the production of gases and acids like 3-methylpropanoic acid, and an acidic polysaccharide containing galacturoac and glucoronic acid moieties (Shimada, et al, 1997, and Kim, et al, 2003 ). The enterobacteriaceae are classical pathogens associated with blood, cerebrospinal fluids, respiratory tract, urinary tract, gastro­intestinal tracts and soft tissue infection in immune-compromised hosts. (Baron and Finegold, 1990), Members of the Enterobacteriaceae include klebsiella pneumonia, Escherichia coli, Salmonella spp, Shigella spp, etc. They could be aerobic and facultatively anaerobic.

The klebsiella genus includes K. Pneumonia, K. oxytoca, K. ozaenae (a rare species). 95% of clinically isolated klebsiella are K. pneumonia. They are responsible for pneumonia, lung abcess, septicemia, meningitis, and infections of the intestinal tracts, bilary tract, urinary tract and soft tissues.

1.11.4.1Pathogenicity of Klebsiella pneumonia

The pathogenicity of K. Pneumonia, is associated with the presence of

(1) Cell wall receptors that enable it to attach to host cells and also protect organism:, against phagocytosis and intracellular killing.

(2) Large polysaccharides capsule which protects it from phagocytosis and also directly suppresses immune response.

(3) Production of endotoxins Ampiciliin and carbenicillin resistants strains have been reported (Baron and Finegold, 1990).

 

 

 

 

1.11.5 CLINICAL SIGNIFICANCE OF SALMONELLA paratyphi (ENTEROBACTERIACEAE)

Salmonella is a genus of the Enterobacterloceae family which includes species like Salmonella typhi (typyhoid samonella) and the non-typhoid salmonella like Salmonella paratyphi, S. choleraesus, etc. Both the typhoid and non-typhoid salmonellae   are   Gram-negative   reds,   mostly   motile.   They   are   aerobic   and facultatively anaerobic.

Salmonella typhi, causes typhoid fever, while the non-typhoid salmonella like Salmonella paratyphi, causes enteric fever which is similar to typhoid fever but milder. Generally, salmonella infections are acquired by humans through ingestion of contaminated foods and water. Diarrhea, due to non-typhoid salmonella infections is believed to be related to the stimulation of adenylcyclase system. The stimulation of adenylcyclase systems is believed to be due to the production of entero-toxins by the Salmonella spp or by prostaglandins released from the polymorphonuclear leukocytes present in inflammatory response (Jiwa, 1981). The Salmonella generally penetrate intestinal mucosa, and invade intestinal epithelium.

Salmonella spp, are generally resistant to penicillin G. Ciprofloxacin, is now recommended for their treatment

1.11.6.0 CLINICAL SIGNIFICANCE OF ESCHERICHIA coli

 (ENTEROBACTERIOCEAE)

Species of the Escherichia genus o the Entorobacterioceae family are non-spore forming Gram-negative bacteria. Escherichia coli is the commonest species. Other species include; E. blattae, E. fergosonil, E. hermanni, and E. vulneris, E. coli, is associated with clinical conditions like; diarrhea, gastroenteritis, cystitis, pyelitis, pyelonephritis, appendicitis peritonitis, gallbladder infections, seplicemia, neo-natal meningitis, pneumonia, and endocarditis (Howard, et al, 1987).

 

 

 

 

1.11.6.1 The Pathogenicity of Escherichia coli

The pathogenicity of E. coli is due to its production of two enterotoxins: LT and ST. LT, is heat labile toxin while ST, is heat stable. Their production is plasmid mediated. (Howard, et al, 1987).

LT is similar to the cholera toxin in structure, antigenicity, and mode of action. LT, like the cholera toxin, binds to specific Gm, gangliosides, or the epithelial cells of the small intestine and stimulates the production of the enzyme, adenylcyclase. Adenylcyclase cause increased production of cyclic Adenosine monophosphate  cAMP which stimulates fluid transport into the lumen of the bowel resulting in diarrhea. ST, like LT, binds to specific receptors on the cell surface of intestinal cells but activates the enzyme Guanylate cyclase which leads to increased production of cyclic guanosine monophosphate cGMP, the mediator responsible for diarrhea.

(Howard, et al, 1987). Ampicillin and Carbenicillin resistant strains of E. coli have been reported.  (Howard, et al, 1987; and Baron and Finegold, 1990).

1.11.7.0 CLINICAL SIGNIFICANCE OF CANDIDA albicans (YEAST)1

Candida is a genus of the non-dermaliaceous fungi imperfecti class of yeasts. Other genera include Blastoschizomyces, Cryptococcus, Rhodotorula, Torulopsis, and Trichosporon. Candida and Cryptococcus are the only pathogenic yeasts though other genera are now associated with opportunistic yeast infections in immuno- compromised individuals. (Howard, et al, 1987)

Candida albicans is a species of the Candida genus. It is part of the normal microflora of both the gastro-intestinal tract and the mucocutaneous areas (Marples, 1995). It is also present in the vagina of about 5% females. “Candidiasis”, a general term used for infections caused by Candida spp, leads to extensive array of clinical symptoms like: thrush, glossitis, stomatitis, vaginitis, esophagitis, diaper disease, eczema, meningitis, asthma, and endocarditis.

Other species of Candida includes: C.glabrata,  C.kefyr, C.guiliermondii, and C.krusei among others.

 

1.11.7.1 The Pathogenicity of Candida albicans

Candida albicans is pathogenic because when it adheres to receptor sites, the glycoproteins on its surface interact with those of the host’s epithelial cells eliciting inflammatory reactions like fever, chemotaxis of leucocytes, release of histamine from most cells, and other effects on humoral and cell-mediated immunity which are observed in candidiasis. (Guentzel, et al, 1985)

Candida albicans is known to be susceptible to the polyene antifugal agents like Amphotericin B, and Nystatin.s

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