In vitro ANTIOXIDANT ACTIVITY AND PHYTOCHEMICAL PROPERTIES OF METHANOL, ETHANOL AND HEXANE SOME EXTRACTS OF Alstoniaboonei LEAVES

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ABSTRACT

In traditional setting, Alstoniaboonei is used for the treatment of different ailments, this has been attributed to its rich phytochemical and antioxidant properties.The objective of the study was to evaluate the phytochemical properties and antioxidant activity of the methanol, ethanol and hexane extracts of Alstoniaboonei leaves. Qualitative analysis of phytochemical constituents such as tannins, saponins, cardiac glycosides flavonoids and phenols were carried out. Quantitative analysis of total phenolics, saponins and flavonoids was performed by well-known test protocol. Antioxidant activity reveals the presence of reactive oxygen species which helps to scavenge free radicals that are present in the body.  The in vitro antioxidant activity was studied by diphenyl-2-picryl-hydrazyl (DPPH), ferric reducing antioxidant power (FRAP), reducing power (RP) and ferrous ion metal chelating. Results obtained for phytochemical studies revealed that flavonoids were  highest in ethanol extract(101.6±1.35) while hexane extract (56.6±1.510) had the lowest content. Also phenol was highest in methanol extract (262±4.93) and lowest in ethanol extract (172.7±1.66). Result for total tannins (271±6) and proanthocyanidin (200.7±2.19) indicates that  methanol and ethanol extract had the highest amount while hexane extract  had and the lowest amount. IC5o values obtained by DPPH for ethanol extracts of Alstoniaboonei (2.362µg/ml), when compared with other extracts, and was significantly (P>0.05) lower than that of the standard vitamin C (12.81µg/ml). Also, the ethanol extract(0.026µg/ml) showed low IC50 value for ferrous ion metal chelating ability when compared with other extract, but this value was non significantly(P>0.05) lower than the standard(4.11µg/ml). Therefore,  Alstoniaboonei has a great potential for use as a natural source of  antioxidant and also help in the treatment of various ailments, or against free radical damage.


 

 

 

 

TABLE OF CONTENTS

Title page    -        -        -        -        -        -        -        -        -        -        i

Certification         -        -        -        -        -        -        -        -        -        ii

Dedication -        -        -        -        -        -        -        -        -        -        iii

Acknowledgement         -        -        -        -        -        -        -        -        iv

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

Abstract      -        -        -        -        -        -        -        -        -        -        vii

CHAPTER ONE

1.0.         Introduction         -        -        -        -        -        -        -        -        -        1

1.1.         Literature review  -        -        -        -        -        -        -        -        3

1.2.         Geographical distribution        -        -        -        -        -        -        -        4

1.3.         Ecology      -        -        -        -        -        -        -        -        -        4

1.4.         Medicinal and non medicinal uses     -        -        -        -        -        4

1.5.         Common names/local names   -        -        -        -        -        -        6       

1.6.         Phenolics    -        -        -        -        -        -        -        -        -        9

1.7.         Flavonoids  -        -        -        -        -        -        -        -        -        11

1.8.         Antioxidants        -        -        -        -        -        -        -        -        18

1.9.         Aimand objectives         -        -        -        -        -        -        -        22     

1.10.    Specific objectives         -        -        -        -        -        -        -        -        22


CHAPTER TWO

2.0.         Materials and methods  -        -        -        -        -        -        -        23

2.1.          Reagents              -        -        -        -        -        -        -        -        23

2.2. Sample collection    -        -        -        -        -        -        -        -        25

Preparation of plant extract    -        -        -        -        -        -        -        25

2.5     Extraction of the leaves using different solvents.          -        -        -        26

2.6     Antioxidants assays      -        -        -        -        -        --       -        26

2.6.2  Reducing power assay   -        -        -        -        -        -        -        28

2.6.3  Ferric reducing antioxidant power (frap assay)   -        -        -        28

2.6.4  Ferrous ion chelating ability              -        -        -        -        -        29

2.7     Quantitative determination of phytochemicals     -        -        -        29

2.7.1  Total phenolic content   -        -        -        -        -        -        -        30

2.7.2  Total flavonoids   -        -        -        -        -        -        -        -        30

2.7.3 Total proanthocyanidin  -        -        -        -        -        -        -        30

2.7.4 Total tannins        -        -        -        -        -        -        -        -        -        30

2.8     Qualitative determination of pytochemicals        -        -        -        -        31

CHAPTER THREE

3.1  Percentage  yield            - -        -        -        -        -        -        -        32

3.2 Qualitative phytochemicals                   -        -        -        -        -        -        33

3.3   Quatitative phytochemicals      -        -        -        -        -        -        33     

3.4 Reducing power       -        -        -        -        -        -        -        -        34

3.5 ferrous ion metal chelating -        -        -        -        -        -        -        35

3.6 diphenyl ̵ 2 ̵ picrylhydrazyl (DPPH)     -        -        -        -        -        36

3.7 ferric reducing antioxidant power ( FRAP) assay    -        -        -        37

 

CHAPTER FOUR

4.2. Discussion     -        -        -        -        -        -        -        -        -        38

4.2. Conclusion    -        -        -        -        -        -        -        -        -        41

References -        -        -        -        -        -        -        -        -        -        42

 









 

CHAPTER ONE


1.0     INTRODUCTION

          A medicinal plant is any plant used for the extraction of pure substances either for direct medicinal use or for hemi-synthesis of medicinal compounds which can be used for therapeutic purpose or as a precursor for the synthesis of useful drugs (Sofwora, 1982). Approximately 10% of these plants are used either as food or for medical purposes (Borris, 1996)

Medicinal plants have been the mainstay of traditional herbal medicine amongst rural dwellers worldwide since antiquity to date. The therapeutic use of plants certainly goes back to the Sumerian and the Akkadian  Civilizations in about the third millennium BC. Hippocrates (ca.460-377BC) one of the ancient authors who described medicinal natural products of plant and animal origins listed approximately 400 different plant species for medicinal purposes. Natural products have been an integral part of the ancient traditional medicine systems e.g Chinese, Ayurvedic and Egyptian (Sarker and Nahar 2007). Over the years they have assumed a very central stage in modern civilization as natural source of chemotherapy as well as amongst scientist in search of alternative sources of drugs.

          According to the World Health Organization, a medicinal plant is any which, in one or more of its organs, contain substances that can be used for therapeutic purpose, or which are precursors for chemo-pharmaceutical semi synthesis. Such a plant will have its parts including leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds employed in the control or treatment of a disease condition and therefore contain chemical components that are medically active.

          Medicinal plants have been found to contain bioactive compounds called phytochemicals (phyto from Greek-phyto meaning plant) or phytoconstituents and are responsible for protecting the plant against microbial infections or infestations by pest (Abo et al., 1991;Lui, 2004;Nwezeet al.,2004; Doughariet al., 2009). They also contain secondary metabolite that can protect humans against diseases (Kumar et al., 2009). The study of natural products on the otherhand is called phytochemistry. Phytochemical have been isolated and characterized from fruits such as grapes and apples, vegetables such as broccoli and onion, spices such as turmeric, beverages such as green tea and red wine as well as many other sources (Doughari and Obidah, 2008; Doughariet al.,2009). Some important groups of thesePhytochemical (secondary metabolites) are involved in many in-vitro studies and assessment of haematological parameters, antioxidant, antimicrobial and analgesic effect (Finar, 1986).

          This indigenous knowledge, passed down from generation to generation in various parts of the world, was significantly contributed to the development of different traditional systems of medicine. This exploration of biologically active products have played an important role in finding New Chemical Entities (NCEs) for example approximately 28% of NCEs n=between 1981 and 2002 were natural products or natural product derived (Newman et al., 2003).

          Alstoniaboonei, a member of Apocynaceae family, is a deciduous plant found abundantly around Africa, from the rain forest of Senegal to Western Cameroon extending to Egypt in North and Uganda and Zaire in the East it is known by several common names in different localities (Amole and llori, 2010). Alstoniabooneiis reported to have adverse uses, it is given to ameliorate toothache and after child delivery, to aid in expelling the placenta. It is applied topically to reduce Oedema and to clear suppurant sores and exposed fractures. It is also used for ulcers and as a remedy for snake bite and arrow poison (Akinmoladunet al.,2007).

          Traditional African medicine has also reported the use of Alstoniaboonei for treatment of chronic diarrhoea, dysentery, fever, pain and intestinal disorders (Amole and llori, 2010).

          Numerous therapeutic properties have been attributed to Alstoniaboonei like antifugal, antibacterial, antiviral antithrombosis, anti-tumor. Anti-inflammatory, analgesic, antioxidant and antipyretic activities (Olayideet al., 2000; Akinloyeet al., 2013).

 

1.1     LITERATURE REVIEW

Scientific classification

Kingdom:Plantae

(unranked):Angiosperms

(unranked):Eudicots

(unranked):Asterids

Order:gentianales

Family:Apocynaceae

Genus:Alstonia

Species: A.boonei

Bionomialname:Alstoniaboonei De wild. Burkhill H.M1985


1.2     GEOGRAPHICAL DISRIBUTION

Alstoniaboonei De Wild (Devil tree) of the family Apocynaceae, is an African evergreen deciduous crude medicinal tree up to 45m tall with bole branchless, fluted at the base with steep buttresses. They are mostly in forest up to 1200m altitude at places like Uganda, Gambia, Western Ethiopia and Senegal (Afolabiet al., 2007). It is found in dry peripheral Semi-evergreen Guineo-Congolian forest and transitional rainforest. It occurs in similar habitats and in swamp and riverine forest. Alstoniaboonei requires large amounts of light and colonizes gaps in the forest. It has plenty of natural regeneration in young secondary forest.

 

1.3     ECOLOGY

          In Nigeria Alstoniaboonei occurs in moist low land and forest but may extend into drier types including gentle to steep, rocky hillsites in Liberia, but most commonly found scattered or in small groups in wet or marshy places that are occasionallyinundated it’s the tree of the swampy high forest in west Africa, it can tolerate a wide range of sites, from rocky hillslides to seasonal swamps in general it prefers damp situations but it grows satisfactorily on well drained slopes.

 

1.4     MEDICINAL AND NON MEDICINAL USES

Alstonia a genus of the family Apocynaceae to which many other medicinally important plants belong like Rauwolfia Serpentine and Vincarosea which have been producing well known remedy for various disorders like Schizophrenia and Cancer (Das et al., 2014). The traditional method of medications has long been known in the developing countries like India and China.

          Previous study detected the presence of secondary metabolites such as alkaloids, tannins, Saponnins, resins, flavonoids, steroids, glycosides and terpenoids in the pulverized dried leaves (Kuceroet al.,1972;Fashola and Egunyemi., 2005; Afolabiet al., 2007). However the various species of Alstonia are highly rich in alkaloids, steroids and triterpenoids and phenolic compounds which contributes to the toxicity of Alstoniaboonei. Moreover the plant was found to contain poisonous alkaloid comprising ditamine, echitamine and echitamidine (Adoteyet al, 2012).

          It’screamy white wood is utilized for light constructions in interior Joineries, Furniture, household equipments, sculptures, boats, boxes, matches, pencils, moldings and plywood. It serve as shade tree for people who want to relax under a shade and take their drinks, as well as in the cultivation of banana plantains. Famous Asante stools in Ghana and sound boxes of musical instruments of Nigerian Yorubasare made from Alstoniaboonei (Olajideet al.,2000). It is not edible as food, it possess roots, stems, banks, leaves, fruits, seeds, flowers and latex which are claimed to have medicinal properties in some cultures and climes. The plants and its latex are applied in traditional medicine for treating many diseases. There are records on the use of alcoholic or aqueous extracts of most parts of Alstoniaboonei. The stem bark is utilized for treating febrile illness, painful urination, rheumatic conditions and Jaundice (Ojewole, 1984; Asuzu  andAnago  1991), malaria fever (Phillipsonet al., 1987; Majekodunniet al., 2008; Bello et al., 2009; Mazekodunmi and Odeku, 2009).Intestinal helminthes (Wescheet al., 1990). Rheumatism, reversible anti-fertility (Rajiet al., 2005), and hypertension (Olajideet al., 2000; Terashima, 2003; Abel and Busia, 2005; Belti, 2007).As an anti-venon against snake bite and antidote against arrowpoisoning.Other pharmacological uses are anti-inflammatory, antipyretic and as an analgesic (Olajide., 2000).

 

1.5     COMMON NAMES/LOCAL NAMES

English (stool wood, cheese wood, pattern wood, alstonia, luganda (mujua, mubaJandalabi, mukoge, musoga), Ghana (sinupo), Cameroon (botuk), Ivory Coast (emien). (Amole and llori, 2010), Yoruba (Ahun), Igbo (Egbu-ora), Edo (Ukhu) and Urhobo (Ukpukunu), (Dalziel., 1997; Bever, 1986).

PLANT DESCRIPTION

Alstoniaboonei is a large deciduous tree, up to 45 m tall and 1.2 m in diameter; bole often deeply fluted to 7 m, small buttresses present; bark greyish-green or grey, rough; slash rough-granular, ochre-yellow, exuding a copious milky latex; branches in whorls.

Leaves in whorls of 5-8, simple, subsessile to petiolate, stipules absent; petiole 2-10 (max. 15) mm long, stout; blade oblanceolate to obovate, rarely elliptic, 7-26 x 3-9.3 cm; apex acute to rounded or sometimes emarginate; base narrowly cuneate; margins entire, sub-coriaceous to coriaceous, dark shiny green top surface, light green on under surface; midrib more prominent below.

Inflorescence terminal, compound with 2-3 tiers of pseudo-umbels;

primary peduncles 0.5-7 cm long, greyish pubescent; bracts ovatetriangular, 1-1.5 mm long, pubescent; pedicels about 5 mm long. Flowers regular, hermaphrodite, pentamerous; calyx cupular tube about 1 mm long; lobes ovate, about 1.5 mm long, spreading; corolla pale green tube up to 14 mm long; lobes slightly obliquely ovate, up to 6 mm long and wide, pubescent outside.

Fruit formed by 2 pendent green follicles up to 60 cm long, longitudinally striate, dehiscing lengthways while on the tree; seeds numerous, flat, about 4 x 2 mm, with tufts of hair at each end 10 mm long. ‘Alstonia’ is named after Dr C. Alston (1685-1760), a professor of botany at Edinburgh University.

 

1.6     PYTOCHEMICALS

Phytochemicals  (from  the  Greek  word  phyto, meaning  plant)  are  biologically  active,  naturally occurring  chemical  compounds  found  in  plants, which provide health benefits for humans further than  those  attributed  to  macronutrients  and Micronutrients (Harvey,2004).  Theyprotect  plants  from disease and damage and contribute to the plant’s colour,  aroma  and  flavour.  In  general,  the  plant chemicals  that protect  plant  cells  from environmental  hazards  such  as  pollution,  stress, drought, UV exposure and pathogenic attack are called  as  phytochemicals (Mathai, 2000).  Recently,  it  is clearly  known  that  they  have  roles  in  the protection  of  human  health,  when  their  dietary intake  is significant.  More  than  4,000 phytochemicals  have  been  catalogued and  are classified  by  protective  function,  physical characteristics and  chemical characteristics (Mueller,1999) and About  150  phytochemicals  have  been  studied  in detail. In wide-ranging dietary phytochemicals are found in fruits, vegetables, legumes, whole grains, nuts, seeds, fungi, herbs and spices (Mathai, 2000).  Broccoli, cabbage, carrots,  onions,  garlic,  whole  wheat bread,  tomatoes,  grapes,  cherries,  strawberries, raspberries,  beans,  legumes,  and  soy  foods  are common  sources[6]. Phytochemicals  accumulate in  different  parts  of  the  plants,  such  as  in  the roots,  stems,  leaves,  flowers,  fruits  or  seeds. Many  phytochemicals particularly  the  pigment molecules,  are  often  concentrated  in  the  outer layers  of  the  various  plant  tissues.  Levels  vary from  plant  to  plant  depending  upon  the  variety, processing,  cooking  and  growing  conditions(Moorachian,2000). Phytochemicals  are  also  available  in supplementary forms, but evidence is lacking that they  provide  the  same  health  benefits  as  dietary phytochemicals .These  compounds  are known  as secondary plant  metabolites  and  have  biological  properties such as  antioxidant activity, antimicrobial effect, modulation  of  detoxification  enzymes, stimulation  of  the  immune  system,  decrease  of platelet  aggregation  and  modulation  of  hormone metabolism  and  anticancer  property.  There are more than  thousand  known  and  many  unknown phytochemicals.  It is  well-known  that  plants produce  these  chemicals  to  protect  themselves, but  recent researches demonstrate  that  many phytochemicals can  also  protect  human  against diseases (Narasinga,2003).


Classification of Phytochemicals

The exact classification of phytochemicals could have not been performed so far, because of the wide variety of them.  In  recent  years Phytochemicals  are  classified  as  primary  or secondary  constituents,  depending  on  their role in plant metabolism. Primary constituents include  the  common  sugars,  amino  acids, proteins,  purines  and  pyrimidines  of  nucleic acids, chlorophyll’s etc. Secondary constituents are  the  remaining  plant  chemicals  such  as alkaloids,  terpenes,  flavonoids,  lignans,  plant steroids,  curcumines,  saponins,  phenolics,

flavonoids and glucosides.  Literature survey  indicate  that  phenolics  are  the  most numerous  and  structurally  diverse  plant phytoconstituents.


 

1.7     PHENOLICS

Phenolic phytochemicals are the largest category of phytochemicals and the most widely distributed in the plant kingdom. The three  most important  groups  of  dietary  phenolics  are flavonoids,  phenolic  acids,  and  polyphenols.

Phenolic  are  hydroxyl  group  (-OH)  containing class  of  chemical  compounds  where  the  (-OH) bonded  directly  to  an  aromatic  hydrocarbon group.  Phenol  (C6H5OH)  is  considered  the simplest  class  of  this  group  of  natural compounds. Phenolic compounds are a large and complex group of chemical constituents found in plants (Walton et al.,2003). They are plant secondary metabolites, and they have an important role as  defence  compounds.  phenolics exhibit several  properties beneficial  to  humans  and  its  antioxidant properties are important in determining their role as protecting agents against free radical-mediated disease  processes.  Flavonoids  are  the  largest group  of  plant  phenols  and  the  most  studied (Dia and Mumper.,2010). Phenolic acids form a diverse group that includes the widely distributed hydroxybenzoic and hydroxycinnamic acids.  Phenolic  polymers, commonly  known  as  tannins,  are  compounds  of high  molecular  weight  that  are  divided  into  two classes: hydrolysable and condensed tannins.


Fig 2. Phenol/chemical compound/Britannica.com


 

1.8    Flavonoids

Flavonoids are polyphenolic compounds that are ubiquitous in nature. More than 4,000 flavonoids have  been  recognized,  many  of  which  occur  in vegetables,  fruits  and  beverages  like  tea,  coffee and fruit drinks. The flavonoids appear to have played  a  major  role  in  successful  medical treatments  of  ancient  times,  and  their  use  has persisted  up  to  now.  Flavonoids  are  ubiquitous   vascular  plants  and  occur  as  aglycones, glucosides and methylated derivatives. More than 4000 flavonoids have been described so far. within the parts of plants normally consumed  by humans  and  approximately  650  flavones  and 1030  flavanols  are  known(Harborne and Baxter, 1999).  Small  amount  of aglycones  (i.e.,  flavonoids  without  attached sugar)  are  frequently  present  and  occasionally represent  a  considerably  important  proportion  of the total flavonoid compounds in the plant . The  six-membered  ring  condensed  with  thebenzene  ring  is  either  -pyrone  (flavones  and flavonols )   or  its  dihydroderivative  (flavanone and flavan-3-ols ). The position of the benzenoid substituent divides the flavonoids into two classes:  flavone (2-position) and isoflavone  (3-position).  Most  flavoniods occur  naturally associated  with  sugar  in  conjugated  form  and, within  any  one  class,  may  be  characterized  asmonoglycosidic, diglycosidic, etc. The glycosidic linkage is normally located at position 3 or 7 and the  carbohydrate  unit  can  be  L-rhamnose,  Dglucose,  glucorhamnose,  galactose  or arabinose (Pretorius et al.,2003).

Quercetin:


Fig 3 Quercetin: A versatile flavanoid

 

1.1     Tannin

From  a  chemical  point  of  view  it  is  difficult  to define  tannins  since  the  term  encompasses  some very  diverse  oligomers  and  polymers.  It might be said that the tannins are a heterogeneous group  of  high  molecular   weight polyphenolic compounds  with  the  capacity  to  form  reversible and  irreversible  complexes  with  proteins (mainly),  polysaccharides  (cellulose, hemicellulose,  pectin,  etc.),  alkaloids,  nucleic acids  and  minerals,  etc(Schofield et al.,2001) .  On  the  basis  of their  structural  characteristics  it  is  therefore possible  to  divide  the  tannins  into  four  major groups:  Gallotannins, ellagitannins,  comtannins, and condensed tannins(Manga,1988).

Gallotannins  are  all  those  tannins  in  which galloyl  units  or  their  meta-depsidic  derivatives are  bound  to  diverse  polyol-,  catechin-,  or triterpenoid units.

Ellagitannins  are  those  tannins  in  which  at least  two  galloyl  units  are  C–C  coupled  to  each other, and do not contain a glycosidically  linked catechin unit.  Complex  tannins  are  tannins  in  which  a catechin  unit  is  bound  glycosidically  to  a gallotannin or an ellagitannin unit. Condensed  tannins  are  all  oligomeric  and polymeric  proanthocyanidins  formed  by  linkage of  C-4  of  one  catechin  with  C-8  or  C-6  of  the next monomeric catechin. Tannins  are  found  commonly  in  fruits  such  as grapes,  persimmon,  blueberry,  tea,  hocolate, legume  forages,  legume  trees  like  Acacia  spp., Sesbania  spp.,  in  grasses  i.e;  sorghum,  corn, etc.  Several  health  benefits  have  been recognized  for  the  intake  of  tannins  and  some epidemiological  associations  with  the  decreased frequency  of  chronic  diseases  have  been established (Serranoet al.,2009).In  medicine,  especially  in  Asian  (Japanese  and Chinese)  natural  healing,  the  tannin-containing plant  extracts  are  used  as  astringents,  against diarrhoea,  as  diuretics,  against  stomach  and duodenal  tumors, and  as  anti-inflammatory, antiseptic,  antioxidant  and  haemostatic pharmaceuticals (Dolaraet al.,2005).  Tannins  are  used  in  the dyestuff  industry  as  caustics  for  cationic  dyes (tannin dyes), and  also  in the production of  inks (iron gallate ink). In the food industry tannins are used to clarify wine, beer, and fruit juices.  Other industrial uses of tannins  include textile dyes, as antioxidants  in  the  fruit  juice,  beer,  and  wine industries,  and  as  coagulants  in  rubber Production (Gyamfi  and Aniya., 2002).  Recently the tannins  have attracted scientific interest, especially  due to the increased incidence  of  deadly  illnesses  such  as  AIDS  and various  cancers.  The  search  for  new  lead compounds  for  the development  of  novel pharmaceuticals  has  become  increasingly important,  especially  as  the biological  action  of tannin-containing  plant  extracts  has  been  well  documented(Muller-Harvey,1999).


Fig 4: Phytojournal.com

 

1.1 Alkaloids

Alkaloids  are  natural  product  that  contains heterocyclic  nitrogen  atoms,  are  basic  in character. The name of alkaloids derives from the “alkaline” and it was used to describe any nitrogen-containing   base.  Alkaloids   are naturally synthesis by a large numbers of organisms, including animals, plants, bacteria and fungi.  Some  of the  fires  natural  products  to  be isolated  from  medicinal  plants  were  alkaloids when they first obtained from the plants materials in  the  early  years  of  19th century,  it  was  found that  they  were  nitrogen  containing  bases  which formed  salts  with  acid.  Hence  they  were  known as  the  vegetable  alkalis  or  alkaloids  and  these alkaloids  are  used  as  the  local  anaesthetic  and stimulant as cocaine. Almost all the alkaloids have a bitter taste.  The  alkaloid  quinine  for example is one of the bitterest tasting substances known  and  is  significantly  bitter  (1x10-5)  at  a molar concentration(Georgeet al.,2002). Alkaloids  are  so  numerous  and  involve  such  a variety  of  molecular  structure  that  their  rational classification  is  difficult.  However,  the  best approach  to  the  problem  is  to  group  them  into families,  depending  on  the  type  of  heterocyclic ring  system  present  in  the  molecule.  For historical  reasons  as  also  because  of  their structural  complexities,  the  nomenclature  of alkaloids  has  not  been  systematized.  The  names of  individual  members  are  therefore generally derived from the name of the plant in which they occur,  or  from  their  characteristic  physiological activity. The various classes of alkaloids according to  the  heterocyclic  ring  system  they  contain  are listed below.

Pyrrolidine alkaloids: they contain pyrrolidine(tetrahydropyrrole)  ring  system. E.ghygrine  found  in erythroxylum coca leaves. Pyridine alkaloids:  they have piperidine (hexahydropyridine)  ring  system.  E.g coniine,  piperine and isopelletierinepyrrolidine-pyridine  alkaloids:  The  heterocyclic  ring system  present  in  their  alkaloids  is  pyrrolidinepyridine.E.gmyosmine, nicotine  alkaloid  found  in pyridine-piperidinealkaloids:This  family  of  alkaloids contains a pyridine ring system join to a piperidine ring system  the  simplest  member  is  anabasine  alkaloid isolated from poisonous Asiatic plant anabasis aphyllan.

Quinoline Alkaloids:  These have the basic heterocyclic ring system quinoline .E.g quinine occurs in the bark of cinchona tree.It has been used for centuries for treatment of  malaria.Synthetic  drugs  such  as  primaquinine   have largely replace quinine as an anti-malarial.

Isoquinoline  alkaloids:  They  contain  heterocyclic  ring system isoquinoline. E.g opium alkaloids like narcotine, papaverine, morphine, codeine, and heroine

Fig 5 LON-CAPA. Botany online: the secondary metabolism of plants-Alkaloids

 

1.1 Saponin

Saponins  are  a  group  of  secondary  metabolites found  widely  distributed  in  the  plant  kingdom They  form  a  stable  foam  in  aqueous  solutions such  as  soap,  hence  the  name  “saponin”. Chemically,  saponinsasa  group  include compounds  that  are  glycosylated  steroids, triterpenoids,  and  steroid  alkaloids.  Two  main types  of  steroid  aglycones  are  known,  spirostan and  furostan  derivatives.  The  maintriterpeneaglycone  is  a derivative  of  oleanane  (Traoreet al.,2000).  The carbohydrate part consists of oneor  more  sugar  moieties  containing  glucose, galactose,  xylose,  arabinose,  rhamnose,  or glucuronic  acid  glycosidically  linked  to  a sapogenin  (aglycone).  Saponins  that  have  one sugar  molecule  attached  at  the  C-3  position  are called  monodesmosidesaponins,  and  those  that have  a  minimum  of  two sugars,  one  attached  to the C-3 and one at C-22, are called bidesmosidesaponins(Lacailleet al.,2000)

 Saponin: Wikipedia,the free encyclopedia

 

1.1 ANTIOXIDANTS

An  antioxidant  is  a  molecule  capable  of  slowing  or preventing  the  oxidation  of  other  molecules.  Oxidation  is a  chemical  reaction  that  transfers  electrons  from  a substance  to  an  oxidizing  agent.  Oxidation   reactions   can produce   free   radicals, which start chain reactions that damage cells.  Antioxidants  terminate  these  chain reactions  by  removing  free  radical  intermediates  and Inhibit  other  oxidation  reactions  by being oxidize themselves.  As a result, antioxidants  are  often  reducing agents such as  ascorbic acid or  polyphenols.Although  oxidation  reactions  are  crucial  for  life,  theycan  also  be  damaging;  hence,  plants  and  animals maintain  complex  systems  of  multiple  types  of  antioxidants,  such  as  glutathione,  vitamin  C  and  vitamin  E,  aswell  as  enzymes  such  as  catalase,  superoxide  dismutase and  various  peroxidases.  Low  levels  of  antioxidants,  orinhibition  of  the  antioxidant  enzymes,  causes  oxidativestress  and  may  damage  or  kill  cells.  As  oxidative  stressmight  be  an  important  part  of  many  human  diseases,  the use  of  antioxidants  in  pharmacology  is  intensivelystudied,  particularly  as  treatments  for  stroke  and  neurodegenerative  diseases.  However, it is unknownwhetheroxidative stress  is  the  cause  or  the  consequence  ofdisease.  Antioxidants  are  also  widely  used  as  ingredientsin  dietary  supplements  in  the  hope  of maintaining  healthand  preventing  diseases  such  as  cancer  and  coronary heart disease. Although  initial  studies  suggested  thatantioxidant supplements might promote health, later largeclinical  trials  did  not  detect  any  benefit  and  suggested instead  that  excess  supplementation  may  be  harmful.  In addition to these uses of natural  antioxidants in medicine, these compounds  have  many  industrial  uses,  such  as  preservatives  in  food  and  cosmetics  and  preventing  thedegradation  of  rubber  and  gasoline.  For  many  yearschemists  have  known  that  free  radicals  cause  oxidationwhich  can  be  controlled  or  prevented  by  a  range  of antioxidants substances (Bjelakovicet al., 2007). It is vital that  lubrication  oils  should  remain  stable  and  liquid should  not  dry  up  like  paints.  For  this  reason,  such  usually  has  small  quantities  of  antioxidants  such  asphenol  or  amine  derivatives,  added  to  them.  Although plastics  are  often  formed  by  free  radical  action,  they  can also  be  broken  down  by  the  same  process,  so  they  too, require  protection  by  antioxidants  like  phenols  or naphthol. Low  density  polythene  is  also  of  protected  by carbon  black  which  absorbs  the  ultraviolet  light  which causes radical production (Sies, 1997).

Sources and origin of antioxidants

Antioxidants  are  abundant  in  fruits  and  vegetables,  as well  as  in  other  foods  including  nuts,  grains  and  some meats,  poultry  and  fish.  The list below describes food sources of common antioxidants.  Beta-carotene  is  found in  many  foods  that  are  orange  in  color,  including  sweet potatoes,  carrots,  cantaloupe,  squash,  apricots,  pumpkin and  mangoes.  Some  green,  leafy  vegetables,  including collard  greens,  spinach  and  kale,  are  also  rich  in  beta-carotene  (Borek, 1991). Lutein,  best  known  for  itsassociation  with  healthy  eyes,  is  abundant  in  green,  leafy vegetables  such  as  collard  greens,  spinach,  and  kale.

Lycopene  is  a  potent antioxidant  found  in  tomatoes,watermelon,  guava,  papaya,  apricots,  pink  grapefruit,blood  oranges  and  other  foods.  Estimates  suggest  85% of  American  dietary  intake  of  lycopene  comes  from tomatoes  and  tomato  products  (Rodriguez-Amaya, 2003; Xianquanet  al.,  2005).

Vitamin  A  is  found  in  three  main  forms:  retinol  (Vitamin A1),  3,4-didehydroretinol  (Vitamin  A2),  and  3-hydroxyretinol  (Vitamin  A3).  Foods  rich  in  vitamin  A  include  liver,sweet  potatoes,  carrots,  milk,  egg  yolks  and  mozzarella cheese  (Baubliset  al.,  2000).  Vitamin  C  is  also  called ascorbic  acid  and  can  be  found  in  high  abundance  in many  fruits  and  vegetables  and  is  also  found  in  cereals,beef,  poultry,  and  fish  (Antioxidants  and  Cancer Prevention, 2007).

Vitamin E, also known as alpha-tocopherol, is  found  inalmonds, in many oils including wheat germ, safflower, corn and  soybean  oils,  and  is  also  found  in  mangoes,  nuts,broccoli, and other foods (Herrera and Barbas, 2001).

Classification of antioxidants

Antioxidants are grouped into two namely;

(1) Primary or natural antioxidants.

(2) Secondary or synthetic antioxidants.

 

Primary or natural antioxidants

They are the chain breaking antioxidants  which react  with lipid  radicals  and  convert  them  into more  stable  products. Antioxidants of this group are mainly phenolic in structures and include the following (Hurrell, 2003):

(1)  Antioxidants minerals.  These   are co-factor of antioxidants enzymes.  Their absence will definitely affect metabolism of many macromolecules such as carbohydrates.  Examples include selenium, copper, iron,  zinc and manganese.

(2)  Anti  oxidants  vitamins  –  It  is  needed  for  most  body metabolic  functions.  They  include-vitamin C   vitamin E.


 

1.2 AIMAND OBJECTIVES

To evaluate the in vitro phytochemical and antioxidant properties of methanol,ethanol and hexane extracts of Alstoniaboonei leaves with view to determine the appropriate solvent that would provide the best yield of the bioactive compounds.

1.3 SPECIFIC OBJECTIVES

To determine  the antioxidant properties ofAlstoniaboonei leaves.

To determine the phytochemical constituentsof Alstoniaboonei leaves.     


 

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