University of Nigeria€¦ · Pharmaceutical Chemistry and with Registration Number PG/M.SC/02/33534, has satisfactorily completed the requirement for the course and research works

University of Nigeria Research Publications

Aut

hor

OSONDU, Nnamdi Nwaorgu

PG/M.Sc/02/33534

Title

Phytochemical and Antimicrobial Properties of the Stem Bark of Detarium Microcarpum (Case

Alpiniaceae)

Facu

lty

Dep

artm

ent

Pharmaceutical Sciences

Dat

e January, 2007

Sign

atur

e

PHYTOCHEMICAL A N D ANTIMICROBIAL PROPERTIES O F THE STEM

BARK OF DETARIUM MICROCARPUM (CAESALPINIACEAE)

OSONDU NNAMDI NWAORGU

PG / M. SC/02!'3 3534

A PROJECT SUBMITTED TO THE FACULTY OF PHARMACEUTICAL

, SCIENCES IN PARTIAL FULFILLMENT O F THE REQUIREMENT FOR T H E

AWARD O F MASTER O F SCIENCE (M. SC) DEGREE O F T H E UNIVERSTY

O F NIGERIA, NSUKKA.

PROJECT SUPERVISORS: MR. EBI, G. C.

DR. (MRS) OSADEBE, P.O.

JANUARY, 2007

CERTIFICATION

NWAORGU, OSONDU NNAMDI, a post-graduated student in the Department of

Pharmaceutical Chemistry and with Registration Number PG/M.SC/02/33534, has

satisfactorily completed the requirement for the course and research works for the

Master of Science in Pharmaceutical Chemistry. The work embodied in this project

work is original and has not been submitted in part or full for any other diploma or

degree of this cr any other university.

Dr. (Mrs.) P. 0. Osadebe

Supervisor

Mr. G. C. Ebi / supervisor

Dr. (Mrs.) P. 0.'0sadebe

(Head of Department).

DEDICATION

. This work, a medical cnterprisc towards eradication of Iiunian diseases. has been

dedicated to niotlier, Mrs. Roscline Nwanyieze Nwaorgu and to "nunty" Adaora

Obiageli Ahanotu for their numerous encouragements.

ACKNOWLEDGEMENTS

I would like to express my gratitude to my able supervisors, Mr. G.C. Ebi and Dr.

(Mrs.) P.O. Oslldebe for- their liclpful suggestions and material supports. My thanks

also go to my postgraduate colleagues, Chidube Abana and Afierolio. 0. Eriarie for

their valuable contributions in thc course of this research and to the typist for a well-

typed job. Of course, my warnlest heart also goes to Mrs. A.O. Ahanotu for all the

cares I rcceivcd from her. Somc persons, too, numerous to mention, have also workcd

tirelessly, while the others have worked behind the scene -just to make this project a

reality. To all those I say, "Thank you".

Nwaorgu, 0. N.

January, 2007.

ABSTRACT

277.779s of powdered L)vrtrriirnl niio.octr~ptrrtt stcm bark was cstractcci witli

95% mctliatiot and evaporatcd to dryness. Thc dark hrown extract ( 2 4 . 4 0 " ~ ~ \vi\v) was

subsequently fractionated into ethyl acetatc soluble and insoluble. fractions.

Phytoclicmical analysis of tlie mcthanolic extract was established by standard

~iietliods. The more active EtOAc - soluble a l l - ac t was separarcd into t\vcl\~c bands

(co~istituents) using preparative TLC on silica gel plates. The mobile phase was MEK:

toluer~e: acetic acid: water (2: 1 v/v: 3 drops: 3 drops). Antimicrobial. pliytocliemical.

as well as UVIVIS spectral analysis of the constituents were done using appropriate

procedures. MeOH was used as the solvent and tlie blank in tlic UV/VIS

spccrropliolo~i~e~ric analysis. Multiplc comparisons of mean values of t I i2 bands anti

tlic statidard drugs \verc clone and analyzed for significant diffcrcnccs by ANOVA at

5% lcvcl of significant.

Results of preliminary screening established i ~ r r~itro anlirl~icrobial activity of

the ~norpliological plant parts. Pliytocliemical analysis indicated [lie prescricc of

tannins, steroids, terpenoids, carbohydrates, glycosides, saponins and rcducing sugars

in tlic mcthanolic fraction. \vliiIe the bands contain terpenoids and steroids.

Furlher, Bards 7 - 12 haw little or no in vitro antibactcrial activity. Bands 1 -

0 exhibited broad spectrum antibactcrial activity. Activity of Barids 1.2.5 and S are

much better than that of penicillin Ci witli respect to S.tjphi. Again, S.trrrrt~rrs is Iiigl-ily

scnsitivc to Bands 1 and 8 compared to ciiloramplicnicol. But Band 1 shows somc

anti-E.cwlr activity. UV/VIS spectral deterinination of tlie twelvc bands i~idicatcd the

prcsc~icc of Iiigli ly conj ilgated conipot~~ids.

TABLE O F CONTENTS

... ... ... ... TITLE PAGE ... ... ...

... ... CERTIFICATION ... ... ... ... &

... ... ... ... ... ... DEDICATION

... ... ... .... ACKNOWLEDGEMENT ...

... ... ... ... ABSTRACT ... .... ...

... ... ... ... TABLE OF CONTENTS ... CHAPTER ONE

INTRODUCTION

. . . 1 .1 Aims of study . . . . + . , . .

1.2 Botanic Profile and Functional Properties of Deltrrrtin~ Micr-octrrprrm

. . . . . . ... ... 1.3 Microorganisnl arid Diseases.. . . ,.

... ... ... ... . . . . . . ... 1.4 Phytochemicals . . .

. . . 1.5 Anti-parasitic Properties of Tcrpenoids . . . . . . . . , ,..

CHAPTER TWO

LITERATURE REVIEW

Phytochemistry and Dcttrr-lrirrr Microcrrrprrrr Analysis . . . . . . . . ,

... ... . . . Metliods of Extraction . . . ,.. . . , . . .

. . . Chromatograph Methods of Isolatior~ ... ... ... . . .

. . . Adsorption Solid - Liquid Colun~n Chron~atography, LC. ...

Thin - Layer Cliro~nalography. TLC. . . . . . . . . . . . .

... Gas Liauid Chromatoemliv. GLC. . . . ... ... ...

i

i i

iii

iv

V

vi

3

3

5

6

14

17

18

19

19

2 0

2 1

. . . . . . . 2.4 Classification of Anitmicrobial Agents

. . . . 2.4.1 Anitbacterial Agcnts ... ... ...

2.4.2 Anti fungal Agents . . . . ... ... ... CHAPTER THREE

EXPERIMENTAL

. . . Materials and Methods . . . . . . . . .

. . . . Apparatus and Materials . . . . . .

Reagents and Solvents . . . . ... ...

. . . . . . . Origin and Preparation of Plant Material

Extraction and Co~icentratio~i of Materials . . . .

Anitmicrobial Screening on Extracts of Morphological Parts and

Standard Dmgs . .. . ... ... . . . . . . ... ...

Micro.organisms . . . . ... ... . . . ... ... ...

Culture Media ... . . . ... ... . . . ... ... ...

Anitiniicrobial Sensitivity Tcst ... . . . ... ... ... ...

Fractionation of Stem Bark Extract into Solvent Fractions . . . . ...

Exhaustive Solvent Extraction of Dettrritrnt Microctrrprnl Stem Bark ..

Exhaustive Washing of Metlianolic Extract . . . . . . . . . . . . .

Anitiniicrobiai Tests on Ethyl Acetate Soluble and I~isoluble FTractions ... 3 1

Phytochemical Test on Stem Bark Extracts of Detarium Microcarpurn ... 32

Thin - Layer Chromatograpliy on Ethyl Acetate- Soluble Fraction ... 3 2

Trial Analytical ?'LC . . . . ... ... ... ... ... 32

Preparative TLC . . . . . . . ... ... ... ... ... 32

... ... ... . . . ... 3.7.3 Isolation of Fractionated Spots .

. . . . . . 3.8 Anitiniicrobial Screening of the TLC Fractions . . . .

3.0 Anitimjcrobial Sct-ccning of Varied Concentration of TLC Fractions

... With Significant Activity . . . . ...

... 3.10 Phytochemical Test on TLC Bands . . . .

3.1 . 1 Ultraviolet and Visible Spectral Determination .

CHAPTER FOUR

RESULTS, DISCUSSION AND CONCLUSION

... ... ... ... Results . . . ...

. . . . ... ... Discussions . . . ...

.... Yield of Methanolic Exlracts of Stem Bark

Pllytochcniical Rcsults of S k m Bark Solvent Extracts .

TLC Fractionation of tlic Most Active Solvent Extract and

. . . . . . . . . Phytochemical Results . . . . . . . . . .

Relative Aniiimicrobial Activiiy of Plant Parts and Solvent Fraction . . .

... . . . Relative MIC's of Signi ficaritly Sensitive TLC Bands

. . . ... . . . ... The Rf Values of the TLC Bands . . . .

... Ultraviolet/Vkible Spectroscopy and Extract Determination . . .

. . . . . . . . . ... . . . ... . . . Conclusion . . .

. . . . . . ... . . . . . . ... APPENDlCES . . . . . . ...

... ... . . . ... ... . . . ... REFERENCE . . . . . .

CHAPTER ONE

INTRODUCTION

"Tropical plants provide I

a rich source of

natural products

which exhibits

strong antimicrobial properties,"

(~andey, ei al, 1983)'

Felix Fontana ( 1720 - 1805)~ performed thousands of experiments on the

toxicity of various crude drugs. His results suggested to hini that a crude drug contains

an active principle which preferentially acts upon one or more discrete parts of the

organism to provide a characteristic effect.

For the first time the active principle was isolated. The German apothecary

Frederick W.A. Serturner ( 1 783 - 1841) in 1806 isolated a white crystalline substance,

morphin:, from opium. This first isolation of an active princip!e of a medicinal plant

stimulated so much enthusiastic research on the vegetable drugs that magendie2 was able

to publish a medical forniulary in 1821which contained only pure chemical agents. I

The natural forests of West and Central Africa are rich in natural resources and

have tremendous biodi\,ersity, particularly in trees that provide food, fuel, fiber and

medicares. Drugs derived from plants fonn the mainstay of medical treatment in the

developing country (~nand) ' . ln fact, grecn plants, i t has been noted, appear to be

reservoirs of effective chemotherapeutants and would constitute inexhaustible sources of

drugs wi:h numerous activities.'."" The remarkable contribution of plants to the drug

industry was possible because of the large number of the phytochemicnl and biological

studies all over the world. This progress resulted in isolation of many active components

used as a good remedy for many diseases. A wide variety of pknt materials have been

shown to have a potentially interesting acti\,ity against a variety cf organisms. lwu7 has

reported on the antimicrobial activity of a number of Nigerian and African plants. Other

useful studies include the works of Heydrick et rr? on acaricidal and antiscabies

activity, the works of Hakizamungu et rr? on antiprotozoan activity, as well as those of

Gafner d 0 o n molluscicidal activity of various other African medicinal plants. In

Nigeria, medicinal plants are an in~portant elen~ent of cultural heritage. They have been

used in herbal remedies, providing a still expanding alternative systems of medicine. In

fact, according to the June 1983 issue of "World Health," it has been estiniated that more

than one - half of the world's population, most of then1 in the developing countries. rely

mainly on traditional remedies.

1 . I Aims of Study

There is a serious need tc provide a readily available, more accessible and less

costly drugs to teeming often poor sickfolks of the developing countries. The ain-is of this

present research into an iridigeneous plant therefore include: I

( I ) To scientifically establish the antimicrobial potentials of Derrrriirt,~

(2) To separate the extract, and

(3) To semi-characterize the rzsultant constituents.

The met hod required the reduction of Deltrrirrnr ruicr-ocut-ptrt~r stern bark to a

coarse powder and extracting with 95% CH30H. The aqueous methanolic extract was

screened against a range of microorganisms and standard antibiotics. The extract was

also evaporated to dryness and extracted with ethyl acetate to obtain soluble and

insoluble fractions. Phytochemical analysis of the ethyl acetate soluble, which exhibited

a stronger antimicrobial activity, as well as the ethyl acetate insoluble, were also carried

out.

1.2 Botanic Profile and Functional Properties of Detarirrr~r Microcarprrnr

Dertrt-~irm trrict.octrtprrtlt, locally called ofor by the Igbo of Nigeria, rrr~rt-u by the

Hausa, and oghogho by the Yorubas, is a leguminous plant of the leg~r/uinosrrc of

flowering plants and member of sub-fanlily C(res(rlpinitrcctre. I t can be found in both the

rain forest zones and tlie drier savannah areas of Africa (Keay cr d)."." Deturirrnr

t~rict-onit-pst,l exists in Nigerian's geographical regions."," The fruits of this lesser

known legume are drupe - like, circular and disc shaped with a distinctive layer of

greenish, meanly pulp that is tangled with numerous network of fibers. The seeds occur

singly and are embedded within the fibrous network of the fruit. I t is hard, disc shaped

and wrinkled, brownish coloured husks. Thc cotyledon is white in colour ( ~ n w e l u z o ) . ' ~

Hopkin et ci l noted that Dei(~rit/ii~ nricrocarprr~~-is a plant "without spines or

..I? thorns or latex .... Dettrt-irrnr n~icroccirpunl, though indigenous to Nigeria, is

traditionally neither used whole, fermented nor used because of its contribution to the /

diet. I t is rather used because of its fi~rictional characteristics i t exhibits such as

emulsification and thickening when used as food ingredients in soups and broths. This

legume has found important application as a soup thickener in parts of Abia, Imo and

Anambra. Soup thickeners. Ezueh " :toied.increase the palatability of the soup and

reduce bulk in the diet. Studies have a!sa shown the seeds could be used as a stabilizer

and emulsifier in food juices and other beverages. 'x'' The thicke~ing and the binding

properties of the gum derivable from the riped seeds have been extensively studied. One

use being advocated is as a possible tablet binder. Okorie and Chukwu have studied the

biophysical properties of such gums'(' 2s w l l as the micrometric properties of the drugs

containing them." Their results suggested that Dercrritrru nricroccit-pun/ gum (DMG)

conipares favorably wi{h the high viscous grade sodium carboxyl methyl cellulose

(SCMC), acacia and gelatin and would be considered replacement for these imported

gums.

Trees like Defot-ilrttr ~4ict~oc~rrp1rnr and ln~i~rgia gtrhorterrsrrs (oghmo and l!jit-i)

are included as indigenous and exotic fruits and seeds in Ononogbu's Li~~it ls in Wlrnitur

~.r isrence. '~ Such tree have remained unexplored and unexploited sources of vegetable

oil. For example, though Dercrr-iiwr Microcnrprrnt produces edible fruits. it is however,

primarily managed for fuel wood production. These exotic trees can, however, be

powerful scurces of oil for nutritional, lipid-based and phamaceutical industries.

Ononogb~i gave the percentage oil as 1 2% for Detrrritrrtr Mio-ocitrprrnr, which compares

favorabl~~ with that of Zca 1trrq.s (niaize)." In Burkina Faso, the fn~i i pulp can be sucked

as snack, fresh or boiled after skin is r e m ~ \ < e d ' ~ . In some regions of Africa, the plant has f

been used in ethno medicinal preparations. In the South of Mali, !here are diversity of

medicinal uses depending on the ethnic group. The main sympton~s and disease treated

are stomach problems, and linked with general wellbeing, diarrhoea, malaria and

19 meningitis. Elsewhere also, decoctions of the leaves and fnrits are drunk to manage

dysentery.::' Again, they have been applied as anti - syphilitic asen:..?'

1.3 Micro-Organism and Diseases

Augustino Bassi (1773 -1856)~' demonstrated in IS35 that a silkworm

disease was due to a fungus infection. Also Louis Pasteur in 1877 reported that animals

injected wi!h an inoculation containing Bocillrrs orr~hrrrcis failed 19 develop anthrax.''

Also Emn:erich (1887) accidentally discovered that guinea pig with Strep~ococcrrs

eq~sipelrtis failed to develop cholera when injected with virulent cultures of Vihrio

cholerue." Anthony van Leeuwenhoek ( 1 632 - 1723) had earlier on observed and

described microorganisms accurately.22 Thcse show gemis can cause diseases.

1.4 PHYTOCHEMICALS

Phytochemicals discovered in plants include:

1. Terpenoids

These, also called terpenes, are hydrocarbons and their oxygen dericfatives. They

are based on the isoprene unit, (C5Hx )n.

Classificatinn:- This is based on the number of isoprene units. Ench class is further

sub - divided into (i) acyclic ( i i ) monocyclic (iii) bicyclical

a Monoterpenoid C 16,n=2

I Simplier monoterpenoids and sesqui terpenoids are major components of essential

oils; they are often sweet -- smelling volatilc oils obtained From saps of plants.

Geraniol A Menthol

( fiom rose oil) (peppermint oils)

Camphor

(carrphor tree)

Farnesol

(oils of ambrelte seeds)

Zingiberene Cadinene

(gingcr oil) (oils of cubabs)

c. diterpenoids - C2rlH27, n =4

The di - and tri - terpeniods are not steam volatile, and are obtained from-plant gums

and resins.

abietic acid (a resin acid from bark of pine trees)

d. triterpenoids, Ci()&, n = 6

These by far constitute [he largest terpenoids class and they uide'y occur in the

vegetable kingdom. Some groups have been recognized

1 1 ambrein and squalene

i i ) the tetracyclic triterpenoids

i i i ) the pentacyclic triterpenoids

The tetracyclic triterpenoids contain the stercids structure arid a number of

compounds related to steroids, e.g. lanosterol and the euphol groups.

The pentacyclic triterpenoids are thc largest group and are subdivided into : dt-

arnyrin. 0 - aniyrin and lupeol groups.

Squalene lanost erol

/3 - amyin

ii) Steroids

These possess a tetracyclic backbone based on cyclo-pentanoper

hydrophenanthrene. They include the sex homiones, the adrenocortical hom:ones, the

sterols, bile acids, steroich1 and triterpenoici sapogenins.

Sex hormones - are responsible for the sexual processes, and for the secondary

characteristics which differentiate males from females. Uses include as anabolic steroids,

anticzncer agents and oral contraceptives.

Sterols

St ignasterol Cholesterol

These contain a carbohydrate (glycone) anri non carbohydrate (aglycone)

structures. The aglycone can be tannin, aldehyde, saponin flavonol groups etc.

Glycosides are susceptible to acid hydrolysis, when they yield their sugAr and non-sugar I

moieties.

OH Amgydaline, benza lde!iyde cyanhydrin aglycone

cyanidin 3,5-diglucosidc, Quercetin-3-rut inoside. /

R=glucose ' Flavon$d L

Vi) Saponins glycosides - saponins

Saponins are glycosides with distinctive foaming characteristic. The aqueous

solutions of these natural detergents were traditionally used as soap. Saponins consist of

a polycylic aglycone (the sapogenin).

Classification

The steroidal saponins (the saraponins) - the aglycone is based on the steroid

nucleus, n~ai!~ly the tetracyclic triterpenoids.

The eriterpenoid saponins -the aglycone is based on the per:tacyclic triterpenoid

nucleus. I

steroid skeleton pent acyclic triterpenoid

skeleton.

digitoxin - conpnents of digitalis, straphanthidin - a cardiac glucoside st&oid alchohol, linkd to a trisacclede.

Tannins

These are water - soluble phenolic compounds having molecu!ar weights between

500 and 300C, giving the i~sual phenolic reaction. They can precipitat.; alkaloids, gelatins

and other prcteins.'"

Classification.

i ) Hydrolgscd tannins - the core nt~oleciiles are esters of gallic acid or glycosides of

these esters. Examples are gallitannins and ellagitannins which on hydrolysis give gallic

acid and ellagic acid.

i i ) Condensed tannins - these are built up from catecliin units. An alternative name is

proanthocyanidins since anthocyanidin monomers rnay be released by acid hydrolysis.

Tannins are used as nietal ion chelators and to convert animal hides to leather. Plant

tissues high in tannins are largely avoided by feeders, because of the astrigent taste they

impact. In plants, tannins act as barrier to hcrbivory, especially when i t is above 2%

(dry weight)'

Vii Flavonoids

These are the largest g o u p ef naturally occurring phenolics'" responsible for

plant pigments in flonws. leaves and fruits. Some ten classes are recognized, including

isoflavones, anthocyanidins, flavans, flavonols, flavones and flavonone. They occur both

in the free state and as mainly bond to sugar as flavonoid glycosides. Many flavonoids I

are polyphenolics and they are structurally related to flavones.

0 Flavone

a. anthocyanidins

These are responsible for flower pigments and are based chemically on cyanidin

and often on glycoside combination. Their glycosides are called anthocyanins.

quercetin, R=R'=H I I

OH 0 kaempferol, R=H

azaleatin, R=H,R1=Me myicet in, R=OH

b. flavones

Appearing as colorless pigments to anthocyanins in petals and leaves of higher I

plants, they differ from flavonols in lacking 3 -hydroxyl substitution.

HO

apigenin, R=H (from parsley seed) luteolin. R=OH (carrot leaves) chrysonol. R=OM E

1.5 Anti -Parasitic Properties of Terpenoids

Terpenoids of many plants have been shown to exhibit a wide range of a ~ t i -

parasitic properties. Jaffer pr ol " have studied plants from Iraq. Total sesquiterpene

lactones of some of these p1ar;ts showed both antibacterial and anticandidal activities.

~rtemisinin" is a sesquiterpene lactose endoperoxide derived from the Chinese weed

qirlg lrcro used as antipyretic and anti-malarial agent. Other synthetic derivatives like

di hydroartemisinin have greater antiparasitic activity in ~lirt-o against several other

protozoa including Leisl~rnclr~itr vrt!jor. and Tosoplc~st~~c~ gotlcltlii and it? viva against

schistosomes.

artemisinins dihydroartemisin

Jamaica qrmsia,'" the stem wood Picrusrmr ~-\-cel.str fan^. Simor-ouhocetre),

contains the terpenoid quassin etc. Tea from various parts of members of this family

have been used to treat cancer, amoebic dysentery arid malaria. The activity is based on

the quassinoid content.These compounds are also k ~ ~ o w n to have antiviral activity.

R, quassin

niollic acid, R I = H , R ~ = r__<

glucoside, RI=D-glucosyl,& = Y==-

Acidic triterpenoids and their glycosides with molluscicidal, antifungal, anti-

microbial activities have been isolated from species of Con~hreti t~~~. Examples, mollic

acid and its glycosides have becn isolated."'." The sodium salt of nioiiic acid glycosides

is toxic to Biomplltrltrr-itr ,pltrhr.trr~r snai Is.

Molluscicidal triterpenoid saponins have been isolated from the root of

Clet-orlerrclr~~n~ \t'iltlii, a medical plant from Malawi to be active against infestal parasites

or in the treatn~cnt of malaria. I t is also active against Phytopathogen fungi."

Finally, the leaves of khat (Cutha edu!i,~. family Celtrstrncmc) are chewed to

alleviate hunger and to produce stimulating efkcts (CNS). These are related to its

alkaloid content^.^^.'^ However, the antibacteiial and broad cytotoxic activity of the

Khtrt Crrll~rs cultures t1ai.e been attributed to 22 0- hydroxytingenone and tinsenone, I

both quinone methide tr i t~r~enes."

CHAPTER TWO

LITERATURE REVIEW

Phytochemistry or plant chemistry studies the variety of organic compounds

accumulated by plants and their possible biological functions. I t involves collection,

botanic identification; preparation of crude extracts; biological, phanacological and

chemical screening of the crude extracts; chromatogr=raphic separation; structure

e11~cidation o f isolated compounds; partial or total synthesi- t ic. (Hostettmann et ~rl):'"

2.1 Phytochemistrl)' and Detarirrnr Microcnrpirnr Analysis

Akpata and Miachi had "evaluated the proximate coniposition and functional I

properties o f Dettr~.ilrn~ rrricr-octrr~irt~r seed flour. The dehulled seed flour contained 3.5%

moisture, 1.5% ash, 2.9% crude fiber, 15% crude fat, 37.1% crude protein and

39%carbohydrate. Functional properties showed bulk density of 0.86sJcm' for dehulled

anr! c.92g/cm3 for undel~ulled flour. The results obtained zbove showed that the seed

flocr has good nutritional quality. Also Onweluzo et 01'~ hzve investigated the suitability

of the seed flour as a binder and partial fat substitute in buffalo (Birhtrl~~s hcrhtrlis) meat

3 0 loaves. Abreu and Angela had analyzed the bark extract of this tree for its

carbohydrate content by GLC - CIMS. Preparative GLC of'the benzoylated carbohydrate

f r a d o n led to the isolation of - quinide (figure below), (-) -bomesitol, sucrose, D-

glucose and D-fructose benzoates, which were characterized by extensive NMR

experiments.

Abreu el ul"' in their evaluation of the bark extracts had isolated the con~pounds

lupeol. P - sitosterol, stigmasterol, campesterol etc. The ethar~ol extract showed

antimicrobrai activity against Pse~tdon~otm c~errruginostr. Klehsiellu prle~inlotliue,

Cirr-ohucrer- fr-errndii, SftrplijYococc~rs clurelrs, Lister-icl nlotlocj.togcnes . I

2.2 Methods of Extraction

During extraction, fresh plant material may be used but often the plant material is

dried and ground before extraction using a Soxhiet apparatus. Jaffer cf crl '' Soxhlet

extracted 1003 of powdered plant material with 90% EtOH and the extract evaporated to

dryness. A single solvent or mixture may be used. Desolventization at appropriate

temperature can be done to recover the solvent. However, the coZd maceration process

have been used in this work. Elhag el (11 '' using this method, macerated 259 of freeze -

dried powdercd leaves of Khat callus with CHIOH and extracted at room temperature.

Quavtitative isolarion is based on extractive processes which involve the use of

immiscible solvents. Estraction is based on distribution coefficient of the solute in the

two media.

Distribution coefficient = CA iCt3 = K

CA = concentration in organic layer, C13 = concentration in aqueous layer, K =

equilibrium constant (also called the partition coefficient).

2.3 Chromatographic Methods of Isolation.

Chromatography I S a scparatory technique based on differential distribution of

components of a mixture between two immiscible phases. The stationary phase may be a

fixed rnicroporo~~s or paflic~llsie adsorbent solid or itself being a liquid immobilized on

an inert microporous support. The mobile phase - a liquid or a gas - is percoiat~d

continuously through the stationary phase.

The speed with \\,hicIi the sample nioves on the fixed phase depends on its /

distribution between the two phases. This distribution is based on differences on

partition, adsorption and solubilities of the solutes. The reversible exchange is given by

the distribution ratio:

concentration of solute in stationary phase = K concentration of solute in mobile phase.

2 3.1 Adsorption Solid-Liquid Colurnn Chromatography, LC

The colunin consists of a metal, glass or even plastlc tube and packed with a

- - polar ads-orbent-solrd (s111ca-gel,-alumina)-as -the stat10naryqhase.A-flo~v-afsample

dissolved in the rnoblle phzse elute con~ponents from the column as pure samples.

Separation I S based on polar~t:es of sample components and the solld states

A modification of LC I S the high performance Ilquld clironiatography HPLC

where an eluent under pressure (e.g. 200 bars) is injected through the column at high

flow rate ( I -5cm3 per minute). Detection and recording of separated components can

readily be automated. HPLC can be used for non - volatile, highly polar samples. HPLC

has been used in sterol analysis, ' 'A ' as well as in the analysis of fatty acidsJ3 which are

major constituents of lipids.

2.3.2 Thin - Layer Chromatography, TLC

Two Russian scientists, Izniailov and Shraibes in 1938 working with tintures

reported the use of layers of alumina on glass plates and called the techniques spot

chromatography (~ammond)."" Later, kirchrier e, a?' developed chromatostrips which

were the forerunner of the technique as we know i t today. Nweke and other

investigators4" working on Nigerian medicinal plants used TLC to separate extracts of

Mo,-irrdo Ilrcitlu and TI-cnta grtitrectrsis into four and eight constituents respectively. r

TLC can also be used to separate lipids4' as well as mixtures of amino acids.4H

The content of TLC is a flat, even layer (0.25mm) of adsorbent material

impregnated on a thin layer of paper, plastic or glass. Mixtures are spotted on the plate

and dried. When the plate is immersed in :I15 moving phase in a closed vessel, the liquid

moves through the system (developrnmt processes) by capillarity and becomes

fractionated. Separation of components is due to their different adsorptions and

solubilities in the mobile phase and interactions with adsorbent. For detection of spots.

the plates are dried and the position of the zones revealed visually for colored substances. - - -- - -

Uncolored spots can be viewed on flo:xent adsorbents under ultraviolet lights at

254mn1 and 365nlm. In preparative TLC, the detected spots can be recovered by

scrapping the adsorbent from appropriate portion of the plate and extracting with a

suitable solvent. Each fraction can be examined by gas chromatography or by HPLC.

In TLC, a conipound is identified by its retardation factor, the RI. value:

R,. = distance moved by substance from origin distance moved by solvent from origin

2.3.3 Gas Liquid Chromatography GLC

~arnes'l~'in his work quoted the work of Martin and ~ ~ ~ e - ; ' % v l i i c h described liquid

/liquid partition chron1:ttography and concluded that "the mobile phase need not be a

liquid .. but a vapour ;by means of which I refined separation may be carried out." By

195 1 , James and Martin were working together and they separated methyla~nines" and

short chain fatty acids5' using GC. By 1952, both men had published the first paper /

describing GC?

In GLC, the moving phase is the carrier gas (N2 or He) and columns contain

either the stationary liqu~d phase (of high boiiing point) immobilized on an i,nert support

(eg celite) or else are themselves narrow tubes on whose wall is a thin layer of the

stationary phase.

The sample is dissolved in the moving phase, passed through i.he column,

fractionated, and appropriately identified. A hyphenated technique can also be used

whereby the colunin is coupled to a mass spectrometer (GS -MS) so that the effluents - - .- --- - - - - - - - - - - - - - - - - . - - - - - - -- - -- -

are identified by their mass fragmentation pattern.

2.4 Classification of An timicrobial Agents

According to Paul Ehrlich, drugs are designed to be selectively more toxic for

invading organisms than for the host. Biological response to drug is a consequence of

the interaction of that drug with the living system, causing some change in the biological

processes. Drugs act on receptor - bion~acroniolecles - to initiate response.

Chemotherapy was coined by Paul Ehrlich (1699). Antimicrobial agents are classified

depending on the parasites they act on: antibacterial, antifungal, antiprotozoan, iurcr

tl lio .

2.4.1 Antibacterial Agents

Bacterial infections are caused by bacteria. Bacteria have been classified by their

staining property. The Gram stain divides bacteria into gram negative and gram

positive and depends on whether the bacteria pick up the Gram's stain used for

microscopic investisation (Prescott cr ol).'" Antibacterials include sulpiionaniides and

antibiotics like tetracyclines, chloramphenicol, penicillin, streptomycin etc. I

No11 pathogenic bacteria have been used to source antibiotics. The genus

r p o c e s (order, trcriuor)~g~ccrcs) are source of streptomycin, Kanamycin,

tetracyclines, chlorarnphcnicol, nystatin, r e t i . Also B(Ic~III,s S , (order.

E~rhac~erials) are sources of baci tran, polymyxin -B etc.

Table 2:l Pathogenic Bacteria

1 Pathogen I Representative Diseases I

Gram -Posi tive bacteria Boils, pneumonia, wound infect ions

~~cohacrer-iurn rhv -cdns i s 1 Tuberculosis

M~~cohtrc ter i~~~n Iept-(re Leprosy

.-

ClostricEiwu telcr~li Tetanus

/ Srreptococcas pj7oge,rrs 1 Scarlet fever I -

B N C L ~ ~ I I S s~rhtilis / Food poisoning, food spoilage

- Gram - negative bacteria Typhoid fever, septicemia,

S~lmo~ie l le rjydii gastro - enteritis

- Neisset-iir gonot-14 oetre

-.

Vihr-io cliolcrc~e Cholera

Whooping cough I

1 Urinary tract and bum infections.

1 pneumonia - -

Pneumonia, urinary tract infections

/ Esciierichia coli / Urinary tract infections

"'0 - HN)7y .c%3 --. 02N<=>CP-:H-CH20H

4- COOH O H YH - C H C b 0

Penicillins fi 0

(Penicillin G)

R=S&CH a (NHS- , anipicillin

2.4.2 Antifungal Agents

Fungi cause various mycoses. Fungi vary in complexity and size. ranging from I

the singlexell microscopic ycasts to rnulticellular, filamentous molds, macroscopic

puffballs, and rni~shroorns. Cutaneous or superficial infections of the skin include

ringworn1 of the feet (athlete's foot or ti;retr petlis), of the nails (titretr ~rtrglrilrnr),and of

the scalp (titrec~ cupitis). Thesc denatophytic infections are caused by the

Epi(let-~~topl~j~totl ~ ~ O C C I ( S I I I I I and cal: be treated with tolnaftate and antibiotics like

griseofulvir, jfulcin in tabletfom). Deep or systemic rnycoses like candidiasis caused by

the yeasts ccrrdih (1lhictr11.s and involving the niucus membranes, gastrointestinal track

-- etccan b - e ~ e a t ~ e d ~ l s i x pp_lye11e~aantibiotics~nystatin and._amphoterin-B, -- .-

The non - pathogenic molds Pcrlicilli~rr~l genus are sources of antibiotics

anipir illiurn, griseofulvin, penicillium F, G, X, K etc. The presence of antifungal

coni~nunds in higher plants has long been recognized as an important factor in disease

res is t rn~e,~"~~ 'Such co~i-~pounds are considered valuable for controlling diseases. 58 -60

CHAPTER THREE

EXPERIMENTAL

"It was rhe knowledge of the great abundance and wide

distribution of actinomycetes ... and the recognition the marked

activity of this group of organisms against other organisms that led f

me in 1939 to undertake a systematic study of their ability to

produce antibiotics ."

3.1 Materials and Methods

3.1 . I Apparatus and Materials

Rotary-vacuum c~apora to r type 34912, water bath, test tubes, conical flasks,

measuring cylinder, beakcrs, pipettes, funnels, filter paper, Sauter KG. D - 7470 mass

balance ,silica gel, spreader, chromatoplates, broth cultures of test organisms, petri

dishes, sterile cork borers, inoculating loop, incubator, autoclave, indelible marker,

UNICO - UV2 102 PC spectropliotoznc-;.er , inter alia

3.1.2 Reagents and Solvents

Methanol, ethyl acetate, dimethyl sulphoxide DMSO, ethanol, acetic acid, methyl I

ethyl ketone MEK (by Sigma-Alchrich Laborchernikalien GmbH), tetraoxosulphate (vi)

acid, potassiuni hydroxide, chlorofornl, butanol, n-hexane (by May and Baker Ltd.,

Dagehhani ,England) , distilled water, inter alia. All solvents are of analytical grade.

3.1.3 Origin and Preparation of Want Material

Leaves, seeds and stems of Dcrcrrilrnr n~icroctrrprrnl were obtained from Nsukka

in Enugu State of Southeastern Nigzria in the month of February. They were collected,

dried and properly identified by the plant analyst, Mr. J.M.C Ekekwe, of the Department

o f Botany, University of Nigeria, Nsukka Campus. From the dried plant materials the

following samples were prepared: stem bark, seeds and leaves. These parts were ground

to powder and stored in separate sealed transparent containers.

3.2 Extraction and Concentration of blaterials

The cold extraction method was used with 95% methanol (analar), prepared by

mixing 5ml of distilled water and 951111 of methanol. Each o f the transparent containers

was filled with solvent just cnough to cover thc plant materials and extraction allowcd to

takc p l x c for tlircc days.

Snmplcs of solution (some few milliliters) Srom each of the containcrs wcrc

scparatcly pipeltcd out and placed in a rotary- vacuum evaporator type 34912 at 45" C

whcn tlic sotvcnt was cvaporatcd. Mass of rcsultin!: matcrinls wcre wciglicd using n

Sautcr KG. D - 7470 mass balancc.

Preparation of Stock Solution in DMSO, (CH& SO

i. Samples

Definitc v o l u ~ ~ i c of DMSO was added and used to dissolve each of thc cvnporatctl

stcm bark, leaf, and seed cxtracts in such a way as to provide a final concentration of 10

mg pc:- ml.

ii. Standard D;ugs

A 20- mg quantity of active drug subsrances wcrc each prcparcd as summa^-izcd

Tablc3:l Derivation of Weight of Drug Equivalent to the Standard Drug

Drug

I

Total Wcight of Drug

EquivaIent to Wciglit of

Wciglit of DI-us

Equivalent to 20 mg of

Standard Drug Standard Drug

~ 0 0 n i ~ =6001iig penicillin G 20 nlg

1 I

Nystatin

27.15 mg Chloramphenicol 1 1.35752 = i g drug

3SS1iig=300niy nystatin 26 nig i

Penicillin G* = Benzyl penicillin sodiuni BP

Chloramphenicol = chloramphenicol sodiuni succinate

Weight of Drug Eciuivalcnt to 20 nig of each of the standard drugs were dissolved

in 2 rnl of DMSO, providing a concentration of 10 rng per nil of solution.

The 10 mg per ml solutions of the three samples and standard drugs which provided the

stock solutions were subsequently screened for anti-microbial activity.

3.3 Antimicrobial Screening on Extracts of Morphological Parts and Standard

Drugs.

3.3.1 Micro - organisms - Gram - Positive (B(rci1lris srihrrllis, Sr~rpl~~locc~is aiit-em), I

Gram - negative (Klchsielltr prreunrorrrire, S~rlnrottelkre fjplti, Escherichiu ccoli)

organisms and jrtlgi (a yeast. Ctrrtclicl(r crlhiccrtts and a mold. Aspcr-gillus niger). These

were used as test niicroorganisn~s.

3.3.2 Culture Media - The r.est organisms were cultured in the Phamiaceutical

Microbiology laboratory of the? Department of Pharmaceutics, in the faculty of

Pharmaceutical Sciences, University of Nigeria, Nsukka. Broth cultures containing I x

10\cells per rnl solutio~is of the required niicroorganisms were used. A serial dilution of

the stock solution of the test organisms was done, as well as perfomling variable count in

the dilution that gave countable colonies. The concentration of the stock solution was

then obtained.

3.3.3 Antimicrobial Sensitivity Test.

The Agar Diffusion Method was used for activity determination. Large plates

(23x 23 cm) were prepared with 21111 of molten nutrient agar and inoculated with Iml of

broth culture of the test organisni. The plates were allowed to dry at appropriate

temperature. Wells (Smrn in diameter) wel-e made in the inoculated agar using a cork

borer and filled with two drops of test samples or standard drugs. Plates were then sealed

and incubated at 3 7 " ~ for 24 hours for bacteria and 2 5 " ~ for 48 hours for fungi. The

inhibitions zone, when prcsent, were measured.

Solutions of penicillin G and chloramphenicol sodium succinate (10 mg per ml)

were used as standard bacterial growth inhibi!ors, while also 10 rng per ml solutions o f

fulcin was used as standard anti - fungal growth inhibitor. The drugs served as positive

controls. The results are shown in appendix I.

3.4 Fractionation of Stem Bark Extract into Solvent Fractions /

3.4.1 Exhaustive Sohen t Extraction of Dctariirnr Microcarprrnt Stem Bark.

Grou ~d stem bask extract with 95% methanol

EtOAc sol;blc EtOAc insoluble I

Chromatographic fract lonat ion

'12 BANDS '

Figure 3.0 Fractionation scheme of aqueous methanolic ex tract o f Det~lr i~ct?~

t~ticrocurpzrni stem bark

277.779.g of powdered stem bark were put in a flat-bottomed flask and

exhaustively extracted with 95% aqueous CH30H by maceration.

Procedure

The flask was filled ivith enough methanol and slightly agitated. The contents

were filtered and the process repeated using fresh portions of solvent until no coiour

was imparted to the extracting medium. All CHjOH extracts were niixed together,

vacuum evaporated to obtain a deep brown reddish sticky liqaid. The resulting extract

was weighed and its mass recorded.

I

3.4.2 Exhaustive Washing of Rlethanolic Estract.

The residue was successively washed with various portions of ethyl acetate

EtOAc, on each occasion decanting the solvent. The process was stopped when the

sample ir-nparted no c o l o ~ ~ r to the extracting solvent. All EtCAc - soluble were niixed

together and concenrrated usi~ig a rotary evaporator, Both this fraction as well as the

EtOAc - insoluble were stored below rooni temperature.

3.5 Antimicrobial Tests on Etfijbl Acetate Soluble and insoluble fractions.

Some quantity of the EtOAc-sol~~ble fraction were taken and conipletely

evaporeted under vacuum and re-dissolved in a particular volume of DMSO as to give a

final coxentration of 1 Omg Iml solution. Furthermore, 2Omg of EtOAc - insoluble were

dissolved in 2ml of DMSO also giving a concentration of IOrng/rnl solution. Anti -

microbial screening was undertaken for these solutions against IOnighl of the standard

drugs penicillin G, fulciii and cl~loramplie~~icol, prepared similarly as before (see table

3.1). The results are shonv in appendix 1 .

3.6 Phytochernical Test on Stem Bark Extracts of Detariitrtr Microcarprrrtt

Both the ethyl acctate soluble and insoluble fractions were each subjected to

phytochemical analysis. Methods used wcrc those outlined by Trease and Evans "' and

by Harborne "' (see 3.10 for details). Results are summarized in table 4.1.

3.7. Thin Layer Chromatography on E t l i ~ ' l Acetate- soluble Fraction

3.7.1 Trial P.n:lytical TLC.

Absorbents used: Commercial grades, 5cm x 1.5cm alumina and silica GF coated

aluminum platzs.

Procedure /

A solution o f EtOAc - soluble in EtOAc was spotted on two d~fferent locations

using a capillary tube and the plate a l l o w d to dry. The plate was immersed in a covered

chromatographic vessel containing cyclohcsai~e: MEX ( 5 : 2 by volunle). When the plate

was developed a rd dried. i t was viewed under UV lights (365 n;n, 254 nm). Other

solvents or :::ixtures were equdly tried. The best system was however, MEK: toluene

(2:2 v ~ v ) , plus 3 drops each o f acetic acid and distilled water.

3.7.2 Preparative TLC

Preparation and Activation

Chror;~atograpliic plates, each measuring 20cm by 20cm by 0.15cm thick, were

prepared by mixing silica gel with distilled water in the ratio o f 1.2 ( w h ) . The slurry

was then poured into a Uniplan TLC Spreader- which was set with a thickness of 0.5 rnm

at 8g per plate. Impregnation was then made on the glass plates, plates placed in an

electric ovum and activated at 1 0 0 " ~ for onc hour before use.

Spotting, Dcvclopnltnt and Detection.

EtOAc - solublc fraction was dilutcd with EtOAc (2: 1 ) and subsequcnt'y. a stcak

of spots was made horizontally aboul 5cm tip thc platc. The spots wcrc allo\vcd to dry

and rc- loadcd thrcc otlicr tirncs. Thc plate was dipped in an approprialcly s i ~ c d

chroma~ographic tank containing MEK: toluene mixture, 2:l vlv, plus 3 drops of acctic

acid and 3 drops of distilled water. After devclopment, tlic platcs wcrc rcmovcd from thc

lank, air- dried and appropriatcly viewed. Twclve bands wcrc revealed.

3.7.3 Isolation of Fractionated Spots

From thc developed plates, identical bands werc collccted. pooled together, and

rcco\*crcd by washing wirh CH30H.

Procedure

-Fhc scrapped spots wcrc scparately soaked in cnough mcthanol and tIic contcnts

csliaustivcly washed. Thc mixtures werc filtered and thc filtrates conccntratcd under

vacuum. Thesc soIutions wcre stored, to 13c used for antimicrobial and phytochcniical

tcsts, as wcll as UV detem~inations.

3.8 Antimicrobial Screening of the TLC Fractior~s

Aliquots of each of the extracted bands wcre separately cvaporatcd and rc-

dissolved in appropriate volume of DMSO, providing 10 111gIml of soliitio~is. I'ositivc

controls, 10 mg/nil solutions cach of penicillin G, fi~lcin. chloramphenicol and nystatin

wcrc similarly prcpared as dcscribcd in tablc 3.1. Thc inhibition zonc diamctcrs, IZD,

ivcrc mcasured and recorded. The results are sl~owu in table 4.3.

3.9 Antimicrobial Screening of Varied Concentrations of TLC Fractions with

Significant ActiviQ.

The bands with significant antimicrobial activity were subjected to further

sensitivity test. The following concentl-ations were prepared for each of the standard

drugs, as well as for tlic bands: 5,2.5, 1.25 and 0.625 mg/nil solutions from the stock

solutioiis by serial dilution in DMSO. Tlicsc solutions were tested against some sensitive

microo!ganisni described earlicr. The inliihition zoncs diar;ie!er, IZD, were measured,

and recorded in appendix 11.

3.10 Phytochemical Test on T1,C Bands I

Phytocheniical test was perforniccl on all the twelve bands for the following

6 1 con~pounds as outlincd by Trcase and E\,ans and Harbome. "' (The solvent was

evaporated from sanlple solution beforc testing.)

Saponins

Extraction - Boil a little quantity of po\\clu- \\:ith 5mI of distilled water for 5 minutes.

The mixture was filtered while hot, allo\\ml to cool and the filtrate used for the following

tests:

a. Frothing Test: An aliquot of filtrntc \\,as diluted with 21211 of distilled water and

shaksn. A stable froth proves the presencc of saponins.

b. Emulsion Test: Another aliquo! of filtrate was mixed with 2ml of water and 1

drop of olive oil and shaken. An emulsion is a positive result.

Carbohydrates

Extraction - About 0. Ig of the po\\,di.l. \vns shaken with water, boiled and filtered.

Filtrate was divided into portions

a. General Test - hlolisch's Test

To thc Iiltratc, a Tcw drops of Molisch's rcagcnl wcrc addccf. Concc~~tratcrl 1-12SO~

was then pourcd down thc tcst tubc. A purple ring in the interface of t l~c two laycrs

intlicatcs thc prcscncc of carbohydrates.

b. Specific Tests for Free Reducing Sugars

Reduction of Fellling's Solution Test - To I ml of filtrate \vas added cclual volunies of

Fchling's solution 1 and 2 (or Bcncdict's solutions) and boiled on watcr bath. The

prcsclicc of a rcducing sugar is shown as a tunling of the blue solution to brick rcd.

c. Specific Test For Combined Reducing Sugars - Hydrolysis Test

1 ml of cstract was hydrolyscd by boiling with 5111l of dilutc HC'I. This was

~;cutralizcct wit11 20% NaOH solution (tcst with l i tn~r~s papcr). Thc Fchling's tcst was

I-cpcatcd. A brick red colour would indicate the presencc of conib~ncd rcducing sugar

(glyconc).

Glycosides - The Hydrolysis Test

Iiydrolysis - About 0. Ig of powder was hydrolysed by boiling with 5ml of dilutc HC1

for I S minutcs, tlic~i filtcrcd. Neutralization was carried using 20% NaOH solution and

thc Fcliling's Test repeated. A densc brick red precipitate would indicatc thc prcscncc of

glycosidcs.

Tcsts for Tannins and Flavonoids.

d. General test for phenolic nucleus.

Extraction - O.lg of powdcrcd samplc was boilcd with 6ml of distilled watcl- for 3

miiiutcs on water - bath. The mixture was filtered and the resulting filtrate divided into

two portions.

i . Ferric Chloride Test - 1 ml of filtrate (or detanned filtrate, see below) was diluted

with distilled water (1:4) and a few drops of FeC13 solution added. A blue or green

coloured precipitate would indicate the prescnce of phenolic nucleus.

ii. Vanillin HCI Test - To another portion of extract was added lO%Vanillin HCI (wlv)

solution. A pink precipitatc would be indicative of phenolic nucleus.

Extraction and Detanning of SampIe

To some quantity of so!id sample were added 6 ml of acetone and heated in a water -

bath. The acetone was e~raporated, the mixture cooled and filtered.

a. Specific Test for Flavonoids - Sllinoda Test. I

0.53 of powdered sample \\ere extracted in EtOH by boiling in a water - bath for 5

minutes, filtered and boiled. To the filtratc was added four pieces of magnesium filings,

followed by few drops of concentrated HCI. '4 pink or red colour would ind~cate the

presence of flavonoid~.

b. Specific Test for Tannins Albumin Test - Aqueous extract of sample were added

about equal voluiiie of egg albumin. A precipitate would indicate the presence of

tannins.

Terpenoids and Steroids - Salkowski Test

Portion of powdered sample was dissolved in chlorofomi and then filtered. To the

filtrate were added aliquot amount of conc. H2SO~ to form a lower layer. A reddish

brown colour at the intcrfxe would indicate the presence of steroid agl ycone.

Lieberrnann - Burchard Test

Portion of powdered sample was a ~ i h t e d in acetic acid. Then a mixture of chilled

acetic anhydride: conc. H2SO4 ( 19: 1 ) was poured down through the side of the test tube.

3 7

The formation of a greenish blue colour in the ct~loroforn~ layer would indicate the

presence of steroidsltriteq3enes.

Alkaloids

Extraction - some quantity of powder was boiled almost for 2 minutes with 5nil

of 1% aqueous HCI.

I ml of this extract were trezted with 2 drops of any of the following reagents and

observed for precipitate:

1

. . I I

I

... 111

I v

Proteins

Mayer's reagent wouId give cream precipitate

Dragendorff s reagent - reddish brown precipitate

Wagner's reagent - reddish brown precipitate,

Hagner's reagent (picric acid) - yellow precipitate.

a. Millon's Test - To a few partion of the sample was added a few niilliliters of

millon's reagent. A white precipitate, which changed to brick red on boiling, would

indicate the presence of proteins.

b. Biuret Test - Addition of conc. NaOH to the sample, followed by a few drops of

CuS04 would yield a violet coloured precipitate.

Oils.

The sample was rubbed on a filter paper. The presence of a translucent patch

would confirm the presence of oils.

Resins

Dried sample was dissolved in acetic anhydride and one drop o f

concentrated H3S04 was added. A purple or violent colour would indicate

the presence o f resins

The result of the phytochemical analysis is presented in table 4.2.

3.1 1 Ultraviolet and Visible Spectral Detcrrnination.

The twelve TLC bands were separately but appropriately diluted with methanol

and put in the sample cell of UNICO - UV2102 PC spectrophoton~eter. These were nin

akainst solvent blank (n1etIia1101) and absorbance and wavelengths of maximum

absorption, h ,,,. detem~ined. Absorption spectra of the twelve bands are given in

spectra ( I -XII).

CHAPTER FOUR

RESULTS, DISCUSSION AND CONCLUSION

4:l Results

This rcsearch focuses on q~~alitativc and antimicrobial analysis of an indigenous

plant, Dctwitrrrt ~~~ic~~ocirrprrnr stem bark. In this investigatior., 277.779g of ground plant

part wcrc extracted with aqucous methanol and scrccned against Gram - ncgativc, Gram

- positivc bacteria, a yeast and a mold, using standard d n ~ g s - penicillin G, fulcin and

chloramphenicol, The mcthanolic cxtract was fi~rther evaporated to dryness and washed

with ethyl acetate to obtain soluble and insoluble fractions. The ethyl acctatc solublc was

s~~bjcctcc l to trial TLC usins thc mobile phase MEK: toluene (2:l vfv) , plus 3 drops cach

of acctic acid and water, on silica GF as thc stationary phase. Prcparativc TLC was also

carricd out to extract thc I-esulting (twclvc) bands which wcrc subscqucntly scrccned

phytochemically and for antimicrobial activity. Graphs were constructed to dctcnninc the

MIC of chromatographic fractions as well as those of the standard dnlgs.

Extraction Yields

From thc above 277.7793 of ground Dcrc~riwr rrricr-occrrprrrrr stcm bark, 67.9393

of me~hanolic cxtract - deep brown reddish sticky lnatcrial - was obtained. Thc

percentage yidd of thc extract is calculated as follows:

Mass of Rcsult Extract =67.939,

Hcncc, pcrccntagc yicld = Mass of Result Extract x 100%

Total Mass of Stem Bark

= 6 7 . 9 3 ~ x 100%

377.779

= 24.46% w/w

The Minimum Inhibitory Concentration, MIC

The concentration, log. concentration, diameters o f the zones of inhibition (IZD)

1

and the IZD -- related parameter, (X-) o f the test organisms and the standard

antimicrobial agcnts given in appendix 11. Graphs ( I - XXII) give thc graphs o f log.

concentraiion vs. x2 and summarized in appendix 11, hence establishing the MIC's

of chromatographic fractions and the standard drugs. The diameters of the zones

of inhibition was determined using a transparent meter rule. The graph of

7 logarithm of the concentration against X- was made. The antilogarithm of the

intercept on the log. concentration axis gave the MIC values. X' is calculated as

X' = ( Inhibition Zone dinmeter - 8)' mm2

2

Statistical Anslysis

From the table of Inhibition Zone Diameter (appendix I I ) , mean values o f four

different readings of the TLC fractions with significant activities, as well as those OF

control antibiotics. were computed. Standard deviations were also evaluated. Multiple

comparisons were done for the different TLC bands and standard drugs and analyzed

statistically for significant differences by one-way classification of analysis of variance

(ANOVA) - the F-Test. Any significant differences found were partitioned with the

Least Significant Difference (LSD). Computed F-values were compared with the

critical value (Fo.05) for the degree o f freedom at 5% level of significance, p <

0.05. See appendix 111.

Phytochemical Analysis of the Stem Bark Extracts

Phytochemical tests on ex tracts of Detarim ;;~icroctrrplin~ stem bark are

presented in table 4.1. Shown are those of the methanolic: fraction, the ethyl acetate

insoluble and soluble fractions, as well as those of the fractionated twelve bands.

I

4.2 Discussions.

4.2.1 Yields of Methanolic Extracts of Stem Bark

The calculated yield was 24.46% wJw. This result would suggest that Deturium

n~iwocctrprcnr stem bark is a possible source of material to work with and that

investigators would be assured of relatively enough working material.

4.2.2 Phytochemical Results of Stem Bark Solvent Extracts

Phytochemical screening of the ethyl acetate soluble and insoluble fractions of

the stem bark identified the presence of the following compounds: tannins,

car5ohydrates, steroids, glycosides, saponins and reducing sugars. See results in table

4 . ) below. While flavonoids were indicated in the ethyl acetate insoluble fraction

but not in the other, terpenoids were only in traces in the former. Work by Abreu

. . . and others4' have isolated sitosterol-3~-0-(~'-0-~almitot~l-2,3 ,4 ,-0-triacetyl-b-

glucopyranoside) ( i ) from the stem bark extract of Deicrr-izrni n~icroctlrptrnr.

This compound is a sapenin glycoside with a steroidal aglycone and a s u g r glycone.

Steroidal saponins are rather widely distributed amongst plants. They occur in the plant

' Table 4.1: Ph!.tochemical Analysis -Dc~orium nricrocnrp~rnr stem bark extracts

SIN Phytochemical RIcthonolic Ethylacetate Ethyl acetate

Fraction insoluble soluble

Alkaloids

Glycosides

Saponins

Carbohydrate

Reducing

sugar

Tannins

Flavonoids

Terpenoids

Steroids

Resins

Proteins

Oils

T+ +

r++

rtt

++

trncc

-

- = absent, +, tt, t t t = degree ofabunda~icc

family Sol~r~rmetre (Agmv spp) and Lilirrcctrc (Yrrcctr and S~lriltrr spp) ctc. These

investigators have also isolated stcroids like luy-20-(29)-en-2u, 3u -- diol, -sitostcrol,

stigmastcrol and carnpestcrol. Abreu and ~ n ~ e l a j ' ' in a latcr work havc isolatcd

cnrbohydratcs including (-) -bomcsitol, D-pitiitol, sucrosc, D-glucose and D-fructose

moieties.

The cthyl acetate solublc is rich in carbohydrates, reducing sugars, tannins,

stcroids, glycosides, saponins, and tcrpenoids. Interestingly, alkaloids, prorcins. resins

and oils arc absent in both fractions. Presence of tannins, saponins and alkaloids havc

been reported to be responsible for most of thc antimicrobial propcrtics cxliibitcd by

plants "'."' Possible ilse of the solubility fractions as a therapeutic agcnt may provc

liclpful, cspccially considering its saponin contents. Saponins arc known LO have

liyl~ocl~olcstrolcniic properties"5~"" and could confer somc chcnioprotcction against heart

problems.

4.2.3 TLC Fractionation of the Most Active Solvent Extract Arid Phytochemical

Rcsults

Chromatographic fractionation of tlic ethyl acetate solublc of thc stcm bark extract

afforded 12 bands. This is given in table 4.2. All the bands contained tcrpcrtoid and

stcroids. Tcrpcnoids of many plants haw also bccn showri to exhibit a modcrate

'7.2')-3 I ail~imicrobial activity.-

Band 1 has all the compounds shown in tlic ctliyl acctatc solublc. This is not

surprising since band 1 is thc immobile fraction. Band 2 behaves likc the 0 t h bands but

i t has some amount of tannins. This is also no surprise since i t is the band closest to the

inlmobile fraction.

Table 4.2: Phytochemical Analysis oi TLC Bands

1 Alkaloids

2 Glycosides ++

3 Saponins ++

4 Carbohydrate ++t - - -

4 sugar +++ -

G Tannins +.T.+- +

7 Flavonoids

8 Terpenoids &++ +- + 2 ++ ++ ++ ++ ++ ++ ++ ++ ++

9 Steroids + f + + + + t + + + + t

10 Resins

11 Proteins -

12 Oils -

- = absent, +. ++. ii+ = degree of abundance

4.2.4 Relative Antimicrobial activity of Plan Parts and Solvent Fractions.

The antimicrobial screening on metl~anolic extracts of the sten1 bark, leaves, seeds,

the ethyl acetate - soluble and insoluble fractions (solvent fractions) of the stem bark as

well as those of the standard drugs are given in appendis 1.

Discussion.

Initial antiniicrohial screening shows that the leaves, the seed and stem bark of

Dctcrriw~ nricroccrlpr/nr exhibit good activity. Their anti-bacterial activities against

B.srrhtilis, S. atrretrs and K. pttetrn~o~ricre, though lower, are however, coniparable to those

of penicillin G. Also the steni bark, leaves and seed have almost about equal activity

against the germs. The methanolic cxtracts show reasonable activity against K.

p~rcrrnrorricre compared to that of penicillin 6, indicating they could be effective in severe

infections like pneumonia. They also compare favorably to that of penicillin G in

diseases elicited by S. crrrrerrs. /

The solvent fractions of methanolic extracts of the steni bark have pathogenic

activities against gram positive and negative bacteria and fungi. The ethyl acetate

fractions have higher anti- K. ptrerrtrroniae, samc anti - srrbtilis and slightly lower anti -

S. mrretrs activities than the positive con*!roi penicillin G. These fractions will, therefore,

be important in the treatment of pncvnionia. urinary tract infections and wound

infections . This research confirms work done hy Abreu and other investigators who

earlier reported on the activity of the stem bark against P. erelrr-trgitroscr, K. prtertr~~orliere

and ~.arrrixs.~' The activity of extracts of Derririlinr n~icrocerrp~rrrr, therefore, explains

why it is useful in folk medicine especiaSy in Africa. Decoctions of the plant have been

used in treatment of stomach problems like diarrl~oea.'~

4.2.5 Relative MIC's of Significantly Sensi ti\-c ' I ' I K Bands.

Fractionation of the ethyl acetate solublc :~Sforded twelve bands. The sensitivity

of these bands (presented in table 4.3) am! cr;nsequent MIC evaluation (given in

appendix 11 but sunimarized in table 4.4) gave intwsting results. Bands 9-1 2 have little

or no in virro anti-bacterial activity. Particularly, bands 7-12 have no activity against E.

coil ord K. pnezrnio~ritre. These organisms are, therefore, resistant. Bands 1-6 have

moderate activity. They exhibited broad-spectrurn antibacterial activity. Their degree of

effectiveness, however, differs.

Further. the TLC fractions would have no potential value in mycosis infections.

Nane showed any anti-fungal activity. Activity, however, had been observed earlier on

t k solubility fractions of the stem bark against C. nlhicarrs. Loss of activity might be

because the fungi used were resistant. Also, Dhnr el crP7 explained that the loss of

wtivity by individual bands of a hitherto antimicrobial plants extract upon fractionation

might be due to a possible synergy between constituents of bands. Nweze cr a?" had

also suggested that possible factors such as elimination of inorganic constituents during

frnc:ionation which stabilize and activate potc;lrially anhicrobia l substances in plant

exiract and loss of sonie labile constituents of t ' c c\!racts d ~ r i n g separation could lead to

lcss of activity.

. -. ' 1 ,

I I .. . ,-.

-.. 1

I I - - t-.

.I .. I I ,-. 1.-

,- >

I

! I % . <<>

8 .

' I ,

I ! - >

'?

-.. 5;.

I ' , 2

.-,

!

I

Table 4.4: Average hlinimum Inhibiton Concentration (mglml) of TLC Bands and Standard D r u ~ s

S

Pcn. G

MIC f S.E.M, pen. G = penicillin G, chloramp. = chloramphenicol, (P < 0.05)

The MIC values of TLC bands that did not show a strong response on germs were

not determined. From table 4.4, the antimicrobial propmy of band 1 , is perhaps, more

perculiar than any other band. Its activities are more dispersed, cutting across the tested

Gr-am-positive and Gram-negative bacteria. Band 1, is therefore, a possible broad-

spectrum anti-bacterial agent. From the results also, the maximum susceptibility for

band 1 was indicated by S ~IIII-c~rs, 0.572 5 0.2 mg/ml, the least being K ,pnerrmortioe.

1.24 + 0.1 5 mglrnl. For band 2, these were P.nenrginosu and S.tjplti, averaging 0.550 5

Q.08 mglml, while the least were K. pmrnror~ic~e and B.s~rhtilis averaging 1.23 + 0.04

mg/ml. Max. susceptibility for band 8 was indicated by 5'. crttrelrs (0.241 If: 0.07 mg/ml).

Anti-typhoid activity of some of the teslcd bands were nwch more higher than that of

penicillin G. Maximum susceptibility indicated by the organism was of the order: Sand

2 > Band S > Band 5 > Band 1 > Band G penicillin G > Band 7 (MIC? = 0.539 + 0.13,

MlC ,,,, i,,,,i,G = 1.16 + 0.1 I , and MIC, . 1.32 + 0.07 mg/ml). Analysis of variance

showed that there were no significant differences (P > 0.05) in the anti-typhoid

activities o f all the bands when compared to themselves on one hand and to

penicillin G on the other. Significant difference, however, exists between bands 2

and 7 (p < 0.05). When the activities of tested bands were compared with respect

to S.aur-em, i t f'c!!owed that the organism was most susceptible tr. bands 1 and S

than to any band, band 8 being twice niore active than band 1. Further, these two

I

bands also possessed higher activity against the organism when compared to

chloramphenicol. The maximum susccptibility at concentration o f 0.241+ 0.07

rngtml for band 8 when the positive control had a MIC value of 0.834 It 0.05 is,

therefore, worthy of note. For band 1, this was 0.572 rtl 0.572. The grcwth inhibitory

activity of bands 1 and 8 were significant at p < 0.05 with respect to each other. This

difference (p < 3.05) was also observed between bands 4 and 8. Very highly significant

differences (p< 9.001) existed between band 1 and chlorarnphenicol and between band 4

and the standard drug. Hcnce, the place of the plant in management of wound infestions.

Again, activity zf band 2 was sIightly better than band 1 with respect to Bmhtilis but

much lower thar, those of penicillin G and chloramphenicol. Band 2 woiild, however, be

expected to show some mild activity. Their microbial growth inhibitory activity are

given as: MIC ,,,, (; = 0.83 0.10, MICchloralnp. = 0.6425 0.1 2, MICl = 1.30, MICz = 1.14

+ 0.04 mg/ml. Finally, the anti-coli activity of the bands especially band 1 (0.75 +

0.17mglmI) is noteworthy. The strain of this Gram-negative g e m appeared

resistance to penicillin G. In summary, therefore, this rcscarch on Detnriltrr~

/~ric~-oc.crrp~rt,r reveals a grcat potential for its use in various systemic iind ion-

stcrnic bacterial infections.

4.2.6 The R, Values of TL C Bands

Table 4.5: Rr Values of TLC Bands and Colours in Ordinary Light

I Band I Colour in ordinary light

r-- I Reddish brown

4

tsp

0.32

7

8

Yellow

0.42

9

Dccp yellow -I 0.55

0.70

~~ Rcvalucs in MEK : toluene (2:l v/v) + 3 drops each of acetic acid and HzO

Brown --

Brown

0.S 1

Brown

0.96

0.99

Thc R r valucs of thc fractionated (TLC) bands arc givcn i n table 4.5 abovc. 'The

Light brown

Light brown

Light green

colours in thc daylight (diTTercnt lcvcls of yellow) as well as I l ~ c R, valucs or batids 3-6

(0.26, 0.32, 0.42, 0.48) might ilidicatc the bands originated from thc samc class of

conipoimds in thc tcrpenoids/ stcroids scrics.

Bands 7-1 1 are brown-coloured compounds and might bc anolll~cr group of

compouncls in the terpenoids / stcroids scries. Tlieir RI. values arc 0.55, 0.70, 0.81, 0.92

and 0.96. Bald 12 is the most mobilc band. This is a light grcenisli compcmund whosc R,

value is 0.99. I t occupics a class of its own.

4. 2.7 UltraviotetNisible Spectroscopy and Extract Determination

Wavelength of niaxin~un~ absorption. ,,,,,, of the fractionated bands arc givcn in

appcndix iV (spectral I-XII). The rangc of h ,,,,, is bctween 239.0111~ to 053.01im. This is

mainly in thc visible region (>400nnl). Wc haw somc few absorption in thc U V rcgion

(200-400nm). Tlicsc absorptions will ccrtainly scrvc as a ground for fi~ture work.

Spccific compounds would, liowevcr, not be assigned to the bands. Inforniation which

will bc dcrivablc from tlic UV/VIS spectroscopy at this lcvcl niiglit bc to sap that tlicsc

compounds arc highly unsaturated with rcspect to dcgrec of cot~jugntio~:. Furtlicr

isolation and purification using chro~natographic, chemical and otlicr spuctroscopic

mcthods will then be ncccssary to uneqnivocally identify the b a ~ ~ d s . ~ " Exsmple, nucleal-

magnctic rcsonancc (NMR) can bc used ro deduce the carbon skclcton of a ~i~olcculc. rllc

infrarcd (IR) spectroscopy used to idcntify the presence of certain fi~nctionnl groups in

thc mo~eculcs, and mass spectroscopy can bc used to identify the molecuiar niasscs of

tllc intlivitlual niolcculcs.

4.2.8 Conclusion

In this invcstigation. tlic following are concluded:

52

( I ) Detcrt-irrnr nlicr-ocrrt-prrt~r is a medicinal plant which may prove useful in the E

control of diseases.

(2) Activity are established in the leaves, seeds and stem bark of this plant.

I

(3) Activity are also established in both ethyl acetate soluble and ethyl acetate

insoluble fractions of the niethanolic extract of the stem bark.

(4) The phytochen~icals indicated in these solvent fractions are tann:ns,

carbohydrates, steroids, glycosides, saponins, reducing sugars, flavonoids and

terpenoids.

, (5) The phytochemicals responsible for antimicrobial activity are terpenoids and

steroids.

(6) Alkaloids are virtually absent in Detcrrirtrlr tnicroccrr-prtrn extracts.

There is need for a !borough study of this indigenous plant, geared towards

isolation, purification and de:erniination of structure

activity. Detcir-irrnr nricr-occrr-jxrnr may prove useful

of the cheniicals responsible for

in the search for drugs against

common diseases.

.4ppendix 1: Results of antimicrobial screening on n~ethanoIic extracts of Detnrium microcarpurn parts and

ethyl Acetate fractions (lOmg/rnI solutions)

11 Test organisms

Sample; standard drugs

1 hlethanolic 1 Leaves 1 Extract Seeds I I Stem bark

I Et0.k fractions Soluble I

I I of stein bark Insoiub!e

I Peniciilin G

1 ~hldrarn~heiicol

1 Fulcin

Inhibitor\. Zone Diameters (mm)

R. slrbfilis S.uvrerrs K pnertn7oiiiue E. coli P. oerr~gi?~osa S. t ph i C: albicam

12 20

16 20 i S 16 1 S 2 0 20

12 13 11 11 13 11 12

I 6 22 15 - - - - 25 . 78 20 - - - - - - - - - - - I

Blank spaces indicate no obsendjle inhibition (lack of sensitivity).

' w APPENDIX 11: VALUES OF X ', LOGARITHMNS OF CONCENTFtAnON AND MIC OF TLC

FRACnOPlS WITH SIGNIFICANT ACTIVITIES

X' MIC (mplml)

- 12.25 1.30~0. 10 4 1

1 3 0.572 M.2 12.25 4

APPENDIX I1

MIC 2 S. C

AKAYSIS OF VARIANCE: DESCRI PTIVES

upper Bound I-- ----A Bound /

/ Total

I

I 'I otni

1 TLC 3 I Pcnici

-- Total

TLC l I-LC 2 T1.C 5 TLC 6 r1,c 7 TLC 8 Pcnici

To131

1 = confident interval for [,lean

OSE-\I'I\Y ANAYSIS OF VATII.4NCE (ANOVA)

N.S = Not significant,

S = significant.

I

I S cr711.e~rs

I K. piizi!inoiiim

I i 1 Pomrginosn I I 1 Total 39.875

I

I I

R. subtilis

sum of / d l ~ e r n i Sig. Squares I I Squnre I

Between Gmups Within Groups Toinl

72.190 ) 3 1 24.063 98.167 / 10 1 9.817 190.357 ( 13 I

2.45 1 N.S 1 I j

TLC 2

TLC 5

TL.C 6

TLC 7

TL.CS

Penici

I 'LC h I 'LC 7 TLC 8 Penici TLC I n,c 5 TLC 6 TLC 7 ?'LC 8 Pcnici TLC I TLC 2 TLC 6 l'LC 7 TLC 8 Penici I'LC 1 TL.C 2 T'LC 5 -r LC 7 TLC S Penici TLC 1 TLC 2 TLC 5 TCC 6 ?'LC 8 Pcnici 'I'1.C 1 '1'1,C Z T1.C 5 TLC 6 TLC 7 Penici 1'LC ! TLC 2 TLC 5 TLC 6 n.c 7 Tic S

The post Hoc Tats was done before the ~nultiple c01np.1.' c I ISOIIS

71'l~? mean clifference is signil:cant at the .05 level.

Graph I: Log. Conc. Vs x2 of band 1 Against f3. ~ u b t i l i s

Log Conc = 0.1 14, MIC = 1.30

x2 (mm)

Graph 11: Log. Conc. Vs x2 of band 1 Against Saureus

Log Conc =-0.243, MJC = 0.572

X* (mm)

Graph 111: Log. Conc. Vs x2

Graph 1V: Log. Conc. Vs x2 of band

Log Conc = -0.125, MIC = 0.750

. I I

10 12 14

Graph V: Log. Conc. Vs X* of band 1 Against S.typhi

Log Conc = -0.125, MIC = 0.750

x2 (mm)

Graph VI: Log. Conc. Vs X'

Log Conc = -0.181, MIC = 0.660

.

I I 1

20 25 30

Graph VII: Log. Conc. Vs x2 of band 2 Against B. Subtilis

X2 (mm)

Graph VTII: Log. Conc. Vs x2 of band 2 Against K. pneumoniae

Graph JX :Log. Conc. Vs x2 of band 2 Against P.aeruginosa

x2 (mm)

Log Conc = -0.251, M1C = 0.561

Graph X: Log. Conc. Vs x2 of band 2 Against S.typhi

Log Conc =-0.268, MTC = 0.539

Graph XI: Log. Conc. Vs x2 of band 3 Against K.pneumoniae

Log Conc = 0.0538, MIC = 1.13

Graph MI: Log. Conc. Vs X' of band 4 Against Saureus

Log Conc = 0.130, MIC = 1.35

Graph XIII: Log. Conc. Vs x2 of band 5 Against S.typhi

I

Log Conc = -0.185, MIC = 0.652

1

18

/

+

x2 (mm)

Graph XIV: Log. Conc. Vs x2 of band 6 Against Styphi

/

/ Log Conc = -0.0735, MIC = 6.844

I 1

6 8 10 12 14 16 18

Graph XV: Log. Conc. Vs x2 of band 7 Against Styphi

Log Conc = 0.119, ,MIC = 1.32

Graph XVI: Log. Conc. Vs x2 of band 8 Against S.aureiw

7 '

Graph XVII: Log. Conc. Vs x2 of band 8 Against S.typhi

Graph XVIII: Log. Conc. Vs x2 of Penicillin G Against B. S~b t i l i s

I

Log Cooc = 0.085, MIC = 0.831

1

f 0

Graph XIX: Log. Conc. Vs X* of Penicillin G Against K.pneumoniae

I

Log Conc = -0.215, MIC = 0.61 0

0 ; I

0 80

I

Graph XX: Log. Conc. Vs x2 of Penicillin G Against S.twhi

Log Conc = 0.0633, MIC = 1.16 +

Graph XSI: Log. Conc. Vs x2 of Chloramphenicol Against ~ . S ~ b t i / i s

Log Conc = -0.192, MIC = 0.642

Graph XXIk Log. Conc. Vs X* of Chloramphenicol Against S.aorcos

Log Conc = -0.0789, MIC = 0.834

Graph XXIII: Log. Conc. Vs x2 of Chloramphenicol Against K.pneurnoniae

Appendix V : Spectra of TLC Bands ( I-XI1 ) Test Date: 811 212005 User Name: Mr. Osondu Spectrum 1 Test Mode: SCANNING Graph's Name: Sample scan of Sample ? in methanol Start Wavelength: 200,O nrn End Wavelength: 800.0 nm Scan Interval : 3nm nm

kbs

peak: WLOl=458.0 Abs=1.056

Spectrum I1 rest Date: 811 212005 User Name: Mr. Osondu Test Mode: SCANNING Graph's Name: Sample scan of Sample 2 in methanol Start Wavelength; 200.0 nni End Wavelength: 800.0 nm Scan Interval : 3nm nm

Abs

peak: WLOI = N 8 .O WL02=269,0 W bO3=290.0 WL04=320.0 WL05=356.0 WL06=374.0 WL07=434,0

WL09=458.0 WL10=494.0

Spectrum 1.11 Test Date: 811 212005 Uccr Name: Mr. Osondu Pest Mode: SCANNING GI-aph's Name: Sample scan of Sample 3 in methanol Start Wavelength: 200.0 nrn End Wavelength: 800.0 nm Scan Interval : 3nm nm

peak: WLOl=239.O Abs=1.766 WL02=284.0 Abs.7 -81 8 WL03=461.0 Abs=1,153

Spectrum TV Teat Date: 811212005 User Name: Mr. Osondu Test Mode: SCANNING Graph's Name: Sample scan of Sample 4 in methanol Start Wavelength: 200.0 nm End Wavelength: 800.0 nrn Scan Interval : 3nm nm

peak: WL01=455.0 Abs=0.384

Spectrum V Test Daten 811 Z O O 5 User Name: MS. Osondu Pest Mode: SCANNING Graph's Name: Sample scan of Sample 5 in methanol Start Wavelength: 206.0 nm End Wavelength: 800.0 nrn Scan Interval : 3nm nm

kbs

peak: WLOl=4!6.O Abs=0.644

Test Date: 08-1 2-2005 User Name: Mr. Osondu Test Mode: SCANNING Graph's Name: Sample scan of Sample 6 Start Wavelength: 200.0 nm End Wavelength: 800.0 nrn Scan Interval : 3nm nm

peak: WLOI=248.0 Abs=2.947 WL02=458.0 Abs=0.724

Spectrum VXI[ Test Date: 08-1 2-2005 User Name: Mr. Osondu Test Mode: SCANNING Graph's Name: Saniple scan of Sample 7 Start \h/a\~elength: 200.0 nni End Wavelength: 800.0 nm Scan Interval : 3nm nm

peak: WLOl=242.O Abs=2.968 WL02=455.0 Abs=I ,298

Spectrum WIT - I es t Date: 08-12-2005 User Narnc: Mr. Osondu Test Mode: SCANNING Graph's Name: Sample scan of Sample 8 Start Wavelength: 200.0 nm End Wavelenglh: 300.0 nrn Scan IntenVal : 3nm nm

peak: V\iLOl=239.O Abs=2.806 WL02=242.0 Abs=2.820 WL03=263.0 Abs=2.780 WL04=455.O Abs=l.272

Test Date: 08-1 2-2005 User Name: Mi-. Osondu Test Mode: SCANNING Graph's Plarne: Sample scan of Sample 9 Start \Navelcnglh: 200.0 nm End Wavelength: 800.0 nn Scan Interval : 3nm nm

Abs

peak: WLOl=242.O A b ~ 2 . 8 8 1 VVL02=302.0 Abs=2.719 WL03=4.46.0 Abs=0.935

Spcctrurn X Test Dalc: 38-1 2-2OO5 User Name: Mr. Osondu Test Mode: SCANNING Graph's Name: Sample scan of Sample 10 in methanol Sla~ t Wr-!vclcng:h. 200.0 ntir End \,Vaveleng'lh: 800.0 nm Scan lnieival : 3nm nm

Abs

peak: WL01=251 .O WL02=257 .O 'INLi)3=265.0 WL04=281.0 WL05=284.0 WL06=305.0 WL07=386.0 WL08=458.0 WL09=479.0

Tesi Dale: 08- 12-7005 User Name: MI-. S)sor1du Test Mode: SCANNING Graph's Name: Sample scan of Sample 11 Start Wavelength: 200.0 nrn End Wavelength: 800.0 nrn Scan lntemal : 3nm nrn

peak: WLOl=374.O Abs=0.599 WL02=485,0 Abs=0.650

Spectrum XI1 T ~ s t Date: 08-1 2-2005 h e r Name: Mr. Osonclu Test Mode: SCANNING Graph's Name: Sample scan of Sample 12 in methanol Start Wavelength: 200.0 nrn End Wavelength: 800.0 nm - <

b. . Scan lntc~val : 3nm nm , a ' A-\

Abs . -vs 49 1 : ". 5%: 3 CCD

peak: WLOl=25l.O A b ~ 2 . 2 5 7 WL02=272.0 Abs=2.256 WL03=374.0 Abs=0.404 WLO4=4lO.O Abs=0.392 WL05=653.0 Abs=0.147

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