THE ANTIMICROBIAL ACTIVITIES

AND PHYTOCHEMICAL
INVESTIGATION OF BIOACTIVE
CONSTITUENTS,ISOLATION
IDENTICATION AND CHEMICAL
CHARACTERIZATION OF
SPONDIAS MOMBIN EXTRACTS ON
CLINICAL AND ENVIRONMENTAL
ORGANISMS
 SCP/13/14/H/2006
BY
Osuntokun Oludare Temitope
SUPERVISOR
DR (MRS) A. O. OLUDURO
CO-SUPERVISOR
DR T.O.IDOWU
 INTRODUCTION

 Medicinal plants are defined as a group of plants

that possess some special properties that qualify
them as articles of drugs and therapeutic agents.
 EXAMPLES OF MEDICINAL PLANTS
Botanical Family Local Name Part used Medicinal uses
Name
1.Acacia albida Del Leguminosae Gawo Stem bark Skin infections

Anchomanes Araceae Chakara Roots Cough &

difformis. Respiratory
infection..

Boscia Capparidac Anza Roots Anti-cancer

(Nelson et al.,2005,Moronkola et al.,2003
senegalensis. eae ,Ulcer swellings
BRIEF DESCRIPTION OF SPONDIAS
MOMBIN
Spondias mombin Linn belongs to the family
Anacardiacae.
It grows in the rain forest and in the coastal area. It
can reach a height of 15 – 22m.
The trunk has deep incisions in the bark, which often
produces a brown resinous substance. .
The fruit, an 1½-inch long oval yellow plum, has a
leathery skin and a thin layer of fruit pulp with a very
exotic taste. It hangs in numerous clusters of more
than a dozen on the tree.
(Wurochekke, 2002)
 SPONDIAS MOMBIN CONTD
Other common names
Bala (Costa Rica),
Jobito (Panama),
Jobo blanco (Colombia),
Jobo corronchoso (Venezuela),
Hoeboe (Surinam),
Acaiba, Caja, Pau da tapera (Brazil),
Ubo (Peru),
Hobo (Mexico),
Iyeye (Yoruba),
Uvuru (Igbo).

Morton et al., 1987, Nelson et al.,2005, Berger-Brandwijk et al., 2002

PLATE 1-SPONDIAS MOMBIN TREE
PLATE 2-SPONDIAS MOMBIN PLANT WITH
DEVELOPED &DEVELOPING FRUITS
 MEDICINAL USES OF DIFFERENT MORPHOLOGICAL PARTS OF SPONDIAS
MOMBIN

hological Medicinal Uses

Decoction as purgative, treatment of lungs infection (tuberculosis)

Decoration as emetic, a remedy for diarrhea, dysentery, haemo

and a treatment for gonorrhoea. it is used to expel calcification f

bladder, powder for healing wounds

 NON MEDICINAL USES OF DIFFERENT MORPHOLOGICAL PARTS
OF SPONDIAS MOMBIN

Morphological part Non-Medicinal Uses

Root It is used for emergency water source

(Rodrignes et al., 2000).
Stem Living fences in farm land. .
(Hesse et al., 2000)
Wood It serves as poles for huts, garden poles and for
axes and hoe handles. (Flerry et al., 2002).
Wood Ashes In Africa, it is used as indigo-dye.
(Singh et al., 2003).
 NON MEDICINAL USES OF DIFFERENT
MORPHOLOGICAL PARTS OF SPONDIAS
MOMBIN CONTD
Bark It is used in carving figures like amulet, statuettes,
cigarette holder and various ornamental objects
and also as dyeing agent .
(Aregheore et al., 2003).

Gum Glue (Hamano et al., 2001).

Leaves The young leaves are cooked as green vegetable

(Marcadante et al., 2001).
 NON MEDICINAL USES OF
DIFFERENT MORPHOLOGICAL
PARTS OF SPONDIAS MOMBIN CONTD
Fruit Edible – eaten out of hand in stewed with sugar,
It is also used in the preparation of ice-cream, cool
beverages and jelly.
It is also used as jam, wine and vinegar.
It is widely valued as feed for cattle and pig.
(Caraballo et al., 2004).
Flowers It is used for decoration . (Ayoka et al., 2005).

Nectar It is worked on intensively by honey bees.

(Flerry et al., 2002).
 BIOLOGICAL ACTIVITES
DEMONSTRATED BY SPONDIAS MOMBIN
EXTRACTS
Abortifacients Akubue et al., 1983; Anyanwu et al.,1989
 
Anti epileptic Ayoka et al., 2006

Anti psychotic Ayoka et al., 2006

Anti viral Corthout et al.,1994

Anti-ageing Corthout et al.,1992;Flerry et al., 2002

 
BIOLOGICAL ACTIVITES DEMONSTRATED
BY SPONDIAS MOMBIN
XTRACTS CONTD
Anti-diarrhoea Akubue et al., 1983
 
Anti-fertility Raji et al., 2006
Anti-helmintic Ademola et al., 2005
Anti-inflammation Akubue et al., 1983; Abed et al.,1996

Anti-malarial Carabolla et al., 2004

 
Anti-microbial Verpoote and Dahal et al., 1987;
Corthout et al., 1994; Abo et al.,1996
Anti-oxidant Shaltes and Raffant et al., 1990;
  Corthor Costner et al., 1988;
  Panly and Flerry, 2002

Anxiolytic Ayoka et al., 2005

 

Beta-lactamase Coattes et al., 1994

Inhibitor  

Haemostatic Kone-Bamba et al., 1987

Function  
 SPECIFIC AIMS AND OBJECTIVES

 To determine the antimicrobial susceptibility of the

identified organisms to various antibiotics and antifungal
drugs.
 To assess the antimicrobial potency of crude extracts of
leave, stem and bark of Spondias mombin on selected
clinical and environmental organisms.
 To assess the antimicrobial activities of the various
partitioned extracts of Spondias mombin leave, stem and
bark on selected clinical and environmental organisms.
 SPECIFIC AIMS AND OBJECTIVES CONTD
 To determine MIC and MBC of both crude and
partitioned extracts of Spondias mombin.
 To evaluate the synergistic effect of crude extracts of
Spondias mombin and reference antibacterial and
antifungal drug on the selected clinical and environmental
organisms.
 To Evaluate the Qualitative and Quantitative
Phytochemical constituents of Spondias mombin.
 To determine the antioxidant properties of Spondias
mombin.
 To evaluate the anti-nutrient and elemental composition of
Spondias mombin
SPECIFIC AIMS AND OBJECTIVES CONTD

 To determine the Proximate composition of Spondias mombin

 To assess the toxicity of Spondias mombin on the various organs of
experimental animals.
 To purify the Spondias mombin extracts through Thin layer
Chromatography(TLC), Column Chromatography (CC).
 To Identify, Isolate and characterize the bioactive compounds of
Spondias mombin using Gas Chromatography & Mass
spectroscopy(GC-MS), Fourier transform-Infrared spectroscopy
(FT-IR), Nuclear Magnetic Resonance (NMR) and Mass
spectroscopy
 To determine the possible statistical correlation, using two-way
anova method of statistical analysis for the data obtained from the
research work.
 PROJECT OUTLINE MODEL A
Organisms- 30 organisms -10 typed strain, 10
locally isolated bacterial strain, & 10 fungal strain.
Organism identification and authentication with
the use of API 20E & ID 32 C Kits.
Antimicrobial susceptibility assay for the 30 test
organisms by the use of antibacterial and
antifungal disc.
 PROJECT OUTLINE MODEL B

Collection of Plants and Authentication of plant collected.


Extraction of Spondias mombin extracts using ethyl acetate ethanol and

distilled water


Antimicrobial assay of the crude extracts on the test organisms by the use

of Agar well diffusion assay


Synergistic antimicrobial assay of crude extract of Spondias mombin and

reference drugs on the test isolates by Agar well diffusion assay.


Antimicrobial assay of purify and various partitioned extracts on the test

organisms, to measure the MIC and MBC of the plant extract by Agar
 PROJECT OUTLINE MODEL C

Colum &Thin layer Chromatography(TLC) to prepare the spondias mombium extracts

for purification and bioactive component screening.

Phytochemical analysis-Quantitative and Qualitative analysis.

Elemental composition, Anti-nutrient, Nutrient and Proximate analysis.

In-vitro Antioxidant assay of the spondias mombium extracts.

In-vivo toxicological assay of the spondias mombium extracts.

Isolation, Identification and characterization of bioactive compounds of the Spondias

mombium extracts using Gas chromatography & Mass spectroscopy. (GC-MS), Fourier

transform-Infrared spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR).

Statistical Analysis where appropriate..

 FRAMEWORK OF PROJECT MODEL
A
PROPOSED ORGANISMS 10 Typed strains
10 Locally isolated
strains
10 Fungi strains
Identification & authentication with the use of API
20E & ID 32C Kits.

Antibiogram assay - by the use of Antibacterial and

Antifungal disc.
FRAMEWORK OF PROJECT MODEL B
Sample collection

Shade drying

Weighing out

Maceration

Extraction with solvent (3-9 days)

Filtration

Concentration of extract with rotary evaporator

Drying or freeze drying

 FRAMEWORK OF PROJECT MODEL B CONTD

Antimicrobial
assay Plant Toxicology Elemental Anti-nutrient Proximate
Extracts
Assay Composition Analysis

In vivo Evaluation
(Synthetic disease condition)
Re-activation of microbes Reconstitution of extracts

Standardization of organisms
Serial dilution of intermediate
concentration
Pour plate method
Introduction of extracts into the bored hole

Measurement of zones of inhibition

 FRAMEWORK OF PROJECT MODEL C
Phytochemical In-vitro- Antioxidant

ISOLATION

IDENT IFICATION I

Crude extracts screening

Coluum chromatography

Thin layer chromatography

Fourier transform-Infrared spectroscopy (FT-IR

Gas Chromatography & Mass spectroscopic(GCMS IDENTIFICATION II

STRUCTURAL
ELUCIDATION

Nuclear Magnetic Resonance(NMR)Spectroscopic

CHARACTERIZATION
Mass spectroscopy (MS)
UMMARY OF PROPOSED PROJECT FRAMEWORK A,B,
Medicinal plant(Spondias mombium)

Extracts

Bioassay Fraction Toxicology

Pure compound

Structural Chemical Total synthesis

elucidation characteristics
 MATERIALS AND METHODS
PROPOSED ORGANISMS
30 organisms will be used for this research work which will include 20
bacteria and 10 fungi.

The organisms will include 10 typed strains, 20 locally isolated strain of

bacteria and fungi.

The clinical isolate will include organisms from Gastro-intestinal

tracts(GIT), Tuberculosis infection , Sexually transmitted disease(STD)
and Upper respiratory tract infection(URT)etc.

The environmental isolate will include organisms from infected water,

food and soil.
 ORGANISM IDENTIFICATION AND
AUTHENTICATION
API 20E kits will be used to identify the test
organisms for bacteria isolates
API ID 32 C kits will be used to identify the
test organisms for fungi isolates
(Ramani et al.,1998).
 ANTIMICROBIAL ASSAY FOR THE TEST
ORGANISMS
 Antimicrobial susceptibility Assay will be
carried out on the test organisms,
 To determine their antibiogram profile using
various Gram negative and Gram positive
antibiotic discs and antifungal drugs.
(Kim et al,. 2008)
 COLLECTION OF SPONDIAS MOMBIN
 Leaves, bark and stem of Spondias mombin will be
collected from Ikare-Akoko,a tropical rain forest of
Ondo State, Nigeria with latitude (7.21692 North)
and longitude(5.21561 East).
 The time of collection will be around 6.30 am in the
morning, into a polythene bag for easy handling.
Ajayi 2014
 AUTHENTICATION OF PLANT SAMPLE COLLECTED

 Authentication of plant will be done at the Department

of Pharmacy, Obafemi Awolowo Univeristy, Ile Ife
,Osun State.
 A voucher number specimen will be issued for the plant
and deposited in the department for future reference.
 PREPARATION OF
SPONDIAS MOMBIN EXTRACTS(PULVERIZATION)

 The leaves, stems and bark will be rinsed with sterile

water and air dried for four days, and then diced into
smaller pieces.
 The plant sample will be blended with a high speed
blender, to pulverized the sample into powdery fine
form.
(Vogel 2007)
 EXTRACTION OF CRUDE SPONDIAS
MOMBIN
Ethyl acetate, Absolute ethanol and Distilled
water will be used as solvents for extraction
400g each of the samples will be soaked in 1200
ml of ethyl acetate, absolute ethanol and distilled
water for 7days in a 2L conical flask.
 Rotary evaporator will be used to concentrate the
extracts.
(Gislene et al,. 2000)
 CHROMATOGRAPHIC
FRACTIONATION

 The various crude extracts of the leaves, stem-bark

will be subjected to chromatographic fractionation
ANTIMICROBIAL ASSAY OF CRUDE
SPONDIAS MOMBIN EXTRACTS

 AGAR WELL DIFFUSION METHOD

(Nastro et al.,2008),(Okigbo et al.,2006),
(Sofowora 2006).
 ANTIMICROBIAL ASSAY OF PURIFIED
SPONDIAS MOMBIN EXTRACTS

 AGAR DILUTION METHOD

(Nastro et al,. 2008),
(Okigbo et al,. (2005) (Cowan 1999).
 SYNERGISTIC ANTIMICROBIAL
ASSAY
Agar well diffusion assay.
 Synergistic antimicrobial assay of crude extracts
of Spondias mombin and reference drugs on the
test isolates by Agar well diffusion assay.

(Nastro et al.,2008),(Okigbo et al.,2006),

(Sofowora (2006).
PHYTOCHEMICAL ANALYSIS OF SPONDIAS
MOMBIN EXTRACTS

Quantitative and Qualitative analysis which will include

Quantitative analysis – (Trease and Evans 2002)
Qualitative analysis - (Laima 2009),(Doughari,et al,.
2009),(Adejumobi et al,.2008). (Edeoga 2001)
Chemical determination
Elemental Composition
Anti-nutrient percentage
Nutrient percentage
Proximate analysis.
(Horwitz 2003, Hussain et al,.2011,
 IN-VITRO ANTIOXIDANT ASSAY OF SPONDIAS MOMBIN

 Determination of total phenol.

 Determination of total flavonoid.
 Determination of ferric reducing property.
 Determination of free radical scavenging ability.
 Determination Fe2+ Chelation.
 ABTS(2,2’-azino-bis(3-ethylbenthiazoline-6-sulphonic acid)
scavenging ability.
 Superoxide anion scavenging activity assay.
(Singleton et al,.1999),
(Benderitter et al,.1998),(Bao et al ,.2005).
 TOXICITY ASSAY OF SPONDIAS
MOMBIN EXTRACTS
Thirty nine albino rats will be used for this research work .


The rats will be divided into four groups labeled A, B, C, and D
with group A, B, and C consisting of 12 rats each, while the
remaining 3 of the rats will served as control which will be placed
in group D.

Group A will be given 200mg/kg and 500mg/kg body weight
doses of aqueous leaf extract of Spondias mombin.
Groups B received 200mg/kg and 500mg/kg body weight doses
of ethanolic leaf extract of Spondias mombin.

Group C received 200mg/kg and 500mg/kg body weight doses of
ethyl acetate leaf extract of Spondias mombin respectively with
the aid of an oral gastric tube. .
(Konate et al,. 2011),(Lorke 1983).
 Effect of Spondias mombin leaf extracts on haematological
indices of rats. e.g, WBC, RBC,MCV,MCH,MCHC.
 Liver biochemical parameters of rats treated with extracts of
Spondias mombin.eg Cholesterol(mmolL-1), TP(mmolL-1),
HDL(mmolL-1,LDL(mmolL-1), TAG(mmolL- 1),ALB(gL-1 ),
AST(μmolL-1),ALT(μmolL-1),ALP(μmolL-1).
 Serum biochemical parameters of rats treated with extracts of
Spondias mombin.eg Cholesterol (mmolL-1), TAG(mmolL-1),
HDL(mmolL-1),LDL(mmolL-1),TP(molL-1),ALB(gL1),AST
(μmolL-1),ALT(μmolL-1,ALP(μmolL -1),Sodium(mmolL-
1),Potassium(mmolL-1),Chloride(mmol L-1),Bicarbonate(mmolL-
1),Urea(mmolL-1),Creatinine(mm lL-1).
Randox Laboratories Ltd, United Kingdom
Alexander and Griffiths et al,1993, Dacie and Lewis et al,.1994
 OTHER PARAMETERS

Effect of Spondias mombin leaf extracts on body

weights of rats Effect of Spondias mombin leaf
extracts on weekly rats’ food consumption.
Effect of Spondias mombin leaf extracts on
weekly water intake in rats.
Effect of Spondias mombin leaf extracts on organ
weight.
 IN-VIVO EVALUATION
(Synthetic Disease Condition)

 Ten albino rats will be used for this research work.

 The albino rats will be divided into nine group, with one group as the
control.
 Each will be administered with 2cc/cfu of choice organism daily for three
days through the oral gastric tube.
 The choice organism will be used to synthesis a disease condition for three
days.
 On the third day, each group will be administered with the nine extracts of
Spondias mombium with 200mg/ml each.
 The albino rats will be placed under observation with regular food and
water intake.
 On the seventh day, the albino rats will be sacrifice and organs will be
harvested for in-vivo evaluation.
Cosa et al,.2006, McGaw et al,. 2007
 FOURIER TRANSFORM-INFRA-RED (FTIR)
SPECTROSCOPIC METHOD
 This method will be used to measure the possible absorbance ,
ligands, compounds present the plants extract of spondias
mombium.
 The dry samples will be ground into powder with over 200 meshes
and then blended with KBr powder, the sample will be pressed into
a tablet of 10 mm in diameter. Each spectrum will be averaged from
64 scans with a resolution of 4 cm -1.
 Two-dimensional infrared correlation spectra will be obtained by
analyzing the spectra series, according to I. Noda’s generalized 2D
correlation method. 2D contour maps will be calculated and
generated by the 2D-IR correlation analysis software 2Dshige
version 1.3.
(Programmed by Shigeaki Morita, Yukihiro Ozaki, Kwansei-Gakuin
University, Japan),(LU 2003).
 GAS CHROMATOGRAHY/MASS
SPECTROSCOPIC (GC/MS)METHOD OF
ANALYSIS
• This will be used to characterized
and profiled the oil content of the
plant
 GCMS Schematic Diagram
Sample port /injector = for introduction of Vap sample

Separation column = metals tubing packed with a solid materials

with stationary absorbing liquid

Carrier Gas =N2 or He

Flow control equipment = to maintain a constant flow of carrier gas

Detector = measuring the quantity of separated

component

Oven and heater = control of temperature of column , detector , and

injector
 
Integrator /integrator/ strip chart recorded =to provide permanent record or
analysis
 MODUS OPERANDI OF GCMS
A sample of analyte introduced by syringe injector

Heated injector tube

The sample Vaporized &mixed with carrier gas

Partition between gas and Liquid phase according to solubility at column operating temp

Equilibrium partitioning continues with carrier gas

NB .the rate at which the sample travels through the column is determined by sample
solubility in stationary phase, carrier gas , temperature
Detector Column emit in ITD mass spill
= Record total number of ions entering the mass
analyzer from column
Result detector =chromatogram / total ion chromatogram & each
print mass spec
Identification =compare the retention time i.e Rt is the length of time that
remains in the column to that standard.
 MASS SPECTROSCOPIC METHOD OF
ANALYSIS (MS)

 Mass spectrometry will be used to

determination of the molecular mass and the
molecular formula of a compound, as well as
certain structural features.
 It will also be used for identification of the
structural formula of the bioactive component
(McMurry, 2000). (Bruice, 2000).
 NUCLEAR MAGNETIC RESONANCE(NMR)
SPECTROSCOPIC METHOD OF ANALYSIS
This method of analysis will be used to characterized the
bioactive compound fro the Spondias mombium
Nuclear magnetic resonance spectroscopy depends on the
absorption of energy when the nucleus of an atom is excited
from its lowest energy spin state to the next higher one.
The two elements that are the most common in organic
molecules (carbon and hydrogen) have isotopes
(1H and 13C) capable of giving NMR spectra that are rich in
structural information.
1H and 13CNMR is used in determining a substance's
molecular structure.
Nikolov &Schmidt (2012)
 EXPECTED CONTRIBUTION TO
KNOWLEDGE
 This study is expected to contribute to data base
for the synthesis of prototype herbal drugs against
drug resistant organisms’ species which is
becoming a challenge worldwide.
 This study will authenticates the plant
antimicrobial agents against selected pathogenic
organisms for the development of new antibiotics.
 It will also provide more information on the
chemistry of the Medicinal plants
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