3.1 SAMPLE COLLECTION AND PREPARATION OF
MATERIALS
The Ipecac roots (Ipecacauanha) were bought from Ogbete
Market Enugu, Enugu State and authenticated by Prof. the author of the work of the Industrial
Chemistry Department, Federal University of Technology Owerri (FUTO), Imo
State.
3.2 APPARATUS
AND REAGENTS
·
Soxhlet extractor
·
Reflux condenser
·
Round bottom
flask (receiver)
·
Anti-bumping
stone
·
Ethanol
·
Stove
·
Sodium hydroxide
solution
·
Sulphuric acid
·
Hydrochloric acid
diluted
·
Conc,
Hydrochloric acid
·
Water
·
Chloroform
·
Filter paper
·
Conical flask
·
Beakers
·
Spatula
·
Separation funnel
3.3 EXTRACTION
OF THE IPECAC ROOT
Soxhlet extraction method using
ethanol as a reagent was used to extract the Ipecac roots (Ipecacuanha).
Procedure
The fresh roots of Ipecac (Ipecacuanha) which was bought from the Ogbete
market Enugu was solar dried for 3-4 days and about 39.2g were used. After
grinding in electric blender. Extraction was done with 370ml of ethanol and anti-bumping
stone was introduced to the round bottom flask to avoid explosion and to
maintain uniform heating for 12 hours, in soxhlet extractor equipped with a
reflux condenser.
The flask was connected to the
timble and the condenser as well, hence the set-up was set in place
appropriately with the source of light on. As the content of the round bottom
flask (receiver) boiled, it produces ethanol vapour, which goes into the timble
as nascent ethanol, it condenses at the condenser and returns back to the
receiver (round bottom flask). After 12 hours the extract solution was removed
and put in an empty open glass bottle for easy evaporation of the nascent
ethanol that might have been constituted in the extract, then the extract was
left for about 3-5 days so that the nascent ethanol will evaporate at room
temperature to give a gel-like solid which was dissolved in 20ml ethanol and
10ml of H2O and filtered.
3.4. PHYTOCHEMICAL ANALYSIS OF IPECAC ROOT
EXTRACTS
AIMS AND OBJECTIVES
These tests are carried out on the
sample to identify the presence of pharmacologically active constituents in the
sample with the view to identify the presence of various classes of organic
compounds/functional groups based on the reactions with some specific reagents
to give coloured compounds or precipitates.19
3.5 SEPARATION
OF THE METABOLITES
3.5.1 Basic Metabolites: This
metabolite was prepared as earlier, according to (Ejele and Alinner, 2010). The
filtrate ethanol extract was treated with dilute HCl and extracted with
chloroform in a separatory funnel the lower chloroform layer was removed (and
reserved for the preparation of neutral metabolites). The HCl layer was treated
with dilcute NaOH solution until the mixture became basic. Then the resultant
solution (with or without precipitate) was allowed to evaporate completely at
room temperature to form a gel-like solid. Which was dissolved in 95% ethanol
and filtered, the filtrate was used for antimicrobial experiment without
further purification. Preliminary phytochemical screening of this filtrate
showed the presence of alkaloids. Amino acids glycosides and saponnins. 20
3.5.2 Neutral metabolites. The chloroform layer obtained above was using
separating funnel and treated with dilute NaOH solution. The layer was removed
and reserved for preparation of acidic metabolites, the chloroform layer was
removed and allowed to evaporate completely at room temperature to produce a
gel-like solid which was dissolved in chloroform and filtered. The filtrate was
used without further purification for antimicrobial
experiments. Preliminary phytochemical screeting of this filtrate showed the
presence of amides, esters, steroids and triterpenoids, according to Ejele A.
E. 2010. 21
3.5.3 Acidic
metabolites: The aqueous alkaline
layer obtained above was treated with dilute HCl until the solution became
acidic the mixture (with or without precipitate) was allowed to evaporate
completely at room temperature to produce a gel-like solide which was dissolved
in ethanol and filtered. The filtrate was used for antimicrobial experiments
without further purification Ejele and Alinner 2010, preliminary phytochemical
tests carried out on this filtrate showed the presence of amino acids fatty
acids, flavonoids, glycosides phenols and saponnins. 20
3.5.4 TEST FOR TANNINS
10ml of the filtrate sample was
diluted with 10ml distilled water and 2 drops of ferric chloride FeCl3
solution was added, followed by the addition of dilute hydrochloric acid. A dark
brownish precipitate indicates the presence of tannins.
3.5.5 TEST FOR ALKALOIDS
10ml of ethanol was added to 10ml of
the extract sample and heated on a boiling water bath for 10 minutes, cooled
and filtered. 10ml of the filtrate was tested with a few drops of
• Mayer’s reagent
• Dragendorff’s reagent.
• Ferric Acid Solution.
The remaining filtrate was placed in
100ml separating funnel and made alkaline with dilute ammonia solution, the
aqueous alkaline solution was separated and extracted. The extract was tested
with a few drops of mayer’s, wagner’s, dragendorff’s reagent. Two drops of
Wagner reagent give light brown prectipitate, two drops of ferric acid gives a
brown precipitate and one drop of Dragendorff’s reagent gives a brick-brownish
precipitate, which indicates the presence of alkaloid.
3.5.6 TEST FOR TRITERPENOIDS
The
filtrate sample was treated carefully with 10ml of chloroform in a separating
funnel. Two layers, were formed, and the chloroform layer (I.e the lower layer)
was removed and tested as follows; 3ml of the lower layer of the chloroform was
tested for presence of triterpenoids by the addition of 5 ml of tetraoxo
sulphate (iv) acid H2SO4. And heated in a water bath for
about 10 minutes. A brown colour observed indicates the presence of
triterpenoids.
3.5.7 TEST FOR
FLAVONOIDS
10ml of the sample was dissolved in
dilute sodium hydroxide solution followed by the addition of 10ml of HCl a
brownish colour was observed indicating the presence of flavonoids.
3.5.8 TEST FOR GLYCOSIDES
10ml of sample was dissolved in 5ml
chloroform, 2ml of 10% teraoxosulphate (vi) acid was added carefully, a dense
brick brown precipitate was observed
indicate the presence of glycosides.
3.5.9 TEST FOR SAPONIN
When saponin are suspected acidify
the filtrate and boil for about 10mins allow it to cool to room temperature,
filter off any precipitate formed. Divide the filtrate obtained into two
portion and perform the tests for frothing and anthraquinones.
3.5.9a FROTHING
TEST:
5ml of the filtrate (obtained in
test 9 above) was diluted with 10ml of distilled water and two drops of FeCl3 solution (dissolve in dilHCl) was added and
shake vigorously. The colour stable froth foam upon standing indicates the
presence of saponins.
3.5.9b TEST
FOR ANTHRAQUIONONES
The filtrate (obtained in test 9
above) with equal volume of chloroform, separate the chloroform layer. Then add
equal volume of ammonia solution to the chloroform layer. Shake well and allow
to settle for 5 minutes, then, violet colour was observed indicating the
presence of anthraquinone.
3.5.12 TEST
FOR POLYPHENOLS
10ml of sample was mixed with 5ml of
water and the mixture was heated on a water bath for 5 minutes allowed to cool,
and then filtered. 5ml of ferric chloride solution was added to the filtrate
and then dark brown colour precipitate observed indicating the presence of
polyphenols.
3.5.13 TEST
OF ESTER FORMATION
10ml of the sample was warmed with
5ml of 90% ethanol and 5ml conc H2SO4 for 5 minutes
allowed to cool and poured carefully into 5ml of sodium carbonate (Na2CO3)
solution contained in an evaporating dish. Fragrance of ester was perceive
showed brown colour, indicating the presence of ester formation.
3.6 ANTIMICROBIAL
ANALYSIS OF EXTRACT OF IPECAC ROOTS (IPECACUANHA).
The antimicrobial experiments were
carried out in Microbiology Department, Federal Medical Center, Owerri, Imo
State of Nigeria. Test microorganisms used were Coliform bacilli, Salmonella
typhi and staphylococous aureus and the method used was Agar disc diffusion
method. An inoculating loop was touched to three isolated colonies of the
pathogen on an agar plate and used to inoculate to tube of culture broth, which
was incubated at 35-37oC until it became slightly turbid and was
diluted to match the turbidity standard. Then a sterile cotton swab was dipped
into the standardized bacterial test suspension and used to evenly inoculate
the entire surface of agar plate. After 5 minutes, the appropriate antibiotic
test disks were placed with a multiple applicator device. The agar plate was
incubated at 35-37oC for 16-18 hours, after which the diameters of
inhibition zones (areas showing little or no microbial growth) were measured to
the nearest mm Garred and O-Graddy, 1983. 22
Determination of Minimum Inhibitory
Concentration (MIC)
The determination of MIC was carried
out to obtain an idea of the antibacterial activities of the plant extracts,
since agar disc diffusion assay is quantitative method based on the method of
European Society of Clinical Microbiology and infectious Diseases (2000) for
evaluation of antimicrobial potentials. Standard solution of the metabolites
were prepared: 1.0mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml and 0.062mg/ml. in
agar nutrient and distributed into sterile test tubes. 1ml of the extract of
metabolite dilution was separately added into the agar plate for the bacteria
and poured into petri-plates. The test microorganism was spotted onto the
surface of the solidified extract-agar mixture and the plates were inoculated,
starting from the lowest concentration to the highest concentration. After
inoculation the plates were allowed to dry for 30 minutes and incubated at 37oC
for 18hrs, after which the samples were examined for microbial growth. The
lowest concentration of the extract which showed little or no visible growth of
the microorganism was taken as the MIC of the extract Garred and O-Graddy,
1983. As a result, further test was carried out using oil extract from the root.
22
3.6.1 PREPARATION OF MEDIA
Materials Used: nutrient aga-Antec diagnostic product U. K. Test
tubes, syringes, petric dish, cork-borer, cotton wool, incubator, autoclave,
DMSO, distilled water, round bottom flash, measuring cylinder, bijou bottles,
test organisms, wire loop, bursen burner22.
3.6.2 PREPARATION OF THE MEDIA:
Using manufacturers specification of
agar preparation, 30g of agar (powder) was weighed out and distilled water of
100ml was measured out using measuring cylinder. All were added to the
volumetric flask and allowed to dissolved. Therefore, the solution was heated
by applying gentle heat for proper homogenization before it was then dispensed
into 20ml capacity of bijou bottles which make up 50 bottles of agar solution.
All were packed into an autoclave and sterilized at 1200C for 15
minutes.
3.6.3 PREPARATION OF SUSPENSION OF TEST
ORGANISM
Exactly 5ml sterile distilled waters
was dispensed into 5 sterile test tubes and a colony of fresh culture of the
test organism was transferred from culture plate. Using sterile loop,
thereafter, 45ml of the test organism were added to the sterile petric dish and
the sterile molten nutrient agar was added and mixed for even distribution of
the organism and labeled accordingly with permanent marker and allowed to
solidify.
3.6.4 EXTRACTION OF OIL
Ipecac oil is a fixed oil obtained
from root of Ipecac. (Ipecacuanha) either by expression or by solvent extraction,
or soxhlet extraction. The oil is brownish in colour, it is refined by steaming
to coagulate the poisonous toxin it contains the oil used pharmaceutically was
extracted using soxhlet apparatus.
3.6.5 DILUTION OF OIL
Using 200mg/ml, the oil was prepared
using dimethylsufoxide (DMSO) as solvent it solubilizes most plant extract that
cannot go into solution with distilled water serial dilution of the oil was
made using two-fold dilution for six tubes. Therefore, holes were bored on the
surface of the solidified agar, using cork borer of 10mm diameter 40ml of each
concentration was dispensed into the holes using sterile syringe and allowed
for 10 minutes for it diffusion It was then packed into the incubator and
incubated for 24 hours at 370C.
CHAPTER FOUR
4.1
RESULTS OF ANTIMICROBIAL ANALYSIS OF IPECAC
ROOT
EXTRACTION
TABLE 1.
ANTIMICROBIAL RESULT
|
MICROORGANISM
|
100mg
|
50mg
|
25mg
|
12.5mg
|
6.25mg
|
|
Candida
spp.
|
-
|
-
|
-
|
-
|
-
|
|
E.Coli
|
20mm
|
13mm
|
-
|
-
|
-
|
|
Pseudomonas
spp
aureginosa
|
15mm
|
9mm
|
-
|
-
|
-
|
|
Streptococcus
spp.
|
20mm
|
14mm
|
8mm
|
-
|
-
|
|
Staphy
aureus
|
-
|
-
|
-
|
-
|
-
|
|
Coliform
bacilli
|
12mm
|
6mm
|
-
|
-
|
-
|
TABLE 2.
ACIDIC METABOLITE OF IPECA ROOT
|
MICROORGANISM
|
100mg
|
50mg
|
25mg
|
12.5mg
|
6.25mg
|
|
Candida
spp.
|
-
|
-
|
-
|
-
|
-
|
|
E.
coli
|
20mm
|
10mm
|
-
|
-
|
-
|
|
Pseudomonas
spp.
|
20mm
|
13mm
|
6mm
|
-
|
-
|
|
Streptococcus
Spp.
|
25mm
|
18mm
|
10mm
|
4mm
|
-
|
|
Staphy
aureus
|
-
|
-
|
-
|
-
|
-
|
|
Coliform
bacilli
|
20mm
|
16mm
|
8mm
|
-
|
-
|
TABLE 3.
BASIC METABOLITE OF IPECAC ROOT
|
MICROORGANISM
|
100mg
|
50mg
|
25mg
|
12.5mg
|
6.25mg
|
|
Candida
spp.
|
-
|
-
|
-
|
-
|
-
|
|
E.
coli
|
30mm
|
25mm
|
20mm
|
10mm
|
-
|
|
Pseudomonas
spp.
|
-
|
-
|
-
|
-
|
-
|
|
Streptococcus
Spp.
|
-
|
-
|
-
|
-
|
-
|
|
Staphy
aureus
|
-
|
-
|
-
|
-
|
-
|
|
Coliform
bacilli
|
10mm
|
-
|
-
|
-
|
-
|
TABLE 4.
NEUTRAL METABOLITE OF IPECAC ROOT
|
MICROORGANISM
|
100mg
|
50mg
|
25mg
|
12.5mg
|
6.25mg
|
|
Candida
spp.
|
-
|
-
|
-
|
-
|
-
|
|
E.
coli
|
32mm
|
20mm
|
13mm
|
4mm
|
-
|
|
Pseudomonas
spp.
|
20mm
|
13mm
|
-
|
-
|
-
|
|
Streptococcus
Spp.
|
15mm
|
8mm
|
-
|
-
|
-
|
|
Staphy
aureus
|
-
|
-
|
-
|
-
|
-
|
|
Coliform
bacilli
|
30mm
|
22mm
|
14mm
|
7mm
|
-
|
TABLE 5. REFERENCE
ANTIBIOTIC DRUGS
|
DRUG
|
Candida
spp.
|
E.
coli
|
Coliform
bacilli
|
Pseudomonas
spp.
|
Streptococcus
spp.
|
Staphylococcus
aureus
|
|
Amoxil
|
-
|
30mm
|
-
|
22mm
|
-
|
10mm
|
|
Oraxone
|
-
|
5mm
|
10mm
|
-
|
-
|
15mm
|
|
Levofloxucin
|
-
|
30mm
|
7mm
|
17mm
|
-
|
20mm
|
Table 6
4.2 RESULT
OF PROXIMATE ANALYSIS
|
Sample
|
%Ash
|
%
Moisture
|
%
Crude Fibre
|
%
Fat
|
%
Crude protein
|
|
Ipecac
Root
|
3.45
|
11.34
|
10.65
|
5.82
|
32.61
|
4.3 DISCUSSION
The phytochemical analysis of the
Ipecac root (Ipecacuanha) shows the presence of alkaloids, saponins, tannins
cardiac glycosides, flavonoids, poyphenols ester formation, triterpenses and
anthraquionones. Alkaloid was present in high concentration while the others
had the least concentration.
The antimicrobial test carries out
using the ethanol extract of IKpeta root (ipecacunhan),
Indicate the presence of E. coli, pseudomonas spp., streptococcus spp. and
coliform bacilli, as shown in table 1. The
acidic and neutral metabolites screening of Ikpeta root extract Indicates the
presence of E. coli, streptococcus spp, Pseudomonas spp and coliform bacilli as
shown in table 2,4.
The greatest antimicrobial activity was against E.
coli with coliform bacilli zone of inhibition of 20mm, while the lowest
antimicrobial activity was against staphylococcus spp. zone of inhibition is
8mm. The proximate analysis indicate that Ipecac root extract contains crude
protein in high percentages of 32.61% and ash content in low percentage of
3.45% as shown in low percentage of 3.45% as shown in table 6. However, Ikpeta root extract is useful in
treatment of certain ailments such as dysentery when taken in adequate amount
but excess intakes of this Ipecac root by individual can lead to serious side
effect such as gastrointestinal inflammation with a burning sensation in
abdomen, vomiting purging and digestive disorder.
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