IPECAC ROOTS (IPECACAUANHA) - PHYTOCHEMICAL ANALYSIS | CHAPTER 3 AND 4 PROJECT WORK



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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