Chapter Two Favour
Chapter Two Favour
Chapter Two Favour
The African elemi seed used in this study was purchased from Ochanja market in
Onitsha. The fleshy part was removed and weighed, then sundried and weighed
again. The shell was cracked and the kernel was removed. The Kernel was
weighed before and after sun drying and further grounded to reduce the particle
size. The endocarp being sample A and the seed being sample B were both fed
differently into the soxhelt apparatus to determine its crude fat content after which
the resulting solutions of both samples were retrieved and heated on the hot plate
which was then poured in a crucible to be oven heated using oven heater. The
liquid was extracted from the retrieved solutions of both samples and kept in a jar
with the addition of the base solvent (petroleum ether) for 24hrs after which both
3.1 Materials:
1. Sample A (endocarp)
2. Sample B (seed)
3. Murfle furnace used for proximate analysis in testing of the ash content of the
samples.
6. Petri-dish.
8. Beakers
9. Weighing balance
1. Petroleum ether
2. Ethanol
3. Potassium hydroxide
4. Hexane
5. Sodium sulphate
6. Di-ethyl ether
7. Sulphuric acid
10.Perchloric acid
11.Pyrogallol
Procedure
The weight of the petri dish and sample was noted before drying
The Petri dish and samples were put in the oven and heated at 1050c for 2hrs.
The result was noted and heated another 1hr until a steady result is obtained
The drying procedure was continued until a constant weight was obtained
Weight of sample
Note: The ash of foodstuff is the inorganic residue remaining after the organic
matter has been burnt away. It should be noted, however, that the ash obtained is
Procedures
Empty platinum crucible was washed, dried and the weight was noted.
Calculations:
W2 -W 1
Where
Procedure:
1. Defat about 2g of material with petroleum ether (if the fat content if more
than 10%)
2. Boil under reflux for 30 minutes with 200ml of a solution containing 1.25g
4. Wash with boiling water until the washings are no longer acid.
5. Transfer the residue to a beaker and boil for 30 minutes with 200ml of a
6. Filter the final residue through a thin but close pad of washed and ignited
The loss in weight after incineration x 100 is the percentage of crude fibre.
This method is carried out by continuously extracting a food with non- polar
1. Dry 250ml clean boiling flasks in oven at 105 - 1100c for about 30 minutes.
4. Fill the boiling flasks with about 300ml of petroleum ether (boiling point 40 -
600c)
6. Assemble the soxhlet apparatus and allow to reflux for about 6 hours
7. Remove thimble with care and collect petroleum ether in the top container of
8. When flask is almost free of petroleum ether, remove and dry at 105 0c - 1100c
for 1hour.
9. Transfer from the oven into a dessicator and allow to cool; then weigh.
Wt of sample
(AOAC, 1984)
Principle: The method is the digestion of sample with hot concentrated sulphuric
Procedures
1. Exactly 0.5g of sample was weighed into a 30ml kjehdal flask (gently to
prevent the sample from touching the walls of the side of each and then the
flasks were stoppered and shaken. Then 0.5g of the kjedahl catalyst mixture
was added. The mixture was heated cautiously in a digestion rack under fire
2. The clear solution was then allowed to stand for 30 minutes and allowed to
cool. After cooling was made up to 100ml with distilled water was added to
was placed added under a condenser of the distillation apparatus so that the
tap was about 20cm inside the solution and distillation commenced
immediately until 50 drops gets into the receiver flask, after which it was
Calculations
1g of sample was weighed and transferred in a test tube and 15ml ethanol and 10ml
of 50%m/v potassium hydroxide was added. The test tube was allowed to react in a
water bath at 600c for 60mins. After the reaction time, the reaction product
contained in the test tube was transferred to a separatory funnel. The tube was
washed successfully with 20ml of ethanol, 10ml of cold water, 10ml of hot water
and 3ml of hexane, which was all transferred to the funnel. This extracts were
combined and washed three times with 10ml of 10%v/v ethanol aqueous solution.
The solution as dried with anhydrous sodium sulphate and the solvent was
evaporated. The sample was solubilized in 1000ul of pyridine of which 200ul was
MXT-1 column (15m x 250um x 0.15um) was used. The injector temperature was
280oC with splitless injection of 2ul of sample and a linear velocity of 30cms -1,
Helium 5.0pa.s was the carrier gas with a flow rate of 40 mlmin -1. The oven
operated initially at 2000c, it was heated to 3300c at a rate of 30c min-1 and was kept
Phytochemicals were determined by the ratio between the area and mass of internal
standard and the area of the identified phytochemicals. The concentration of the
Vitamin A was determined by the calorimetric method of kirk and sawyer (1991).
A measured weight (1g) of the sample and standard was mixed with 30ml of
absolute alcohol and 3ml of 500 KOH solution was added to it and boiled gently for
30minutes under reflux. After washing with distilled water, vitamin A was
extracted with 3x50ml of diethyl ether. The extract was evaporated to dryness at
low temperature and then dissolved in 10ml of isopryl alcohol. 1ml of standard
vitamin A solution prepared and that of the dissolved extract were transferred to
Calculation:
vitamin chemist’s (kirk and sawyer 1991). 1g of the sample was mixed with 10ml
of ethanoic sulphuric acid and boiled gently under reflux for 30mins. It was
transferred to a separating funnel and treated with 3x30ml diethyl ether and
recovering ether layer each time, the ether extract was transferred to a dessicator
and dried under for 30mins and later evaporated to dryness at room temperature.
The dried extract dissolved in 10ml of pure ethanol. 1ml of the dissolved extract
and equal volume of standard vitamin E were transferred to separate tubes. After
continous addition of 5mls of absolute alcohol and 1ml of concentrated nitric acid
solution, the mixtures were allowed to stand for 5mins and the respective
zero.
This was determined by the titrimetric method reported by (Kirk and Sawyer). A
was filtered and used for analysis. 20ml of 30% KI solution was added to it and it
was titrated against 0.1M CUSO4 solution. The end point was marked by a black
This was shaken thoroughly and heated for 5 minutes and allowed to cool
and filtered.
The filtrate was poured into cuvette and their respective wavelength for the
Vitamin B1 = 261nm
Vitamin B2 = 242nm
Calculations:
DF = dilution factor
3.2.13 Determination of vitamin B3(Nicotinamide)
warmed slightly.
Calculation:
C8H11NO3HCL
Calculation:
Conc Mg% = AX D.F x volume of cuvette(s)
E
Where A = absorbance
E = extinction coefficient = 25
Association)
on the sample being aspirated into the flame and atomized when the AAS's light
beam is directed through the flame into the monochromator, and onto the detector
that measures the amount of light absorbed by the atomized element in the flame.
Since metals have their own characteristic absorption wavelength, a source lamp
composed of that element is used, making the method relatively free from spectral
sample.
transferred into a glass beaker of 250ml volume, to which 5ml of conc. nitric acid
is added and heated to boil till the volume is reduced to about 15-20ml, by adding
conc. nitric acid in increments of 5ml till all the residue is completely dissolved.
The mixture is cooled, transferred and made up to 100ml using metal free distilled
water. The sample is aspirated into the oxidising air-acetylene flame. When the
1. Weigh out 2g of the dried sample in to a digestion flask and add 20ml of the
acetic acid mixture (650ml conc HNO3; 80ml perchloric acid; 20ml conc
H2SO4)
Principle: The assay of SOD is based on the inhibition of the formation of NADH
end of the reaction can be extracted into butanol and measured at 560nm.
Reagents:
4. NADH (780M)
6. n-butanol
Procedure
Preparation of enzymatic contract: The different samples, 0.1ml, were mixed with
3.0ml of potassium phosphate buffer, centrifuged at 2000g for 10 minutes and the
Assay: The assay mixture contained 1.2ml of sodium pyrophosphate buffer, 0.1ml
of PMS, 0.3ml of NBT, 0.2ml of the enzyme preparation and water in a total
volume of 2.8ml. The reaction was initiated by the addition of 0.2ml of NADH.
The mixture was incubated at 30C for 90 seconds and arrested by the addition of
1.0ml of glacial acetic acid. The reaction mixture was then shaken with 4.0ml of n-
butanol, allowed to stand for 10 minutes and centrifuged. The intensity of the
One unit of enzyme activity is defined as the amount of enzyme that gave 50%
Sample OD
X% inhibition
whose absorbance decreases when degraded by the enzyme catalase. From the
Reagents:
Procedure:
in phosphate buffer. The homogenate was centrifuged and the supernatant was
followed by the rapid addition of 40μl of enzyme extract and mixed thoroughly.
The time required for a decrease in absorbance by 0.05 units was recorded at
240nm in a spectrophotometer (Genesys 10-S, USA). The enzyme solution
One enzyme unit was calculated as the amount of enzyme required to decrease the
The method proposed by Reddy et al. (1995) was adopted for assaying the activity
of peroxidase.
spectrophotometrically at 430nm.
Reagents:
3. Glutathione (1mM)
Procedure
Preparation of enzyme contract: A 20% homogenate was prepared in 0.1M
phosphate buffer (pH 6.5) from the sample, clarified by centrifugation and the
Assay: To 3.0ml of pyrogallol solution, add 0.1ml of GSH, 0.1ml of the enzyme
extract was added and the spectrophotometer was adjusted to read zero at 430 nm.
To the test cuvette, 0.5ml of H2O2 was added and mixed. The change in absorbance
at 430nm.
Foam Formultion