CHAPTER THREE

MATERIALS AND METHODS

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

samples where filtered.

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.

4. Soxhelt apparatus for crude fat experiment.

5. Oven heater for heating the samples.

6. Petri-dish.

7. Dessicator for cooling

8. Beakers

9. Weighing balance

10.Kjehdal flask. Etc.

The following chemicals, purchased from were also used;

1. Petroleum ether

2. Ethanol

3. Potassium hydroxide

4. Hexane

5. Sodium sulphate

6. Di-ethyl ether

7. Sulphuric acid

8. Anhydrous glacial acetic acid

9. Mercury II acetate solution

10.Perchloric acid

11.Pyrogallol

12.Phosphate buffer etc.

3.2 Methodology

3.2.1 Determination of Moisture Content

Procedure

A petri-dish was washed and dried in the oven

 Approximately 1-2g of the sample was weighed into petri dish

 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

and the weight was noted.

 The drying procedure was continued until a constant weight was obtained

% moisture content = w1-w2 x100

Weight of sample

Where w1 = weight of petridish and sample before drying

W2 weigh of petridish and sample after drying.

3.2.2 Determination of Carbohydrate

Using differential method:

100 – (%Protein + %Moisture + %Ash + %Fat + %Fibre)

3.2.3 Determination of Ash content

(AOAC, 1984)

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

not necessarily of the composition as there may be some from volatilization.

Procedures

 Empty platinum crucible was washed, dried and the weight was noted.

 Approximately 1- 2g of sample was weighed into the platinum crucible and

placed in a muffle furnace at 5500c for 3 hours.

 The samples were cooled in a dessicator after burning and weighed.

Calculations:

% Ash content = W3 -W 1 x 100

W2 -W 1

Where

W1 = weight of empty platinum crucible

W2 = weight of platinum crucible and sample before burning

W3 = weight of platinum and ash.

3.2.4 Crude Fibre Content

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

of H2SO4 per 100ml of solution

3. Filter the solution through linen

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

solution containing 1.25g of carbonate free NaOH per 100ml

6. Filter the final residue through a thin but close pad of washed and ignited

asbestos in a Gooch crucible

7. Dry in an electric oven and weigh

8. Incinerate, cool and weigh

The loss in weight after incineration x 100 is the percentage of crude fibre.

% crude fibre = weight of fibre x 100

Weight of sample

3.2.5 Crude Fat Content

Soxhlet Fat Extraction Method:

This method is carried out by continuously extracting a food with non- polar

organic solvent such as petroleum ether for about 1 hour or more.

Procedure:

1. Dry 250ml clean boiling flasks in oven at 105 - 1100c for about 30 minutes.

2. Transfer into a dessicator and allow to cool

3. Weigh correspondingly labeled, cooled boiling flasks.

4. Fill the boiling flasks with about 300ml of petroleum ether (boiling point 40 -

600c)

5. Plug the extraction thimble lightly with cotton wool

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

the set – up and drain into a container for re – use.

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.

% fat = wt of flask + oil - wt of flask x 100

Wt of sample

3.2.6 Crude Proteins content

(AOAC, 1984)

Principle: The method is the digestion of sample with hot concentrated sulphuric

acid in the presence of a metallic catalyst. Organic nitrogen in the sample is

reduced to ammonia. This is retained in the solution as ammonium sulphate. The

solution is made alkaline, and then distilled to release the ammonia. The ammonia

is trapped in dilute acid and then titrated.

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

until a clear solution appeared.

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

avoid caking and5ml was transferred to the kjedahl distillation apparatus,

followed by 5ml of 40% sodium hydroxide.

3. A 100ml receiver flask containing 5ml of 2% boric acid and indicator

mixture containing 5 drops of Bromocresol blue and 1 drop of methlene blue

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

titrated to pink colour using 0.01N hydrochloric acid.

Calculations

% Nitrogen = Titre value x 0.01 x 14 x 4

% Protein = % Nitrogen x 6.25

3.2.7 Extraction of phytochemicals

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

transferred to a vial for analysis.

3.2.8 Quantification by GC-FID

The analysis of free steroids was performed on a BUCK M910 Gas

chromatography equipped with a flame ionization detector. A RESTEK 15 meter

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

at this temperature for 5min. the detector operated at a temperature of 3200c.

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

different phytochemicals expressed in ug/g.

3.2.9 Determination of vitamin A

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

separate curettes and their respective absorbance were read in a spectrophotometer

meter at 325nm with a reagent blank at zero.

Calculation:

Conc of vitamin A in sample = Abs of sample x conc of standard

Abs of std
3.2.10 Determination of vitamin E

This was determined by the futter – mayercolometric method with association of

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

absorbance measured in a spectrophotometer meter at 410nm with blank reagent at

zero.

3.2.11 Determination of Vitamin C

This was determined by the titrimetric method reported by (Kirk and Sawyer). A

weighted sample was homogenized in 6% EDTA/TCA solution. The homogenate

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

coloration. A reagent blank was also titrated.

Vitamin C content was calculated based on the relationship below. 1ml of 0.1 mole

CuSO4 =88mg vit. C

Vitamin C mg/100 = 1 x .88 x titre – blank

W

3.2.12 Determination of vitamin B1 and B2

 1g of sample was weighed into a conical flask

 This was dissolved with 100ml of deionized water

 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

vitamins set to read the absorbance using spectrophotometer

Vitamin B1 = 261nm

Vitamin B2 = 242nm

Calculations:

Concentration (mg%) = A x D.F x volume of cuvette (5)

E
Where A = absorbance

E = extinction coefficient = 25 for B1 and B

DF = dilution factor
3.2.13 Determination of vitamin B3(Nicotinamide)

 5g of sample was dissolved in 20ml of anhydrous glacial acetic acid and

warmed slightly.

 5ml of acetic anhydride was added and mixed.

 2 -3 drops of crystal violet solution was added as indicator.

 Titrate with 0.1M perchloric acid to a greenish blue colour.

Calculation:

VitaminB3 = titre value x 0.0122

0.1

3.2.14 Determination of vitamin B6

 5g of sample was dissolved in a mixture of 5ml of anhydrous glacial acetic

acid and 6ml of 0.1m mercury II acetate solution.

 2 drops of crystal violet was added as indicator.

 Titrate with 0.1m perchloric acid to a green colour end point.

Calculation: each meal of 0.1M perchloric acid is equivalent to 0.02056g of

C8H11NO3HCL

3.2.15 Determination of vitamin B12

25mg of sample was dissolved in 250ml of deionized water

Calculation:
Conc Mg% = AX D.F x volume of cuvette(s)
E
Where A = absorbance

E = extinction coefficient = 25

D.F = dilution factor = 5

3.2.15 Methods for the Elemental Analysis of samples

Elemental analysis was conducted using Agilent FS240AA Atomic Absorption

Spectrophometer according to the method of APHA 1995 (American Public Health

Association)

Working principle: Atomic absorption spectrometer's working principle is based

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

or radiational interferences. The amount of energy of the characteristic wavelength

absorbed in the flame is proportional to the concentration of the element in the

sample.

Procedure: The sample is thoroughly mixed by shaking, and 100ml of it is

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

aqueous sample is aspirated, the sensitivity for 1% absorption is observed.

Sample Digestion (Adrian, 1973)

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)

2. Heat the flask until a clear digest is obtained.

3. Dilute the digest with distilled water to the 100ml mark.

4. Appropriate dilutions are then made for each element.

ASSAY OF SUPEROXIDE DISMUTASE (SOD)

SOD was assayed according to the method of Kakkar et al. (1984).

Principle: The assay of SOD is based on the inhibition of the formation of NADH

phenazine methosulphate-nitroblue tetrazolium formazon. The colour formed at the

end of the reaction can be extracted into butanol and measured at 560nm.

Reagents:

1. Sodium pyrophosphate buffer (0.025M, pH 8.3)

2. Phenazine methosulphate (PMS) (186M)

3. Nitroblue tetrazolium (NBT) (300M)

4. NADH (780M)

5. Glacial acetic acid

6. n-butanol

7. Potassium phosphate buffer (50mM, pH 6.4)

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

supernatants were used for the assay.

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 30C 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

chromogen in the butanol layer was measured at 560nm in a spectrophotometer

(Genesys 10-S, USA).

One unit of enzyme activity is defined as the amount of enzyme that gave 50%

inhibition of NBT reduction in one minute.

SOD% = sample OD – control OD x 100 = X% inhibition

Sample OD

50% inhibition = 1unit enzyme

X% inhibition

ASSAY OF CATALASE (CAT)

Catalase activity was assayed following the method of Luck (1974).

Principle: The UV absorption of hydrogen peroxide can be measured at 240nm,

whose absorbance decreases when degraded by the enzyme catalase. From the

decrease in absorbance, the enzyme activity can be calculated.

Reagents:

1. Phosphate buffer : 0.067 M (pH 7.0)

2. Hydrogen peroxide (2mM) in phosphate buffer

Procedure:

Preparation of enzyme extract: A 20% homogenate of the sample was prepared

in phosphate buffer. The homogenate was centrifuged and the supernatant was

used for the enzyme assay.

Assay: H2O2-phosphate buffer (3.0ml) was taken in an experimental cuvette,

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

containing H2O2-free phosphate buffer served as control.

One enzyme unit was calculated as the amount of enzyme required to decrease the

absorbance at 240nm by 0.05 units.

Catalase umol/ml = abs change x total reaction volume

Sample volume x extinction coefficient
Extinction coefficient = 0.394umol-1ml-1

ASSAY OF GLUTHIONE PEROXIDASE (POD)

The method proposed by Reddy et al. (1995) was adopted for assaying the activity

of peroxidase.

Principle: In the presence of the hydrogen donor pyrogallol or dianisidine,

peroxidase converts H2O2 to H2O and O2. The oxidation of pyrogallol or

dianisidine to a colored product called purpurogalli can be followed

spectrophotometrically at 430nm.

Reagents:

1. Pyrogallol : 0.05 M in 0.1M phosphate buffer (pH 6.5)

2. H2O2 : 1% in 0.1M phosphate buffer, pH 6.5

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

supernatant was used for the assay.

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

was recorded every 30 seconds up to 3 minutes in a spectrophotometer (Genesys

10-S, USA). One unit of peroxidase is defined as the change in absorbance/minute

at 430nm.

Gluthione Peroxidase (umol/ml) = abs change x total reaction volume

Sample volume x extinction coefficient
Extinction coefficient = 0.266umol-1ml-1

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