EXTRACTION AND PARTIAL CHARACTERIZATION OF MUSTARD SEED (BRASSICA SPP.) OIL

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177

Emmanuel et al. World Journal of Pharmaceutical Research

EXTRACTION AND PARTIAL CHARACTERIZATION OF MUSTARD

SEED (BRASSICA SPP.) OIL

Aloh,G.S.1, *Obeagu, Emmanuel Ifeanyi

2, Odo Christian Emeka

1, Kanu, Stella

Ngozika3, Okpara,kingsley Ezechukwu

4, Onyekwere Chioma

5,

Obeagu Getrude Uzoma6

1Department of Biochemistry, Michael Okpara University of Agriculture, Umudike, Abia

State, Nigeria.

2Diagnostic Laboratory Unit, University Health Services, Michael Okpara University of

Agriculture, Umudike, Abia State, Nigeria.

3Abia State University Teaching Hospital, Aba, Abia State, Nigeria.

4Rivers State College of Health Technology, Port Harcourt.

5Department of Biochemistry, Ebonyi State University, Abakaliki, Nigeria.

6School of Nursin Science,ESUT Teaching Hospital,Parklane,Enugu,Nigeria.

ABSTRACT

The extraction (with soxhlet apparatus) and partial characterization of

Brassica juncea oil, its phytochemical and physiochemical analyses

were carried out to investigate the extractable oil of the seed, to

classify the oil and to evaluate its pharmacological and industrial

potentials. The extraction gave a percentage yield of 22.93% while the

partial characterization results (which include the physicochemical and

phytochemical analyses), were specific gravity - 0.8286; acid TOlue-

11.781%; free fatty acid value-5.922%; peroxide value-7.0%; iodine

value -103.499%, saponification value 315.563%, unsaponifiable

matter - 5.0%. The Rf values from the TLC gave 0.92, 0.94, 0.84, and

0.89 for stearic, linoleic, cleic and palmitic ands respectively niiich

corresponded to the Rf values of the sample, while the phytochemistry

revealed the presence of alkaloids, flavonoids, tannins, saponins,

glycosides and proteins. These suggest good, stable, edible and semi-

drying extractable industrial oil with pharmacological properties.

KEYWORDS: Brassica juncea oil, semi- drying extractable, phytochemical analyses.

World Journal of Pharmaceutical Research

SJIF Impact Factor 5.045

Volume 4, Issue 3, 177-195. Research Article ISSN 2277– 7105

Article Received on

23 Dec 2014,

Revised on 17 Jan 2015,

Accepted on 11 Feb 2015

*Correspondence for

Author

Obeagu, Emmanuel

Ifeanyi

Diagnostic Laboratory

Unit, University Health

Services, Michael Okpara

University of Agriculture,

Umudike, Abia State,

Nigeria.

Phone: +2348037369912

obeagu.emmanuel@mouau

[email protected]

mailto:obeagu.emmanuel@mouau

mailto:[email protected]


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INTRODUCTION

Vegetable or plant oils which are often essential and non-essential oils have been known and used

by man from pre-historic times. Indeed, apart from water, there is perhaps no liquid chemical mat

the common is more familiar with its uses in the home than vegetable oils

(Ibemesi, 1992), including essential oils (also referred to as ethereal and volatile oil), which are

volatile plant oils obtained by other means than by direct steam distillation (Bryan, 1996). Oils

obtained by enzymatic action followed by steam distillation example, mustard oils isolated by

simple pressing example, lemon and orange oils, and oils Dtained by solvent extractions are

included among essential or ethereal oils.

The major difference between these oils and fatty oils is their volatility (Bryan, 1996).

Vegetable or plant oils are used in many industries for diverse purposes which are necessities

in advanced civilization, thereby contributing directly to health, happiness and general well

being. The various industries that use these products include pharmaceuticals, animal feeds,

baked foods, canned foods, chewing gum, condiments and confectioneries, beverages, soft drinks,

cosmetics, soaps, adhesives, insecticides, paints, paper and textile processing industries (Ibemesi,

1992).

Although the extraction and analyses of the chemical composition of vegetable oils has been the

subject of numerous investigations, relatively very few reports contain comparative data on samples

from different methods of extraction.

Since vegetable (plant) oils are such important industrial (especially pharmaceutical, in the case of

this work) raw materials, there is need to evaluate and analyze the lipid content of this planVegetable

le oils are water-insoluble substances of plant origin which consist predominantly of long-chain

fatty acids esters derived from the single alcohol, glycerol (HOCH2 CHOHCH2 OH), and are known as

trigtycerides (Ibemesi, 1992).common usage considers as oils, triglycerides des that are liquid at

room temperature and as fats, those that are solid or semi-solid under the ne conditions. This

difference in their physical state arises from their chemical composition: oils being composed of low-

melting fatty acids (mostly unsaturated) while fats are famed from high-melting fatty acids (mostly

saturated) (Ibemesi, 1992)However, at higher temperatures, fats (solid) become oils (liquid) so

that both terms can , and will be used interchangeably in.

The word and this will introduce us to the term "lipids". Lipids are naturally occurring compounds, which

are long chain fatty acid esters They are insoluble in water but soluble in "fat" (organic) solvent such as


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acetone, alcohol, chloroform, or benzene (David, 1987).

Lipids are the only macromolecules that are defined, not by their chemical composition, but

rather by their solubility, hence were defined by Abaelu (2004) as a group of chemical substances

insoluble in water but soluble in organic solvents. They are the waxy, greasy and oil substances that

are found in living systems. They are also considered as polymers of monomeric units known as

fatty acids (Nwachukwu et al., 2002). According to Onwurah et al. (2001), the term lipids refer to

a wide variety of naturally occurring substances including fatty acids and their derivatives,

steroids, terpenes, vitamins etc, which have in common the ability to solubilize in organic

solvents such as diethyl ether, hexane, benzene, chloroform and acetone. Thus, they are water

insoluble organic biomolecules that are extracted from cells and tissue by non-polar solvents.

The alkaline hydrolysis of lipids, known as saponification, leads to release of its constituent alcohol

and acids, which are usually, water soluble (Nwachukwu, et al, 2002). Lipids are classified based on

the hydrocarbon nature of the major portion of their backbone structure (Onwurah et al, 2001).

Chemically, they are classified into two major groups as simple lipids and compound lipids. Steroids,

and the fat-soluble vitamins are also considered as lipids because of their similar soluble

characteristics, and are known as "derived lipids" (David, 1987.)

Lipids are also classified based on their state at room temperature, as seen earlier, into fats and

oils (the storage forms of lipids called triglycerides). Fats are triglycerides that are solid at room

temperature while oils are triglycerides that are liquid at room temperature (Raven et al., 1992).

Lipids can be found in plants and animals as food sources. In plants,they can be found as

secondary metabolites (plant products) such as essential oils (which includes cyanogenic

substances and saponins), alkaloids, quinines, flavonoids and even raphids (needle-like crystals

of calcium oxalate) (Raven et al., 1992). For example, the mustard family - Brassicaceae,

which is our subject plant, is characterized by the presence of mustard enzymes (myrosinases) that

break down these glycosides (into isothiocyanate, ITC ) to release the pungent odours associated

with cabbage, horseradish, and mustard (Raven et al.,1992).

Oils are also found in mature pollen grains since they are packed with starch or oils depending on

the group, and are a very nutritious source of food for animals. Other plants like soybean,

sunflower, olive, oil palm, groundnut etc. are important sources of oil.


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In animal systems, they are found as fat deposits in the adipose tissues, muscles, subcutaneous

layers of the skin etc. They are also found in fish, liver, dairy products, cereals, green vegetables etc.

Although most oils are obtahed from plants (about 60%), a sizeable quantity (36%) comes from

animal sources and the rest (4%) from marine life. Some well-known animal fats and oils are

butter (milk), lard (Hog) and tallow (cow, sheep or goat) and cord-liver oil. These animal and

marine fats and oils find extensive uses in the industry. However, the present acute shortage of

milk, meat and fish in the country makes meaningless any talk of oil extraction from these

sources for commercial or individual purpose (Jemesi, 1992).

AIMS OF THE RE SEARCH

Vegetable oils abound in their great numbers, each with its own definite characteristics and

"claimed" compositions (constituents). Mustard seeds have been claimed to possess some medicinal

qualities by some individuals.

Also, It has been known that mustard seed contains some percentage of oil, which could be extracted,

characterized and harnessed into the vegetable oil industries for tlieir fats and oil or rather lipid

content in the edible and non-edible uses of vegetable oil. It is the objective of this work to extract

this oil, evaluate its constituents and ascertain or disprove the "claimed" composition of the mustard

seed using specific extraction and characterization method (though our characterization will be

limited to the available resources). This would reduce the pressure that has been mounted on the

edible and non-edible uses of other vegetable oils like; groundnut oil, castor oil, cashew nut oil,

olive oil etc.There are also reports that mustard seed has some medicinal/pharmacological properties

as some claim that when ifflicted with some ailments they have their ailments lifted after

chewing and swallowing the mustard seed. Analyzing for the phytochemical constituents of the

mustard seed could only prove this allegory. Hence, this project was undertaken to achieve the

following ibjectives.

- To extract the mustard seed oil

- To evaluate it's percentage yield;

- To ascertain the pharmacological importance of the seed by determining it's phytochemical

components

- To characterize the oil in terms of it's physicochemical properties.


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MATERIALS AND METHODS

MATERIALS

TEST SAMPLES

The test material for the research work is brown mustard seed (Brassica juncea). The mustard

seeds, packaged in Asia, were collected from the St. Theresa's Catholic Church, Ogoja

road, Abakaliki, Ebonyi State. Oil was extracted using diethyl ether (37 -40°C) in a soxhlet

apparatus, and the crude oil was used to carry out the analysis.

OIL EXTRACTION (Pearson, 1973)

The soxhlet extraction unit of Pearson (1973) was used for extraction of lipids fro m the material.

The soxhlet extraction was set up with pre- weighed soxhlet flask and the extraction procedure

followed. A quantity (116g) of the dried milled sample was put into the thimble and the materials

were continuously extracted for 6 hours using diethyl ether (37°C - 40°C) as solvent. , the end of

the extraction, the thimble was removed and the solvent allowed to evaporate, the flask and the

content were dried in the oven at 60°C for 10 minutes. The flask containing the oil was cooled in

the desiccators, weighed and drying process repeated until a constant weight was obtained.

LIPID CONTENT DETERMINATION

The lipid content of the sample was estimated using the difference between the weight of the sample

and the defatted sample divided by the original weight of the sample multiplied by 100. This gave the

percentage lipid content of the sample. The formular is written.as; % Lipid content =

Weight of sample - weight of defatted sample x 100.….equation (5)

Weight of sample 1

CHARACTERISATION OF MUSTARD SEED

(BRASSICA JUNCEA) OIL

Characterization of Brassica juncc oil was carried out to assess the quality of the oil extracted,

which ss measurable by suitable physical and chemical techniques.

PHYSICAL PROPERTIES

The physical tests carried out include the specific gravity, appearance at room temperature,

organoleptic properties like colour, odour and taste, solubility and miscibility. The colour, odour

and taste including physical state at room temperature, solubility and miscibility were

physically evaluated by visual inspection, smelling, tasting and mixing the oil sample with water, and

then, organic solvent.


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PHYSICAL STATE AT ROOM TEMPERATURE

A 50ml beaker was first washed with a detergent, rinsed in water and allowed to dry, 10ml of the

oil was poured into it and allowed to stand for 24 hours. The physical state of the oil after 24 hours

at an ambient temperature was noted. Generally, oils are liquid at room (ambient) temperatures.

SPECIFIC GRAVITY

A 50ml specific gravity bottle was first washed with a detergent, rinsed in water and then with

diethyl ether. The bottle was dried and weighed empty, after which it was filled with distilled

water and then weighed again. The crude oil was used to fill the bottle and also weighed. The

specific gravity was calculated with the formula: Specific gravity =

Weight of Xcm3 of oil - - equation (6)

Weight of Xcm3 of water

ORGANOLEPTIC PROPERTIES

i. Colour: The determination of colour and appearance of the sample was by the visual

determination .and the yellow colour was noted,

ii. Odour: The odour was determined by smelling the oil, and the slight pungent odour was noted,

iii. Taste: The taste bud of the tongue was employed, and the taste of the oil noted.

SOLUBILITY AND MISCIBILITY

A 250ml conical flask was washed and then dried 2g of the oil was weighed and poured into

the flask, and 20ml of distilled water subsequently. The mixture was shakened vigorously for 2

minutes and was observed for miscibility. This was noted in the result.

CHEMICAL PROPERTIES AND TESTS

The chemical tests carried out were the acid value; free fatty acid value; iodine value peroxide

value; saponification value and the amount of unsaponifiable matter.

ACID AND FREE FATTY ACID VALUES (AOAC, 1980)

A quantity (25ml) of ethanol (95%) was mixed with 25ml of diethyl ether (equal volume) and

1ml of phenolphtlialein indicator (1% in ethanol) was added. The mixture was neutralized by

titrating with 0.1 N KOH from a burette and wanned. A quantity (50ml) of this solution was added

to 2g of the oil weighed into a 250ml conical flask. The mixture was boiled for about 5 minutes

and titrated while hot with a standard aqueous solution (standardized by titrating with standard

HCL solution) of potassium hydroxide while shaking vigorously during the titration until a pink

colour, which persisted for 15 seconds appeared. The acid value was calculated using the formula;


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Acid value = 56.1 x V xN - - - equation (7)

W Free fatty acid value

28.2 x V x N - - -equation (8)

Where V- Volume ofKOHtitre i&

N-Normality of KOH ( W = Weight of the oil sample

PEROXIDE VALUE (Hamilton et al., 1992)

The wheeler method as described by Hamilton et al, 1992, was used. A quantity (Ig) of the oil

was dissolved in 25ml of a solvent mixture consisting of 60% glacial acetic acid and 40%

chloroform and 1ml of 10% saturated solution of potassium iodide was added. The flask was

shaken and allowed to stand in the dark for 5 minutes, after which 75ml of distilled water was

added. The mixture was then titrated with 0.1N standard solution of sodium thiosulphate using 2ml

of 1% starch solution as indicator. A blank determination was carried out at the same time and

the peroxide value was calculated using the formular.

Peroxide value = (Vs-Vb)xNx 100 - - - equation (9)

W

Where, Vs = Volume of sodium thiosulphate used in sample Vb = Volume of sodium thiosulphate

used in blank N = Normality of sodium thiosulphate W = Weight or Mass

IODINE VALUE (Ikwuagwu et al., 2000). The Wij's method as described by Hendriske and

Harwood (1986) was used (also cited in Ikwuagwu et al., 2000).

Wij's reagent (Iodine trichloride solution) was prepared by dissolving 2g of iodine trichloride in

50ml of glacial acetic acid and was mixed with 2.25g iodine, dissolved in 100ml of glacial

acetic acid. The mixture was then made up to 250ml with glacial acetic acid and stored in a brown

glass bottle and kept out of light until use.

A quantity of the oil (0.5g) was weighed and transferred into a 250ml glass-stoppered bottle. A

quantity, 15ml of chloroform was added to dissolve the oil and 25ml of Wij's solution was added

from a burette.

The flask was closed and the content mixed and allowed to stand in the dark for 30 minutesAfter

standing, 20ml of 15% potassium iodide solution was added and the bottle stoppered and shaken

thoroughly and the sides of the bottle and the stopper was washed with 100ml of recently boiled and

cooled water. The solution was titrated with standard solution (0.1N) sodium thiosulphate, the reagent

being added with constant shaking until the yellow colour of the iodine has almost disappeared. The


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bottle was stoppered and shaken vigorously for the remaining iodine in the organic layer to pass

into the water layer. Two blank determinations with the same quantities of reagent were carried out at

the same time under the same conditions. The iodine value was calculated with theformula:

Iodine value =

= 12.69 x M f V - V ) - - - equation (10) W

Where

M = Molarity of sodium thiosulphate used

W = Weight or mass of oil in grams

V = Volume of Na2S2O3 used for the blank

VI = Volume of NaiSiOa used for the sample

SAPONIFICATION VALUE (AOAC, 1980)

A quantity (2g) of oil was weighed into a 250ml conical flask and 25ml of 0.5m ethanoic

potassium hydroxide solution was added. The flask was connected to a reflux condenser and the mixture

heated on a hot plate for 1 hour (by which time the sample was completely saponified as

indicated by the absence of oil matter and appearance of clear solution). The inside of the condenser

was washed down with 10ml of hot ethanol neutral to phenolphthalein. 1ml of phenolphthalein

indicator solution was added and the solution titrated with 0.5M standard hydrochloric acid. A

blank determination using the same quantity of potassium hydroxide was carried out at the same

time. The saponification value was calculated using the formula:

Saponification value = 56.1 x (V - V1) x M - - - equation (11)

Where M = Molarity of KCH

V = Titration volume the bl

VI = Titration volurr-- i the on sample

W = Mass of oil used i!i grams

UNSAPONIFIABLF MATTER (AOAC 1980)

After the titration of the saponification value he neutralized liquid was made alkaline with 1ml

of aqueous 3M potassium hydroxide solutions and transferred to a separator and washed with

20ml of water. While still warm, the solution was extracted 3 times with 50ml quantities of diethyl

ether. Each ether extract was poured into another separator containing 20ml of water. The

combined ether extract was shaken first with 20ml of distilled water and then vigorously with two

further 20ml quantities of distilled water.

The ether extract was further washed twice with 20ml quantities of aqueous potassium


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hydroxide sol MS on and with 20ml quantities of distilled water until WR ng were no longer alkaline

to phenolphthalei'1 The ether tract was then poured into a weighed flask and was 'vapora; to

constant weight at 80°C. The unsaponifiable mr or was u nnined using the formula: Unsaponifiable

matter =

M x 100 - - -equation (12)

W 1

Where M =Mass of residual matter in grams

W = Mass overwight of oil in grams

PHYTOCHEMICAL ANALYSIS

Phytoclntmstry deals mainly with secondary plant metabolites. It involves plant growth and

metabolism and the study of phytohormones or growth regulators. It deals with the chemistry and

metabolism of complex plant constituents - phytocemicals, their physiological effects and their

medicinal usefulness.

Phytochemical analysis were carried out to determine the presence or absence of phytochemicals

such as alkaloids, flavonoids, glycosides and many other steroids which occur as glycosides

such as cardiac glycosides, cyanogenic glycosides, steroidal aglycones, anthracenes glycosides

and O - and C - glycosides. Others include proteins,saponins and tannins.

TEST FOR THE PRESENCE OF ALKALOIDS

A quantity (0.2g) of the milled sample (Brassica junced) was used,and boiled with 5ml of 2%

hydrochloric acid on a steam bath.The mixture was filtered and 1ml portion of the filtrate was

treated with 2 drops of the following reagents.

A. Wagner's reagent (Iodine in potassium iodide solution); a reddish-brown precipitate

indicates the presence of alkaloids.

Picric acid (1%); a yellow precipitate indicates the presence of alkaloids.

B. TEST FOR THE PRESENCE OF FLAVONOIDS

A quantity (0.2g) of the milled sample was heated with 10ml of ethyl acetate in boiling water for

3 minutes. The mixture was filtered and the filtrate used for the following tests:

a. A quantity (4ml) of the filtrate was shaken with 1 ml of 1% aluminium chloride solution

and observed for light yellow colouration in the ethyl acetate layer. A yellow colouration

indicates the presence of flavonoids. b. A quantity (4ml) of the filtrate was shaken with 1ml of


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dilute ammonia. The layers were allowed to separate. A yellow colouration indicates the presence of

flavonoids.

C. TEST FOR THE PRESENCE OF GLYCOSIDES

A quantity (2.0g) of the milled sample was mixed with 30ml of distilled water and heated on a

water bath for 5 minutes, filtered and used for the following tests:

a. A quantity (5ml) of the filtrate was added to 0.2ml of Fehlings solution. A and Periling solution

B until it turned alkaline (tested with litmus paper) and heated on a water bath for 2

minutes. A brick red precipitate indicates the presence of glycosides.

b. Using 15ml of dilute sulphuric acid instead of water, the above

process was repeated and the similar result as in (a) indicates the presence of glycosides.

D. TEST FOR THE PRESENCE OF CARDIAC GLYCOSIDES

A quantity (O.lg) of the sample was warmed with 5ml of chloroform in a water bath and decanted. The

decanted solution was evaporated to dryness on a water bath and the residue dissolved in 3ml of

glacial acetic acid containing a drop of ferric chloride. The solution was carefully poured into the

second tube containing 1ml of concentrated sulphuric acid. A reddish-brown ring observed at the

interface indicates the presence of cardiac.

E. TEST FOR THE PRESENCE OF O - AND C – GLYCOSIDES

A quantity (O.lg) of the sample was heated with 5ml of water in a boiling water bath for 15

minutes, cooled and filtered. The filtrate was treated with 5ml of 25% hydrochloric acid and the

solution heated again for 15minutes, while 10ml of diethyl ether was added in a separating

funnel. Both phases were examined for the presence of a purple red colour in the aqueous layer. The

combined ether extract was shaken with 5ml of 3.5% dilute ammonia solution and the mixture

was observed. The acid phase was treated with 0.5g ferric chloride heated for 30 minutes on a

water bath and then cooled. The solution was extracted with 10ml of chloroform washed with 5ml of

water and then 3ml of 3.5% dilute ammonia solution added. A dark red precipitate indicates the

presence of O - and C - glycosides while a colourless solution indicates absence.

F. TEST FOR THE PRESENCE OF SAPONINS

A quantity (0.lg) of the sample was boiled with 5ml of distilled water for 5 minutes. The mixture was

filtered while hot and used for the following tests:

a. Emulsion test: A quantity (1ml) of filtrate was added to 2 drops of olive oil. The mixture was

shaken and observed for the, w formation f emulsion.


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b. Frothing test: A quantity (1ml) of the filtrate was diluted with 4ml of distilled water. The

mixture was shaken vigorously and then observed for stable froth.

G. TEST FOR THE PRESENCE OF TANNINS

A quantity (2g) of the sample was boiled with 5ml of 45% ethanol for 5 minutes. The mixture was

cooled and filtered. The filtrate was used for the following test:

a. Ferric Chloride: A quantity (1ml) of filtrate was diluted with distilled water and 2 drops of

ferric chloride solution was added. A transient greenish to black colour indicates the presence of

tannins.

TEST FOR THE PRESENCE OF PROTEINS

A quantity (5ml) distilled water was added to O.lg of the sample and left to stand for 3 hours and

filtered. To 2ml portion of the filtrate was added O.lml of millions reagent, shaken and kept for

observation. A yellow precipitate indicates the presence of protein.

I. TEST FOR THE PRESENCE OF REDUCING SUGAR

A quantity (O.lg) of the sample was shaken vigorously with 5ml of distilled water and filtered. 1ml

portion of the filtrate was added to equal volumes of Fehlings solution A and B, and heated for 2

minutes while it was shaken vigorously. A brick-red precipitate indicates the presence of reducing

sugar.

THIN LAYER CHROMATOGRAPHY (TLC).

A prepared thin layer plate of silica gel was used. The plate was activated, by heating in an oven

at 110°C for one hour, after which it was allowed to cool. A 2cm mark was made from one end of the

plate, and a spot (about 20ul-50ul) of approximately 1% w/v of each lipid sample in the solvent, was

made on the 2cm mark.

The plate was vertically placed in a chromatographic tank containing the solvent, a mixture of

petroleum ether, diethyl ether and glacial acetic acid, in the ratio of 80:20:1 respectively. The tank

was closed and the solvent allowed to run up the plate as it separates the lipids on

the basis of adsorption and partition. When the solvent front is about 2cm to the other end of the

plates, the plate was withdrawn and the chromatogram allowed to develop. The spots (fatty acids)

were located (visualized) by spraying the plate with 50% v/v sulphuric acid followed by heating it in

the oven at 110°C for 10 minutes, after which the were taken.


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4.0 RESULTS

4 . 1 RESULT OF THE EXTRACTION OF BRASSICA JUNCEAOIL

Table 5: Shows the result of oil extraction from the seed.

Table 5: Extraction of oil

Sample

Initial mass of the

milled sample

before extraction (g)

Mass of the

sample after

extraction (g)

Mass of the

extracted

oil (g)

Percentage extraction

(lipid content

determination)

Brassica

juncea 116g

89.4g

26.6g

22.93%

4.2 RESULT OF THE CHARACTERIZATION OF BRASSICA JUNCEA OIL.

Physical and Chemical Properties

The physical and chemical analyses were carried out on the crude mustard seed oil. The result was used

as an index of quality.

Table 6; The Physical Properties of The Crude Oil of Brassica juncea**

PHYSICAL ANALYSIS, CRUDE OIL Physical state at room temperature (28°C) Liquid Organoleptic properties:

a. Colour b. Odour

c. Taste

Yellow Slightly pungent

Pungent acrid taste

Solubility/iniscibility (in water) Insoluble/immiscible Specific gravity 0.8286

The result in table 4.2.1 gives the physical properties of the sample. The sample was observed to

be liquid at room temperature. The crude oil was observed to be yellow in colour. The specific

gravity was also determined and the result obtained is 0.8286. The organoleptic properties were

also determined and the results were as in above.

Table 7: The Chemical Properties of The Crude Oil

CHEMICAL ANALYSIS OIL

Acid value 11.781

Free fatty acid value 5.922

Peroxide value 7.000

Iodine value 103.499

Saponification value 315.563

Unsaponifiable matter 5.000

The result represented in table 4.2.2 show the acid value, the free fatty acid value, the peroxide

value, iodine value, the saponification value and the amount of unsaponifiable matter present in the

crude oil. »™


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4.3: THE RESULT OF PHYTOCHEMICAL ANALYSIS.

Table 8; The Phytochemka J Screening Qf Brassicajuncea Seed

PHYTOCHEMICALS RESULT

Alkaloids +ve Flavonoids +ve Glycosides +ve Anthracene glycosides -ve

Cardiac glycosides +ve O - and C - glycosides +ve

Tannins +ve Saponins (i) Emulsion test (ii) Frothing

test

+ve

+ve Proteins +ve Reducing sugar -ve

Note: +ve = Positive = Present

-ve = Negative = Absent

Results in table 4.3 shows that the Brassica Juncea seed contains alkaloids, flavonoids, and

glycosides like cardiac and O- and C- glycosides, tannins, saponins and even proteins.

4.4 THIN LAYER CHROMATOGRAPHY OF THE CRUDE OIL

Table 9; The Results of TheThin layer chromatography of the oil

I*.

SAMPLE SAMPLE FRONT RF. VALUE Stearic acid 7.7 .,,,. 0.92 Linoleic 7.9 0.94 Oleic acid 7.1 0.84 Palmitic acid 7.5 0.89 Cholesterol 6.1 0.73 N - hexadecane 8.1 0.96 Crude mustard oil 7.0,7.5,7.7,8.0 0.92, 0.83, 0.94, 0.89

The solvent front = 8.4cm. The results in table 5 suggests that the sample has components

that corresponded to stearic acid, palmitic acid, oleic acid and linoleic, This is observed from the

close Rf values of 0.92, 0.89, 0.83 and 0.94 respectively.

4.5 RESULT OF THE ALLYL-ISOTHIOCYANATE TEST

The abundant sulphur contained by this active principle in mustard seed oil discoloured silver

plate, after leaving it to stand for a long time, forming black sulphuret of silver.


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Fig 10: Thin layer chromatography result of the mustard seed oil Key:

S= Mustard seed Oil

St= Stearic acid

C= Cholesterol

L Lenoleic acid acid

P= Palmitic acid

n-H= n-Hexadecane

DISCUSSION

This study tried within the limits of available resources, to extract, characterize the

components of the oil, and analyze the phytochemical constituents of the seeds of Brassica juncea.

The partial characterization of mustard seed oil was undertaken by looking at its physical and

chemical properties and phytochemical analysis after soxhlet extraction.

The liquid state of the oil extracted makes it a good nutritional and industrial material because

of ease of use.

Its organoleptic properties namely, colour, odour, and taste, serve the purpose of distinguishing

the genuine from adulterated oil as well as its degree of purification or refinement. It has

yellow colour, penetrating odour and slightly pungent acrid taste which can be attributed to

the presence of such substances as chlorophyll, carotenoids, oxidative products resins and

other impurities since a pure fat and oil and their constituent fatty acids are generally

colourless, odourless and tastefess.

Its solubility and miscibility revealed that it is readily soluble and miscible with certain organic

solvents like diethyl ether, but insoluble in water. This observation could be due to the fact that


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fats and oils are generally insoluble in water since they contain a predominance of non- polar

groups, but are soluble in certain organic solvents.

Its specific gravity satisfies the regulatory requirements for transportation and storage. It value

is 0.8286. The specific gravity does not describe any particular performance characteristic but

can be used to indicate approximately the components concentration and adulteration in the

oil. It is comparable to the 0.900 - 0.960 reported in the survey of plant oil engines and fuel

specifications for running on rapeseed oil (Krause, 1998). It is also comparable to 0.920 -

0.930 reported for rubber seed oil (Eckey and Miller, 1954), 0.9012 -0.9027 by Njoku (1993)

and 0.922 - 0.918 by Ikwuagwu et Al. (2000). It is an essential facior for the estimation of the

quality of the product (though an indirect indicator of quality).

Its acid and free fatty acids values are 11.781% and 5.922% respectively. These values

are related to presence of acid and free fatty acid in the oil, and also an indicator of the presence

and exten of hydrolysis by lipolytic enzymes and oxidation (Gordon, 1993). The value is

comparable to the free fatty acid value of 5% in cruel oils (Hammond, 1993) and < 10

reported in the survey of plant oil engines and fuel specifications for running on rape

seed oil (Krause,1998). The acid value is comparable to the range 9.0-21.6, reported by

Ikwuagwu et al.(2000).These values also show that the oil has a high acid value comparable to

3.0, 12.2 0.8,2.land 2.5 as reported for sunflower, cotton seed, groundnut, olive, and coconut

oils respectively ( Engler and Johnson, 1983). The low free fatty acid value is an indication that

the components are predominantly triacylglycerols.

Its peroxide and iodine values of 7.0 and 103.499 respectively show that oil would be stable

and has a low degree of unsaturation (iodine values increase with the degree of unsaturation).

This stability could be attributed to the presence of natural antioxidants in the oil, which are

quite effective in slowing down the rate of oxygen absorption by reacting with the fatty acid

peroxy free radicals (Hoffinann, 1986b). It could also be due to presence of hydro peroxides

which could initiate or propagate further oxidation of the oil thereby improving the stability of

the oil. The peroxide value is also comparable to < 14 for sunflower reported by Engler and

Johnson, (1983) , but higher than < 3meq/ kg reported in the survey of plant oil engines and

fuel specifications for running on rape seed oil ( Krause,1998). The iodine value is comparable

to 105 for brown mustard seed oil reported by Ensminger, (1986). The value indicates that

the oil is an edible and semi- drying oil, since the iodine value of edible oils range from 7-

200, and 100-150 for semi -drying oils.


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The saponificalion value and the unsaponifiabie matter which are both high (315.563% and 5%)

, are indicative of the presence of more of lower molecular weight fatty acids and hence an

increase in the volatility of the oil as saponification value is inversely proportional to molecular

weight , and the presence of some type of foreign matter that are non- volatile at 103°C which

includes sterois, high aliphatic alcohols, tocopherols, pigments ( Hoffinann, 1986a), since the

unsaponifiabie matter of more than 2% suggests with presence of foreign matters and probably,

adulteration (Jacobs, 1958).

Its thin layer chromatography (TLC) revealed that sample has components whose Rf values

correspond to those have stearic, linoleic, oleic and palmitic acid. The results were

comparable to the report that the mustard seed oil consists of the glycerides of oleic, stearic

and erucic (brassic) acids (Fortin Francois, 1988). It is also comparable to the report that the

brown mustard seed oil contains palmitic, oleic, erucic, linoleic, linolenic, and eicosanoic acids

(Ensminger, et al., 1986). It is important to note that erucic acid (though not tested for), is

harmful to human health (Ensminger et al., 1986).

Its phytocksmical screening which showed the results of the investigation of the nutritional and

anti-nutritional factors, as shown in table 8, revealed that the Brassica juncea seed contains

tannins, which are anti-nutritional factors, alkaloids; flavonoids; glycosides like cardiac

glycosides, O - and C - glycosides; saponins and proteins. These secondary plant metabolites

have a lot of pharmacological properties. The alkaloids, saponins and flavonoids are said to

have medicinal properties in animals. However, high concentrations of these substances are

toxic and may impair body metabolism. The cardiac and cyanogenic glycosides have the

properties of stimulating heart muscle and affect oxidative phosphorylation. Therefore, plant

materials used, as foodstuff in humans should be processed to eliminate some of the harmful

phytochemical and anti-nutritional factors.

Allyl isothiocyanate (AITC), the active principle in Brassica spp. that is formed in the presence of

water, contains abundant sulphur. The result of this test shows a discoloration of silver plate,

which was due to the formation of black sulphuret of silver. This could be detrimental to human

health.

CONCLUSION

The mustard seed was shown to be a good source of extractable vegetable oil. The physical

properties showed that the oil was liquid at room temperature, non-volatile and viscous, the


193


specific gravity comparable to that of commercial diesel, while the organoleptic properties -

colour, odour and taste showed a possible vegetable oil that could be harnessed and refined for

edible and industrial uses.

The chemical properties showed a moderate acid and free fatty acid values expected for crude

vegetable oils, an acceptable (low) peroxide value which shows that the oil was relatively stable,

an indication of little or no unsaturation. The iodine value which was between 100 and 150 showed

that Brassica juncea oil is a semi-drying oil with very low degree of unsaturation which will

make it stable against oxidation when exposed to the atmosphere.

The saponification value showed that there were lower molecular weight fatty acids in the oil than

higher molecular weight fatty acids. This saponification value showed an increase in the

volatility of the oil as saponification value is inversely proportional to molecular weight of the oil.

The higher amount of unsaponifiable matter revealed the presence of non-volatile components

and proved that the oil was not made up of only triacylglycerols, but contains foreign matters,

and probably adulteration. This shows that the oil will not burn evenly, and will leave particulate

organic matter behind, hence non-detrimental to the ecosystem.

The phytochemical studies revealed that it has some pharmacological and medicinal values and even

some anti-nutritional components. The TLC suggests the presence of fatty acids like stearic acid,

linoleic acid, oleic acid and palmitic acid.

Apart from the reported erucic acid (a fatty acid of mustard seed), which is harmful to human

health, traces of isothiocyanate (ITC) may be found in the oil, which accounts for its, acidity in taste.

Therefore, in spite of high fraction of unsaturated fatty acids (iodine index 105) (Ensminger, et al,

1986), mustard oil cannot be recommended for human consumption, except proper measures are

taken during refining to eliminate the harmful components.

The major problem of the utilization of mustard seed is the difficult terrain in which they grow

which sometimes is inaccessible to harvesters. Mustard seeds have their habitat throughout

Europe, except in the northeastern part. It has not been found in this country Nigeria, since we lack

the rich, alluvial soil on which it grows. Therefore, one obstacle to mustard seed oil from

regenerative raw materials is the lack of good soil and area for cultivation and the non-discovery of

the usefulness of the whole plant parts and products. Mustard seeds are being cultivated for

the sake of the seeds used partly as a condiment, and partly for its oil. Research is necessary to


194


discover better breeding conditions that will favour and improve the oil yield of the crop. Also,

more research institutes with sophisticated equipments and up-to-date reagents are necessary to

facilitate further research work on vegetable oils, especially mustard seed oil.

Further research will offer an opportunity to understand and control gaseous emissions which

constitute some of the pollutants that lead to global warming, flooding and drastic climatic changes.

Furthermore, it will create a platform that will help the Government, as Government all over the

world have established Environmental Protection Agencies, and World Health Organization, to

advice on and enforce quality standards designed to protect human health and prevent crop and

structural damage.

However, these studies extracted and partially characterized the components of the oil, as well as

the phytochemicl analysis of Brassica juncea seed, with a view to furnishing and updating

baseline data for further systematic experimentation.

Meanwhile, it is only through further research and development as well as additional measures

would there be an opportunity for mustard seed oils to find their rightful place in the oil industries

in countries with agricultural over production or with sufficient and rich alluvial soil, if given the

political will.

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3. Eckey, E. W. and Miller, L. P. Vegetable fats and oils, Reinhold Publishing Corp. New

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4. Engler, C. R. and Johnson, L. A. Effects of Processing and chemical characteristics of plant

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5. Ensminger, A. H. and Ensminger, M. K. J. Food for Health. A nutrition Encyclopedia Clovis,

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EXTRACTION AND PARTIAL CHARACTERIZATION OF MUSTARD SEED (BRASSICA SPP.) OIL

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