Biochemistry and microbiology
Nwaoguikpe R N. et al. / JPBMS, 2012, 20 (09)
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Research
article
JPBMS
ISSN NO- 2230 – 7885
CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES
Phytochemical and biochemical compositions of African Walnut
( Tetracarpidium conophorum)
®Nwaoguikpe R N1, Ujowundu CO1, Wesley B 2
1Department
2Department
of Biochemistry, Federal University of Technology, P.M.B. 1526,Owerri,Imo State,Nigeria
of Microbiology, Federal University of Technology, P.M.B.1526,Owerri,Imo State, Nigeria.
Abstract:
The phytochemical and biochemical compositions of African walnut (Tetracarpidum conophorum) were determined for
two samples of the variety-the boiled and mashed wet nuts, and the dried powdered nuts. Results of phytochemical
analysis revealed the presence of the following expressed as percentage. For the mashed wet nuts: Saponins (8.37±0.1);
flavonoids (3.20 ±0.2); phenols (1.90±0.1), tannins (0.51±0.0) and alkaloids (0.41±0.1). For the dried and powdered
variety, the results are- Saponins (5.03±0.2); flavonoids (2.70 ±0.1), tannins (0.45±0.1), phenols (1.51±0.1) and alkaloids
(0.350±0.1). Proximate compositional analysis expressed as percentage shows the following results: moisture
(45.01±0.1)WM,(2.88±0.02)DP; Crude fat (13.81±0.01) WM, (32.21±0.01) DP; Crude protein (18.75±0.01) WM,
(13.72±0.02 ) DP;CHO (18.43±0.1) WM,(46.38±0.2) DP; Crude fiber (1.95±02) WM,(1.72±0.02 ) DP; Ash (1.91±0.01)
WM,(3.11±0.01) DP, FE( 273.01±0.1) WM, (530.29±0.02) DP. The values of the vitamins are expressed in mg/ml as
shownbelow:VitaminA
is
expressed
in
µg/ml(1283.33±1.18)DP,
(285.60±0.2)WM;Vit.C
(14.80±0.02)DP,(17.57±0.01)WM;riboflavin (0.12±0.02)DP,(0.12±0.02)WM;Thiamine(B1) (0.20±0.01)DP,(0.12±0.01)WM;
Niacin(2.81±0.01)DP,(2.91±0.01).The mineral composition of the nut is very outstanding and not significantly affected by
the processing methods. The values of the minerals are shown as follows: Mg (0.36±0.01) DP.,(0.31±0.01)WM; Ca
(2.10±0.2)DP, (1.88±0.1)WM; P(0.35±0.01)DP, (0.36±0.01)WM and others. The phytochemical and nutrient compositions
of this tropical nut are expository and depicts the role of the seeds in nutrition and health.
Keywords:Biochemical composition, phytochemicals, African walnut.
Introduction:
One of the most unexpected nutritional discoveries of the
1990’s was that frequent eating of nuts greatly lower the
risk of heart disease[1] .Walnut is an edible seed of any tree
of the genus, Juglans, especially , the Persian walnut. A
study has suggested that consumption of walnut increases
fat oxidation and reduces carbohydrate oxidation without
affecting total consumption, suggesting that walnut
consumption may improve the use of body lipids in over
weight adults. Walnuts are shown to decrease endothelial
dysfunction associated with high fat diets [2]. Scientists are
not yet convinced whether walnuts act as chemoprotective
agents, an effect which may show the result of the fruit’s
high phenolic content [3], an antioxidant activity to be used
in in vitro antiproliferative therapy[3] .Walnuts have been
found in pre-historic deposits dating from the iron age in
Europe. In the middle ages, walnuts were thought to ward
off witchcraft, the evil eye and epileptic fits. Black walnuts
are applied in certain skin conditions such as eczema,
pruritus, psoriasis, warts, and parasitic skin conditions [4].
Extracts of black walnut are used to dye the hair, skin and
cloths. Black walnut contain juglone (5-hydroxy-1,4naphthoquinone,alpha(α)hydroxyjuglone and its glycoside,
beta(β) hydrojuglone, caffeic acid, plumbagum, hyperin,
kaempferol and tannin. Ellagic acid is also present .
The study of oil extract from Tetracarpidium conophorum
seed showed the fatty acids and triacyl content with
1
linoleic acid[4] .Walnuts have sufficiently higher amounts of
omega-3-fatty acids as compared to other nuts[1]. Raw
walnuts contain glyceryltriacylates of the n-3 fatty acid,
alpha linolenic acid (AlA). Roasting reduces the antioxidant
quality of the seeds. Many authors/researchers have
identified some uses of the seeds and leaves of the plant.
For example, an extract of the leaf is used to mitigate
prolonged and constant hiccups, improvement of fertility
in both men and women. It can also be used to improve
spermatozoa count in men. It has equally been used to
reduce the incidence of tumor and cancer cells [5]. It is used
to regulate menstrual flow and to treat dysentery.
Preparations of the leaf, stem bark, kernel and root
extracts as well as the hexane, ethylacetate and methanol
fractions of the leaf are able to inhibit the activities of gram
negative bacteria such as Staphylococcus aureus , Bacillus
subtilis, Pseudomonas aeruginosa and Escherichia coli [6]
.The heavy metal content of the walnuts are within the
WHO permissible limits of heavy metals. Cardiac glycosides
were relatively absent. Compared to certain other nuts
such as almonds, peanuts, and hazelnuts; walnuts,
especially in their raw form, contain the highest total level
of antioxidants, including both free antioxidants and
antioxidants bound to fiber [7,8].
The research work is geared towards elucidating the
phytochemical, biochemical, minerals and vitamins
resident in this all important nut. We were intrigued by the
avidity with which Nigerians purchase and consume the
Journal of Pharmaceutical and Biomedical Sciences ©(JPBMS), Vol. 20, Issue 20
Biochemistry and microbiology
Nwaoguikpe R N. et al. / JPBMS, 2012, 20 (09)
nuts during the harvest period especially people of the
South Eastern region, as it is not always there throughout
the year. The result of such research work would
nonetheless, place this nut in its actual position of
relevance in the nutritional and medicinal needs of
Nigerians and by extrapolation to other people of other
nations.
Determination of phenol
Determination of phenol content is done using the methods
of the Association of Official Analytical Chemists [12 ] .
Determination of the energy value
The gross food energy value is estimated according to the
method [13].
Materials and methods:
Plant materials.
The seeds of viable African walnuts were purchased from a
local market within Owerri metropolis. Five hundred
(500g) grams of recently plugged seeds removed from
seed coat while fresh were prepared for the studies. Before
hulling, the seeds were authenticated by a technologist, Mr.
Francis Iwunze at the department of Forestry and Wild Life
of the School of Agriculture and Agricultural Technology of
the University.
Sample preparation:
The seed samples are into two groups of two hundred
grams (200 g} each. Sample A consists of 200g of
completely hulled seeds, boiled and sliced into small bits to
expose the surface area and finally dried in an oven at 65
0C for six (6) hours. The oven dried seeds were grinded to
powder using a manually operated machine (LANDERA
CORONA).The grinded seeds sieved with a sieve of mesh,
30mm pore size to obtain flour of the seed. The second
group B, consists of two hundred grams (200 g) of fresh
seeds, mashed and sun-dried for two days. The macerated
sun-dried seeds were powdered using a grinding machine
(LANDERA CORONA), manually operated. The grinded
seeds were then sieved with a sieve of the same mesh size
to obtain flour, which would then be used for analysis.
Phytochemical Analysis:
Phytochemical analysis is carried out with the two
prepared samples – the mashed wet sample (B) and the
dried powdered sample (A) respectively.
Determination of alkaloids
The analysis is done using the alkaline precipitation
methods [9][10] . Five grams (5 g) of each of the samples
are weighed into 250 ml beaker and 200 ml of 20 % acetic
acid in ethanol was added. The flask was then covered and
allowed to stand at room temperature. The mixture is
shook for 30 minutes, using a magnetic shaker. The
mixture was then concentrated using rotor evaporator
maintained at 60 0C to obtain ¼ of the original volume.
The extract is treated by drop wise addition of
concentrated ammonium hydroxide solution until
precipitation was complete. The solution is allowed to
settle and filtered with Whatman filter paper (No 42).The
precipitated alkaloid is dried at 60 0C and weighed.
Determination of tannins
The tannin content of the sample is estimated by the Folin–
Dennis spectrophotometric method. The methods of [9]
were also used for analysis.
Determination of flavonoids
Ten grams (10 g) of each of the samples was extracted
repeatedly with 100 ml of 80 % aqueous methanol at room
temperature. The entire solution is filtered through
Whatman No 1 filter paper. The filtrate was then
2
transferred into a crucible, evaporated to dryness over a
water bath and weighed to a constant weight [11].
Proximate analysis
The proximate compositions of the wet and dry samples
are analyzed for the moisture content, carbohydrate, crude
lipids, protein, ash and crude fiber by the methods of the
Association of Official Analytical Chemists [12].
Determination of Vitamin A (β-Carotene )
The determination of Vitamin A (β-Carotene) content of
the samples is estimated using the methods described [14].
Quantitative determination of vitamin b 1 (thiamine)
Five grams (5 g) of samples are homogenized with 50 ml of
ethanolic sodium hydroxide solution. This was filtered into
a 100 ml flask. Ten milliliters (10 ml) of the filtrate was
pipetted into a beaker and color developed by the addition
of 10ml potassium dichromate . The absorbance is read at
360nm. A blank sample was also prepared and read at the
same wavelength. The values are extrapolated from a
standard curve [11].
Determination of riboflavin (Vitamin B 2)
Five grams (5 g) of each of the samples was extracted with
100 ml of 50 % ethanol solution shaken for 1 hr. This was
filtered into a 100 ml of 30 % hydrogen peroxide (H2O2)
and allowed to stand over hot water bath for 30 minutes.
Two milliliters (2 ml) of 40% sodium sulphate added to
make up the 50 ml mark and absorbance read at 510 nm in
a spectrophotometer [11].
Determination of niacin
Five grams (5 g) of the sample is treated with 50 ml of 1N
sulfuric acid and shaken with magnetic shaker for 30 mins.
Three drops of ammonia solution added to the mixture and
filtered. Ten milliliters (10 ml) of the filtrate was pipetted
into a 50 ml volumetric flask, and 5 ml potassium cyanide
added .The mixture acidified with 0.02 MH2SO4, and the
absorbance read at 470 nm in a spectrophotometer
(Unicam Spectronic 20 ).
Determination of Vitamin C
The ascorbic acid content of the samples were estimated
by the Barakat titrimetric method. Twenty (20 g) grams of
the samples homogenized in 100 ml EDTA/TCA extracting
solution by bleaching for 5 mins. The homogenate filtered
and the filtrate analyzed by the methods of [11].
Determination of Vitamin E
The vitamin E content of the samples is quantifiable using
the method described [11].
Determination of mineral composition
The determinations of the mineral compositions of the
samples done using the methods [13] involving the
determination of sodium and potassium by flame
Journal of Pharmaceutical and Biomedical Sciences© (JPBMS), Vol. 20, Issue 20
Biochemistry and microbiology
Nwaoguikpe R N. et al. / JPBMS, 2012, 20 (09)
photometric method. Phosphorus is determined the
Vanado–Molybdate
Yellow
method
using
spectrophotometer. Calcium and Magnesium were
determined by the EDTA titration method.
Statistical analysis.
All data analyzed using one way ANOVA and results
presented as Mean ± SD from triplicate determinations
.The level of significance is set at p≤ 0.05
Results:
Results of various analyses are shown in tables 1-4 below.
Table 1 shows the quantitative phytochemical composition
of the wet and dry samples of African walnut
(Tetracarpidium conophorum Values shown as percentage
composition .Table 2 shows the proximate composition of
samples of seed of the African walnut. Table 3 depicts the
vitamin content of the seeds of Tetracarpidium
conophorum. Values expressed in mg/100g and µg/100g of
samples respectively. Table 4 shows the mineral
composition of the samples of African walnuts.
Table1. Phytochemical composition of both the dried and the wet samples of Tetracarpidium conophorum. Values are expressed as percentages.
Samples
Flavonoids
Alkaloids
Saponins
Tannins
Phenols
Dried and Powdered
2.70±0.1b
0.35±0.01b
5.03±0.01b
0.45±0.01b
1.51±0.01b
Wet and Mashed
3.20±0.0a
0.41±0.01a
8.37±0.12a
0.51±0.00a
1.90±0.01a
The values in the table are the Mean ± SD from triplicate determinations. Values with the same superscript are
significantly related along the row at p≤0.05 and different from others.
Table 2. Proximate composition of samples of the Seed of Tetracarpidium conophorum. Values are expressed as percentage (%) composition.
Samples
MC
Crude Fat
CF
Ash
CP
CHO
FE (J)
Dried and
Powdered
Wet and Mashed
2.88±0.02b
32.21±0.01a
1.72±0.02a
3.11±0.01a
13.72±0.02a
46.38±0.02a
530.29±0.2a
45.01±0.01a
13.81±0.01b
1.95±0.02a
1.91±0.01b
18.75±0.01a
18.43±0.01b
273.01±0.2b
The values in the table are the Mean± SD from triplicate determinations. Values having the same superscript are
significantly related along the columns at p≤0.05.
MC=Moisture content CP=Crude protein CHO=Carbohydrate FE=Food energy CF=Crude fiber.
Table 3. Vitamin composition of the seeds of Tetracarpidium conophorum .Results are expressed in mg/100g or µg/100g
Samples
Vitamin A
Vitamin C
Riboflavin
Thiamin
Dried and
Powdered
Wet and Mashed
1283.33±1.18
285.60±9.95
a
b
14.80±2.43
a
17.57±0.02
a
0.12±0.01
a
0.13±0.01
a
0.20±0.01
a
0.12±0.01
b
Niacin
Vitamin E
2.81±0.13
a
0.22±0.02
a
2.91±0.10
a
0.27±0.02
a
The values in the table above are the Mean ± SD from triplicate determinations .Values with the same superscript are
significantly related at p≤ 0.05 across the rows and columns.
Table 4.The Mineral compositions of Samples A and B OF Tetracarpidium conophorum . Values are expressed as percentage of sample.
Samples
Mg (%)
Ca (%)
K (%)
Na (%)
Dried and
Powdered
Wet and Mashed
0.36±0.01a
2.10±0.20
0.31±0.01a
1.88±0.11b
a
The values in the table are the Mean± SD from triplicate
determinations. Values with the same superscript are
significantly related at p≤0.05 along the rows and columns.
The minor difference observed in the data was due to the
effect of heat on drying, leading to concentration effect on
the different minerals present in the samples.
Discussion:
The findings or results from the analyses of different
samples revealed fascinating observation. The results of
phytochemical analysis revealed high preponderance of
phytochemicals especially saponins and flavonoids in both
the dried and wet samples. The high level of antioxidants in
this nut is reported severally by many workers[15]. Many
researchers have equally reported on the level of
polyphenolic compounds such as Ellagic and Gallic acids;
apart from these, other phenolic acids have been found in
African walnuts such as phenylacetic acid, a strong
antisickling agent [16], protocatechoic acid, syringic, vanillic
acid and caffeic acid. These phenolic acids found in foods
have been associated with astringency, discoloration and
inhibition of some enzyme activity; they are also known to
3
1.02
P (%)
±0.02a
0.26±0.01b
0.35±0.01a
0.87±0.01b
0.39±0.01a
0.36±0.01a
provide the human body with extra line of defense against
bacterial and viral attacks, thereby, boosting the immune
system [16-18].
The high protein content vis-à-vis, the high carbohydrate
and food energy values are reminiscent of high rate of
consumption during the harvest period. From our findings,
it can be seen in table 2, that the nuts are rich in protein
with values-13.27 % for the dried and powdered sample to
18.75% for the wet and mashed sample. This finding is in
line with what other workers reported for other nuts like
Hazel nuts [19], Almonds and peanuts respectively .The nut
is also rich in nutrients which lower the risk of gaining
unwanted weights [20]. For example, some workers have
reported the presence of resveratrol[20-22],a phytochemical
found in grapes and walnuts, which activates STRT 1 gene,
which in turn increases the body’s metabolic rate, also
implicated in positive correlation with longevity[23,24].
Several studies have highlighted or unveiled a gamut of
substances present in walnuts[25]. Several vitamins are
resident in the African walnuts, whose compositions agree
with our findings. Some of the vitamins and other nutrients
reported by other workers and their values include-
Journal of Pharmaceutical and Biomedical Sciences© (JPBMS), Vol. 20, Issue 20,
Biochemistry and microbiology
Nwaoguikpe R N. et al. / JPBMS, 2012, 20 (09)
vitamin A (β-Carotene) 380 IU/100g, thiamin 0.48-0.53mg
/100g,riboflavin 0.11-0.14mg/100g, nicotinic acid 0.61.2mg/100g; when compared with our findings/results
,the dried and powdered /wet and mashed results are
shown :vitamin A 1283.3/285.6 IU per 100g; vitamin C
:14.80/17.57 per 100g;it has been found that this value of
vitamin C is lower than that reported for some nuts like
Hazel nuts and peanuts[22,23],but results of Vitamin A
estimation, showed higher than what some workers
reported . Essential amino acids are present in walnuts,
such as Arginine-2.3mg/100g; Histidine-0.4mg/100g;
Isoleucie 0.73mg/100g; others include-leucine1.3, lysine
0.3, methionine 0.25, phenylalanine 0.8, threonine o.5,
tryptophan 0.18 and valine 0.9, all expressed in mg/100g.
The mineral content of Tetracarpidium conophorum is
very outstanding. The presence of Mg, Ca, K, Na and P
cannot be under rated because of the role of these metal
ions in health and nutrition. Magnesium is a cofactor for
many enzymes. Calcium and phosphorus are very essential
for bone metabolism. Sodium and potassium are very
important for nerve transmission and osmolality of body
fluids and moreover Zinc is an antioxidant, as it is a
cofactor for many antioxidant enzymes like catalase. The
phytochemical and nutrient composition of the walnut is
worth investigating. The results from our work have far
reaching importance as this could support the numerous
nutritional and medicinal roles attributed to this all
important nut[26,27]. It is reviewed extensively, the roles of
this nut. The arginine content of the nut has been
attributed the role of an antihypertensive drug by
producing Nitric oxide in vivo, whose function is to act as a
vasodilator of the smooth muscles and blood vessels[12].
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Source of funding:- None
Conflict of Interest:- Not declared
4
Journal of Pharmaceutical and Biomedical Sciences© (JPBMS), Vol. 20, Issue 20
Biochemistry and microbiology
Nwaoguikpe R N. et al. / JPBMS, 2012, 20 (09)
Corresponding Author:Dr. Nwaoguikpe RN.,
Department of Biochemistry, Federal University of Technology,
P.M.B. 1526,Owerri,Imo State,Nigeria.
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