1. Art and Diseño

Advances in Biochemistry & Biotechnology
RESEARCH ARTICLE
Open Access
Bioscience Research
Phytochemical, proximate and antimicrobial
screening of neglected and underutilized Dioscorea
species (Landrace) in Boki Local Government Area,
Cross River State - Nigeria
1
Agbor R.B., 1Okoi E.P., 2Uno U.U and 3Iyam S.O
1
Department of Genetics and Biotechnology, University of Calabar, Calabar,
Cross River State, Nigeria
2
Department of Biological Sciences, Cross River State College of Education
Akamkpa, Nigeria
3
Department of Microbiology, University of Calabar, Calabar, Cross River State, Nigeria
E-mail: [email protected]
ABSTRACT
This research work was carried out to determine the phytochemical, proximate
composition and antimicrobial activity of Dioscorea sp obtained from Boki Local
Government Area. The antimicrobial effect of Dioscorea sp plant was examined on some
bacteria (Serratia, Pseudomonas, E. coli, Salmonella) and fungi (Penicillium, Candida,
Aspergillus, Rhizopus ) species. The solvents used for the extraction of plants were
water, ethanol, and methanol. The antimicrobial activity was performed by agar disc
diffusion method. The result shows that all the microbial species used for this
investigation were susceptible to the plant extracts. But the susceptibility of the
microorganisms was mostly observed at 50% and 100% of the plant extract. The results
of the phytochemical screening shows that tannin, alkaloids, flavonoids, glycosides,
saponins, polyphenols and reducing compounds were of significantly high quantity in the
tuber than the leaves of Dioscorea sp. The proximate constituent such as crude protein,
crude fibre, ash, moisture, dry matter, ether extract and carbohydrate were significantly
high (p<0.05) in the tuber than the leaves of the plant.
Key words: Proximate, phytochemical, antimicrobial, Dioscorea, species
Received: 21 January 2015
Accepted: 27January 2015
Available Online: 29 January 2015
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Advances in Biochemistry & Biotechnology
INTRODUCTION
Naturally occurring substances are of plants, animals and mineral origin. They are
organic substances and could be obtained in both primary and secondary metabolic
process; they also provide a source of medicine since the earliest time. The plant
kingdom has proven to be the most useful in the treatment of diseases and they provide
an important source of all the world’s pharmaceuticals (Takagi et al. 1980). The most
important of these bioactive constituents of plants are steroids, terpenoids, carotenoids,
flavonoids, alkaloids, tannins and glycosides. Plants in all facet of life have served a
valuable starting material for drug development (Feroz et al. 1993). Antibiotics or
antimicrobial substances like saponins, glycosides, flavonoids and alkaloids etc are found
to be distributed in plants, yet these compounds were not well established due to the lack
of knowledge and techniques (Nagata et al., 1985). The phytoconstituents which are
phenols, anthraquinones, alkaloids, glycosides, flavonoids and saponins are antibiotic
principles of plants . From these phytoconstituents, saponins have been reported to
exhibit hemolytic and foaming activity Shi et al., (2004), antifungal, anti-inflammatory,
fungistatic, molluscidal (Zehavi and Segel 1986). Plants are now occupying important
position in allopathic medicine, herbal medicine, homoeopathy and aromatherapy.
Medicinal plants are the sources of many important drugs of the modern world. Many of
these indigenous medicinal plants are used as spices and food plants; they are also
sometimes added to foods meant for pregnant mothers for medicinal purposes (Okwu
1999, Okwu 2001). Many plants are cheaper and more accessible to most people
especially in the developing countries than orthodox medicine, and there is lower
incidence of adverse effects after use. These reasons might account for their worldwide
attention and use (Ayitey-smith and Addae-Mensah 1977). The medicinal properties of
some plants have been documented by some researchers (Gill and Saleem 1982, and
Banso and Adeyemo 2007). This study looks into the fundamental scientific bases for the
use of some medicinal plant seeds by determining the crude phytochemical constituents
present in these plants. The yam species grows in the wild and in some forest area in
Cross River State. The stem diameter ranges from 10-15 meters long, it has oval shape
leaves with a leaf length range of 9-13.5cm, the leaf area of the plant ranges from
98.2cm2 – 136.8cm2, the leaf width ranges from 6.5cm -10.8cm. This species of yam
grows with spines on its stems while the root of the plant produces large thorns that
served as defense against predators. The thorns in the root grow in circle and form a heap.
According to local farmers these species of plant is of two type, one eaten by humans and
the other eaten by wild animals that can withstand the thorns. Harvesting is normally
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done with the use of hand digger and the harvester wears thick gloves to avoid injuries.
The plant grows well in both dry and wet season.
MATERIALS AND METHODS
Experimental laboratory
The phytochemical and proximate analysis were carried out in the Department of
Biochemistry while the antimicrobial analysis was carried out in the Department of
Microbiology, University of Calabar, Calabar. The yam sp was obtained from the forest
zone of Bashua village in Boki Local Government Area. The plant identification was
done in the Herbarium unit of Botany Department, University of Calabar., University of
Ibadan and Cross River State University of Technology. However, no proper
identification has been made on the plant.
Preparation of plant materials
The plant samples were washed thoroughly, cut into small parts and then air-dried
for 14 days. The dried plant samples were blended using Master chef electric blender
(Model: MC-BL 1242) and the powdered samples were stored in an airtight sterile
containers.
Preparation of the plant extract
Aqueous extraction
Ten (10) gram of air dried powder was added to distilled water and boiled on
slow heat for 2 hours. It was then filtered through 8 layers of muslin cloth and
centrifuged at 5000g for 10minutes. The supernatant was collected. This procedure was
repeated thrice. After 6 hours, the supernatant was collected at an interval of every 2
hours and was pooled together, concentrated to make the final volume one-fourth of the
original volume. It was then autoclaved at 1210C and at 15 Ibs pressure and stored at
400C.
Methanol extraction
Ten (10) g of air-dried powder was taken in 100 ml of methanol in a conical flask,
plugged with cotton wool and then kept on a rotary shaker at 190-220 rpm for 24 h. After
24 hours the supernatant was collected and the solvent was evaporated to make the final
volume one fourth of the original volume (12) and stored at 40 C in airtight bottles.
Phytochemical screening
Chemical tests were carried out on the aqueous extract and on the powdered
specimen using standard procedure to identify the constituents.
Test for tannins:
One gram of each powdered sample was separately boiled with 20 ml distilled
water for five minutes in a water bath and was filtered while hot. 1 ml of cool filtrate was
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Advances in Biochemistry & Biotechnology
distilled to 5 ml with distilled water and a few drops (2-3) of 10 % ferric chloride were
added and observed for any formation of precipitates and any colour change. A bluishblack or brownish-green precipitate indicated the presence of tannins.
Test for saponin:
One gram of each powdered dried stain was separately boiled with 10ml of
distilled water in a water bath for 10minutes. The mixture was filtered while hot and
allowed to cool. The following tests were then carried out. Demonstration of frothing: 2.5
ml of filtrate was diluted to 10ml with distilled water and shaken vigorously for 2minutes
(frothing indicated the presence of saponin in the filtrate). Demonstration of emulsifying
properties: 2 drops of olive oil was added to the solution obtained from diluting 2.5 ml
filtrate to 10 ml with distilled water (above), shaken vigorously for a few minutes
(formation of a fairly stable emulsion indicated the presence of saponins).
Test for phlobatannins:
Deposition of a red precipitate when an aqueous extract of each plant sample was
boiled with 1 % aqueous hydrochloric acid was taken as evidence for the phlobatannins.
Test for terpenoids:
Five milligram of each extract was mixed in 2 ml of chloroform. 3 ml of
concentrated H2SO4 was then added to form a layer. A reddish brown precipitate
colouration at the interface formed indicated the presence of terpenoids.
Test for flavonoids:
One gram of the powdered dried leaves of each specimen was boiled with 10 ml
of distilled water for 5 minutes and filtered while hot. Few drops of 20 % sodium
hydroxide solution were added to 1 ml of the cooled filtrate. A change to yellow colour
which on addition of acid changed to colourless solution depicted the presence of
flavonoids.
Test for cardiac glycosides:
Five milligram of each extract was treated with 2 ml of glacial acetic acid
containing one drop of ferric chloride solution. This was underplayed with 1ml of
concentrated sulphuric acid. A brown ring at the interface indicated the deoxysugar
characteristics of cardenolides. A violet ring may appear below the ring while in the
acetic acid layer, a greenish ring may be formed.
Test for combined anthraquinones:
One gram of powdered sample of each specimen was boiled with 2 ml of 10 %
hydrochloric acid for 5 mins. The mixture was filtered while hot and filtrate was allowed
to cool. The cooled filtrate was partitioned against equal volume of chloroform and the
chloroform layer was transferred into a clean dry test tube using a clean pipette. Equal
volume of 10% ammonia solution was added into the chloroform layer, shaken and
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Advances in Biochemistry & Biotechnology
allowed to separate. The separated aqueous layer was observed for any colour change;
delicate rose pink colour showed the presence of an anthraquinone.
Test for free anthraquinones:
Five milligram of chloroform was added to 0.5 g of the powdered dry seeds of
each specimen. The resulting mixture was shaken for 5 mins after which it was filtered.
The filtrate was then shaken with equal volume of 10 % ammonia solution. The presence
of a bright pink colour in the aqueous layer indicated the presence of free anthraquinones.
Test for carotenoids:
One gram of each specimen sample was extracted with 10 ml of chloroform in a
test tube with vigorous shaking. The resulting mixture was filtered and 85 % sulphuric
acid was added. A blue colour at the interface showed the presence of carotenoids.
Test for reducing compounds:
To about 1 g of each sample in the test tube was added 10 ml distilled water and
the mixture boiled for 5 mins. The mixture was filtered while hot and the cooled filtrate
made alkaline to litmus paper with 20 % sodium hydroxide solution. The resulting
solution was boiled with an equal volume of Benedict qualitative solution on a water
bath. The formation of a brick red precipitate depicted the presence of reducing
compound.
Test for alkaloids:
One gram of powdered sample of each specimen was separately boiled with
water and 10ml hydrochloric acid on a water bath and filtered. The pH of the filtrate was
adjusted with ammonia to about 6-7. A very small quantity of the following reagents was
added separately to about 0.5 ml of the filtrate in a different test tube and observed.
Proximate composition analyses of Dioscorea sp:
Fats contents were determined by using AOAC (1990) 22.034 and protein content
AOAC, PN-75/A-04018. Crude fiber was determined by treating oil-free sample by
sulphuric acid (0.26 N) and potassium hydroxide (0.23 N) solution in refluxing systems,
followed by oven drying and muffle furnace incineration (AOAC, 1984). For the
determination of ash content 3 g of grounded Dioscorea sp leave and tuber were taken in
desiccated china dishes. The samples were then charred and ashed by using muffle
furnace in two shocks first at 5500C for 30 min and then at 8500C for 30 min. The dishes
were removed and when cool to room temperature each dish was reweighed containing
white appearing ash. By difference the weight of ash was calculated.
Where
(W2- W) x100
Ash (%) =
W 1- w
W2 = Weight in gm of the dish with the ash
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W = Weight in gm of empty dish
W1 = Weight in gm of the dish with the dried material taken for test.
Moisture Contents were determined according to the method used by Khan and Kulachi
(2002). For this purpose leaves were taken and their fresh weight was determined. Then
they were placed in oven at 720C for 48h and were again weighted. The moisture content
was determined according to the formula:
Weight of fresh seeds-weight of dry seeds x 100
Moisture content (%) =
Weight of fresh
The digestible carbohydrates were calculated by difference. Total energy values were
calculated by multiplying the amounts of protein and carbohydrate by the factor of 4
kcal/g and lipid by the factor of 9 kcal/g as described by Bazi Yabani et al. (2009).
Antimicrobial activity of the plant
Antimicrobial activity was examined for aqueous, ethanol and methanol extracts
from leaves and tuber of Dioscorea sp. The microbial isolates were collected from
University of Calabar Teaching Hospital (UCTH). The antibacterial activity of Dioscorea
sp plant extract was evaluated by disc diffusion method (Chung et al. 1990). Muller
Hinton agar medium was prepared and poured into the petri dishes and allowed to
solidify. Then it was inoculated with a swab of culture and spread throughout the medium
uniformly with a sterile cotton swab. A sterile filter paper disc was prepared and dipped
with plant tuber and leaf extracts (water, ethanol, methanol) at concentration of 25%,
50% and 100% and then placed on the surface of agar plates. The test was performed in
triplicate and their efficiency was determined by measuring the diameter of zone of
inhibition around the well
Statistical analysis
Data collected for the antimicrobial activities of the plant were subjected to a
2x3x4 factorial CRD experiment and significant means were separated using Least
Significant difference (LSD) Test at 5% probability level. The data for proximate and
phytochemical analysis of the plant were subjected to Complete Randomized Design.
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Plate 1: Tuber of Kekor (Dioscorea sp)
Plate 2: Kekor (Dioscorea sp) root and stem with sharp thorns
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Plate 3: Leaves of Kekor (Dioscorea sp)
Plate 4: Stem of Kekor (Dioscorea sp)
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Plate 5: Harvesting of yam tuber of Kekor (Dioscorea sp)
RESULTS AND DISCUSSION
Phytochemical properties of Dioscorea sp
The alkaloids, polyphenols and reducing compounds in the leaves of Dioscorea sp
were significantly higher (p<0.05) than those of the tuber of the plant. Saponins, tannins
in the leaves and tuber were not significantly different. The flavonoids and glycosides in
the tuber of Dioscorea sp were significantly higher (p<0.05) than those of the leaves of
the plant. Phytochemicals are non-nutritive plant chemicals that have protective or
disease preventive properties. These bioactive compounds are naturally found in food,
leaves, stem and roots or other parts of plants that interplay with nutrients and dietary
fiber to protect them (Ekpo et al., 2012). The presence of alkaloids, polyphenols,
reducing compounds, saponins, tannins, flavonoids, and glycosides in significantly high
amount in both leaves and tuber of Dioscorea sp is an indication that the plant possesses
medicinal properties that could be used in the abetment of infectious diseases (Table 1).
Traditionally saponins have been extensively used as detergents, as piscicides and
molluscicides, in addition to their industrial applications as a foaming and surface active
agent and also have beneficial health effects Shi et al., (2004). However, with the
significantly high amount of flavonoid in the tuber of the plant, Dioscorea sp can then be
seen as a good dietary source for flavonoid. Alkaloids in the leaves of Dioscorea sp were
significantly higher (P<0.05) than those of the tuber. This implies that extraction of high
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amount of alkaloid in the plant can be achievable using the leaves. Alkaloid is an
important bioactive compound that could be used in stimulating the nervous system as
well as lowering blood pressure in patients. Okaka et al., (1992) reported that high levels
of alkaloids in foods could cause gastro-intestinal upset and neurological disorders.
However, based on the result of this study the tuber of Dioscorea sp with low level of
alkaloids could be of more medicinal use than the leaves. Tannins have been reported by
Osuntogun et al., (1987) to affect the nutritive value of food products by forming
complex with protein (both substrate and enzyme) therefore inhibiting digestion and
absorption. Tannins in leaves and tuber of Dioscorea sp were found to be of considerably
equal amount.
Proximate composition of Dioscorea sp
The proximate composition of the tuber such as crude protein, crude fibre, ether
extract, ash, dry matter and carbohydrate contents were significantly higher (p<0.05) than
those of the leaves (Table 2). The significantly high amount of carbohydrate and crude
protein in the plant is an indication that consumers will gain energy needed for the
biochemical and biological reactions with the body systems which would promote growth
and development. Crude fibre content in the plant was found to be significantly high
(p<0.05) in the tuber than the leaves this could be due to the presence of waxy
substances. Alabi et al., (2005) also found that P. biglobosa had high amounts of lipid,
protein, carbohydrate, soluble sugars and ascorbic acid. Interestingly, the amount of
protein in the tuber of Dioscorea sp would play an important part in human nutrition.
Protein not only support growth but is also important for maintenance and repair of body
tissues.
Table 2: Proximate composition of the tuber and leaves of Dioscorea sp
Parameters
Crude protein (%)
Moisture (%)
Crude fibre (%)
Ether extract (%)
Ash (%)
Dry matter (%)
Carbohydrate (%)
Leaves
11.80b±0.02
8.00a±0.02
19.20b±0.07
4.5b±0.01
2.67b±0.02
92b±0.11
26.3b±0.0
Tuber
23.65a±0.02
5.00a±0.01
23.19a±0.06
7.0a±0.04
3.18a±0.01
95a±0.12
76.8a±0.03
Means with the same case letter along the horizontal arrays indicate no significant
difference (p>0.05
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Effect of concentration of Dioscorea sp on zones of inhibition of microorganisms
It has been observed over time that different ethanol, water, and methanol extract
of the leaves have demonstrated in-vitro antibacterial activities against E.coli,
Staphylococcus, Pseudomonas, Salmonella, Streptobacillus and Streptococcus. The result
of this study shows that the zone of inhibition of bacteria using Pseudomonas sp at 25%
of the extract and Salmonella sp at 100% of the extract had significantly high zones of
inhibition with no significant difference (P>0.05) between them (Table 3). This was
followed by E.coli (at 25%, 50%, 100%), Pseudomonas sp at 50%, 100% and Serratia sp
at 100% had no significant difference (P>0.05), this was also followed by Salmonella sp
at 25% of the extract while Serratia sp at 25% and 100% of the extracts produces the
lowest zones of inhibition. Agatemor (2009) reported that the ethanol extract of Ocimum
gratissium showed antibacterial activity against S. aureus and E.coli.
In Fig 2 Proteus sp produces a high zone of inhibition at 100% of the extract
followed by 50% and 25%. Serratia sp at 25% of the extract was found to produce a high
zone of inhibition followed by 100% and 50% respectively. Pseudomonas sp at 25% and
100% produces no significant difference (p>0.05) but had a high zone of inhibition than
50% of the extract. E. coli at 50% and 25% produces a high zone of inhibition followed
by 100% of the extract. Salmonella sp had a high zone of inhibition at 100% of the
extract, followed by 25% of the extract while 50% of the extract produces no zone of
inhibition. In Fig 1. The methanolic and ethanolic extract of Dioscorea sp were found
effective in the inhibition of bacteria growth than the cold and hot water extract.
The tuber methanolic extract produces significantly high (p<0.05) zone of
inhibition using Penicillium sp followed by tuber methanolic extract and leaf methanolic
extract that had no significant difference (p>0.05), this was then followed by the leaf
ethanolic extract while leaf hot water extracts had the lowest zone of inhibition. The leaf
cold water extract, tuber hot and cold water extract had no zones of inhibition. It was
observed that the Candida sp using tuber ethanolic extract produces a high zone of
inhibition followed by the tuber methanolic extract, also followed by leaf ethanolic
extract, and tuber hot water extract that had no significant difference (p>0.05) between
them. This was also followed by the leaf methanolic extract, then tuber, cold water
extract and the leaf hot water extract. The result obtained using Aspergillus sp on leaf
ethanolic extract and tuber methanolic extract of Dioscorea sp with no significant
difference (p>0.05). This was also followed by tuber cold water extract and leaf
methanolic extract that had low zone of inhibition. It was observed the Rhizopus sp on
tuber methanolic extract and leaf ethanolic extract plate of kekor produces significantly
high zones of inhibition followed by tuber ethanolic extract, leaf methanolic and leaf hot
water extract with no significant difference (p>0.05) which was also followed by the
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tuber hot and tuber cold water extract. The leaf cold water produces zero zones of
inhibition. The observed difference of the plant extracts may be as a result of insolubility
of the bioactive components in the various solvent or the presence of the inhibitors to the
antimicrobial components Okigbo and Ogbonnanya (2006). Dioscorea sp with the
numerous inhibitory properties could be regarded as an important food crop that could be
utilized as antifungal agent.
Table 3: Zone of inhibition of bacteria sp using Kekor (Dioscorea sp) extract
Concentration
Serratia sp
Pseudomonas
E.coli
Salmonella sp
Of extract
sp
e
25%
0.24 ±0.01
1.67a±0.02
1.40b±0.02
0.78c±0.01
50%
0.63d±0.02
1.06b±0.01
1.27b±0.02
NZ
b
b
b
100%
1.52 ±0.01
1.16 ±0.01
1.39 ±0.01
1.68a±0.01
LSD value = 0.11
3.5
Zones of inhibition (mm)
3
2.5
2
1.5
1
0.5
0
Proteus sp
Serratia sp
Pseudomonas sp
Fig.1: Effect of Dioscorea sp extract on bacterial activity Key: LHW-Leaf hot water.,
LCW- Leaf cold water., LETH- Leaf Ethanol., LMETH- Leaf methanol., THW- Tuber hot
water., TCW- tuber cold water., TETH- Tuber ethanol., TMETH- Tuber Methanol
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Zones of inhibition (mm)
Advances in Biochemistry & Biotechnology
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
25%
50%
100%
Fig.2: Effect of extract concentrations on bacterial activity
Table 4: Zone of inhibition of fungi species using Kekor (Dioscorea sp) extract.
Concentration
Penicillium sp Candida sp
Aspergillus sp Rhizopus sp
Of extract
25%
NZ
1.60a±0.02
0.78b±0.02
1.26c±0.01
50%
1.26d±0.02
1.15b±0.01
0.66b±0.02
1.53a±0.01
100%
2.25b±0.01
2.8a±0.01
1.50a±0.01
2.03b±0.01
LSD value = 0.09., NZ- No Zones
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Table 1: Phytochemical composition of Dioscorea sp
Means with the same case letter along the horizontal array indicate no significant difference (p>0.05).
SAMPLES
Alkaloids
Saponins
Tannins
Flavonoids
Glycosides
Polyphenols
Leaves
Tuber
2.20a±0.02
1.40b±0.01
0.40a±0.01
0.20a±0.01
0.51a±0.02
0.42a±0.03
3.0b±0.01
3.6a±0.01
1.57b±0.03
2.16a±0.02
17.65a±0.02
14.67b±0.01
Reducing
compounds
27.88a±0.01
26.46b±0.02
Table 5: Zones of inhibition of fungi species
Fungi
species
LHW
LCW
LETH
LMETH
THW
TCW
TETH
TMETH
Pencillium
1.53d±0.02
NZ
2.03c±0.01
2.44b±0.02
NZ
NZ
2.37b±0.02
2.87a±0.02
Candida
1.08f±0.01
NZ
2.27c±0.02
1.59d±0.01
2.17c±0.02
1.40e±0.02
3.30a±0.03
2.99b±0.02
Apergillus
NZ
NZ
2.33a±0.01
0.74d±0.01
NZ
0.90c±0.01
1.97b±0.02
1.9b±0.01
Rhizopus
1.49b±0.01
NZ
3.09a±0.03
1.42b±0.02
1.06c±0.01
1.10c±0.01
1.54b±0.01
3.14a±0.01
LSD0.05 = 0.13., NZ - No zones
Key: LHW-Leaf hot water., LCW- Leaf cold water., LETH- Leaf Ethanol., LMETH- Leaf methanol., THW- Tuber hot
water., TCW- tuber cold water., TETH- Tuber ethanol., TMETH- Tuber Methanol
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Zones of inhibition (mm)
3
2.5
2
1.5
1
0.5
0
25%
50%
100%
Fig. 3: Effect of extract concentrations on fungi activity
Key: LHW-Leaf hot water., LCW- Leaf cold water., LETH- Leaf Ethanol., LMETHLeaf methanol., THW- Tuber hot water., TCW- tuber cold water., TETH- Tuber
ethanol., TMETH- Tuber Methanol
Conclusion
The phytochemical contents of the stem, leaves, root and bark of plant serve as
supplements for food and also have the potential to improve the health status of its users as a
result of the presence of various compounds vital for good health. Their fiber content
provides bulk in the diet and this helps to reduce the intake of starchy foods, enhances
gastrointestinal function, prevents constipation and may thus reduce the incidence of
metabolic diseases like maturity onset diabetes mellitus and hypercholesterolemia. Some of
the plants are also potent antibiotics, antihypertensives and blood building agents and also
improves fertility in females when used.
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