Advances in Zoology and Botany 5(4): 50-54, 2017 http://www.hrpub.org
DOI: 10.13189/azb.2017.050403
Comparative Screening of Phytochemical Compounds in
Scent Leaf Ocimum gratissimum Linn. (Family: Lamiaceae)
and Bitter Leaf Vernonia amygdalina Del.
(Family: Asteraceae) Extracts
R.N. Oladosu-Ajayi1,*, H.E. Dienye2, C.T. Ajayi1, O.D. Erinle1
1Department of Fisheries Technology, Federal College of Freshwater Fisheries Technology, New-Bussa (FCFFT), Nigeria
2Department of Fisheries, Faculty of Agriculture, University of Port Harcourt, Nigeria
Copyright©2017 by authors, all rights reserved. Authors agree that this article remains permanently open access under the
terms of the Creative Commons Attribution License 4.0 International License
Abstract The study on Phytochemical screening of the
leaves of Ocimum gratissimum and Vernonia amygdalina
was carried out to determine the compounds present. The
result revealed the presence of alkaloids, flavonoids, steroid,
tannins, and carotenoid. The best solvent of extraction for
both plants recorded was the hot water followed by the cold
water even though they were unable to liberate flavonoids
from bitter leaf extracts. The washed bitter leaf extracts
contained more alkaloids than the extracts made from the
unwashed leaf though the differences were not significant
(washed bitter leaf- 7.32% from hot water and 6.83% from
cold water, unwashed bitter leaf- 6.12% from hot water and
5.32% from cold water). The ethanolic extracts of bitter leaf
liberated the flavonoids while the hot water was also able to
liberate it from the scent leaf. Carotenoids were liberated
from the extracts of both plants though the quantities were
not significantly different. The study showed that bitter leaf
and scent leaf contain similar antimicrobial compounds but
former contains more quantity. It can be concluded that the
different solvents of extraction have varying abilities to
liberate these compounds and the quantities that each
liberated has been ascertained.
Keywords Phytochemical, Scent Leaf, Bitter Leaf,
Extract, Ethanol, Water
1. Introduction
Phytochemical screening is a process of tracing the
medicinal value of plants constituents in some chemical
substance that produce a definite physiologic action on the
human body (Jigna et al., 2006; [7] Allero and Afolayan,
2006[2]). The most important of these bioactive compounds
of plants are alkaloids, flavonoids, tannins, saponins,
glycosides, cardenolides, bufadionolides and polyphenolic
compounds (Erinle, 2012[5]). Knowledge of the chemical
constituents of plants is desirable not only for the discovery
of therapeutic agents but also because such information may
be of value in disclosing new sources of such economic
materials as tannins, oils, gum, precursors for the synthesis
of complex chemical substances (Erinle, 2012[5]). Bitter leaf
(Vernonia amygdalina) is gotten from the leaves attached to
the stalk of a small ever-green shrub found all over Africa
belonging to the family Asteraceace. The leaves of
Vernonia amygdalina are green with a characteristic odour
and bitter taste It is well known as a medicinal plant for
diabetes and fever (Adenuga et al., 2010[1]). Vernonia
amygdalina (Del.) commonly called bitter leaf is the most
widely cultivated species of the genus Vernonia which has
about 1,000 species of shrubs (Muanya, 2015[9]). It is
vegetatively propagated by stem cutting at an angle of 450
and popular in most of West Africa countries including
Nigeria, Cameroon, Gabon and Congo Democratic
Republic. Although most popularly used for food, it has
also, been traditionally used for its medicinal properties.
True to its name, bitter leaf is bitter to taste but surprisingly
delicious in meals. Bitter leaf is called Omjunso in East
Africa especially Tanzania, Onugbo in Igbo-Eastern Nigeria
and Orugbo among the Itsekiri and Urhobo tribes in Nigeria,
Ewuro (Yoruba), Etidot (Ibibio), Ityuna (Tiv), Oriwo (Edo),
Chusa-doki Shiwaka (Hausa). Scent leaf, Ocimum
gratissimum is an aromatic perennial herb, with erect stem,
much branched, glabrous and woody at the base often with
epidermic peeling in strips. Ocimum gratissimum is grown
for the essential oil in its leaves and stems while engenol and
to a lesser extent thymol extracted from the oil which are
substitutes of clove oil and thyme oil. The essential oil
possesses antibacterial properties and is also an important
Advances in Zoology and Botany 5(4): 50-54, 2017 51
insect repellent so also are the leaves when left dry and burnt
(Health Facts, 2015[7]). They are used as vegetables
(Sulistiarini, 1999[12]), as spice due to its aromatic nature to
spice various kinds of soup (e.g. pepper soup) and other
delicious meals like porridge (Health Facts, 2015[7]). The
whole plant has many applications in traditional medicine
especially in Africa and India. The applications include in
the treatment of ringworms, gout and fungal infections,
malaria, catarrh, aches, colon pain. The juice gotten from
squeezing its leaf can be used to cure several stomach related
illnesses like cholera, diarrhea, dysentery, vomiting and
convulsion (Health Facts, 2015[7]). Ocimum gratissimum
and Vernonia amygdalina plants are known to have common
phytochemical compounds which are used in traditional
medicine for the treatment of several ailments and the
extracts have been evaluated for their ability to stall the
activities of organisms responsible for spoilage of fresh
catfish (Clarias gariepinus) by extending its shelf
life .(Oladosu-Ajayi et al., 2016[11]).It is against this
background this study was carried to determine the quantity
of the phytochemical compounds present in the extracts of
scent leaf (Ocimum gratissimum) and bitter leaf (Vernonia
amygdalina) and also to know which solvent of extraction
contains more of the phytochemical compounds.
2. Materials and Methods
2.1. Collection of Plant Materials
The study site was located in a home stead garden inside
the National Institute of Freshwater Fisheries Research
(NIFFR) residential quarters in Kainji, New-Bussa, Niger
state. Fresh leaves of Ocimum gratissimum and Vernonia
amuglidana were collected early in the morning from a
garden. The leaves were cut from a non-flowered stalk and
were identified at the herbarium of the Institute
2.2. Preparation of Bitter and Scent Leaf Extracts
The bitter leaf (Vernonia amygdalina) and scent leaf
(Ocimum gratissimum) were first separated from the stalk,
rinsed with clean water to remove the dust and dirts. This
was macerated and divided into two portions, one part of the
bitter leaf was unwashed and the other part was washed to
remove part of the juice that is responsible for the bitter taste.
The extraction was done as follows according to Azu and
Onyeagba (2007). [3]
1. Bitter leaf (300g) was soaked in 150ml of 95% ethanol
for 24hours. The pulp obtained was left in clean, sterile glass
container and vigorously shaken to allow for proper
extraction. Filtration was done using a sterile muslin cloth
after which the extract obtained was air-dried and store for
use.
2. Bitter leaf (300g) was soaked in 150ml of cold water for
24hours and the resultant juice extracted was air dried and
stored as done in (1) above.
3. Bitter leaf (300g) was soaked in 150ml of hot water for
24hours and the resultant juice extracted was air dried and
stored as done in (1) above.
The scent leaf extracts were also prepared using the above
procedure.
2.3. Qualitative Analysis of Phytochemical Compounds
Qualitative analysis was carried out to ascertain the
presence of the different phytochemical compounds in the
leaves. The method used and their corresponding inferences
are shown below.
Tab le 1. Qualitative Analysis of Phytochemical Compounds
Phytochemical Test Observation Inference
1 Alkaloid Wagner dragendroffs Brown precipitate which turns intense yellow with the picnic acid Alkaloid present
2. Tannins Ferric chloride Greenish – black precipitate Tannin present
3. Flavonoid
Ammonium and sodium
hydroxide acid
Yellow colour which turns colourless on addition of acid Flavonoid present
4. Steroids
Liberman Burchard and
Salkowkis
Brownish colour Red colour at interference Steroid present
Source: Harborne (1973)
52 Comparative Screening of Phytochemical Compounds in Scent Leaf Ocimum gratissimum Lnn.
(Family: Lamiaceae) and Bitter Leaf Vernonia amygdalina Del. (Family: Asteraceae) Extracts
3. Quantitative Analysis of
Phytochemical Compounds
Determination of Alkaloids: This was done by the
alkaline precipitation gravimetric method described by
Harborne, (1973) [6]. A measured weight of the sample was
dispersed in 95% acetic acid solution in ethanol to form a
ratio of 1:95(95%). The mixture was allowed to stand for
24hours. The filtrate was concentrated to one quarter of its
original volume by evaporation and treated with drop wise
addition of concentrated aqueous NH4OH until the alkaloid
was precipitated. The alkaloid precipitated was received in
weighed filter paper, washed with 1% ammonia solution
dried in the oven at 80oc. Alkaloid content was calculated
and expressed as a percentage of the weight of sample
analysed.
Determination of Flavonoids: This was determined
according to the method of Harborne (1973)[6]. 300gram of
the same was boiled in 150ml of 2m HCL solution for
30mins under reflux. It was allowed to cool and then filtered
through Whatman No 42 filter paper. A measured volume of
the extract was treated with equal volume of ethyl acetate
starting with a drop. The flavonoids precipitated were
recovered by filtration using weighed filter paper. The
resulting weight difference gave the weight of flavonoids in
the sample.
Determination of Carotenoids: A measured weight of
each sample was homogenized in ethanol using a laboratory
blender. A 1:10 (1%) mixture was used. The homogenate
was filtered to obtain the initial crude extracts. 20ml of ether
were added to the filtrate to take up the carotenoids mixed
well and then treated with 20ml of distilled water in a
separating funnel. The other layer was recovered and
evaporated to dryness at low temperature (35 -50c) in a
vacuum dessicator. The dry extract was then saponified with
20ml of ethanolic potassium hydroxide and left over night in
a dark cupboard. The next day, the carotenoid were taken up
in 20ml of ether and then washed with two portions of 20ml-
distilled water. The carotenoid extract (ether layer) was dried
in a dessicator and then treated with a light petroleum
(petroleum spirit) and allowed to stand overnight in a freezer
(-10oc). The next day the precipitation steroid was removed
by centrifugation and the carotenoid extracts was evaporated
to dryness in a weighed evaporation dish, cooled in a
dessicator and weighed. The weight of carotenoid was
determined was expressed as a percentage of the sample
weight (Okafor, 1987) [10].
4. Results
The phytochemical compounds present in the extracts of
washed bitter leaf are shown in table 2. Flavonoids were
absent in the hot water extracts while alkaloids, tannins and
steroids are present. Also flavonoids and tannins were absent
in the cold water extract while steroids and alkaloids were
present. Tannins were not present in the ethanolic extract
while the alkaloids, steroids and flavonoids were present.
The screening of unwashed bitter leaf extract, flavonoids and
steroids were absent in the hot water extract while it was only
the flavonoids that were absent in the cold water extract. The
ethanol extract was able to liberate flavonoids, alkaloids and
tannins with the exception of the steroids. The
phytochemical compounds present in the extract of washed
scent leaf as shown in table 3, steroids and tannins were
absent in the ethanolic extract while tannins were absent in
the hot water extract. The cold water extract was able to
liberate the alkaloids and steroids. The phytochemical
compounds present in the extract of unwashed scent leaf.
Steroids were absent in the hot water extract while only
alkaloids were present in the cold water extract. The
ethanolic extract showed the presence of alkaloids and
tannins. The summary of the result of quantitative screening
of phytochemicals present in washed bitter leaf extracts as
shown in table 4.Alkaloid has the highest quantity 17.08%
also flavonoids as 2.32% present in ethanolic extract only
while carotenoids has 0.135% in ethanolic, cold and hot
water extracts. The quantity of phytochemicals found in
unwashed bitter leaf extracts was also summarizes. Alkaloids
has the highest amount 14.56% in ethanolic, hot and cold
water extract and flavonoids has 2.42% in ethanolic, cold and
hot water extracts only followed by carotenoid which has
0.24% in ethanolic, cold and hot water extracts. Table 8
shows the quantity of phytochemical compounds in washed
scent leaf extracts. Alkaloids has 5.86% in ethanolic, cold
and hot water extract which flavonoids has 3.28% in
ethanolic, and hot water and not found in cold water extract
and carotenoids has 0.2% in ethanolic hot and cold water
extracts. The quantity of phytochemical compounds in the
extracts of unwashed scent leaf is shown below in table 5.
Alkaloids has 7.82% in ethanolic, cold and hot water extracts
and flavonoids 2.11% in hot water extract and not found in
ethanolic and cold water extracts while carotenoids has
0.22% in both ethanol, hot and cold water extracts.
Tab le 2. Qualitative Screening of Washed Bitter and Unwashed Leaf Extracts
WASHED BITTERLEAF EXTRACT UNWASHED BITTERLEAF EXTRACT
Alkaloids Flavonoids Steroids Tannin Alkaloids Flavonoids Steroids Tannin
HWE + - + + + - - +
CWE + - + - + - + +
EE + + + - + + - +
Advances in Zoology and Botany 5(4): 50-54, 2017 53
Tab le 3. Qualitative Screening of Washed Scent Leaf and Unwashed Scent Leaf Extracts
WASHED SCENTLEAF EXTRACT UNWASHED SCENTLEAF EXTRACT
Alkaloids Flavonoids Steroids Tannin Alkaloids Flavonoids Steroids Tannin
HWE + + - - + + - +
CWE + + + - + - - -
EE + - + - + - - +
Tab le 4. Quantitative Screening of Washed Bitter Leaf and Unwashed Bitter Leaf Extract in % (G/100g)
WASHED BITTER LEAF EXTRACT UNWASHED BITTER LEAF EXTRACT
Alkaloids Flavonoids Carotenoids Alkaloids Flavonoids Carotenoids
EE 2.93 2.32 0.045 3.12 2.42 0.14
CWE 6.83 -- 0.04 5.32 -- 0.06
HWE 7.32 -- 0.05 6.12 -- 0.07
TOTAL 17.8 2.32 0.135 14.56 2.42 0.26
Tab le 5. Quantitative Screening of Washed and Unwashed Scent Leaf Extract in % (G/100g)
WASHED SCENT LEAF EXTRACT UNWASHED SCENT LEAF EXTRACT
Alkaloids Flavonoids Carotenoids Alkaloids Flavonoids Carotenoids
EE 2.56 1.88 0.10 1.75 -- 0.11
CWE 1.88 -- 0.04 1.43 -- 0.03
HWE 1.40 1.40 0.06 4.64 2.11 0.08
TOTAL 5.86 3.28 0.2 7.82 2.11 0.22
KEY
EE = Ethanolic extract BW = Bitter leaf washed
HWE = Hot water extract BUN = Bitter leaf unwashed
CWE = Cold water extract SW = Scent leaf washed
SUN = Scent leaf unwashed + = Present - = Absent
5. Discussion
The results obtained from this study showed that hot water,
cold water and ethanolic extracts of the plants have varying
contents of the phytochemical compounds. It also shows that
though the two plants contain similar phytochemical
compounds, the quantities differ. The aqueous extracts of the
bitter leaf contained the highest quantities of alkaloids
(Tables 4 and 5). This was obvious in the washed and
unwashed bitter leaf (washed: Cold water extract-6.83% and
Hot water extract-7.32%; unwashed: Cold water
extract-5.32% and Hot water extract-6.12%). Therefore, it
can be concluded that alkaloids are more revealed in bitter
leaf extract when it is washed, although an appreciable
quantity can also be gotten when it is not washed. It can also
be deduced from this experiment that alkaloids are not heat
labile since the highest quantity was liberated from the hot
water extract. The ethanolic extracts contained the least
quantities (washed: 2.93% and unwashed: 3.12%). This
shows that ethanol is an organic solvent and an excellent
phytochemical compound liberator; it was unable to exhibit
those characteristics in the extraction of alkaloids from bitter
leaf extracts. This is unlike the findings of Ephraim (2010) [4]
who got more alkaloids from the ethanolic extracts of black
pepper seeds and pawpaw seeds. The study also discovered
that the hot water extracts of the two plants (black pepper and
pawpaw seeds) liberated more alkaloids than the coldwater
extracts. The carotenoids were also not well liberated by the
aqueous extracts (cold and hot water). Bitter leaf (washed
and unwashed) extracts contained low content of flavonoids
compared to the alkaloids as shown in this result. Hot water
extract retained more of the compounds compared to cold
water extracts. Carotenoid levels increased in hot water
extracts 0.05g/100g while the content was reduced in cold
water extracts 0.04g/100g. The resistance of carotenoid to
the effect of heat is in line with the earlier report of Anderson
(2002) that phytochemical compounds are not affected by
processing. It can thus be said about bitter leaf that the
phytochemical compounds are better liberated after washing
using either the hot or cold water as solvents of extraction
while the flavonoids content can only be revealed in the
ethanolic extract.
This study reveals that more of the compounds were found
in both scent leaf and bitter leaf.. Unlike the bitter leaf, the
aqueous extracts did not liberate much alkaloids (washed:
Cold water extract-1.88% and Hot water extract-1.40%;
unwashed: Cold water extract-1.43% and Hot water
extract-4.64%) while the ethanolic extract liberated 2.56%.
54 Comparative Screening of Phytochemical Compounds in Scent Leaf Ocimum gratissimum Lnn.
(Family: Lamiaceae) and Bitter Leaf Vernonia amygdalina Del. (Family: Asteraceae) Extracts
This means that to use hot water as solvent of extraction for
flavonoids, the scent leaf should not be washed while it
should be washed and ethanol used as solvent of extraction
when alkaloids are been screened for. Erinle (2012)[5]
reported that the pigment taste of scent leaf is contributed by
its high content of alkaloid. The hot water extract of the
unwashed scent leaf can also be depended upon for
flavonoids even though the ethanolic extract of the washed
sample can also be screened for it. This result thus infers that
flavonoids in the scent leaf are not well liberated and are not
heat labile.
Generally, heating did not significantly affect the
carotenoid content of the leaf. This indicates that carotenoid
(colored pigment) remained stable during heating, though
there was slight reduction in carotenoids content in ethanolic
extracts and hot water extracts in the result of this study.
6. Conclusions
The results from this work has shown that the ethanolic,
hot and cold water extracts of the plants under study contains
phytochemical compounds which include alkaloids, steroids,
flavonoids, carotenoids and tannins, which are responsible
for their various activities and for medicinal benefits. It can
be concluded that the different solvents of extraction have
varying abilities to liberate these compounds and the
quantities that each liberated has been ascertained. The
selected plants contain substantial amount of phytochemical
which are helpful in the prevention of deadly diseases. Scent
leaf, (Ocimum gratissimum) and bitter leaf (Vernonia
amygdalina) leaves could help fulfill the growing demands
of plant based foods for human nutrition.
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