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South Asian Research Journal of Natural Products
4(4): 8-17, 2021; Article no.SARJNP.69706
Comparative Phytochemical and Antimicrobial
Analyses of Leaves of Pterocarpus mildbraedii
Harms and Xylopia aethiopica (Dual) A. Rich
C. E. Anarado
1*
,
C. J. O. Anarado
1
,
E. E. Okechukwu
1
,
F. M. Chukwubueze
1
and G. E. Kenechukwu
1
1
Department of Pure and Industrial Chemistry, Nnamdi Azikiwe University, P.M.B 5025, Awka,
Anambra State, Nigeria.
Authors’ contributions
This work was carried out in collaboration among all authors. Author ACE designed the study and
supervised the work. Authors OEE and KGE carried out the analysis. Authors ACJO and CFM wrote
the literature and edited the work. All authors read and approved the final manuscript.
Article Information
Editor(s):
(1) Prof. Mohamed Fawzy Ramadan Hassanien, Zagazig University, Egypt.
Reviewers:
(1) Asdren Zajmi, Management and Science University, Malaysia.
(2) Alfred Ngenge Tamfu, University of Ngaoundere, Cameroon.
(3) Manjunatha H, Bangalore University, India.
Complete Peer review History:
http://www.sdiarticle4.com/review-history/69706
Received 10 April 2021
Accepted 16 June 2021
Published 21 June 2021
ABSTRACT
Aim:
To compare the phytochemicals and antimicrobial activities of Pterocarpus mildbraedii
Harms and Xylopia aethiopica(Dual) A.Rich
Methodology: The leaves of P. mildbraedii and X. aethiopica were collected, washed, air-dried,
ground and each extracted with n-hexane, ethyl acetate and methanol. The extracts were analysed
for the presence of phytochemicals. Antimicrobial analyses were also carried out on the extracts.
Results: Alkaloids, saponins, flavonoids, phenols and resins were found in all the extracts of both
plants. As the polarity of the solvents used increased, the abundance of saponins, tannins and
phenols increased in both plants. Also the abundance of steroids decreased as the polarity of
solvents used increased in both plants. Saponins, tannins and phenols were found to have high
percentage composition in P. mildbraedii while alkaloids and flavonoids were very high in X.
aethiopica. Generally extracts of X. aethiopica showed more activity against the bacteria than the
P. mildbraedii. S. aureus was only susceptible to ethyl acetate leaf extract of X.aethiopica. The
Original Research Article
Anarado et al.; SARJNP, 4(4): 8-17, 2021; Article no.SARJNP.69706
9
ethyl acetate extract of both plants showed inhibition to the growth of E. coli. N-hexane extract of
X. aethiopica was the only extract which showed against the one of two fungi used.
Conclusion: The two plants contained many metabolites which have been attributed to the
antimicrobial activities exhibited by the two plants. These metabolites should be isolated and the
subsequent development of the metabolites in formulation of drugs.
Keywords: Pterocarpus mildbraedii; Xylopia aethiopica; phytochemicals; antibacterial; antifungal.
1. INTRODUCTION
Plants serve as an important therapeutic agents
as well as valuable raw materials for
manufacturing numerous traditional medicine [1].
The traditional medicine system has continued to
be practiced in different parts of the world
especially in developing countries. However, the
prohibitive cost of treatment, side effects of
several synthetic drugs and development of
resistance to the currently used drugs for
infectious diseases have led to an increased
emphasis on the use of plant materials in
developing countries as source of
medicine[2,3,4,5,6,7]. According to WHO, about
three-quarter of the world’s population currently
use medicinal plants as primary health care
system[1,3]. They also estimated that about
21,000 plant species have potentials of being
used as medicinal plants, whereas only 30% of
the entire plant species are already in use [8,9].
The large diversity of medicinal plant species is a
huge source of potentially active phytochemicals
with novel structures. Approximately 119 pure
chemical substances isolated from higher plants
are used as medicine throughout the world [1].
These chemical substances can be classified
into alkaloids, flavonoids, terpenoids, tannins,
steroids, etc which are known as the secondary
metabolites. The secondary metabolites act as
antibiotics and helps in sustaining the overall
health and functional status of the cells within
organ systems of the body [10]. In the course of
this study, we will be comparing the
phytochemical and antimicrobial properties of the
leaves of P. mildbraedii and X. aethiopica.
P. mildbraedii Harms belonging to the family of
Papilionaceae is majorly found in African
countries like Nigeria, Ghana, Liberia,
Cameroon, Sierra Leone, Equatorial Guinea and
Tanzania. The Pterocarpus genus includes some
species like P. tinctorius Welw., P. osun Craib, P.
mildbraedii Harms., P. santalinus L. F. and P.
erinaceus Poir distributed in Africa. In
ethnomedicine reports, showed that Pterocarpus
genus is used to treat inflammation, pain,
infectious, cardiovascular, gastrointestinal and
skin diseases in Africa, Asia and Latin America. It
commonly known in Nigeria with different local
names such as “Oha” in Igbo, “Madobiyar rafi” in
Hausa, “Urube” in Edo, “Geneghar” in Ijaw and
“Kakupupu” in Urhobo[11,12]. In Eastern Nigeria,
Pterocarpus has two more different species:
Soyauxii Taub (Oha) and Santalinoides L`Herit
(Uturukpa)[13]. P. mildbraedii leaves are
nutritional vegetables often harvested from the
wild and consumed in Southern Nigeria.
According to [14], the level of sodium, hydrogen
cyanide and oxalate in the vegetables are low
while the amino acid profile indicates that the
leaves is rich in essential amino acids. The tree
is also local source of wood, they are rarely
exploited for their timbers in Tanzania and used
in making mortars. In Ghana, the trees are
planted in cocoa plantation to provide shades
[15]. The leaves extracts are used in the
treatment of headache, pains, fever, convulsion,
respiratory disorders and as antimicrobial agents
[11]. The leaves of P. mildbraedii have been
recommended for consistent use to prevent
diabetics [16,17]. The result of analysis carried
by Ezekwesili et al, 2016 [17], showed that P.
mildbraedii is also good source of beneficial
chemicals having antioxidant,
hypocholesterolemic, chemoprotective and
antibacterial properties. The leaf extract of P.
mildbraedii was reported to have no apparent in
vivo toxicity on the kidney and heart. Hamzah et
al, 2018)’s [11] work also showed that the
hepatoprotective compounds present in the plant
can be exploited for the prevention and treatment
of liver damage. Proximate, mineral, anti-nutritive
and phytochemical screening of leaf of P.
mildbraedii revealed that the leaf contained 20.63
± 0.03% ash, 13.33 ± 0.01% moisture, 26.45 ±
0.03% crude protein, 8.66 ± 0.01% fat, 12.33 ±
0.02% crude fibre, 18.61 ± 0.44% Carbohydrate,
Ca, Na, Mg, Zn, K, P, Fe, Mn, 0.47 ± 0.47%
tannic acid, 0.23 ± 0.00% polyphenol, 5.49 ±
0.02% saponin, 4.65 ± 0.02% alkaloids, 3.66 ±
0.01% flavonoids, 3.33 ± 0.09mg/g oxalate, 6.38
± 0.58mg/g phytin phosphorus and 22.65 ± 2.06
mg/g phytic acid[18]. Some compounds like
fagasterol or lupeol, oleic acid, palmitic acid,
1,2,3,4 Butanetetrol or Erythritol, N,N Dimethyl-
Anarado et al.; SARJNP, 4(4): 8-17, 2021; Article no.SARJNP.69706
10
2-propyn-1- amine, 1,2-Benzenediol, 4-
Hydroxypiperidine, and n-Hexadecanoic acid
have been isolated from ethanol leaf of P.
mildbraedii [19].
X. aethiopica (Dunal) A.Rich belonging to the
family of Annonaceae, is commonly known as
‘Negro pepper’, ‘grains of Selim’, 'Ethiopian
pepper', 'Guinea pepper' or 'Negro pepper'. In
Nigeria, it is known as ‘Uda’ in Igbo, ‘Eeru’ in
Yoruba, and ‘Chimba’ in Hausa. It grows
naturally in the savanna region of Africa,
particularly in Ghana, Nigeria, Cameroon,
Ethiopia, Sudan, Angola and Senegal [20,21]. X.
aethiopica has played a key role in African
traditional medicine for several countries owing
to its wide array of therapeutic indications.
Almost every morphological part of the plant is
used as medicine especially the fruits. They are
used in the treatment of cough, stomachache,
dizziness, amenorrhea, bronchitis, dysentery,
headache, neuralgia, female sterility, purgative,
rheumatism, biliousness, malaria, hemorrhoids,
uterine fibroid, diabetes, boils, diarrhea, stomach
disorder, menstrual disorder, naso-pharyngeal
infections, arthritis, sores, wounds and cuts
among others [20,21,22,23,24]. They are also
used as medicine for managing various ailments
including skin infections, candidiasis, syphilis,
dyspepsia etc [25].
According to [20], the decoction of the seeds is
used by traditional birth attendants to induce
placental discharge postpartum due to its
abortificient effects. The powdered roots are
used in local treatment of cancer. The decoction
of the leaves is used as anti-emetics [26]. The
stem bark is used in combination with other
medicinal plants for treatment of postpartum
breast infections. In Ivory Coast, X. aethiopica is
taken to promote fertility and ease of childbirth
[27]. Studies have shown that the plant possess
antibacterial, antifungal, antiplasmodial,
analgesics, anti-inflammatory, antidiabetic and
antimicrobial properties, anti-hypertensive and
diuretic effects [28]. [25,29,23] reported that the
essential oil of the plant consist of mainly
monoterpenoids and sesquiterpenoids like β-
pinene, 1,8-cineol, α-terpineol, terpinene-4-ol,
bisabolene. [20] also isolated oxoaporphine
alkaloids and lysicamine from the methanol and
ethyl acetate extracts of the plant respectively.
The two plants are used in South Eastern part of
Nigeria in many soups and decoctions used in
various traditional medicines. The work aims to
compare the antimicrobial activities of the plants
and also compare the metabolites in some
extracts of the plants which could be responsible
for the antimicrobial activities.
2. METHODOGY
The Leaves of Pterocarpus mildbraedii and
Xylopia aethiopica were collected from Awka,
Anambra State and were identified by
taxonomists in the Department of Botany,
Nnamdi Azikiwe University, Awka. Both were air-
dried for 3 weeks and pulverized to powder. The
powdered samples were stored to be used for
analysis.
Qualitative and Quantitative tests were carried
out on the samples to determine the presence of
the phytochemicals in the powdered samples.
Antimicrobial analysis was also carried out on the
sample.
2.1 Extraction of the Phytochemicals
10 g of the powdered samples were soaked
separately in 100 ml of each of the three
solvents: methanol, ethyl acetate and n-hexane.
Each of the three solutions was shaken and the
mixtures were left to stand at room temperature
for 48 hours after which they were filtered with
Whatman No. 1 filter paper. The filtrates were
collected and concentrated by heating on a
rotary evaporator. The concentrated extracts
were then used for the analysis.
Qualitative and quantitative analyses were
carried out using the standard methods
described by [30,31,32], to ascertain the
presence and quantity of metabolites such as
tannins, alkaloids, flavonoids, steroids,
terpenoids, saponins, cardiac glycosides,
proteins, phenols and resins.
2.2 Determination of Antimicrobial
Activity
2.2.1 Antimicrobial screening tests
The crude extracts and fractions of the leaves of
the two plants were tested against 24 hours broth
cultures of Escherichia coli, Staphylococcus
aureus, Streptococcus faecalis, Candida albican,
Aspergillus niger and Salmonella app by
following the procedure of [33] Nester et al.,
2002.
2.3 Bacterial Susceptibility Test
Susceptibility test were performed on the crude
extracts to ascertain their activity or not against
Escherichia coli, Staphylococcus aureus,
Streptococcus faecalis, Candida albican,
Anarado et al.; SARJNP, 4(4): 8-17, 2021; Article no.SARJNP.69706
11
Aspergillus niger and Salmonella typhi. Higher
concentrations of extracts were used (50 mg/ml,
using methanol as solvent). In the test tube, 20
ml nutrient agar (in a test tube) was melted at
100°C and stabilized at 45°C for about 15
minutes. About 0.1 ml inoculums were added
from culture tubes to the agar in the test tube by
the use of a loop. The test tube containing the
agar and the inoculums was then rolled in
between the palms gently to mix the inoculums
thoroughly with the agar. The loop was flamed
before it was used each time.
The content of the test tube was poured into a
Petri dish and allowed to set. The Petri dishes
were then labelled with the respective organisms
(inoculums) and dates. By means of a 10 mm
cork borer, three cups were bored, well
separated and equidistant from each other in the
agar. The cups were labelled with three crude
extracts. Each cup was filled with its
corresponding extract to about three-quarters
full. They were kept on a bench at room
temperature for about 60 minutes (for the
extracts to diffuse into the agar). The plates were
then incubated aerobically at 37°C and examined
for any zone of inhibition after 24 hours.
2.4 Determination of Minimum Inhibitory
Concentration
Four different concentrations of the antimicrobial
agents were prepared (40, 20, 10 and 5 mg/ml)
from the crude extracts, aqueous and also from
the chloroform fractions of the various extracts.
The working area was disinfected using phenol
before the start of the work.
20 ml nutrient agar was melted at 100°C and
stabilized at 45°C for about 15 minutes in a test
tube. About 0.1 ml staphylococcus aureus was
added from culture tubes to the agar in the test
tube by the use of a loop. The test tube
containing the agar and the inoculums was then
rolled in between the palms gently to mix the
inoculum thoroughly with the agar. The loop was
flamed before it was used each time.
The content of the test tube was poured into a
Petri dish (which was previous autoclaved at a
pressure of 15 lb/in2 for 20 minutes) to set. The
Petri dish was then labelled with the name of the
inoculum and date. By means of a 10 mm sterile
cork borer, four cups were bored well separated
and equidistant from each other in the agar. The
cups were labelled with the four concentrations
of the crude aqueous extract. Each cup was filled
with its corresponding extract to about three-
quarters full.
The Petri dish was quickly covered and then kept
on a bench at room temperature for about 60
minutes (for the extracts to diffuse into agar).
The same procedure was followed for the
different extracts and fractions, with the same
microorganism and the other organisms. Thus,
each extract was tested against each of the test
organism, using chloramphenicol as the control
for each organism.
The plates were incubated aerobically at 37°C
for 24 hours and examined for any zone of
inhibition. The reading was done against a dark
background under reflected light. The diameters
of the zones of inhibition of growth were
measured with the help of a pair of dividers and
rule from the underside of the covered plates for
spots with inhibitions. The average of the
diameters was taken. The actual zones were
calculated by subtracting the diameter of the
cups (10 mm) from the total zone of growth.
The zones of inhibition obtained were plotted
against the log of concentrations to determine
the minimum concentrations at which these
extracts can inhibit the growth of the test
organisms. The minimum inhibitory
concentrations were obtained by determining the
concentration at which the zone of inhibition was
zero.
3. RESULTS AND DISCUSSIONS
The results of the phytochemical screening of the
leaves of P. mildbraedii and X. aethiopica are
summarized in the tables.
4. DISCUSSION
The results of the qualitative phytochemical
analysis showed that alkaloids, flavonoids,
phenols, resins and saponins were present in all
the extracts of both Pterocarpus mildbraedii and
Xylopia aethiopica plants. The presence of
alkaloids in X. aethiopica could be the basis of
Anarado et al.; SARJNP, 4(4): 8-17, 2021; Article no.SARJNP.69706
12
Table 1. Results of qualitative phytochemical Analysis of leaf of Pterocarpus mildbraedii
Phytochemical constituents Hexane Ethylacetate Methanol
Alkaloids + + +++
Saponins + ++ ++
Tannins - - ++
Flavonoids +++ ++ ++
Steroids + + -
Terpenoids - - -
Cardiac glycosides ++ - +
Proteins - - -
Phenols + + +++
Resins + + +
Table 2. Results of qualitative phytochemical analysis of leaf of Xylopia aethiopica
Phytochemical
Hexane
Ethylacetate
Methanol
Constituents
Alkaloids + + ++
Flavonoids +++ +++ +
Saponins + ++ ++
Cardiac glycosides + + ++
phenols ++ ++ +++
steroids ++ + -
Terpenoids + - +
Tannins _ + ++
Resins + + ++
Proteins + + -
Key: + = low abundance, ++ = moderate abundance, +++ = high abundance and – = absent.
Table 3. Result of quantitative determination of phytochemcial constituents of Leaf of P.
mildbraedii
Phytochemical constituent Quantity (%)
Flavonoids 2.52
Phytate 0.580
Alkaloids 2.76
Saponins 14.44
Tannins
9.1
Phenolics 9.25
Oxalate 1.20
Table 4. Results of quantitative determination of phytochemical constituents of leaf of X.
aethiopica
Phytochemical constituents
Quantity (%)
Alkaloids 6.38
Flavonoids 9.27
Saponins 4.12
Tannins 4.96
Phenolics 0.61
Phytate 0.35
Cardiac glycosides 2.84
Anarado et al.; SARJNP, 4(4): 8-17, 2021; Article no.SARJNP.69706
13
Table 5. Results of antimicrobial analysis of leaf extracts of P. mildbraedii
Organism
n-hexane extract
Ethyl acetate
Methanol extract
control
Staphylococcus aureus - - - ++ (10mm)
S. faecalis - ++ (11mm) + (9mm) ++ (11mm)
E. coli - ++ (10mm) - + (8mm)
Salmonella typhi - - + (8mm) ++ (10mm)
Candida albicans - - - ++ (10mm)
Aspergillus niger
-
-
-
++ (10mm)
Table 6. Results of antimicrobial analysis of leaf extracts of X. aethiopica
Organism
n-hexane extract
Ethyl acetate
Methanol extract
control
Staphylococcus aureus - ++(12mm) - ++ (10mm)
S. faecalis - +(8mm) - ++ (11mm)
E. coli ++ (11mm) ++ (13mm) - + (8mm)
S. typhi - - ++ (10mm) ++ (10mm)
Candida albican ++ (15mm) - - ++ (10mm)
Aspergillus niger - - - ++ (10mm)
Values are DZI = Diameter of inhibition zone; ++ = high activity (highly Sensitive); += low activity (slightly
Sensitive); - =No activity or growth (Resistant)
Table 7. Result of MIC analysis of leaf extracts of P. mildbraedii
Extract
Organism
MIC VALUE(µg/ml)
Methanol S. faecalis 1
Ethylacetate E.coli 1
Ethylacetate S. faecalis 2
Table 8. Result of MIC analysis of leaf extracts of X. aethiopica
Extract
Organism
MIC value (µG/ML)
Ethyl-acetate Staphylococcus aureus 2
Ethyl acetate E. coli 1
N-hexane E. coli 1
Methanol S. typhi 2
the therapeutic use of the plant as an antimalaria
drug [34]. Flavonoids were more abundant in the
n-hexane which is against the report of Chanda
et al, [35] that flavonoids were found more in
polar solvent, and phenols were more in polar
solvent which is in line with Ghasemzadeh et al,
[36] and Widyawati et al, [37] reports. The
presence of saponins which increased with
increase in the polarity of the solvent used is in
line with the report of Üstündağ and Mazza, [38],
that polar solvents are the common extraction
solvents for saponins. Tannins were found
present only in the methanol extract of P.
mildbraedii and methanol and ethyl acetate
extracts of Xylopia aethiopica, which is not
surprising since it has been reported that
solubility of phenolic compounds increased with
increase in solvent polarity [39]. The anti
diarrhea, anti diabetic and wound healing
properties shown by X. aethiopica could be
attributed to the presence of tannins found in
ethyl acetate and methanol extracts of the plant
[40,41,42]. Steroids were present in the n-
hexane and ethyl acetate extracts of both plants
and absent in the methanol extracts, which is not
surprising because steroids are essentially
hydrophobic [43]. Terpenoids and Proteins were
absent in all the extracts of P. mildbraedii but
present in the n-hexane and ethyl acetate
extracts of X. aethiopica. The presence of
terpenoids in the extracts of X. aethiopica is in
line with findings of [20,23,44] that many
monoterpenoids and sesquiterpenoids were
found in the plant. Cardiac glycosides were
present in all the extracts of both plants except
the ethyl acetate extract of P. mildbraedii.
Cardiac glycosides have been reported to have
antibacterial, antifungal, analgesic, anti
inflammation, antihypertensive, muscle relaxation
and anticancer activity, as heart tonic, diuretics
Anarado et al.; SARJNP, 4(4): 8-17, 2021; Article no.SARJNP.69706
14
and emetics, and many of the ethno medicinal
uses of both plants could be as a result of the
presence of the cardiac glycosides [45,46].
The results of the quantitative analysis showed
that Alkaloids were more present in the
X.aethiopica sample (6.38%) than the P.
mildbraedii sample (2.76%). Flavonoids are also
more present in X. aethiopica (9.27%) than P.
mildbraedii (2.52%). Saponins however, are
more present in the P. mildbraedii (14.44%) than
X. aethiopica (4.12%). The very high quantity of
saponins in P. mildbraedii is in line with high
quantity reported by Akinyeye et al [18] and
could be the basis of the pharmacological use of
the plant as hepatoprotective, anti diabetic agent
[11,16,17,38,47]. Tannins are also more present
in P. mildbraedii sample (9.1%) than X.
aethiopica (4.96%). P. mildbraedii sample has
more percentage of phytate (0.58%) than X.
aethiopica (0.35%). Phenols are also more
present in P. mildbraedii (5.987%) than X.
aethiopica (0.61%). Oxalate are only present in
P. mildbraedii (0.243mg/kg) while cardiac
glycoside in X. aethiopica (2.84%). This shows
that X. aethiopica has greater amount of
alkaloids and flavonoids while P. mildbraedii has
greater amount of saponins and tannins.
The results of the Antimicrobial activity showed
that S. aureus was resistant to all extracts of P.
mildbraedii. However, it was highly sensitive to
the ethyl acetate extract of X. aethiopica, which
is in line with the report of Padalia et al, [48] that
S. aureus was susceptible to semi polar extracts.
Streptococcus was resistant to the n-hexane
extract of P. mildbraedii but was highly sensitive
and slightly sensitive to the ethyl acetate and
methanol extracts respectively. For X. aethiopica
sample, streptococcus was slightly sensitive to
the ethyl acetate extract and resistant to the rest.
E. coli was highly sensitive only to the ethyl
acetate extract of P. mildbraedii whereas it was
highly sensitive to the n-hexane and ethyl
acetate extract of X. aethiopica. The
susceptibility of the gram-negative E. coli to three
extracts was in line with findings of Tamfu et al,
[49] and against the report of Singariya et al, [50]
who reported that extra lipo-polysaccharide and
protein cell wall of gram negative bacteria
provides a permeability barrier to the
antibacterial agent, and thus makes them less
sensitivity to plant extracts. S. typhi was only
sensitive to the methanol extracts of both plant
samples (high activity in X. aethiopica and low
activity in P. mildbraedii) which supported the
findings of Ibrahim and Kebede, [51], that
methanol extracts showed high inhibition to
growths of many bacteria including S. typhi.
Candida albican was resistant to all the extracts
of P. mildbraedii. However, it was highly sensitive
to the n-hexane extract of X. aethiopica and
resistant to the other extracts. Aspergillus niger
was resistant to all the extracts of the two plant
samples. The inhibition against some of the
bacteria and could be attributed to the presence
of metabolites like alkaloids, tannins and
flavonoids [50]. The sensitivity of S. aureus to
extract of X. aethiopica can be said to be as a
result of greater percentage of alkaloids and
flavonoids in the plant sample as identified in the
quantitative analysis. According to Compean and
Ynalvez [52], alkaloids and flavonoids have
antibacterial activities against Staphylococcus
aureus. The high activity of P. mildbraedii on
Streptococcus may be attributed to greater
percentage of tannins in P. mildbraedii. Its
activity in X. aethiopica can be attributed to the
presence of terpenoids. Studies have shown that
tannins and terpenoids have antibacterial
activities against streptococcus. X.aethiopica
showed greater antimicrobial activities than P.
mildbraedii and this can be attributed to greater
phytochemicals present as identified in the
qualitative analysis.
5. CONCLUSION
It is evident that plant cells produce variety of
phytocompounds for defense mechanism against
bacteria. The two plants were shown to have
contained many metabolites which have been
attributed to the antimicrobial activities exhibited
by the two plants. These metabolites should be
isolated and the subsequent development of the
metabolites in formulation of drugs.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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