Measurement: Food 5 (2022) 100018

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Measurement: Food
journal homepage: www.elsevier.com/locate/meafoo

Phytochemical constituents and assessment of crude extracts from

Boerhavia diffusa L. and Lonchocarpus sericeus (Poir.) Kunth ex DC. leaves
for antioxidant and antibacterial activities
Emmanuel Adeku, Oluwatooyin F. Osundahunsi, Sunday A. Malomo∗, Idowu I. Asasile,
Olajumoke M. Owolabi, Ganiyat Oyewole
Department of Food Science and Technology, Federal University of Technology, Akure,
Nigeria

a r t i c l e i n f o a b s t r a c t

Keywords: Medicinal plants have been used by humans for therapeutic purposes since ancient times. Hence, this study
Medicinal plant investigated the human health benefits of aqueous and ethanolic extracts of Boerhavia diffusa and Lonchocar-
Lochocarpus sericeus pus sericeus leaves after these solvents’ extraction. The ethanolic L. sericeus extract (ELS), aqueous L. sericeus
Boerhavia diffusa
extract (ALS), ethanolic B. diffusa extract (EBD) and aqueous B. diffusa extract (ABD), were respectively eval-
Antioxidant
uated for their phytochemical constituents, antioxidant and antibacterial properties. Results showed that both
Antibacterial
Extracts plant species have various constituents such as flavonoid (7.11–21.05%), alkaloid (2.00–8.00%), oxalate (1.98-
3.51 mg/g), saponin (3.00–16.00%), tannin (9.70–18.10 mg/100 g), total phenol (7.36- 18.26 mg/100 g) and
phytate (225.64–394.88 mg/100 g), respectively. The ethanolic extracts of the leaves showed higher phytochem-
icals values than the aqueous extracts, with the highest values in the L. sericeus species, which might be due to
the less polarity nature of ethanol solvent when compared to the aqueous solvent. The antioxidant results showed
that the ethanolic extracts of the L. sericeus species had higher DPPH (78.61%), ABTS (75.57%), Fe2+ chelation
(72.13%), FRAP (2.89 mg AAE/g) and hydroxyl radical scavengers (79.81%) than the ethanolic extracts of the B.
diffusa species (58.20%, 71.46%, 61.03%, 2.72 mg AAE/g, 66.58%), respectively. The leaves extracts possessed
remarkable antibacterial effects against the selected bacterial strains such as Bacillus cereus, Staphylococcus aureus,
Pseudomonas aeruginosa, Escherichia coli and S. typhimurium. For instance, the ethanolic leaves extracts showed
better inhibitory performances (2–14 mm) than the aqueous extracts (1–8 mm). Therefore, the study recorded the
best activities from the ethanolic extracts of Lonchocarpus sericeus plant. Hence, the study substantiated the fact
that the effective antibacterial performances of the ethanolic leaves extract is dependent on its high antioxidant
capacities as well as the species of plants being used.

1. Introduction
Plant and plant materials have been utilized by human beings, not
only for food but also to treat infectious diseases, since the time of their
existence. For instance, the ever-longing scientific investigations of these
plant materials have shown and proven their effective exhibition of ther-
apeutic efficacy [1]. Moreso, in today’s modern world, many countries
use plants to treat different maladies including infectious diseases of the
gastrointestinal, urinary, biliary and respiratory systems [2].
Medicinal plants, which constitute an effective source of both tradi-
tional and modern medicine, have the genuine utility with ∼80% of the
rural population dependency on them as mean of their primary health
care [3]. These ones have been used as sources of remedies in the treat-
ment of many diseases since ancient times and people of all continents
especially Africa, have this old tradition [4]. Despite the remarkable
progress in synthetic organic medicinal products of the twentieth cen-
tury, there are over 1300 medicinal plants used in Europe, out of which
90% are harvested from wild resources and ∼118 of the top 150 pre-
scription drugs are based on natural sources in the United States [5].
Furthermore, ∼80% of people in developing countries are totally de-
pendent on herbal drugs for their primary healthcare, and over 25% of
prescribed medicines in developed countries are derived from wild plant
species [6]. Statistically, the annual global market value of medicinal
plant products has exceeded $100 billion [5]. Such wild plant species
being used for the medicinal purposes are but not limited to Boerhavia
diffusa L. and Lonchocarpus sericeus (Poir.) Kunth ex DC.

∗
Corresponding author.
E-mail address: samalomo@futa.edu.ng (S.A. Malomo).

https://doi.org/10.1016/j.meafoo.2021.100018
Received 5 October 2021; Received in revised form 7 December 2021; Accepted 16 December 2021
2772-2759/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
E. Adeku, O.F. Osundahunsi, S.A. Malomo et al.
B. diffusa is a well-known medicinal plant in traditional Indian
medicine as well as other parts of world, like Southern America and
Africa. Its various parts and especially roots have been used for gas-
trointestinal, hepatoprotective, and gynecological indications in India
[7]. In Nigeria, the whole plant has been used in the treatment of threat-
ened miscarriage [1]. It belonged to the family Nyctaginaceae, which is
naturally occurring as weed and commonly known as hogweed or red
spiderling.
L. sericeus (Poir.) Kunth ex DC. is a leguminous plant that belonged
to the family Leguminosea, which is also known as Senegal lilac or Cube
root [8]. It is used in the treatment of epilepsy, laxative, to stimulate
appetite and to treat stomach disorder in Africa. It also has common
application in the treatment of convulsions and backaches [8–10].
One of the most serious global public health threats in this century
is antimicrobial resistance [11], which is due to the ongoing resistant
of bacterial strains against antibiotics, thereby leading to increase in
treatment failure ratio [12]. Several past studies [13–15] also linked
the exponential increase in bacterial infection in Nigeria to the available
poor health system, low income, expensive medication and limited data,
that contributed to the high death rates in the country.
Medicinal plants are first aid resources for a large number of African
population (∼80% of the population uses medicinal plants as remedies)
so as to treat a variety of ailments [16,17]. The growing resistance of
bacteria against antibiotic and their negative side effects demanded for
the discovery of new organic antibiotics [18,19]. However, until now,
there is still no single solvent that is regarded as standard for extracting
bioactive compounds from medicinal plants [20,21]. Hence, the present
study is aimed at evaluating chemical composition, antioxidant and an-
tibacterial potentials of B. diffusa and L. sericeous leave extracts from
two solvents (aqueous and ethanol). This would ensure production of
bioactive compounds of organic status with no health side effects while
showcasing the effects of polarity and non-polarity level of the solvents
being employed in the food processing and human health systems.
2. Materials and methods
2.1. Sample Collection and chemicals reagents
Freshly harvested Boerhavia diffusa and Lonchocarpus sericeus leaves
were obtained from Teaching and Research Farm, Federal University of
Technology, Akure Nigeria. All chemicals used were of analytical grade
and obtained from Sigma-Aldrich, London, United Kingdom.
2.2. Identification of plant leaves
The fresh leaves were collected from the farmland of the Federal
University of Technology, Akure, Nigeria in the early morning hours
by one of the authors. The plants were then identified and authenti-
cated by the Botanists of the Department of Crop, Soil and Pest Manage-
ment and Department of Forestry and Wood Technology, Federal Uni-
versity of Technology, Akure, Nigeria through their catalogues of plant
origins. The plants were then, given voucher numbers FUACFW11134
and FUACFW11176, respectively, after identification.
2.3. Source of microorganisms
Microorganisms that were used in this study, which include Bacil-
lus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli
and Salmonella typhimurium, were obtained as stock cultures from the
Department of Microbiology, Federal University of Technology, Akure,
Nigeria.
2.4. Methods
2.4.1. Sample preparation
The Boerhavia diffusa and Lonchocarpus sericeus leaves were prepared
by a modified method described by Olabinri et al. [22]. The leaves were
2
Measurement: Food 5 (2022) 100018
sorted, washed with distilled water, dried under room temperature,
milled into a fine particle size using Pyramid electrical stainless-steel
blender (PM-B999, Pyramid Electrical Coy, Beijing, China) and sieved
using 2 mm sieve.
2.4.2. Preparation of leaves extracts
The extracts from Boerhavia diffusa and Lonchocarpus sericeus was
prepared according to the previously reported method [21] by weighing
100 g of each leaf sample into two different sterile glass bottles (A and
B). Ethanol (500 mL) was added to glass bottle A and distilled water
(500 mL) was added to glass bottle B. Each bottle was tightly covered,
shaken vigorously and kept for 24 h to enhance proper dissolution of
the bioactive compounds in the samples after which it was filtered with
Whatman filter paper 125 mm (Whatman Int. Ltd., Maidstone, U.K) at
room temperature. The extracts were further concentrated, dried under
room temperature, and stored at 4 °C in a refrigerator until required for
further analysis.
2.5. Phytochemical Analysis of the leaves extracts
2.5.1. Determination of tannin content
This was done using the method of Medoua et al. [23]. Two (2 g)
of each sample extract was weighed into a 250 mL flask followed by
addition of 200 mL of 0.004 M K3 Fe(CN)6 and 10 mL of 0.008 M FeCl3
in 0.008 M HCl. The flask was allowed to stand for 20 min and stirred
occasionally at 10 min interval and 1 mL aliquot was removed. To this
aliquot, 2 mL of 0.008 M FeCl3 was added in 0.008 M HCl and 10 mL
of 0.0015 M K3 Fe(CN)6 . After adding the final reagent, the absorbance
was read at 720 nm after 30 s against a blank.
𝑇 𝑎𝑛𝑛𝑖𝑛 (𝑚𝑔∕𝑔 )
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 𝑐 𝑜𝑛𝑐 𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓 𝑎𝑐𝑡𝑜𝑟
= (2.1)
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝑠𝑎𝑚𝑝𝑙𝑒 𝑠𝑖𝑧𝑒
2.5.2. Determination of flavonoid
Total flavonoid content was determined by aluminum chloride col-
orimetric assay [24] with slight modification. About 500 𝜇L of methanol
was added to 10 mL flask containing 500 𝜇L of the leaves’ extracts. To
this 50 𝜇L, 10% AlCl3 and 50 𝜇L of 1 M CH3 COOK was added, respec-
tively. The total volume was made up to 2500 𝜇L with distilled water.
The solution was then incubated at room temperature for 30 min. The
absorbance was read against blank at 415 nm with spectrometer. The
flavonoid was calculated using quercetin as standard. The same process
was repeated for the ethanolic extracts.
( ) 𝐴𝑏𝑠.𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 𝐶𝑜𝑛𝑐 .𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (𝑚𝑔∕𝑚𝑙)
𝑇 𝑜𝑡𝑎𝑙 𝑓 𝑙𝑎𝑣𝑜𝑛𝑜𝑖𝑑 𝑐 𝑜𝑛𝑡𝑒𝑛𝑡 𝑚𝑔 𝑄𝐸∕𝑔 =
𝐴𝑏𝑠.𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝐶𝑜𝑛𝑐 .𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔∕𝑔 )
(2.2)
2.5.3. Determination of oxalate content
The method described by Ukpabi and Ejidoh [25] was used for the
determination. Two gram (2 g) of each extracts was digested with 10 mL
6 M HCl for 1 h and made up to 250 mL in a volumetric flask. The pH
of the filtrate was adjusted with concentrated NH4 OH solution until the
color of the solution changed from salmon pink color to a faint yellow
color. Thereafter, the filtrate was treated with 10 mL of 5% CaCl2 solu-
tion to precipitate the insoluble oxalate. The suspension was centrifuged
at 2500 rpm for 10 min, after which the supernatant was decanted. The
precipitate was dissolved in 10 mL of 20% (v/v) H2 SO4 and the solution
was made up to 300 mL. An aliquot (125 mL) was heated until near boil-
ing point and then titrated against 0.05 M standardized KMnO4 solution
to a faint pink color which was persisted for about 30 s after which the
burette reading was taken and used to estimate the oxalate content.
( )
𝑡𝑖𝑡𝑟𝑒 𝑣𝑎𝑙𝑢𝑒 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 KMnO4 𝑥 𝑑𝑖𝑙𝑢𝑠𝑖𝑜𝑛 𝑓 𝑎𝑐𝑡𝑜𝑟 ∕5
𝑂𝑥𝑎𝑙𝑎𝑡𝑒 (𝑚𝑔∕𝑔 ) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑠𝑖𝑧𝑒
(2.3)
E. Adeku, O.F. Osundahunsi, S.A. Malomo et al.
2.5.4. Determination of total phenol content
The total phenol content (TPC) was determined by Folin–Ciocalteu
assay [26] using gallic acid as standard. Fifty microliter of aqueous ex-
tract solution containing 0.5 mg of aqueous extract was dispensed into
a test tube and also repeated for the ethanolic extract sample, 50 𝜇L of
distilled water and 500 𝜇L of Folin–Ciocalteu reagent was added respec-
tively into the test tube and shaken thoroughly, after 3 min, 400 𝜇L of
7.5% sodium carbonate solution was added and the mixture was incu-
bated at 45 °C in a water bath for 40 min. Absorbance was measured at
765 nm against blank. The same procedure was repeated to all standard
gallic acid solution (0.1 mg/mL). The blank was a mixture of 100 𝜇L of
distilled water, 500 𝜇L of Folin-Ciocalteu reagent and 400 𝜇L of 7.5%
sodium carbonate. The total phenolic content was expressed as gallic
acid equivalent per gram of sample (mg of GAE/g sample) through the
calibration curve of gallic acid and calculated as follows;
( ) 𝐴𝑏𝑠.𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 𝐶𝑜𝑛𝑐 .𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (𝑚𝑔∕𝑚𝑙)
𝑇 𝑜𝑡𝑎𝑙 𝑝ℎ𝑒𝑛𝑜𝑙𝑖𝑐 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑚𝑔 𝐺𝐴𝐸∕𝑔 =
𝐴𝑏𝑠.𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝐶𝑜𝑛𝑐 .𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔∕𝑔 )
(2.4)
2.5.5. Determination of phytate content
The determination of phytate in sample was done using the method
described by Abulude [27]. Two (2 g) of each leave extracts samples was
dispersed in 200 mL of 2% HCl and extracted. Following extraction, the
dispersion was filtered and 50 mL of the filtrate was mixed with 10 cm3
of 0.3% ammonium cyanide (NH4 SCN) and diluted with 107 mL of dis-
tilled water. The extract was titrated against 0.00195 g/mL of Ferric
chloride solution until a brownish yellow color persisted. Phytate con-
tent was estimated with the expression
𝑃 ℎ𝑦𝑡𝑎𝑡𝑒 𝑃 ℎ𝑜𝑠𝑝ℎ𝑜𝑟𝑜𝑢𝑠 = (𝐼𝑟𝑜𝑛 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑥 1.95 𝑔 𝑜𝑓 𝑡𝑖𝑡𝑟𝑒) 𝑥 3.65 𝑔. (2.5)
2.5.6. Determination of saponin content
The method described by Obadoni and Ochuko [28] was used to de-
termine the saponin content of the leave extracts samples. Two (2 g) of
each sample was put into a conical flask and 100 mL of 20% aqueous
ethanol was added. This was heated over a hot water bath for 4 h with
continuous stirring at about 55 °C. The mixture was filtered and the
residue re-extracted with another 200 mL 20% aqueous ethanol. The
combined filtrate was concentrated to 40 mL with the water bath at
about 90 °C. The concentrate was transferred into a 250 mL separator
funnel and 20 mL of diethyl ether was added and shaken vigorously.
The aqueous layer was recovered while the ether layer was discarded.
This purification step was repeated. 60 mL of n-butanol was added, the
combined butanol extract was washed twice with 10 mL of 5% aqueous
sodium chloride. The remaining solution was heated in a water bath, af-
ter evaporation the samples were dried in the oven to a constant weight;
the saponin content was calculated in mg/g.
2.5.7. Determination of alkaloid content
The method described by Makkar et al. [29] was used to determine
the alkaloid content. Two (2 g) of each sample was weighed into a
250 mL beaker and 200 mL of 10% acetic acid in ethanol was added,
covered and allowed to stand for 48 h. After filtration, the extracts were
concentrated on a water bath to 1/4th of the original drops to the extract
until the precipitation was complete. The whole solution was collected,
washed with dilute ammonium hydroxide and then filtered. The residue
obtained was dried in an oven and weighed and the percentage of alka-
loid is expressed mathematically as
𝑤𝑒𝑖𝑔𝑡ℎ 𝑜𝑓 𝑎𝑙𝑘𝑎𝑙𝑜𝑖𝑑
% 𝐴𝑙𝑘𝑎𝑙𝑜𝑖𝑑 = 𝑥 100 (2.6)
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
2.6. Evaluation of antioxidant properties of the leaves extracts
2.6.1. Determination of 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
The scavenging effect of the samples on 2, 2- diphenyl-1-
picryhydrazyl (DPPH) free radical was measured according to the
3
Measurement: Food 5 (2022) 100018
method of Malomo et al. [30]. Appropriate dilution of the extracts
(1 mL) was mixed with 1 mL of buffer (0.1 M sodium phosphate buffer,
pH 7.0 containing 1% (w/v) Triton X-100). DPPH was dissolved in
methanol to a final concentration of 100 𝜇M. The sample (100 𝜇l) which
was mixed with 100 𝜇L of the DPPH solution in the 96-well plate to a fi-
nal assay concentration of 1 mg/mL and incubated at room temperature
in the dark for 30 min. The absorbance values of the blank, Glutathione
(GSH) (control) and samples were measured at 517 nm. The control is
made up of sodium phosphate buffer in place of the extract samples
while Glutathione (GSH) was used as the positive control. The percent
DPPH radical scavenging activity of the samples was determined using
the following equation
( )
A 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
DPPH radical scavenging act ivit y (% ) = 1 − 517 x100 (2.7)
A517 𝑜𝑓 𝑏𝑙𝑎𝑛𝑐
2.6.2. Determination of ABTS radical scavenging activity
The ABTS scavenging ability of the extracts was determined accord-
ing to the method described by Re et al. [31]. The ABTS was generated
by reacting a (7 mM) ABTS aqueous solution with K2 S2 O8 (2.45 mM/L,
final concentration) in the dark for 16 h and adjusting the absorbance at
734 nm to 0.700 with ethanol. About 0.2 mL of the appropriate dilution
of the extract was added to 2.0 mL of ABTS solution and the absorbance
was read at 732 nm after 15 min. The ABTS scavenging activity was
calculated using the following equation:
𝐴𝑏𝑠.𝑟𝑒𝑓 − 𝐴𝑏𝑠.𝑠𝑎𝑚𝑝𝑙𝑒
𝐴𝐵 𝑇 𝑆 ∗ 𝑠𝑐𝑎𝑣𝑒𝑛𝑔 𝑖𝑛𝑔 𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝑥100 (2.8)
𝐴𝑏𝑠.𝑟𝑒𝑓
TMW = Molecular Mass of Trolox (264.32 g/mol)
2.6.3. Iron (Fe2+) chelation
The metal chelating activity of the extracts was determined using
a modified method of Malomo et al. [30]. Experimental samples and
Glutathione (GSH) solution (final assay concentration of 1 mg/dL) were
combined with 0.05 mL of 2 mM FeCl2 and 1.85 mL double distilled wa-
ter in a reaction tube. Ferrozine solution (0.1 mL of 5 mM) was added
and mixed thoroughly. The mixture was allowed to stand at room tem-
perature for 10 min from which an aliquot of 200 μL was removed and
added to a clear bottom 96-well plate. A blank experiment was also con-
ducted by replacing the sample with 1 mL of double distilled water. The
absorbance of blank (Ab) and sample (As) at 562 nm was measured us-
ing a spectrophotometer and the metal chelating activity of the sample
was compared to that of GSH. The percentage chelating effect (%) was
calculated using the following equation:
( )
( ) A 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
Metal 𝐹 𝑒2+ chelat ing act ivit y (% ) = 1 − 517 X 100 (2.9)
A517 𝑜𝑓 𝑏𝑙𝑎𝑛𝑐𝑘
2.6.4. Determination of ferric reducing antioxidant potential (FRAP)
The ferric reducing power of the leave extracts was determined ac-
cording to the modified method of Malomo et al. [30]. Experimental
sample and Glutathione (GSH) was dissolved in 0.2 M phosphate buffer,
pH 6.6; an aliquot (250 𝜇L) was mixed with 250 𝜇L of the buffer and
250 𝜇L of 1% potassium ferricyanide solution. The mixture was thor-
oughly mixed using a vortex machine and heated at 50 °C for 20 min.
After incubation, 250 𝜇L of 10% trichloroacetic acid (TCA) was added
followed by 50 𝜇L of 0.1% ferric chloride dissolved in double distilled
water and then 200 𝜇L of double distilled water was added. The solu-
tion was allowed to stand for 10 min at room temperature, after which
it was centrifuged at 1000 × g for 10 min. An aliquot (200 𝜇L) of the
supernatant was transferred to a clear bottom 96-well plate and the ab-
sorbance was measured at 700 nm.
2.6.5. Hydroxyl radical scavenging
The hydroxyl radical scavenging activity of the samples was deter-
mined according to the method reported by Malomo et al. [30]. Exper-
imental samples, Glutathione and 1, 10-phenanthroline (3 mM) were
E. Adeku, O.F. Osundahunsi, S.A. Malomo et al.
Table 1
Phytochemical constituent of leaves extracts using aqueous and ethanolic solvent.
Samples Flavonoid (%) Alkaloid (%) Oxalate (mg/g)
a a c a
ELS 21.05 ± 0.05 8.00 ± 0.04 1.98 ± 0.01
EBD 14.14 ± 0.03b 7.00 ± 0.02b 3.51 ± 0.03a
ALS 12.00 ± 0.01c 2.00 ± 0.01c 3.15 ± 0.03a
ABD 7.11 ± 0.01d 2.00 ± 0.01c 2.97 ± 0.02b
Means within the same column with different superscripts are significantly different at p ≤ 0.05.
Keys: ELS (ethanolic extracts of L. sericeus), ALS (aqueous extracts of L. sericeus), EBD (ethanolic extracts of B. diffusa) and ABD (aqueous extracts
of B. diffusa).
each separately dissolved in 0.1 M phosphate buffer (pH 7.4) while
FeSO4 (3.0 mM) and 0.01% hydrogen peroxide were dissolved sepa-
rately in distilled water. An aliquot (50 μL) of samples or GSH (equiv-
alent to a final assay concentration of 1 mg/mL) or buffer (blank) was
first be added to a clear, flat bottom 96-well plate followed by 50 μL of 1,
10-phenanthroline and then 50 μL of FeSO4 . To initiate the Fenton reac-
tion in the wells, 50 μL of hydrogen peroxide was added to the mixture,
covered and incubated at 37 °C for 1 h with shaking. The absorbance
was measured using a spectrophotometer at 536 nm at 10 min intervals
for 1 h. The hydroxyl radical scavenging activity was calculated using
the reaction rate (DA/min) equation below
( ( ) )
(ΔA536 ∕min)𝑏 − (ΔA536 ∕min s
OH radical scavenging act ivit y (% ) =
(ΔA536 ∕min)b
2.7. Antibacterial assay
Antibacterial activity of the leave extracts was carried out on the
bacterial isolated strains (such as Bacillus cereus, Staphylococcus aureus,
Pseudomonas aeruginosa, E. coli and S. typhimurium) using agar well dif-
fusion method previously described by Oloyede et al. [32]. The bacterial
isolates were cultured for 16–18 h in Muller Hinton broth at 37 °C. The
culture plates were inoculated with the different selected strains of test
organisms using streak plate method. These plates were left for 30 min
for the test and typed organisms to be well fixed to the agar plates.
Sterilized 6 mm cork borer was used to bore wells in the media. The
concentrated plant extracts were reconstituted using 30% v/v Dimethyl
sulfoxide (DMSO), which was dispensed into the well. 250 mg/ml of the
extracts was dispensed into the agar wells 30% aqueous DMSO was used
as the negative control and the plates will be incubated at 37 ºC ± 2 ºC
for 24 h. The sensitivity of the test plates was observed for the zone clear-
ance (area without growth) around the wells after 24 h of incubation.
The zone of inhibition was calculated by measuring the diameter of the
inhibition zone around the well (in mm) including the well diameter.
2.8. Statistical analysis
Data of phytochemical quantification and antioxidant assays were
carried out in triplicates while the Statistical Package for Social Sciences
(SPSS) version 21.0 for Windows was used for the statistical analyses.
The results were presented as mean (±SD), and statistical difference be-
tween the means was determined using one-way analysis of variance
(ANOVA). Duncan’s Multiple Range Test (DMRT) was used to separate
the means at p ≤ 0.05 significant difference level.
3. Results and discussion
3.1. Phytochemical properties of the leaves extracts
The level of phytochemicals constituents in the leave extracts were
presented in Table 1. The extraction of bioactive components from plant
materials have been the first important step during any phytochemistry
analysis of food [33] . For instance, the result confirmed the presence of
flavonoids, which ranged from 7.11% in aqueous extracts of Boerhavia
Measurement: Food 5 (2022) 100018
Saponin (%) Tannin (mg/100 g) Total phenol (mg/100 g) Phytate (mg/100 g)
b a
16.00 ± 0.04 18.10 ± 0.06 18.26 ± 0.06 282.21 ± 0.07b
14.00 ± 0.03b 20.20 ± 0.07a 11.86 ± 0.04b 225.64 ± 0.05c
5.00 ± 0.02c 13.19 ± 0.05c 9.34 ± 0.02c 394.88 ± 0.09a
3.00 ± 0.02d 9.70 ± 0.03d 7.36 ± 0.01d 282.21 ± 0.07bc
diffusa (ABD) to 21.05% in ethanolic extracts of Lonchocarpus sericeus
(ELS). The alkaloids contents were given as 2.00% in both aqueous ex-
tracts of the plant leaves, ALS and ABD as well as 8.00% in ELS, re-
spectively. The oxalate and tannin contents of the extracts ranged from
1.98 mg/g in ELS to 3.51 mg/g in EBD and 9.70 mg/100 g in ABD to
20.20 mg/100 g in EBD, respectively.
Notably, the phytochemical constituents of the leave extracts re-
vealed much extraction (leaching out) of the phytochemicals upon ap-
plication of ethanol solvents than the aqueous solvents. This could be as
a result of the level of polarity of the solvents used during this extraction
process since alcohol is less polar than water hence more bioactive com-
ponents are extracted from the leaves than in aqueous solvents [34,35].
For instance, phytochemicals, especially the polyphenols, are more sol-
x100
uble in less polar compounds than water. This scientific phenomenal
(2.10) of polarity of solvents to play a key role in increasing phenolic solu-
bility of plant materials have been widely reported [36]. The presence
of total phenol was also confirmed in the extracts which ranged from
7.36 mg/100 g in ABD to 18.26 mg/100 g in ELS as well as the phytate
constituent that ranged from 225 mg/100 g in EBD to 394.88 mg/100 g
in ALS, respectively.
The present result also agreed with past findings on these phyto-
chemicals in the two plants as reported by other researchers. For in-
stance, the total phenol contents of 101.09 mg/100 g was reported
for Lonchocarpus sericeus ethanolic leaves extracts [37]. Meanwhile, the
total phenol contents of ∼37, ∼193 and ∼163 mg/ 100 g were re-
ported for the Boerhavia diffusa aerial part after extraction with hexane,
ethyl acetate and methanol solvents, respectively [38]. However, Kaur
[7] confirmed the presence of flavonoids, alkaloids, saponins, steroids
but no tannins in the root powders of Boerhavia diffusa after extrac-
tion with chloroform, ethanol and aqeous solvents, respectively. Fur-
thermore, Muthulingam and Chaithanya [39] also confirmed the pres-
ence of flavonoids, phenol, alkaloids, tannins and saponins in methano-
lic extract of Boerhavia diffusa leaves. In the same vein, Addotey et al.
[40] reported the significance presence of tannins, flavonoids, saponins
and alkaloids in the leaves, barks and roots of Lonchocarpus sericeus.
The current finding disagreed with the previous report [7] that rated
plant leaves to be abundant in phytochemicals than plant’ roots and stem
bark, due to their probable much exposure to stressors. For instance,
higher alkaloids (16.94 mg/g), saponins (16.90 mg/g) and flavonoids
(21.14 mg/g) were reported for ethanolic extracts of Solanum torvum
immature fruits [11] when compared to the present results (2–8, 3–16
and 7–21 mg/ 100 g), respectively. Moreso, the fact that the composi-
tions of these compounds were dependent on the different locations of
the plant body have been fully established in this study. However, the
amount of these compounds in the plant body was insignificantly re-
lated to their bioactivity. This is because the present study still reported
high antioxidant (Figs 1–5) and antibacterial (Table 2) activities even
with low amount of the phytochemicals obtained in the leaves’ extracts
when compared to the matured fruits [11].
3.2. Antioxidant properties of the leaves extracts
The antioxidant properties exhibited by the leaves extract samples
is shown in Figs. 1–5. The ability of the extract to scavenge free rad-
icals against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay is presented
4
E. Adeku, O.F. Osundahunsi, S.A. Malomo et al. Measurement: Food 5 (2022) 100018

Table 2
Antibacterial properties of the leaves extracts.

Samples Staphylococcus aureus Pseudomonas aeruginosa Bacillus cereus S. typhimurium E. coli

ELS 7 (mm) 14 (mm) 3 (mm) 3 (mm) 2 (mm)

EBD 4 (mm) 8 (mm) —— 12 (mm) 7 (mm)
ALS 5 (mm) 7 (mm) 2 (mm) —— 1(mm)
ABD 1 (mm) 8 (mm) ——– 6 (mm) 4 (mm)

Keys: ELS (ethanolic extracts of L. sericeus), ALS (aqueous extracts of L. sericeus), EBD (ethanolic extracts
of B. diffusa) and ABD (aqueous extracts of B. diffusa).

Fig. 1. DPPH Radical Scavenging Activities of the Leaves Extracts
Means within the same column with different superscripts are significantly dif-
ferent at p ≤ 0.05
Keys: ELS (ethanolic extracts of L. sericeus), ALS (aqueous extracts of L. sericeus),
EBD (ethanolic extracts of B. diffusa) and ABD (aqueous extracts of B. diffusa).
Fig. 3. Fe2± Chelation activities of the leaves extracts
means within the same column with different superscripts are significantly dif-
ferent at p ≤ 0.05
Keys: ELS (ethanolic extracts of L. sericeus), ALS (aqueous extracts of L. sericeus),
EBD (ethanolic extracts of B. diffusa) and ABD (aqueous extracts of B. diffusa).

Fig. 2. ABTS Radical Scavenging Activities of the Leaves Extracts
Means within the same column with different superscripts are significantly dif-
ferent at p ≤ 0.05
Keys: ELS (ethanolic extracts of L. sericeus), ALS (aqueous extracts of L. sericeus),
EBD (ethanolic extracts of B. diffusa) and ABD (aqueous extracts of B. diffusa).
in Fig. 1. The result ranged from 51.05% in ABD to 78.61% in ELS ex-
tracts, respectively. The result showed invariable dependency between
plant species/materials and the DPPH scavenging power. For instance,
the ELS and ALS extract samples had significant (P < 0.05) higher DPPH
scavenging capacities (78.61 and 70.88%) than the EBD and ABD (58.20
and 51.05%) extract samples, respectively. Past study on the ethanolic
and aqueous extracts of the dried root powder of B. diffusa showed them
as good DPPH free radical scavengers as well as being a good ferric re-
ducer (Shisode and Kareppa, 2011). The ability of the ethanol extracts
having the strongest radical scavenging ability is in agreement with the
present study on the higher ability of ethanol extracts EBD than the
aqeous extracts ABD. However, the present result (51–79%) is higher
than 15–56% reported for B. diffusa root powder [41] but similar to
78% reported for the B. diffusa leaves [42] aqueous and ethanolic ex-
tracts, respectively. Moreso, another study [40] reported low DPPH abil-
ity (65%) for Lonchocharpus sericeus leaves ethanolic extracts than the
present findings (79%). The differences in the DPPH abilities of these
extracts could be attributed to differences in the plant sources, plant
parts and solvents being used. For instance, past work had reported the
embedment of more polyphenols in the plant leaves than in the roots
[43].
The ability of the leaf extracts samples to scavenge free radical
against 2, 2-Azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)
assay is presented in Fig. 2. The ABTS free radical scavenging ability of
the leaf extracts showed ELS and ABD samples as the highest (75.57%)
and least (66.73%) free radical scavengers, respectively. The result pre-
sented in Fig. 2 also followed the same trend with those reported for
DPPH radical scavengers (Fig. 1), because both aqueous and ethanolic
extracts from L. sericeus had the significant (P < 0.05) highest (69.44–
75.57%) when compared to those extracts (66.73–71.46%) from B. dif-
fusa plants, respectively. A previous study reported a corresponding
ABTS value (∼72%) for the plants’ ethanolic extracts [40]. The little
difference in the outcome of this present study and past findings could
be attributed to different genetic constituents of the plants, location of
harvesting, maturity stage and method of extraction as previously high-
lighted [44]. The mechanism of phytochemicals as ABTS free radical

5
E. Adeku, O.F. Osundahunsi, S.A. Malomo et al.
Fig. 4. Ferric reducing antioxidant power (FRAP) Activities of the leaves ex-
tracts
means within the same column with different superscripts are significantly dif-
ferent at p ≤ 0.05
Keys: ELS (ethanolic extracts of L. sericeus), ALS (aqueous extracts of L. sericeus),
EBD (ethanolic extracts of B. diffusa) and ABD (aqueous extracts of B. diffusa).
Fig. 5. Hydroxyl radical scavenging activities of the leaves extracts
means within the same column with different superscripts are significantly dif-
ferent at p ≤ 0.05
Keys: ELS (ethanolic extracts of L. sericeus), ALS (aqueous extracts of L. sericeus),
EBD (ethanolic extracts of B. diffusa) and ABD (aqueous extracts of B. diffusa).
scavengers included the donation of electron to the unstable compounds
so as to make the free radicals stable.
The Fe2+ chelation ability of the leaf extracts was obtained in the
ranges of 48–72% with the ELS and ABD samples showed the highest
(72.13%) and least (48.00%) chelating ability, respectively (Fig. 3). The
ethanolic extract samples, as usual with the DPPH and ABTS radicals
scavenging activities, had the significant (P < 0.05) higher metal chela-
tion activities than the aqueous extracts. Earlier studies [45,46] reported
the values of 81 and 76.21% for the Fe2+ chelating ability of Boerhavia
diffusa methanolic leaves extracts, respectively, which were higher than
the present result.
The ferric reducing antioxidant power of the extracts showed no sig-
nificant (P > 0.05) difference as it ranged from (2.66–2.89 mg AAE/g)
with highest and least power obtained with those extracted with ethanol
and aqueous, respectively (Fig. 4). The FRAP value of this study for EBD
samples were lower than 19.48, 14.67 and 11.67 μg trolox/100 μm re-
6
Measurement: Food 5 (2022) 100018
ported [47] for ethanolic, chloroform and petroleum ether solvent ex-
tracts, respectively. Phytochemicals have been found to suppress reac-
tive oxygen species formation by inhibiting the enzymatic processes in-
volved in their production, through chelation and reduction of metal
ions, also known as the process co-factors [48,49].
The results of the hydroxyl radical scavenging capabilities of the
extracts (Fig. 5) also tended towards ethanolic extracts of the plants
species, ELS showing the significant (P < 0.05) highest activity (79.81%)
when compared to EBD (66.58%), ALS (59.46%) and the ABD (55.64%).
The result of hydroxyl radical scavenging capabilities of EBD extract
obtained in this study was similar to 63.72 [39] but lower than 83.7%
[45] reported for Boerhavia diffusa methanolic leaf extracts, respectively.
However, this result was higher than 43% reported by another study for
Boerhavia diffusa aqueous acetone extract [44]. The differences observed
in the various studies being reported might be due to differences in the
experimental model, type of solvents used and/or plant species [50].
Notably, the oxidative stress, arose as a result of an imbalance between
free radical production and antioxidant defenses, and these radicals are
associated with damage to a wide range of molecular species including
lipids, proteins, and nucleic acids [51,52]. Therefore, when the body
is under a lot of stress and during infection, the production of reac-
tive oxygen species thus, overwhelmed the primary endogenous antiox-
idants, which invariably cause health problems [53]. Hence, the medic-
inal plants, such as the ones used in this study, are natural sources of
several phytochemicals and polyphenols that possess these antioxidant
activities, free from side effects and less expensive [54].
3.3. Antibacterial properties of the leaves extracts
The antibacterial activities of the leaf extracts, after testing them
against some selected bacteria, is shown in Table 2. The zone of inhibi-
tion of the leaf extract against Staphylococcus aureus, ranged from 1 mm
in ABD extract to 7 mm in ELS but with measurement of EBD and ALS
extracts at 4 mm and 5 mm, respectively. Meanwhile, the zone of inhi-
bition against Pseudomonas aeruginosa and Bacillus cereus ranged from
7 to 14 mm and 2–3 mm, respectively. However, no zone of inhibition
against B. cereus was observed in both the EBD and ABD. The zone of
inhibition against S. typhimurium are 12 mm in EBD and 3 mm in ELS.
Although, there exists no observable inhibition in ALS but a measure-
ment of 6 mm was obtained for the ABD. Similarly, the zone of inhibition
against E. coli was given as 1 mm in ALS and 7 mm in EBD while the
values of ELS was measured at 2 mm and ABD at 4 mm, respectively.
The antibacterial activities of the ELS in this study agreed to the
previous study [40] that reported similar values for the leaves of Lon-
chocharpus sericeus after ethanolic extractions. The past study showed
the zone of inhibition in the leaves against E. coli. (3 mm), Staphylococcus
aureus (9 mm), Bacillus cereus (11 mm), respectively. Another previous
study revealed the antibacterial activities of B. diffusa aerial parts using
methanol as extract against the activity of Staphylococcus aureus (9 mm).
The difference in variation could be probably attributed to variation of
collection place and time and the extraction process of the plant [38].
Different authors have reported the mechanism of polyphenols in
their role as antibacterial agents. For instance, Singh et al. [55] and
Majdanik et al. [56] reported that polyphenols found in the plants
worked against both gram-negative and gram-positive bacteria through
the breakdown of the cell wall and membranes of microorganisms,
which led to the release of cellular content, protein binding domain dis-
ruption, enzyme inactivation and ultimately death of cell.
4. Conclusion
The phytochemicals quantification of the leaves extracts undergone
in this study revealed more phytochemicals present in the ethanolic ex-
tract of the leaves than the aqueous extract, which might be due to po-
larity of the solvent and the solute. This is because phytochemicals, es-
pecially the polyphenols, are more soluble in less polar solvents than
E. Adeku, O.F. Osundahunsi, S.A. Malomo et al.
water. The findings further revealed the dependency of phytochemical
levels and the type/species of plants involved, wherein the leaves of
Lonchocarpus sericeus plant had higher phytochemicals than Boerhavia
diffusa. Moreover, the aqueous and ethanolic leaves extracts of the two
plants have good antioxidant properties, as well as antibacterial proper-
ties with the ethanol extracts performing slightly better than the aqueous
extracts. However, the Lonchocarpus sericeus plant recorded better an-
tioxidant and antibacterial properties than the Boerhavia diffusa plant.
The study also provided novel information on the property of extrac-
tion solvent (e.g. polarity, extractive power, etc.), to obtain the targeted
bioactive compounds. Therefore, the inclusion of these plants in the food
industry could help to reduce body metabolic inflammations, prevent
lipid oxidation-related food spoilage and serve as potential agents to
give functional benefits in food and human health applications.
Abbreviations: ABD = aqueous boerhavia diffusa; ALS = aqueous
lonchocarpus sericeus; BD = boerhavia diffusa; LS = lonchocarpus sericeus;
EBD = ethanolic boerhavia diffusa; ELS = ethanolic lonchocarpus sericeus;
DPPH = 2, 2-diphenyl-1-picrylhydrazyl (DPPH); FRAP = ferric re-
ducing antioxidant power; GAE = gallic acid equivalents; HCl = hy-
drochloric acid; HPLC = high performance liquid chromatography;
TCA = trichloroacetic acid
Funding
N/A.
Availability of data and materials
Data are available upon request by contacting the authors.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Emmanuel Adeku: Visualization, Formal analysis, Data curation,
Writing – original draft, Writing – review & editing. Oluwatooyin F.
Osundahunsi: Visualization, Supervision, Writing – review & editing.
Sunday A. Malomo: Formal analysis, Data curation, Writing – review
& editing. Idowu I. Asasile: Visualization, Writing – review & edit-
ing. Olajumoke M. Owolabi: Visualization, Writing – review & editing.
Ganiyat Oyewole: Visualization, Writing – review & editing.
Acknowledgment
We acknowledge the support of the laboratory staff of the Depart-
ments of Food Science and Technology, Biochemistry and Microbiology
of The Federal University of Technology, Akure, Nigeria.
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