Nisebj 22 1 2022

Volume 22, Numbers 1/2 ISSN 1595-6938
March/June, 2022
NISEB JOURNAL
Journal of Society for Experimental

Biology of Nigeria
KLOBEX
NISEB Journal
Journal of the Society for Experimental Biology of Nigeria
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Prof. C.C. Osubor. Dept of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City
EDITOR
Dr. A.M. Petu Ibikunle. School of Science & Technology, National Open University of Nigeria, Lagos
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Prof. (Mrs.) Joy Okpuzor, Department of Cell Biology & Genetics, University of Lagos, Lagos, Nigeria
Dr. S.M.C. Ezeukwu, Microbiology Division, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria
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Nigeria
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NISEB JOURNAL
Volume 22, Numbers 1/2 Contents March/June, 2022
Proximate and Phytochemical Analyses of Vinca minor L Leaf Extracts 1

Osibote, Elizabeth Adejoke, Abiose, Modupeola Mopelola and Olasupo, Idris Abiodun
Antioxidant Activities of Leaf Extracts of Vinca minor L

Osibote, Elizabeth Adejoke, Abiose, Modupeola Mopelola and Olasupo, Idris Abiodun 10
Effect of pH Modifications of PCB-Contaminated Soil on Growth and Leaf Characteristics of

Maize (Zea mays) Plant 14
Oyeyemi Adeyemi and Olalekan Adeyemi
Hibiscus Sabdariffa L. Calyx Ethanol Extract Ameliorates Thioacetamide-Induced Hepatic

Oxidative Stress in Male Wistar Rats 25
Ebhohon, Shirley O., Asoya, Ekene V. and Iyare, Harrison E.
Comparative Studies on the Phytochemical Content and In Vitro Antioxidant Capacity of 34

Methanol Extract of Spondias mombin and Chasmanthera dependens
Omorede Ikponmwosa-Eweka1 and Ehimwenma Sheena Omoregie
Changes in Some Biochemical Parameters in Red Meat Tendered with Paracetamol and 41
Extract of Ocimum basilicum
Ugbeni, O.C. and Uanseoje, S. O.
iv
Osibote et. al.
NISEB Journal Vol. 22, Nos. 1/2. March/June, 2022 1595-6938/2022

Printed in Nigeria (2022) Society for Experimental Biology of Nigeria
http://www.ojs.klobexjournals.com/index.php/nisebj
Proximate and Phytochemical Analyses of Vinca minor L Leaf Extracts
Osibote, Elizabeth Adejoke*, Abiose, Modupeola Mopelola and Olasupo, Idris Abiodun
Department of Chemistry, Faculty of Science, University of Lagos, Akoka, Nigeria
Abstract
Tropical plants are used to manage various ailments, Vinca minor L. is an herb used for antimicrobial,
leukamia, diabetes, depression, inflammation and cancer. Extraction was carried out on the pulverized leaves
with 50 % methanol/water. Serial solvent partitioning was done on aqueous mixture of crude extract with n-
hexane, ethyl acetate and butanol according to polarity of constituents. Thin layer chromatography was carried
out to further purify the fractions with column chromatography and analyse with FTIR. Phytochemical and
proximate analyses were carried out. Three components were obtained from n-hexane, four from ethyl acetate
and one from butanol fraction. The nutritional content of the leaves from proximate analysis are 7.23 %
moisture, 7.32 % ash, 5.05 % fat, 9.07 % fibre, 4.65 % protein and 66.70 % carbohydrate. Phytochemicals
showed saponins (19.13 mg/100 g), reducing sugars (42.85 mg/100 g), cardiac glycoside (9.16 mg/100 g),
steroids (20.05 mg/100 g), phenolic compounds (27.33 mg/100 g), flavonoids (46.92 mg/100 g) and tannins
(12.39 mg/100 g). The functional groups identified with FTIR were consistent with the metabolites obtained
from phytochemical screening and proximate analysis. The presence of high phenolics may support the use of
the plant as a herb for antimicrobial agent. The results are scientific indications of potentials of extracts from V.
minor for synthesis of potent drugs against microorganisms.
Keywords: Antimicrobial agents, Ethyl acetate fraction, phytochemicals, V. minor (Apocynaceae)
Introduction
The natural products often used as starting points for drug discovery can be divided into two major classes,
primary and secondary metabolites [1]. Many natural products can serve as chemical models or templates for the
design, synthesis and semi synthesis of novel substances for treating human diseases. Although there are some
new approaches to drug discovery, such as combinatorial chemistry and computer-based molecular modeling
design [2]. Secondary metabolites are typically organic compounds produced through the modification of
primary metabolite synthases. Many of the identified secondary metabolites have a role in three ecological
function, including defense mechanism(s), serving as antibiotics and producing pigments. Examples of
secondary metabolites with importance in industrial microbiology include atropine, a secondary metabolite with
important use in the clinic and antibiotics such as erythromycin and bacitracin are also considered to be
secondary metabolites. Bacitracin, derived from organisms classified under Bacillus subtilis, is an antibiotic
commonly used as topical antibiotic, in the clinic [3].
Secondary metabolites serve as competitive weapons used against other bacteria, fungi, amoebae, plants, insects
and large animals. They are also used as metal transporting agents and as agents of symbiosis between microbes,
plants, nematodes, insects and higher animals. Although antibiotics are not obligatory for sporulation, some
secondary metabolites used as differentiation effectors (including antibiotics) stimulate spore formation and
inhibit or stimulate germination [4].
V. minor is widely used in traditional medicine in Africa and Asia, gathered from the wild and also widely
cultivated. Recent discovery of vincristine, an alkaloid in the plant has led to its commercial cultivation,
especially in Spain, China and the USA, since the compound has proved itself useful in the treatment of
leukaemia. A popular garden ornamental, it is grown as a perennial in the tropics, as an annual in temperate
regions, it can be planted as a pot plant in a conservatory. It is valued for its bushy habit and many large flowers
carried above dark green foliage. It can also be kept as a cut flower; the branches last for weeks, producing new,
smaller, flowers all the time. It exhibited strong antimicrobial activity against Gram-positive bacteria in acetone
and antifungal effect in ethyl acetate extracts of V. minor L. collected in Balkan Mountains (Dinaric Alps,
Serbia). The plant has antibiofilm, antioxidant and antimicrobial activity, total phenolic content [5]. Vinca minor
has been listed among Vinca species that synthesize natural products with effective antibacterial, antifungal,
antiparasitic and antitumor activities, it is listed in The United States Department of Agriculture [6,7].
Materials and Methods

Extraction of the plant materials
Vinca minor l was collected from Ota in Ogun State in September 2021, identified and authenticated at the
Herbarium in Botany Department, University of Lagos. The voucher number LUH 8940 was assigned. The leaf
was dried; 225 g of pulverized plant was extracted by maceration with one litre of 50 % methanol-water (ratio of
plant to solvent being 1:4) for 72 hours with frequent agitations in conical flask. The conical flask containing the
Corresponding Author’s Email: eosibote@unilag.edu.ng
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Osibote et. al.
mixture was made air-tight by covering with foil paper; mixture was filtered with a sieve of fine-sized mesh
after 72 hours and re-soaked for another 24 hours for thorough extraction. The two extracts obtained were
pooled together and concentrated by rotary evaporator at 40 oC. The concentrated crude extract was freeze dried,
35.5 g was obtained and had a brown colour. Procedure was carried out as in the previous work [8,9].
Serial Solvent Partitioning [8,9]
Aqueous mixture of the crude extract was obtained by dissolving in water in ratio of extract to water being 1:1.
The mixture was then partitioned between the aqueous and organic solvents n-hexane, ethyl acetate and butanol.
Non polar n-hexane was used first for the separation followed by slightly polar ethyl acetate and highly polar
butanol, was used last on the same mixture. About 35 ml of water was used to dissolve 35.5 g of crude extract
and the partitioning was done by shaking the aqueous mixture with 30 ml of n-hexane four (4) times in a
separating funnel to remove the non-polar compounds. The mixture of aqueous and n-hexane was left to stand
for few minutes each till two distinct layers were formed in the separating funnel with the n-hexane fraction at
the top while aqueous fraction was below. The n-hexane fraction was collected carefully. The colour of n-
hexane fraction was light brown.
The ethyl acetate fraction was obtained next by shaking the remaining aqueous solution with 30 ml of ethyl
acetate four (4) times in a separating funnel to remove the slightly polar compounds. The same procedure as in
n-hexane fraction was carried out. The ethyl acetate fraction was collected and the colour was dark brown.
The butanol fraction was obtained by shaking the remaining aqueous solution with 30 ml butanol fraction four
(4 times) in the separating funnel then left to settle till two distinct layers were seen each time and butanol
fraction was collected at bottom. The colour was dark brown. The fractions were evaporated to dryness by air
drying the n-hexane and ethyl acetate fraction and concentrated the butanol fraction by rotary evaporation.
Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out on each fraction to obtain different components. UV lamp
with wavelength in the 254 nm region was used to identify components on the TLC plates. Various solvent
systems were used until the one that was able to separate the fractions in the extract was resolved. For the n-
hexane fraction, the solvent system (H for hexane, E for ethyl acetate) was H: E 2:3, giving three (3)
components (Rf 0.42, 0.78 and 0.92).
For the ethyl acetate fraction, the solvent system (M for methanol, E for ethyl acetate, H for hexane) was M: E:
H 2:2:1, giving two (2) components (Rf 0.65 and 0.88).
For the butanol fraction, the solvent system (E for ethanol for chloroform B butanol) E:C: B 0.1:2.5:2.5 was
established, giving two (2) components (Rf 0.76 and 0.88)
Column Chromatography
A column with 10 mm radius was used for the separation. Cotton wool was inserted down the bottom to ensure
no air bubble is trapped to prevent the slurry running out of column. 30 g of silica gel 60 - 120 mesh size by
Merck was mixed with the appropriate solvent system determined by analytical TLC to make a homogenous
slurry. The cotton wool was soaked with the solvent system and the slurry was poured into the column.
Separation of n- Hexane fraction: After the column had been packed, 0.10 g of this fraction was blended with
1 g of silica gel and introduced to the topmost layer of packed column. The mobile phase was prepared using the
solvent system resolved by TLC and was introduced into the column by the side of the wall carefully. The
solvent system was allowed to run through the column and more solvent system mixture was added
intermittently making sure that the column never runs dry. Components eluted were collected into twenty (20)
labelled test-tubes, TLC was carried out on each component to batch before it was evaporated. Three
components were obtained from this fraction (Rf: 0.72, 0.48 and 0.21).
Separation of Ethyl Acetate fraction: After the column had been packed, 0.32 g of this fraction was blended
with 3.2 g of silica gel and was introduced to the topmost layer of the packed column. The mobile phase was
prepared using the solvent system resolved by TLC and was introduced into the column by the side of the wall
carefully. The column was allowed to run and more solvent system mixture was added intermittently making
sure that the column never runs dry. Components eluted were collected into 25 labelled test-tubes, TLC was
carried out on each component to batch before it is evaporated. Four components were obtained from this
fraction (Rf: 0.13, 0.21, 0.38 and 0.80).
Separation of Butanol fraction: After the column had been packed, 0.24 g of this fraction was blended with 2.5
g of silica gel and was introduced to the topmost layer of the packed column. The mobile phase was prepared
using the solvent system resolved by TLC and was introduced into the column by the side of the wall carefully.
The column was allowed to run and more solvent system mixture was added intermittently making sure that the
column never runs dry. Components eluted were collected into 20 labelled test-tubes, TLC was carried out on
each component to batch before it is evaporated. One component obtained from this fraction (Rf :0.51)
Phytochemical Screening
Phytochemical screening of the secondary metabolites in the plant was carried out. Some of these
phytochemicals are flavonoids, alkaloids, phytosterols, terpenoids, tannins, antioxidants, phenolic compounds.
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Osibote et. al.
Proximate composition
The moisture content, total ash, and crude protein of Vinca minor were measured and calculated using the
standard methods [10,11,12]. The crude protein content was calculated by multiplying the nitrogen content as
determined by standard procedures [10] with the total nitrogen multiplied by a conversion factor of 6.25 (%
Protein = % nitrogen x 6.25, where 6.25 is the protein-nitrogen conversion factor for fish and fish byproducts).
Qualitative and Quantitative Phytochemical Analyses
The preliminary phytochemical procedure used in a previous study was followed to identify the phytochemicals
and supported by other standard procedures [13,14,15,16]. The qualitative and quantitative estimation of each
phytochemical was done according to the standard protocols [15]. Total content of terpenoids, tannins, phenolic
compounds, flavonoids, alkaloids, phlobatannins, cardiac glycosides, steroids and reducing sugars was assessed
according to the standard procedures [14,16,17] and as reported [18]. The estimation of alkaloid was done by
alkaline precipitation-gravimetric method [16]; total phenolic compound content was estimated using the Folin
Ciocalteau reagent [19,20] and total flavonoid determined where the content was expressed as Rutin equivalents
(mg Rutin Equivalents /g extract [20]. Reducing sugars, steroids, saponin, tannins, and cardiac glycosides were
determined [21,22,23]. The results are presented in (Tables 4, 5, 6).
e.g; Total phenolic content in extract were reported as mg equivalents of gallic acid / 100 g of dry sample).
Total flavonoid content in extract expressed as mg equivalent of rutin / 100 g of dry sample.
Total steroid content in extract (mg equivalent of cholesterol / 100 g of dry sample.
Total reducing sugars content in extract expressed as mg equivalent of glucose / 100 g of sample.
Total tannin content in extract (mg equivalent of tannic acid / 100 g of dry sample.
Total cardiac glycosides content in extract expressed as (mg equivalent of Digoxin / 100 g of dry sample.
Results
The masses of wet, dried and moisture content of 50 % methanol/water extracts and the colours of the plant,
extracts and fractions are presented in table 1.
Table 1: Masses of wet, dried and moisture content of 50 % methanol/water extracts of
Vinca minor
Plant materials V. minor

Mass of wet plants materials (g 390.2
Mass of dry plants materials (g) 260.6
Amount of water lost (g) 109.6
% Moisture content 28.09
Weight of sample after pulverizing(g) 225.4
Colour of the pulverized plant Light brown
Colour of crude extract Brown
Colour of n-Hexane fraction Dark brown
Colour of Ethyl acetate fraction Light brown
Colour of Butanol fraction Light brown
Percentage moisture content is (28.09 %)
Yield of Extracts
The yield of the 50 % methanol/water (crude) and other extracts obtained from the n-hexane, ethyl acetate and
butanol fractions of V. minor after the serial solvent partitioning are presented in Table 2
Table 2: The yield of crude extract and fractions

Plant V. minor
Mass of the dried 50 % MeOH/H2O extract (g) 35
Volume of water used to dissolved (ml) 35
Volume of hexane used (ml) 30x4
Mass of hexane fraction (g) 0.084
% Yield of hexane fraction 0.24
Volume of ethyl acetate (ml) 30x4
Mass of ethyl acetate fraction (g) 0.322
% Yield of ethyl acetate fraction 0.92
Volume of butanol used 30x4
Mass of butanol fraction(g) 0.235
% Yield of butanol fraction 0.67
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Osibote et. al.
Column Chromatography
The chromatograms of the fractions after TLC are presented in Figure 1.
Three components with Rf values of 0.78, 0.48 and 0.21 respectively were eluted from n-hexane fraction, three
components with Rf values of 0.21, 0.38 and 0.80 respectively were eluted from ethyl acetate fraction and one
component with Rf value of 0.50 eluted from butanol fraction of V. minor .
Rf = 0.78 Rf = 0.80
Rf = 0.50
Rf = 0.48
Rf = 0.38
Rf = 0.21 Rf = 0.21
Rf = 0.00 Rf = 0.00
HVM EVM BVM
Figure 1: TLC chromatograms of fractions
Retention Factor, Rf of column chromatography of test-tubes batched, components obtained from separation for
n-hexane, ethyl acetate and butanol fractions of V. minor, the solvent system used and the colours of the
components are presented in Table 3.
Table 3: The components and Rf values of the components from n-hexane, ethyl acetate and butanol
fractions
Number of Test-tube Retention TLC result Solvent system Colour of
test-tubes S/N Factor used component
batched
n-Hexane fraction
3 1-3 - No spot H:E 2:3
2 3-4 0.72 Single spot Dark brown
2 5-6 - No spot
1 10 - No spot
7 14-20 - No spot
Ethyl acetate fraction
4 1-4 - No spot M:E:H2:2:1
3 5-7 0.21 Single spot Light brown
6 13-18 - No spot
5 21-25 - No spot
Butanol fraction
2 1-2 - No spot E:C:B 0.1:2.5:2.5
15 6-20 - No spot
The three components with Rf values of 0.78, 0.48 and 0.21 respectively were eluted from n-hexane fraction.
Three components with Rf values of 0.13, 0.21, 0.38 and 0.80 respectively were eluted from ethyl acetate
fraction and only one component of Rf value 0.50 was eluted from the butanol fraction.
The results of the qualitative phytochemical analyses are presented in Table 4
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Osibote et. al.
Table 4: Qualitative analysis results

Phytochemical Standard Test Presence
Alkaloids Mayer’s Test -
Dragendorff’s Test +
Wagner’s Test -
Saponins Frothing Test ++
Reducing Sugar Fehling’s Test +++
Anthraquinones Borntrager’s Test -
Cardiac glycosides Keller Killani’s Test +
Terpenoids Liebermaan-Burchard ++
Triterpenoids Liebermaan-Burchard -
Steroids Salkowski’s Test +++
Phenolic Compounds Lead acetate Test ++
Tannins Ferric chloride Test ++
Flavonoids Shinoda’s Test +
Note: (+++) Highly detected, (++) Moderately detected, (+) Lightly detected, (-) Not detected. Alkaloid is only
slightly detected with the Dragendorff’s test.
The results of the quantitative phytochemical analysis of the crude extract of V. minor are presented in Table 5.
Table 5: Quantitative Phytochemical analysis of Vinca minor
Phytoconstituents (mg/g extract)
Saponins 19.13
Reducing Sugar 42.85
Cardiac Glycosides 9.16
Steroids 20.05
Phenolic Compounds 27.33
Flavonoids 46.92
Tannins 12.39
The phytochemical analysis carried out on the plant revealed the presence of saponins, reducing sugar, cardiac
glycosides, steroids, phenolics, tannins and flavonoids. The phenolics, tannins and flavonoids are known to be
strong antioxidants and anti inflammatory agents and their high content support bioactive activities responsible
for medicinal claim of the plant.
Proximate analysis of the 50 % methanol/water extract of V. minor
The result of the proximate analysis carried out on the leave extracts of V. minor is presented in Table 6.
Table 6: Results of Proximate analysis n=2 Composition (g in 100g or %)
Parameter Vinca minor

Moisture content 7.23
Total ash 7.32
Crude fat 5.05
Crude fibre 9.07
Crude protein 4.65
Carbohydrate 66.70
The proximate analysis of Vinca minor showed that the leave is made up of about 7 % moisture content, 5 % fat,
4.7 % protein and 66.7 % of carbohydrate. This result makes the plant an average source of carbohydrate in
human diet.
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Osibote et. al.
Table 7 : FTIR results for Vinca minor
Fraction Wavenumber (cm-1) Assignment Likely functional group present

n-Hexane fraction
component 1 3328 OH stretching Alcohol
2958 CH2 stretching Aliphatic
Asymmetrical or alicyclic hydrocarbon
2853 CH3 stretching Aliphatic

symmetrical or alicyclic hydrocarbon
1464 CH2 stretching

1335 CH3 stretching
1251 C-O Assymetrical C-O bond primarily as an
stretching ether functional group
1113 C-O-C stretching ether functional group
990 C=C bending Alkene
Likely compound present Steroids

n-Hexane fraction
component 2 3009 C=C stretching Alkene
2923 CH2 stretching
Asymmetrical Alkane
2853 CH3 stretching
symmetrical Alkane
1709 C=O stretching Carboxylic acid
1412 O-H Bend Carboxylic acid
1280 C-O stretching Ether
936 O-H Bend Carboxylic acid
Likely compound present Flavonoids
n-Hexane fraction
component 3 2954 CH 2 stretching
Asymmetrical Alkane
2868 CH3 stretching
symmetrical Alkane
1665 C=C bending Alkene
1493 CH stretching Aromatic
1311 OH stretching Phenol
Likely compound present Phenolic
Flavonoids
component 1 3393 OH stretching Hydroxyl
2957 CH2 stretching
asymmetrical Alkane
2872 CH3 stretching
symmetrical Alkane
1721 C=O stretching Ketone
2359 COO stretching Ester
1642 C=C stretching Alkene
Likely compound present Tannins
2955 CH2 stretching
Asymmetrical Alkane
2854 CH3 stretching
symmetrical Alkane
1690 N-H bending Amine
1605 C=C stretching Aromatic
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Osibote et. al.
Likely compound present Alkaloids

2956 CH2 stretching
Asymmetrical Alkane
2872 CH3 stretching
symmetrical Alkane
1658 C=C stretching Aromatic
1161 C-O stretching Ester
Likely compound present steroids
2959 CH2 stretching
asymmetrical Alkane
2873 CH3 stretching
asymmetrical Alkane
1715 COO stretching Carboxylic acid
1433 OH stretching Carboxylic acid
Likely compound present Saponins
Butanol Fraction
2958 CH2 stretching
Asymmetrical Alkane
2873 CH3 stretching
symmetrical Alkane
Likely compound present Terpenoid
The FTIR analysis of the components eluted from column chromatography for all the fractions of Vinca minor
showed that n-hexane fraction revealed functional groups that are present in phenolics/flavonoids, ethyl acetate
fraction gave many components which showed bands corresponding to C-O, C=C, C=O, OH and COO-
functional groups that may be present in steroids, saponins and tannins (highly phenolics and very strong
antimicrobial activity, anti-inflammatory) the presence of these functional groups in the phytochemicals support
the bio-activities claim in the plant in its use as antimicrobial agent.
Discussion
The phytochemical analyses carried out on aqueous methanol extracts of Vinca minor revealed the presence of
tannins, reducing sugar, saponins, cardiac glycosides, terpenoids, flavonoids, phenolic compounds these are also
reported in [11]. The partitioned fractions for V. minor gave three, four components and one component with the
retention factors (Rf) for the n-hexane (Rf: 0.72, 0.48 and 0.21), ethyl acetate (Rf: 0.13,0.21,0.38 and 0.80) and
butanol (Rf: 0.50) fractions respectively. The results obtained from FTIR analyses revealed also the presence of
functional groups related to steroids, flavonoids, phenolic compounds saponins and terpenoids. The presence of
these was consistent with the results of phytochemicals present from the qualitative and quantitative
phytochemical analyses carried out on the extracts of V. minor. The phenolic compounds and terpenoids are
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Osibote et. al.
very good antimicrobial agents. This is corroborated by [6] which reported that the least familiar Vinca species
have a great antioxidant, antitumoral property.
The proximate and elemental analyses showed that the plant possesses nutritional properties; with 7.23 %
moisture, 7.32 % ash, 5.05 % fat, 9.07 % fibre, 4.65 % protein and 66.70 % carbohydrate. Phytochemicals found
in V. minor were saponins (19.13 mg/100 g), reducing sugars (42.85 mg/100 g), cardiac glycoside (9.16 mg/100
g), steroids (20.05 mg/100 g), phenolic compounds (27.33 mg/100 g), flavonoids (46.92 mg/100 g), tannins
(12.39 mg/100 g) some of these have strong medicinal properties. Hence the leaves of the plant can contribute
to the nutritional and energy requirement of humans.
Conclusion
Vinca minor can be used as important source of new bioactive compounds because of presence of bioactive
phytochemicals such as saponins, reducing sugar, cardiac glycosides, terpenoids, steroids, phenolic compounds,
tannins and flavonoids which had positive results to the qualitative and quantitative phytochemical analyses
carried out. It has a substantially high content of phenolics and so will be good source of lead compounds for
antibiotic drugs. This plant has great potential to be developed to synthesize potent antibiotic drugs by
pharmaceutical industries
Acknowledgement
The authors acknowledge the contribution of Dr. Solayide Adesida in the preparation of the manuscript.
Conflict of Interest
All authors disclose that there is no actual or potential conflict of interest with other people or organizations
within three years of beginning the submitted work that could inappropriately influence, or be perceived to
influence the work.
References
[1]. Roberts, JD and Caserio, MC . Natural Products and Biosynthesis. California Institute of Technology. 2021.
https://chem.libretexts.org/@go/page/21994.
[2]. Veeresham, C. Natural products derived from plants as a source of drugs. Journal of advanced
pharmaceutical technology & research, 3(4):200–201. 2012. https://doi.org/10.4103/2231-4040.104709
[3]. Bourgaud, F, Gravot, A, Milesi, S and Gontier, E. Production of plant secondary metabolites: a historical
perspective. Plant science 161(5): 839-851. 2001.
[4]. Demain, AL and Fang, A. The natural functions of secondary metabolites. History of modern
biotechnology I, pp.1-39. 2000.
[5]. Grujić, SM, Radojević, ID, Vasić, SM, Čomić, LR and Topuzović, MD Antimicrobial and antibiofilm
activities of secondary metabolites from Vinca minor L. Applied biochemistry and microbiology 51(5), pp.572-
578. 2015.
[6]. Ciorîță A, Zăgrean-Tuza C, Moț AC, Carpa R, Pârvu M. The phytochemical analysis of Vinca L. species
leaf extracts is correlated with the antioxidant, antibacterial, and antitumor effects. Molecules. 19;26(10):3040.
2021.
[7]. PLANTS. United States Department of Agriculture. PLANTS Database. Available online:
https://plants.sc.egov.usda.gov (accessed on 14 May 2021).
[8]. Sezer, ENŞ and Uysal, T . Volatile and phenolic compositions of the leaves of two Vinca L. species from
Turkey. Current Perspectives on Medicinal and Aromatic Plants (CUPMAP) 1(2): 103-110. 2018.
[9]. Osibote, EA, Olasupo, IA and Chilaka L. Proximate, Phytochemical And FTIR Analyses of The Fruit
Extracts Of Kigelia africana And The Antioxidant Activity. FUW Trends in Science & Technology Journal 6(3)
890 – 896. 2021. www.ftstjournal.com e-ISSN: 24085162; p-ISSN: 20485170.
[10]. AOAC (Association of Official Analytical Chemists), 2006. Official Methods of Analysis of the AOAC
(Horwitiz W, Editor), 18th Education., Washington DC, U.S.A.
[11]. Iniaghe OM, Malomo SO and Adebayo JO. Proximate composition and phytochemical constituents of
leaves of some Acalypha species. Pakistan Journal of Nutrition 8(3), pp.256-258. 2009.
[12]. Ayuba VO, Ojobe TO and Ayuba SA. Phytochemical and proximate composition of Datura innoxia leaf,
seed, stem, pod and root. Journal of Medicinal Plants Research 5(14), pp. 2952-2955. 2011. Available online at
http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011
[13]. Osibote EA, Nwafor SP and Iluobe HO. Chemical and Phytochemical Analyses of Extracts from the
Leaves of Acalypha wilkesiana, “an Herbal Plant used for the Treatment of Various Skin Disorders” Annals of
Science and Technology – A. 5 (2): 40-48. 2020
[14]. Sofowora A. . Medicinal plants and Traditional Medicine in Africa. Spectrum Books, Ibadan. 1998
[15]. Osabor VN, Bassey FI and Umoh UU. Phytochemical Screening and Quantitative Evaluation of
Nutritional Values of Zingiber officinale (Ginger) American Chemical Science Journal 8(4): 1-6, 2015.
Article no.ACSj.16915 ISSN: 2249-0205 DOI: 10.9734/ACSj/2015/16915
8
Osibote et. al.
[16]. Sodamade, A, Bolaji, OS and Adeboye, OO. Proximate Analysis, Mineral Contents and Functional
Properties of Moringa oleifera Leaf Protein Concentrate. IOSR Journal of Applied Chemistry (IOSR-JAC) 4(6):
47-51. 2013. e-ISSN: 2278-5736. www.iosrjournals.org
[17]. Harbone, JB Phytochemical methods. London. Chapman and Hall, Ltd., 49-188. 1973
[18]. Trease, G.E and W.C Evans. Phenols and phenolic glycosides in Trease and Evans pharmacognosy and
Biliere Tindall London, pp: 832 1996
[19]. Shad MA, Nawaz H, Rehman T, Ikram, N. Determination of some biochemicals, phytochemicals and
antioxidant properties of different parts of Cichorium intybus L: A comparative study. J. Anim. Plant Sci. 23 (4),
1060–1066. 2013.
[20]. Singleton, VL, Orthofer, R. and Lamuela-Raventós, RM. Analysis of total phenols and other oxidation
substrates and antioxidants by means of folin-ciocalteu reagent. In: Methods in Enzymology. Academic Press.
299: 152-178. 1999.
[21]. Ahmad R, Parveen G, Gauri NA and Wal N. Phytochemical screening, sugar content, total protein and
antimicrobial activity of three important medicinal plants. International Journal of Fauna and Biological Studies
5(6): 125-139. 2018.
[22]. El-Olemy, MM, Al Muhtadi, FJ and Afifi, AFA. Experimental phytochemistry: A laboratory manual. King
Saud University Press, Saudi Arabia. 21-27. 1994. 26, 3040. https://doi.org/10.3390/molecules26103040
[23]. Koel M, Kuhtinskaja M, Vaher M. Extraction of bioactive compounds from Catharanthus roseus and
Vinca minor. Separation and Purification Technology. 1;252:117438. 2020.
9
Osibote et. al.

Antioxidant Activities of Leaf Extracts of Vinca minor L
Osibote, Elizabeth Adejoke*, Abiose, Modupeola Mopelola and Olasupo, Idris Abiodun
Department of Chemistry, Faculty of Science, University of Lagos, Akoka, Nigeria
Abstract
Tropical plants are used to manage various ailments, Vinca minor L. is an herb used in the management of
diabetes, depression, inflammation and cancer. Extraction was carried out on the pulverized leaves with 50 %
methanol/water. Serial solvent partitioning was done on aqueous mixture of crude extract with n-hexane, ethyl
acetate and butanol according to polarity of constituents. The antioxidant assays carried out at concentration of
200 ug/ml in DPPH assay gave (33.23 %), Nitric oxide assay (55.75 %) free radical inhibition and the total
antioxidant capacity (TAC) (1.023) for ethyl acetate fraction which indicated the highest antioxidant activity of
the three fractions. The functional groups identified with FTIR were consistent with the metabolites obtained
from phytochemical screening and the antioxidant assays. The high antioxidant activities indicate presence of
phenolics, flavonoids, tannins and steroids in extracts. These results are scientific indications of potentials of
extracts from V. minor for synthesis of potent drugs against diseases implicated in reactive oxygen species.
Keywords: Antioxidants, Ethyl acetate fraction, Phytochemicals, V. minor (Apocynaceae)
Introduction
The natural products may act as active components not only for traditional medicine but also for modern
medicines [1] and are often used as starting points for drug discovery. They can be divided into two major
classes, primary and secondary metabolites [2]. Secondary metabolites are typically organic compounds
produced through the modification of primary metabolite synthases. Many secondary metabolites are classified
as alkaloids which have a diverse array of pharmacological actions including analgesia, local anesthesia, cardiac
stimulation, respiratory stimulation and relaxation, vasoconstriction, muscle relaxation and toxicity, as well as
antineoplastic, hypertensive and hypotensive properties. Many alkaloids are sufficiently toxic to animals to
cause death if eaten. Several (nicotine and anabasine) are used as insecticides [3,4]. Others are phenolics which
constitute the largest group of plant secondary metabolites. They share the presence of one or more phenol as a
common characteristic and range from simple structures with one aromatic ring to highly complex polymeric
substances. They contribute significantly to the color, taste and flavor of many herbs, foods and drinks. Some
phenolics are valued pharmacologically for their anti-inflammatory activities such as quercetin or
antihepatotoxic properties such as silybin, phytoestrogenic activity as genistein and daidzein, while others are
insecticidal as naringenin [5,6]. Many of the phenolic molecules are also effective antioxidants and free radical
scavengers, especially flavonoids.
The plant has antibiofilm, antioxidant and antimicrobial activity, total phenolic content, DPPH and reduction
potential of the aquatic extract of V. minor was significantly stronger compared to the acetone and ethyl acetate
extracts [7,8]. Antihyperlipidemic, hypoglycaemic, hypolipidemic and antioxidant activities were discovered in
Vincamine extract in diabetic rats and showed it has a protective role with effective antidiabetic effects [9].
Vicamine has antioxidant and antiapoptotic properties, it is also neuroprotective and cerebral metabolic
enhanser. The alkaloids showed cytotoxic effect on the cell lines [10,11]. Volatile oil and phenolic compounds
from the plant revealed that the leaf extracts contain very valuable compounds that have anticancer and
antiproliferative effects [11]. Vinca minor has been listed among Vinca species that synthesize natural products
with effective antibacterial, antifungal, antiparasitic and antitumor activities, it is listed in The United States
Department of Agriculture [12,13,14].

Extraction of the Plant Materials
The fresh leaf of Vinca minor L was collected from Ota in Ogun State in September 2021, identified and
authenticated at the Herbarium in Botany Department, University of Lagos. The voucher number LUH 8940
was assigned. The leaf was dried, 225 g of pulverized plant was extracted by maceration as reported by Osibote
et al, [15]. The concentrated crude extract was frozen, dried and had a brown colour. The crude extract was
partitioned into n-hexane, ethyl acetate and butanol fractions. The n-hexane fraction was light brown, ethyl
acetate and butanol fractions were dark brown.
The Antioxidant Activity
The antioxidant activity of n-hexane, ethyl acetate and butanol fractions of Vinca minor extracts were
determined by different in vitro methods; 2,2-Diphenyl-1-
Corresponding Author’s Email: eosibote@unilag.edu.ng

10
Osibote et. al.
picyrlhydrazyl (DPPH) free radical scavenging assay, Nitric oxide scavenging and Total antioxidant capacity.
[16,17,18,19,20,21,22].
DPPH (1,1-diphenyl-2-picrylhydrazyl) Free Radical Scavenging Assay
The antioxidant activity of n-hexane, ethyl acetate and butanol fractions of the V. minor extracts were evaluated
on the basis of the radical scavenging effect of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH)- free radical
activity, in comparison with Ascorbic acid standard in different concentrations prepared from stock solution in
triplicates using suitable dilution. 0.1 mM of DPPH was prepared in methanol. These solutions were shaken and
absorbance was measured at 517 nm using UV-VIS Spectrophotometer. Methanol with DPPH solution was used
as control. Methanol was used as blank. The same procedures with the Ascorbic acid standard were carried out
with the n-hexane, ethyl acetate and butanol fractions samples.
The percentage inhibition was calculated as follows:
Inhibition of DPPH = (Ab – Aa)/Ab x 100 %
Where Ab is the absorbance of the Blank sample and Aa is the absorbance of the fraction sample.
Nitric Oxide Scavenging Assay
The nitric oxide scavenging activity was estimated using sodium nitroprusside in phosphate -buffered saline,
mixed with different concentrations of the n-hexane, ethyl acetate and butanol fractions and incubated at 25 oC
for 150 minutes. Each fraction of V. minor was mixed with Griess reagent. The absorbance of the chromophore
formed during the diazotization was read at 546 nm using a UV-Vis spectrophotometer. The inhibition of nitric
oxide formation was determined with respect to potassium nitrite equivalent used as a standard.
Total Antioxidant Capacity (Phosphomolybdate Assay)
Total antioxidant capacity assay (TAC) was assessed spectrophotometrically by the phosphomolybdenum
method. Some n-hexane, ethyl acetate and butanol fractions were dissolved in methanol and sonicated for 5
minutes to get a homogeneous mixture. Some volume of each of n-hexane, ethyl acetate and butanol fraction
was mixed with reagent solution (sodium phosphate and ammonium molybdate). The blank solution contained
reagent solution only. Ascorbic acid was used as a standard and the stock solution was prepared in distilled
water, from which dilutions of various concentrations were made. The mixtures were incubated at 95 °C for 90
minutes and cooled to room temperature, after which absorbance was measured at 695 nm.
Total antioxidant capacity (TAC) was expressed as ascorbic acid equivalent (AAE).
Results
The results of the antioxidant assays are thus presented in tables 1-3.
Table 1 shows that at concentration of 200 ug/ml, the HVM, EVM and BVM, decreased the DPPH signal by
18 %, 33 % and 26 % respectively compared to 94 % decrease with 200 ug/ml of ascorbic acid. Nitric oxide %
Inhibition of n-Hexane, Ethyl Acetate and Butanol fraction at different concentrations are presented in Table 2.
The Total Antioxidant Capacity of n-Hexane, Ethyl Acetate and Butanol Fractions for Vinca minor are
presented in Table 3
Table 1: Free radical scavenging activity of Vinca minor using 2, 2-Diphenyl-1-picyrlhydrazyl (DPPH)
Assay
Conc. (ug/ml) 10 25 50 100 200

Hexane 8.67 ± 0.058 10.07 ± 0.051 13.54 ± 0.084 16.27 ± 0.055 18.07 ± 0.058
Ethyl acetate 16.75 ± 0.050 19.34 ± 0.057 24.65 ± 0.073 28.64 ± 0.039 33.23 ± 0.062
Butanol 11.04 ± 0.051 14.26 ± 0.055 20.27 ± 0.060 22.04 ± 0.053 26.04 ± 0.064
Ascorbic acid 81.16 ±0.05 90.23 ± 0.024 92.32 ± 0.020 93.26 ± 0.017 94.41 ± 0.014
% Inhibition of n-Hexane, Ethyl Acetate and Butanol fraction of Samples (Values are expressed as Mean ±
SEM and n = 3)
Table 2: Nitric oxide free radical scavenging activity (NOSA) of V. minor
Sample
Fraction 10 ug/ml 25 ug/ml 50 ug/ml 100 ug/ml 200 ug/ml
n-Hexane 8.37 ± 0.052 12.87 ± 0.051 22.79 ± 0.019 28.06 ± 0.018 37.24 ± 0.027
Ethylacetate 10.48 ± 0.039 25.20 ± 0.058 34.84 ± 0.060 42.08 ± 0.047 55.76 ± 0.058
Butanol 9.35 ± 0.040 18.73 ± 0.44 26.63 ± 0.084 33.47 ± 0.049 41.89 ± 0.042
Ascorbic
Acid (control) 62.57 ± 0.018 68.28 ± 0.014 72.42 ± 0.020 78.36 ± 0.029 84.42 ± 0.042
11
Osibote et. al.
Table 2 shows that at concentration of 200 ug/ml, the HVM, EVM, BVM, decreased the nitric oxide signal by
37.24 %, 55.76 % and 41.89 % as compared to 84.42 % decrease with 200 ug/ml ascorbic acid. Total
Antioxidant Capacity of n-Hexane, Ethyl Acetate and Butanol Fractions for Vinca minor are presented in Table
3
Table 3: Total Antioxidant Capacity (TAC) (Phosphomolybdate Assay) of Vinca minor with different
fractions
Extract Concentration of TAC (mg AAE/g of extract)

Hexane 0.697 ± 0.059
Ethyl A 1.023 ± 0.048
Butanol 0.994 ± 0.057
Discussion
There is an earlier report on the phytochemical analyses on aqueous methanol extracts of Vinca minor which
revealed the presence of tannins, reducing sugar, saponins, cardiac glycosides, terpenoids, flavonoids, phenolic
compounds and steroids, these are also reported in [10,11,12] and some of these have strong medicinal
properties. Several recent reports have confirmed that phytochemicals including alkaloids, glycosides,
terpenoids, saponins, phenols and steroids have enormous antioxidant and free radical scavenging activities
[23,24]. Plant extracts rich in polyphenols and essential phytoconstituents have been shown to display potency
antioxidant and free radical scavenging activities in many antioxidant assays [23,24] The flavonoids and
phenolic compounds are very good antioxidant agents because of the presence of -OH which can scavenge
radicals formed from oxidative products of ROS. This is corroborated by [12] which reported that the least
familiar Vinca species have a great antioxidant, antitumor properties. The two form the greater part of the
phytochemicals detected and as such gave the appreciable antioxidant ability of the extract especially from the
crude extract.
The antioxidant assay indicates that the V. minor extracts have potent antioxidant activity relative to the
antioxidants present in ascorbic acid. The potency of the fractions were tested and it was observed that of all the
extracts, the ethyl-acetate fraction at concentration of 200 ug/ml showed the best antioxidant and free radical
scavenging activity, in DPPH assay with (33.23 % inhibition); The Nitric oxide assay with (55.75 % inhibition).
The total antioxidant capacity (TAC) had antioxidant activities with (1.023), the activity is concentration
dependent. The antioxidant activities and the inhibition activity in the extracts from the plant will serve as a
good platform for their usefulness in the food industry.
Conclusion
Vinca minor can be used as important source of new bioactive compounds because of presence of bioactive
phytochemicals such as saponins, reducing sugar, cardiac glycosides, terpenoids, steroids, phenolic compounds,
tannins and flavonoids which had. It has a higher content of phenolics and flavonoids and so will be good source
of antioxidant lead compounds for drug delivery. The antioxidant assays support this also. This plant has great
potential to be developed together to synthesize potent drugs by pharmaceutical industries for the management
diseases caused by reactive oxygen species.
Acknowledgement
The authors acknowledge the contribution of Dr. Solayide Adesida in the preparation of the manuscript.
Conflict of Interest
All authors disclose that there is no actual or potential conflict of interest including any financial, personal or
other relationships in any way with other people or organizations within three years of beginning the submitted
work that could inappropriately influence, or be perceived to influence the work.
References
[1]. Chintoju, N., Konduru, P., Kathula, R. L. and Remella, R. Importance of natural products in the modern
history. Research and Reviews: Journal of Hospital and Clinical Pharmacy. 1: 5-10. 2015
[2]. Roberts, JD and Caserio, MC . Natural Products and Biosynthesis. California Institute of Technology. 2021;
https://chem.libretexts.org/@go/page/21994.
[3]. Hoffmann, D. Medical herbalism: the science and practice of herbal medicine. Simon and Schuster 2003.
[4]. Veeresham, C. Natural products derived from plants as a source of drugs. Journal of advanced
pharmaceutical technology & research 3(4): 200–201. 2012. https://doi.org/10.4103/2231-4040.104709
[5]. Goławska, S., Sprawka, I., Łukasik, I. and Goławski, A. Are naringenin and quercetin useful chemicals in
pest-management strategies? Journal of pest science 87(1): 173-180. 2014.
[6]. Bourgaud, F, Gravot, A, Milesi, S and Gontier, E. Production of plant secondary metabolites: a historical
perspective. Plant science 161(5): 839-851. 2001.
12
Osibote et. al.
[7]. Grujić, SM, Radojević, ID, Vasić, SM, Čomić, LR and Topuzović, MD Antimicrobial and antibiofilm
activities of secondary metabolites from Vinca minor L. Applied biochemistry and microbiology 51(5), pp.572-
578. 2015.
[8]. Kim, JS, Yu, IH, Joo, JH, Nam, GH, Jung, KH, Chung, YS et.al., Biological Effects of Vinca minor extract;
Tyrosinase inhibition, stimulation of
ROS generation and increasement of cell migration activity in keratinocytes. Journal of the Korean Applied
Science and Technology 33(4): 788-794. 2016.
[9]. Aboelnaga SM. Evaluation of the antihyperlipidemic and antioxidant effects of Catharanthus roseus
extracted from Vinca minor in diabetic rats. Evaluation. 33(2). 2021.
[10]. Khanavi, M, Pourmoslemi, S, Farahanikia, B, Hadjiakhoondi, A and Ostad, SN. Cytotoxicity of Vinca
minor. Pharmaceutical biology 48(1): 96-100. 2010.
[11]. El-Dessouki AM, El Fattah MA, Awad AS, Zaki HF. Zafirlukast and vincamine ameliorate tamoxifen-
induced oxidative stress and inflammation: Role of the JNK/ERK pathway. Life sciences. 1;202:78-88. 2018.
[12]. Ciorîță A, Zăgrean-Tuza C, Moț AC, Carpa R, Pârvu M. The phytochemical analysis of Vinca L. species
leaf extracts is correlated with the antioxidant, antibacterial, and antitumor effects. Molecules. 19;26(10):3040.
2021.
[13]. PLANTS. United States Department of Agriculture. PLANTS Database. Available online:
https://plants.sc.egov.usda.gov (accessed on 14 May 2021).
[14]. Sezer, ENŞ and Uysal, T . Volatile and phenolic compositions of the leaves of two Vinca L. species from
Turkey. Current Perspectives on Medicinal and Aromatic Plants (CUPMAP) 1(2): 103-110. 2018.
[15]. Osibote, EA, Olasupo, IA and Chilaka L. Proximate, Phytochemical and FTIR Analyses of The Fruit
Extracts Of Kigelia africana And The Antioxidant Activity. FUW Trends in Science & Technology Journal 6(3)
890 – 896. 2021. www.ftstjournal.com e-ISSN: 24085162; p-ISSN: 20485170.
[16]. AOAC (Association of Official Analytical Chemists), Official Methods of Analysis of the AOAC
(Horwitiz W, Editor) 2006. 18th Education., Washington DC, U.S.A.
[17]. Ayuba VO, Ojobe TO and Ayuba SA. Phytochemical and proximate composition of Datura innoxia leaf,
seed, stem, pod and root. Journal of Medicinal Plants Research 5(14), pp. 2952-2955. 2011. Available online at
http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011
[18]. Trease, G.E and W.C Evans. Phenols and phenolic glycosides in Trease and Evans pharmacognosy and
Biliere Tindall London, pp: 832 1996
[19]. Shad MA, Nawaz H, Rehman T, Ikram, N. Determination of some biochemicals, phytochemicals and
antioxidant properties of different parts of Cichorium intybus L: A comparative study. J. Anim. Plant Sci. 23 (4),
1060–1066. 2013.
[20]. Singleton, VL, Orthofer, R. and Lamuela-Raventós, RM. Analysis of total phenols and other oxidation
substrates and antioxidants by means of folin-ciocalteu reagent. In Methods in Enzymology. Academic Press.
299: 152-178. 1999.
[21]. El-Olemy, MM, Al Muhtadi, FJ and Afifi, AFA. Experimental phytochemistry: A laboratory manual. King
Saud University Press, Saudi Arabia, 21-27. 1994. 26, 3040. https://doi.org/10.3390/molecules26103040
[22]. Koel M, Kuhtinskaja M, Vaher M. Extraction of bioactive compounds from Catharanthus roseus and
Vinca minor. Separation and Purification Technology. 252:117438. 2020.
[23]. Farhan H, Malli F, Rammal H, Hijazi A, Bassal A, Ajouz N, Badran B. Phytochemical screening and
antioxidant activity of Lebanese Eryngium creticum L. Asian Pac J Trop Biomed. 2:S1217–S1220. 2012.
[24]. Amari NO, Bouzouina M, Berkani A, Lotmani B. Phytochemical screening and antioxidant capacity of the
aerial parts of Thymelaea hirsuta L. Asian Pac J Trop Dis. 4:104 109.2014.
13

http://www.ojs.klobexjournals.com/index.php/niseb
Effect of pH Modifications of PCB-Contaminated Soil on Growth and Leaf
Characteristics of Maize (Zea mays) Plant
Oyeyemi Adeyemi* and Olalekan Adeyemi
Department of Environmental Management and Toxicology,
Federal University of Petroleum Resources, PMB 1221, Effurun, Delta State, Nigeria
*Corresponding Author: adeyemi.oyeyemi@fupre.edu.ng
Abstract
This study involved the extraction of crude polychlorinated biphenyls (PCBs) from soil obtained from dump sites
located in three areas of Warri Delta State, Nigeria: Niger CAT (longitude 5°47' 56.497''E, latitude 5°29'
31.403''N), OLU TEE (longitude 5°46' 11.546''E, latitude 5°28' 40.537''N), and DSC (longitude 5°47' 7.631''E,
latitude 5°31' 48.995''N and 5.60S). These extracted PCBs were used to contaminate separate groups of soil, which
were labeled as A, B, and C. The pH levels of the soils were adjusted to 5.5, 7.0, and 8.5, respectively, resulting in
designations such as A5.5, A7.0, A8.5 for Niger CAT, B5.5, B7.0, B8.5 for OLU TEE, and C5.5, C7.0, C8.5 for DSC.
Maize seeds were then planted in each group and allowed to grow for duration of 4 weeks. Throughout this period,
various growth parameters such as plant height and stem girth were monitored. Additionally, standard enzyme
assays were conducted on the leaves and stems of maize to measure catalase (CAT), superoxide dismutase (SOD),
glutathione reductase (GR), ascorbate peroxidase (APX), malondialdehyde (MDA), and soluble protein content
(SPC). The results indicated that the height of maize in PCBs-contaminated soil was significantly lower compared
to the control group (p<0.05). The data from this study suggested that PCBs caused stunted growth in maize, but
adjusting the pH of the contaminated soil to 7.0 reduced the toxicological effects of PCBs on maize. Elevated levels
of MDA and antioxidant enzymes, along with reduced soluble protein content, indicated the presence of oxidative
stress induced by the PCBs.
Keywords: Toxicological, PCBs, Soil, Leaves, Maize
Introduction
Polychlorinated biphenyls (PCBs) are synthetic organic compounds that were widely utilized in various industrial
and commercial applications, including transformers, capacitors, and plasticizers [1]. Despite being banned for
several years, PCBs remain a significant concern due to their persistence in the environment and their detrimental
effects on human health, wildlife, and ecosystems [1].
Studies have identified several sources of PCB contamination, including buildings, paints, and outdated appliances
[2-4]. Additionally, contaminated soil and water serve as crucial secondary sources, leading to the release of PCBs
into the atmosphere [5-6]. It is important to note that PCB production and use were predominant in developed
countries. However, the global movement of electronic and electric waste (e-waste) from developed regions to
developing areas, coupled with the improper and unsafe recycling or disposal of this waste, has garnered increased
attention [7]. As a result, these e-waste sites have become significant reservoirs and potential emission sources of
PCBs.
The presence of vegetation has a significant impact on the behavior of semi-volatile organic compounds (SOCs) in
the environment, including their transport, cycling, and elimination [8]. Over the past few decades, extensive
research has focused on the exchange of SOCs between the air and vegetation, as well as the interactions between
plants and soil in relation to various SOCs [9]. Apart from the uptake of these compounds by plant tissues, the
metabolism of xenobiotic substances within plants also plays a crucial role in the remediation of environmental
pollutants. Notably, the presence of plants in soil has been observed to lead to the attenuation of PCBs through
microbial degradation in the rhizosphere [10]. Laboratory experiments have demonstrated that PCB congeners can
be metabolized by plant cell cultures [11-12], with a significant mechanism involving the oxidation of PCBs to form
various hydroxylated metabolites [13]. Furthermore, studies have indicated the involvement of cytochrome P-450
(CYP-450) enzymes, which are commonly found in animals, plants, insects, and microorganisms, in the
transformation of PCBs in plants [14]. This suggests the possibility of enantio-selective metabolism of atropisomeric
PCBs in plant tissues due to the unique chirality of the enzyme's active site.
*Corresponding Author’s Email: adeyemi.oyeyemi@fupre.edu.ng

14
Soil pH has emerged as a significant environmental stressor with evident impacts on plant germination,
development, and overall performance [15-16]. The soil's acidity or alkalinity, also known as soil reaction or pH,
reflects the concentration of active hydrogen ions within the soil solution [17-18]. pH is defined as the negative
logarithm of the hydrogen ion concentration and was introduced by Danish Biochemist Soren Peter Lauritz Sorensen
in 1909 [19-20]. For crop production, the pH of the soil is a crucial soil characteristic as it represents the activity of
hydrogen (H+) ions in the soil solution, typically determined by shaking soil with distilled water [21-24]. In pure
water, there is a nearly balanced concentration of positive and negative ions [19, 25-28].
Soil pH has direct and indirect effects on seed germination and plant growth by influencing the availability of
nutrients, disrupting water uptake, potentially increasing levels of certain elements to toxic concentrations, and
impacting microbial activity and other soil properties [29-31]. The pH of the soil can also affect the movement of
hormones across cellular membranes, the ability of roots to modify their micro-environment and extract nutrients
and water from the soil, thereby affecting the germination process and early seedling development [29, 32-33]. Soil
pH is a crucial environmental factor that significantly influences maize germination, seedling establishment, growth,
and development [15, 31]. Maize producers can manipulate soil pH through various management practices to
promote fast and vigorous seed germination and seedling development, aiming for higher yields. This is particularly
important as the initial stages of maize development have a substantial impact on later growth stages [34]. By
understanding the developmental processes of maize plants, farmers can implement appropriate soil management
practices to achieve higher yields and profitability [24, 31, 35]. The variation in soil pH is primarily influenced by
natural biological processes related to the nitrogen, carbon, and sulfur cycles. However, human activities, such as
agricultural and industrial practices like fertilizer and lime application, irrigation, and acidic inputs, can accelerate
the rate of pH changes in soils [36-37]. The objective of the current study is to investigate the impact of altering the
pH of PCB-contaminated soil on the growth and leaf characteristics of cultivated maize (Zea mays) plants.

Reagents
The reagents and solvents used in this study were of analytical grade and sourced from British Drug House, Poole,
England.
Study Area
The study area for this research consisted of three dumpsites located in Warri Delta State. The selected dumpsites
were Niger Construction Company (Nigercat), Olutee Engineering International Limited, and Delta Steel Company
(DSC). Olutee Engineering International Limited is involved in engineering design, civil construction, intensive and
integrated waste management and disposal services, environmental services, marine maintenance service and
logistics, and technical support services. It is situated at longitude 5°47' 56.497''E and latitude 5°29' 31.403''N along
Udu Road in Warri (Figure 1). Delta Steel Company (DSC) is engaged in the construction of iron and steel and the
production of iron rods. It is located at longitude 5°46' 11.546''E and latitude 5°28' 40.537''N along the Warri-Port
Harcourt expressway. Niger Construction Company is situated at longitude 5°47' 7.631''E and latitude 5°31'
48.995''N and 5.60S along Enheren Road in Warri.
Sampling and Crude PCBs Extraction
Soil samples were collected for the study, and the extraction of crude polychlorinated biphenyls (PCBs) from the
soil was performed following standard methods with slight modifications, as described [38]. Glassware used in the
extraction process was thoroughly washed, rinsed with distilled water, cleaned with acetone, and dried in an oven at
100°C for 30 minutes.
To initiate the extraction, 40 grams of soil sample was carefully measured and placed into a 250 ml glass bottle.
Subsequently, 80 ml of a cyclohexane/ethyl acetate mixture in a ratio of 1:1 was added to the bottle. In addition, 10
grams of sodium sulfate, which acts as a deliquescent substance, were also included in the mixture.
The glass bottles containing the soil sample, solvent mixture, and sodium sulfate were placed on a rotator shaker and
agitated at a speed of 200 revolutions per second (rps) for a duration of 30 minutes. This process facilitated the
extraction of crude PCBs from the soil samples. The resulting crude PCB extract obtained from this procedure was
then utilized for the subsequent toxicological studies.
15
Figure1: Map of Warri Showing the Sample Areas (Nigercat, Olutee and DSC)
Experimental Design and Agronomic Details

The experiment was conducted in the Greenhouse of the College of Science, Federal University of Petroleum
Resources, Effurun, Nigeria. The methodology employed in the study was based on the approach described by
Adewole and Aboyeji [39], with slight modifications.
Bulk surface soil samples, obtained from a designated area within the University, were collected from a depth of 0-
15 cm. These soil samples were air-dried for a period of seven days and subsequently sieved using a 2 mm sieve.
The sieved soil samples were then subjected to analysis using standard methods to determine their physical and
chemical properties.
For the experimental setup, a total of thirty polythene pots were utilized, with each pot containing 10 kg of surface
soil. The pots were randomly placed on a table in the Greenhouse. The experiment was designed as a factorial
combination, involving thirteen treatment levels.
The experimental groups were categorized as follows:
Control: Soil with no pH adjustment and no crude PCBs contamination.
A: Soil with no pH adjustment and contaminated with crude PCBs extracted from the NigerCat dumpsite sample.
A5.5: Soil with pH adjusted to 5.5 and contaminated with crude PCBs extracted from the NigerCat dumpsite
sample.
sample.
16
sample.
B: Soil with no pH adjustment and contaminated with crude PCBs extracted from the Olutee dumpsite sample.
B5.5: Soil with pH adjusted to 5.5 and contaminated with crude PCBs extracted from the Olutee dumpsite sample.
C: Soil with no pH adjustment and contaminated with crude PCBs extracted from the DSC dumpsite sample.
C5.5: Soil with pH adjusted to 5.5 and contaminated with crude PCBs extracted from the DSC dumpsite sample.
The soil inside the pots was thoroughly mixed by stirring with a glass rod. It was then moistened with distilled water
and allowed to equilibrate for duration of two weeks. Following the equilibration period, the pH of the soil was
adjusted as per the experimental groups, and the corresponding extracted crude PCBs were added to the soil.
Three seeds of maize, obtained from Effurun market in Effurun, Nigeria, were planted in each pot. Throughout the
growth stage, the maize stands were regularly watered to ensure optimal moisture levels. At two weeks after
planting (WAP), the maize plants were thinned to two stands per pot. The thinned stands were kept within their
respective pots, allowing them to decompose and return any nutrients they had absorbed during the initial two weeks
of growth back into the soil.
Growth parameters of the maize plants, including plant height and stem girth, were measured every four days until
the termination of the experiment at four WAP.
Soil pH Adjustment
To adjust the pH of the experimental soil, 1M NaOH and 1M HCl solutions were utilized. The appropriate volumes
of these solutions were added to the soil samples to achieve the desired pH levels. For alkaline pH, the 1M NaOH
solution was added incrementally until the target pH level was reached. Conversely, for acidic pH, the 1M HCl
solution was added gradually to the soil samples until the desired pH level was attained. The pH levels that were
targeted in this study were pH 5.5, pH 7, and pH 8.5.
Relative Water Content (RWC) Determination
At four weeks after planting (WAP), the relative water content (RWC) of the leaves was measured using the
standard method [40].
Soil Analysis
The pH, temperature, moisture content, soil particle size, phosphorus, potassium, sodium, calcium, and magnesium
content of the soils were analyzed using conventional standard methods as referenced in Black [41] and AOAC [42],
Preparation of Homogenate and Biochemical Analysis
The fresh leaf and stem tissues from each plant pot were collected and weighed (0.5g). The tissues were then finely
chopped into small pieces and homogenized using a pre-cooled pestle and mortar in a bowl containing ice cubes. To
dilute the homogenates, normal/physiological saline solution (9 g of NaCl in 1 liter of distilled water) was used. The
diluted homogenates were stored at a temperature of -8 °C until they were required for further analysis.
To determine the protein content in the plant tissues, the method reported by Gornal et al. [43] was followed. The
MDA (malondialdehyde) concentration in the serum and tissues of the experimental plants was determined using the
method described by Bird et al. [44]. The GSH (glutathione) concentration in the plant tissues was determined
according to the method described by Jollow et al. [45]. The SOD (superoxide dismutase) activity in the plant
tissues was determined using the method described by Misra and Fridovich [46]. The catalase activity in the tissue
homogenate was determined following the method described by Sinha [47]. The activity of ascorbate peroxidase
was assayed using the method of Nakano and Asada [48]. Finally, peroxidase activity was determined specifically
using the method described by Putter [49].
Statistical Analyses
The numerical results obtained from the four groups (control and treated) were presented as mean ± SEM (standard
error of the mean). These data were then subjected to statistical analysis using a one-way analysis of variance
(ANOVA) method. To determine significant differences between the treatment means, Duncan's Multiple Range
Test [50] was employed at a 95% confidence level. This statistical test allows for the comparison of multiple
treatment groups to identify significant variations among them.
Results
Table 1 presents results of soil analysis. The pH of 5.66 suggests slightly acidic soil. The temperature of 28.9°C
indicates the measurement of soil temperature at the time the sample was taken. The total organic carbon content of
0.07% represents the percentage of organic matter in the soil. Organic matter contributes to soil fertility, moisture
17
retention, and nutrient availability. Generally, these results provide valuable insights into the soil's chemical
properties and can help determine its suitability for maize.
Figure 1 illustrates the height of maize plants grown in Crude PCBs-contaminated soil compared to the Control
group. The results indicate that the height of maize plants in the Crude PCBs-contaminated soil was significantly
lower than in the Control group (p<0.05). Additionally, within the Crude PCBs-contaminated soil groups, the height
of maize plants in Group C7.0 was significantly higher compared to Groups A7.0 and B7.0 (p<0.05). However,
there was no significant difference in the height of maize plants between Groups A7.0 and B7.0 (p>0.05). These
findings suggest that the presence of Crude PCBs in the soil negatively affected the height of maize plants, and
adjusting the soil pH to 7.0 in the contaminated soil (Group C7.0) led to relatively better plant height compared to
the other contaminated soil groups. Figure 2 displays the impact of pH on the stem girth of maize plants grown in
Crude PCBs-contaminated soil. It is observed that the stem girth of maize plants in Group A (no pH adjustment) was
the lowest among all the groups. Furthermore, the stem girth of maize plants in the Crude PCBs-contaminated soil
groups was significantly lower compared to the Control group (p<0.05). This suggests that the presence of Crude
PCBs in the soil adversely affected the stem girth of the maize plants.
In Figure 3, the impact of soil pH on the relative water content (RWC) of maize leaves planted in Crude PCBs-
contaminated soil is presented. The RWC of maize plants in groups A, B, and C, which were exposed to Crude
PCBs contamination, was significantly lower compared to the Control group (p<0.05). However, it is noteworthy
that at pH 5.5 and 7.0, the RWC of maize plants was significantly higher compared to maize plants planted in soil
groups A, B, and C (p<0.05). This suggests that adjusting the soil pH to 5.5 and 7.0 had a positive impact on the
water content of the maize leaves, mitigating the adverse effects of Crude PCBs contamination on water retention.
In Table 2, the effect of soil pH on selected enzymes of maize leaves planted in Crude PCBs-contaminated soil over
a 4-week period is presented. The superoxide dismutase (SOD) activity of maize leaves in Groups B7.0 and C7.0
was comparable to that of the Control group. However, the SOD activity of maize leaves in the other treatment
groups was significantly higher compared to the Control group (p<0.05). This suggests that adjusting the soil pH to
7.0 had a positive effect on maintaining SOD activity in the presence of Crude PCBs contamination. The catalase
(CAT) activity of maize leaves in Groups A and B was approximately 200% higher than that of the Control group.
This indicates that the Crude PCBs contamination and soil pH adjustments influenced the CAT activity in maize
leaves. The peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) activities of the
treatment groups were significantly higher compared to the Control group (p<0.05). This suggests that the Crude
PCBs contamination and soil pH adjustments had a significant impact on the activities of these enzymes in maize
leaves. Overall, the results indicate that the presence of Crude PCBs contamination and soil pH adjustments affected
the activity of selected enzymes in maize leaves, with variations depending on the specific treatment group.
Table 3 presents the effect of soil pH on soluble protein content (SPC) and malondialdehyde (MDA) levels in maize
leaves planted in Crude PCBs-contaminated soil. The soluble protein content (SPC) of maize leaves planted in
Crude PCBs-contaminated soil was significantly lower compared to the Control group (p<0.05). Specifically, the
SPC levels in Groups A5.5 and A7.0 were approximately 2 and 3 times higher than that of Group A, respectively.
Similar trends were observed in Groups B and C, indicating that the Crude PCBs contamination and soil pH
adjustments had an impact on the SPC levels in maize leaves. The malondialdehyde (MDA) levels in the leaves of
maize planted in Crude PCBs-contaminated soil were significantly higher than those in the Control group (p<0.05).
This suggests that the presence of Crude PCBs contamination led to increased lipid peroxidation, resulting in
elevated MDA levels in maize leaves. In summary, the results demonstrate that Crude PCBs contamination and soil
pH adjustments affected the soluble protein content and malondialdehyde levels in maize leaves, with significant
differences observed compared to the Control group.
Table 1: Some physico-chemical parameters of the experimental soil

PARAMETERS RESULT
pH 5.66
TEMPERATURE (OC) 28.9
TOTAL ORGANIC CARBON (%) 0.07
TOTAL NITROGEN (%) 0.22
PHOSPHOROUS (mg/kg) 0.10
POTASSIUM (mg/kg) 0.18
SODIUM (mg/kg) 0.72
CALCIUM (mg/kg) 0.23
MAGNESIUM (mg/kg) 0.67
TOTAL ACIDITY (mg/kg) 3.60
18
Figure 1: Effect of soil pH on height of maize planted in Crude PCBs contaminated soil. Results are means of
3 determinations ± SEM.
Figure 2: Effect of soil pH on stem girth of maize planted in Crude PCBs contaminated soil. Results are
means of 3 determinations ± SEM.
19
Figure 3: Effect of soil pH on relative water content of leaves of maize planted in Crude PCBs contaminated
soil. Results are means of 3 determinations ± SEM.
Table 2: Effect of soil pH on activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD),
ascorbate peroxidase (APX)and glutathione reductase (GR) activities of maize leaves
CAT (µmol H2O2 APX (µmol GR (µmol
SOD (units min-1 POD (Units ascorbate min-1 NADPH min-1
GROUPS mg-1 protein) mg-1 protein) mg-1 protein) mg-1 protein) mg-1 protein)
Control 74.40±2.70a 95.50±3.10a 355.60±8.70a 390.20±5.20a 64.60±4.90a
A 126.70±3.10b 208.60±3.40b 477.90±6.70b 438.20±4.30b 98.70±3.60b
A5.5 98.30±2.80c 156.80±2.90c 432.50±5.80c 417.40±3.70c 84.60±3.10c
A7.0 82.50±2.70d 113.50±3.90d 387.20±5.40d 406.90±3.40de 72.40±2.60d
B 121.40±4.10b 214.20±5.20b 469.60±6.20b 429.60±2.60e 98.60±3.70b
B5.5 101.70±2.90c 187.90±2.80f 400.0±7.70e 418.20±2.10c 83.40±3.50ce
B7.0 78.50±2.50a 125.40±3.10e 384.10±5.80d 403.10±2.30d 73.20±2.80d
C 104.30±3.10c 176.30±4.60g 419.40±5.30f 416.10±2.30c 86.90±2.70c
C5.5 86.60±2.80d 139.60±3.80h 395.70±5.40e 408.30±2.10d 80.10±2.40e
C7.0 75.70±3.60a 107.50±3.70d 369.60±5.80g 398.60±2.40a 70.40±3.10d
a,b,c
Values are means ± SEM of six determinations. Column values with different superscripts are significantly
different (p<0.05).
20
Table 3: Effect of soil pH on soluble protein content (SPC) and malondialdehyde (MDA) of maize leaves
GROUPS SPC (mg g-1FW) MDA (µmol g-1FW)
Control 4.50±0.40a 5.20±0.50a
A 1.20±0.10b 11.70±0.80b
A5.5 2.50±0.20c 8.80±0.60c
A7.0 3.80±0.10d 6.70±0.40d
b
B 1.40±0.20 12.30±0.90b
B5.5 2.60±0.20c 9.60±0.70c
B7.0 4.00±0.20d 7.40±0.80e
C 1.90±0.10b 9.10±0.80c
c
C5.5 2.70±0.20 7.30±0.50e
C7.0 4.10±0.20d 6.10±0.30d
Values are means ± SEM of six determinations. a,b,c Column values with different superscripts are significantly
different (p<0.05).
Discussion
Indeed, the present study contributes to the expanding knowledge regarding the toxicological effects of soil PCBs on
maize biochemistry and the influence of changes in soil pH on the underlying mechanisms. By investigating the
impact of Crude PCBs contamination and pH adjustments on various parameters such as plant height, stem girth,
relative water content, selected enzymes, soluble protein content, and malondialdehyde levels, the study provides
valuable insights into the potential toxic effects of PCBs on maize plants.
Maize prefers well-drained soils with a medium to fine texture. Maize grows best in slightly acidic to neutral soils.
Maize is a warm-season crop and prefers soil temperatures between 10°C (50°F) and 35°C (95°F) for optimal
growth. The soil should be sufficiently warm at planting time to promote seed germination and early root
establishment. Soil analysis results shows that it is suitable for maize.
The observed growth retardation in maize plants exposed to Crude PCBs-contaminated soil aligns with previous
studies, indicating the negative impact of PCBs on plant development [39, 51-52]. The partial reversal of this effect
by pH change suggests that altering the soil pH can influence the availability and behavior of PCBs in the soil,
leading to potential mitigation of their toxic effects. The favorable effect of pH 7.0 on growth parameters indicates
that this pH level may promote the immobilization of soil PCBs or enhance microbial degradation processes, leading
to reduced bioavailability and toxicity. The significant reduction in stem girth observed in maize plants grown in
Crude PCBs-contaminated soil further supports the detrimental effects of PCBs on plant growth and development.
The result suggests that PCBs may interfere with the normal physiological processes involved in stem development
and cell expansion, leading to reduced stem girth. The significant decrease in relative water content (RWC) of maize
plants exposed to Crude PCBs-contaminated soil highlights the impact of PCBs on water conduction and nutrient
uptake in plants. Impaired water conduction through the vascular tissues can disrupt the efficient transport of
nutrients, leading to poor plant growth and physiological stress. This finding indicates that the presence of PCBs in
the soil can disrupt the water balance in plants and contribute to their overall physiological imbalance.
Stress conditions, such as those induced by Crude PCBs and reduced water availability, can trigger stomata closure
in plants. This response restricts the entry of carbon dioxide (CO 2) into the leaves, leading to a reduced availability
of CO2 for photosynthesis and carbon fixation. Consequently, the chloroplasts in the leaves may be exposed to
excessive excitation energy, which can result in the generation of free radicals and oxidative stress. This
phenomenon has been documented in previous studies [53-54]. Maize plants, although considered moderately
drought-tolerant, may have limitations in their reactive oxygen species (ROS) scavenging system and other stress
tolerance mechanisms. ROS, such as superoxide radicals and hydrogen peroxide, are natural byproducts of
metabolic processes in plants. Under stress conditions, the production of ROS can increase, overwhelming the
plant's antioxidant defense mechanisms. This imbalance between ROS production and scavenging can lead to
oxidative stress, which can cause damage to cellular components and impair plant growth and development.
CAT is known as the most efficient enzyme in eliminating hydrogen peroxide. It has a high catalytic rate and can
efficiently break down H2O2 into water and oxygen. Unlike peroxidase, CAT is not saturated by H 2O2 at any
concentration. This enzyme is capable of handling high levels of H2O2 and plays a crucial role in the rapid
detoxification of this reactive molecule. GPx, on the other hand, also contributes to the elimination of H 2O2,
21
particularly when it is present in low concentrations. GPx catalyzes the reduction of H 2O2 using glutathione (GSH)
as a co-substrate, forming oxidized glutathione (GSSG) in the process. In situations where H 2O2 levels are low or
the GPx pathway is not saturated with substrate, GPx can effectively scavenge H 2O2. The GSH-dependent
antioxidative system involves both GPx and GR. GR plays a vital role in maintaining glutathione in its reduced form
(GSH) by catalyzing the reduction of GSSG back to GSH. This reaction helps to regenerate the pool of reduced
glutathione, which is essential for antioxidant functions. GSH acts as a scavenger of free radicals and participates in
various cellular processes related to redox balance and detoxification. The interactions and interplay between these
antioxidant enzymes can be complex, making the interpretation of results challenging. The activities and
contributions of CAT, GPx, and GR can vary depending on the concentration of H 2O2 and the overall redox state of
the system. Understanding the dynamics and coordination of these enzymes is crucial for comprehending the overall
antioxidative defense system in response to stress conditions.
In this study, the observed increase in activities of the antioxidant enzymes in the leaves of plants grown in Crude
PCBs-contaminated soil suggests the presence of impending oxidative stress. However, when the pH of the
contaminated soil was adjusted, there was a partial restoration of enzyme activities, although the underlying
mechanism remains unclear. It is worth noting that the MDA level was elevated while the SPC level was reduced
(Table 2). Oxidative stress becomes detrimental to cellular integrity when the antioxidant defense system becomes
overwhelmed by reactive oxygen species (ROS). ROS can react with unsaturated fatty acids in cellular and
subcellular membranes, leading to lipid peroxidation. The level of MDA is often used as an indicator of oxidative
stress caused by various xenobiotics.
Conclusion
This study provides compelling evidence of the toxicological effects of Crude PCBs in soil on maize and
demonstrates the potential of soil pH adjustment to partially reverse these effects. The findings reveal that Crude
PCBs led to stunted growth in maize, while adjusting the pH of the contaminated soil to 5.5 and 7.0 mitigated the
toxicological impact to some extent. However, it should be noted that further increasing the pH of the Crude PCBs-
contaminated soil resulted in the death of maize plants, indicating the inability to support any growth.
Ongoing research aims to quantify and characterize the PCBs present in the soil, as well as investigate the
underlying mechanisms involved in the partial reversal of the toxicological effects of PCBs on maize following pH
adjustment.
References
[1] Cok I, Hakan SM: Polychlorinated biphenyl levels in adipose tissue of primiparous women in Turkey.
Environ. Int. 30: 7-10. 2004.
[2] Diamond ML, Melymuk L, Csiszar SA, Robson M: Estimation of PCB stocks, emissions, and urban fate:
Will our policies reduce concentrations and exposure? Environ. Sci. Technol. 44:
2777-2783. 2010.
[3] Robson M, Melymuk L, Csiszar SA, Giang A, Diamond, ML, Helm P A: Continuing sources of PCBs: The
significance of building sealants. Environ. Int. 36: 506-513. 2010.
[4] Jartun M, Ottesen R T, Steinnes E, Volden T: Painted surfaces - important sources of polychlorinated
biphenyls (PCBs) contamination to the urban and marine environment. Environ. Pollut. 157: 295-302.
2009.
[5] Zhang L, Lohmann R: Cycling of PCBs and HCB in the surface ocean-lower atmosphere of the open
Pacific. Environ. Sci. Technol. 44: 3832-3838. 2010.
[6] Li YF, Harner T, Liu LY, Zhang Z, Ren NQ, Jia HL, Ma JM, Sverko E: Polychlorinated biphenyls in
global air and surface soil: Distributions, air-soil exchange, and fractionation effect.
Environ. Sci. Technol. 44: 2784-2790. 2010.
[7] Breivik K, Gioia R, Chakraborty P, Zhang G, Jones KC: Are reductions in industrial organic contaminants
emissions in richcountries achieved partly by export of toxic wastes? Environ. Sci.
Technol. 45: 9154-9160. 2011.
[8] Barber JL, Thomas GO, Kerstiens G, Jones KC: Current issues and uncertainties in the measurement and
modelling of airvegetation exchange and within-plant processing of POPs. Environ. Pollut. 128: 99-138.
2004.
[9] Collins C, Fryer M, Grosso A: Plant uptake of non-ionic organic chemicals. Environ. Sci. Technol. 40: 45-
52. 2006.
[10] Gerhardt, K. E.; Huang, X. D.; Glick, B. R.; Greenberg, BM: Phytoremediation and rhizoremediation of
organic soil contaminants: Potential and challenges. Plant Sci176: 20-30. . 2009.
22
[11] Mackova M, Macek T, Ocenaskova J, Burkhard J, Demnerova K, Pazlarova J: Biodegradation of

polychlorinated biphenyls by plant cells. Int. Biodeter. Biodegr. 39: 317-325. 1997.
[12] Chroma L, Moeder M, Kucerova P, Macek T, Mackova M: Plant enzymes in metabolism of
polychlorinated biphenyls. Fresen.Environ. Bull. 12: 291-295. 2003.
[13] Coleman, J.; Blake-Kalff, M.; Davies, E. Detoxification ofxenobiotics by plants: chemical modification and
vacuolar compartmentation. Trends Plant Sci. 2: 144-151. 1997.
[14] Singer AC, Crowley DE, Thompson IP: Secondary plantmetabolites in phytoremediation and
biotransformation. Trends Biotechnol. 21: 123-130. 2003.
[15] Heidari H: Yield Components and Seed Germination of Maize (Zea mays L.) at
Different Defoliation and Tassel Removal Treatments. Yield. 96(1): 42–43. 2013.
[16] Janmohammadi M, Dezfuli PM, Sharifzadeh F: Seed Invigoration Techniques To
Improve Germination and Early Growth of Inbred Line of Maize Under Salinity and
Drought Stress. Gen. Appl. Plant Physiology. 34(3-4): 215–226. 2008.
[17] McCall WW, Watanabe RT: Soil Reaction (pH). General Home Garden Series.
Hawaii: University of Hawai. 1980.
[18] Roy R, Finck A, Blair G, Tandon H: Plant nutrition for food security- A guide for
intergrated nutrient management. FAO Fertilizer and Plant Nutrition Bulletin (Vol. 16).
Rome: FAO (Food and Agricultural Organization). http://doi.org/10.1017/S0014479706394537 2006.
[19] Kohlmann F: What is pH, and how is it measured. A Technical Handbook for Industry,
86(2), 94–99. http://doi.org/10.1016/S0031-9406(05)61211-4 2003.
[20] YOKOGAWA: pH and ORP Learning Handbook (1st ed.). Tokyo, Japan: Yokogawa
Electric Coporation. Retrieved from http://www.yokogawa.com/2014.
[21] Albrecht G, Ketterings QM, Beckman J: Soil pH for Field Crops: Agronomy Fact
Sheet Series. New York. Retrieved from http://nmsp.css.cornell.edu.2005.
[22] MAFC: Tanzania National Variety List. Dar es salaam. 2014.
[23] NRCS: Soil Quality Indicators : pH. Natural resources Conservation Science (NRCS).
Washington. Retrieved from www.nssc.nrcs.usda.gov.1998.
[24] Plessis J: Maize production. Pretoria: Republic of South Africa. Retrieved from
www.nda.agric.za/publications.2003.
[25] Bier A: Electrochemistry: Theory and Practice. Loveland: Hach Company. Retrieved from
www.hach.com/smartprobes.2010.
[26] Hoskins BR: Soil Testing Handbook for Professionals in Agriculture, Horticulture,
Nutrient and Residuals Management. Soil Testing Handbook for Professional
Agriculturalists (3rd ed.). Maine: University of Maine. http://doi.org/10.1007/s13398-014- 0173-7.2.1997.
[27] Murray D: Measuring pH in Water or CaCl2 Using a pH Meter. pH Meter Procedure. SFU Soil Science
Lab. Retrieved from http://www.sfu.ca/soils/lab_documents/pH_Meter_Procedures.pdf.2011.
[28] Whitfield M, Butler R, Covington K: The determination of pH in estuarine waters: I. Definition of pH
scales and the selection of buffers. Oceanologica Acta. 8: 423–432. 1985.
[29] Deska J, Jankowski K: Effect of growing medium pH on germination and initial
development of some grassland plants. Acta Scientiarum Polonorum Seria Agricultura,
10(4), 45–56. Retrieved from http://www.agricultura.acta.utp.edu.pl/uploads/pliki/10(4)2011_5.pdf. 2011.
[30] FAO: The importance of soil organic matter: Key to drought-resistant soil and sustained
food production. (A. Bot & J. Benites, Eds.). Rome: FAO (Food and Agricultural
Organization). Retrieved from http://www.fao.org.2005.
[31] Mallarino AP: Corn and soybean response to soil pH level and liming. Integrated Crop
Management Conference. 93–102. 2011.
[32] Miransari M, Smith DL: Plant hormones and seed germination. Environmental and
Experimental Botany, 99, 110–121. http://doi.org/10.1016/j.envexpbot.2013.11.005.2014.
[33] Steed S, Reed J: Measuring pH of Soils. Florida: University of Florida. 2014.
[34] Fernández F, Hoeft R: Managing soil pH and crop nutrients. Illinois Agronomy
Handbook, (8), 91–112. Retrieved from http://www.ebooksmagz.com/pdf/managing-soilph-and-crop-
nutrients-129919.pdf. 2009.
[35] Darby H, Lauer J: Plant Physiology: Critical Stages in the Life of a Corn Plant. Plant
Physiology, 15–20.2004.
23
[36] Adeoye E, Lewis L, Whitehair R, Zitricki B: The Effects of Simulated Acid Rain on Corn Seed
Germination. Maryland, USA: Maryland University. Retrieved from
http://mda.maryland.gov/Documents/ag_brief/AgBrief_Agriculture.pdf.2013.
[37] Merry R H Acidity and Alkalinity of Soils. Environmental and Ecological Chemistry, II.Retrieved from
http://www.eolss.net/Eolss-sampleAllChapter.aspx. 2004.
[38] AOAC: Official Methods of Analysis.Association of Official Analytical Chemists.Washington DC P14.
1996.
[39] Adewole MB, Aboyeji AO: Yield and quality of maize from spent engine oil contaminated soils amended
with compost under screenhouse conditions. J Agrobiol 30: 9-19. 2013
[40] Schonfeld MA, Johnson RC, Carver BF, Mornhinweg DW: Water relations in winter wheat as drought
resistance indicator. Crop Sci. 28: 526-531. 1988.
[41] Black CA: Methods of soil analysis. Amer. Soc. Agron. Madison Wisconsin. Agronomy Part 2 No: 9.
1982.
[42] APHA/AWWA: Standard methods for the examination of water and waste water quality. J Environ Toxicol
Water quality 11: 72-82. 1985.
[43] Gornal AG, Bardawill JC, David MM: Determination of serum proteins by means of biuret reaction J. Biol.
Chem. 177: 751-760. 1949.
[44] Bird RP, Drapper HH, Valli VE: Toxicological evaluation of Malondialdehyde: a 12-month study of mice,
J. Toxicol. Environ. Health. 10: 897-905. 1982.
[45] Jollow DJ, Mitchell JR, Gillette JR: Bromobenzene induced liver necrosis: Protective role of glutathione
and evidence for 3,4-Bromobenzene oxide as the hepatotoxic metabolite. Pharmacol. 11: 151-169. 1974.
[46] Misra HP, Fridovich I: The role of superoxide anion in the antioxidation of epinephrine and a simple
assay of superoxide dismutase. J. Bio1. Chem. pp 2417-3170. 1972.
[47] Sinha KA: Colorimetric assay of Catalase. Anal. Biochem. 47: 389-394. 1971.
[48] Nakano G, Asada, K: Hydrogen peroxidase is scavenged by ascorbate specific peroxidase in spinach
chloroplasts. Plant cell Physiol. 22: 867-880. 1981.
[49] Putter J: Peroxidase. In methods of Enzymatic Analysis, 2, H.U. bergmeyer, Ed., Academic press /
Verlag chemie Weinheim. p 685. 1974.
[50] Duncan DB: Multiple range and multiple F test. Biomet. 11:1-10. 1955.
[51] Adeyemi O: The Effect of Palm Kernel Oil (PKO) Biodiesel-Contaminated Soil on Morphological and
Biochemical Properties of Zea mays. J. Plant Biochem. Physiol. 138 (2): 1-6. 2014.
[52] Okon IE, Udofot EE: Response of Telfairia occidentalis (Hook) to arbuscular mycorrhizal fungi and
Gliricidia sepium leaves manure in spent engine oil contaminated soil. World J Agric Sci 8: 20–25. 2012.
[53] Islam MR, Hu Y, Mao S, Jia P, Eneji AE: Effects of water-saving superabsorbent polymer on antioxidant
enzyme activities and lipid peroxidationin corn (Zea maysL.) under drought stress. J Sci Food Agric 91:
813-819. 2011b.
[54] Islam MR, Xue X, Mao S, Ren C, Eneji AE: Effects of water-saving superabsorbent polymer on
antioxidant enzyme activities and lipid peroxidation in oat (Avena sativaL) under drought stress. J Sci Food
Agric 91: 680-686. 2011a.
24
Ebhohon et al.

Hibiscus Sabdariffa L. Calyx Ethanol Extract Ameliorates Thioacetamide-
Induced Hepatic Oxidative Stress in Male Wistar Rats
*1 Ebhohon, Shirley O., 2Asoya, Ekene V., 2Iyare, Harrison E.

1
Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike.
Nigeria
2
Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City. Nigeria
Abstract
This study investigated the protective effect of Hibiscus sabdariffa L. calyx ethanol extract against hepatotoxic effect
of thioacetamide (TAA) in male Wistar rats. Twenty-five rats were randomly divided into five groups of five rats
each. Group 1: control, Group 2: received a single dose of TAA (300 mg/kg b.w.t) intraperitoneally, Group 3: was
pre-treated with TAA prior to oral administration of silymarin (100 mg/kg b.w.t), while Groups 4 and 5 were
administered TAA and extract at varying doses of 250 and 500 mg/kg b.w.t respectively for one week. Liver marker
enzymes (ALT, AST and ALP) as well as its products (bilirubin and total protein) were determined in serum.
Malondialdehyde (MDA) and reduced glutathione (GSH) level, as well as superoxide dismutase (SOD), catalase
(CAT) and glutathione peroxidase (GPx) activities were assayed in liver homogenate. Exposure to TAA induced
significant (p˂0.05) increases in ALT, AST, ALP and total bilirubin, while total protein was significantly (p˂0.05)
decreased. Significant (p˂0.05) decreases in SOD, CAT, GSH and GPx accompanied TAA treatment while MDA
level was significantly (p˂0.05) increased. The extract significantly restored liver function indices and antioxidant
status of the liver to near normal levels suggesting that the extract has a protective effect against TAA-induced
hepatic toxicity.
Keywords: Hibiscus sabdariffa L. Calyx, Liver, Oxidative stress, Rats, Thioacetamide.
Introduction
Hibiscus sabdariffa is an herbaceous shrub belonging to the family of Malvaceae, native to Asia (India to Malaysia)
or Tropical Africa (1, 2). In Ayurvedic literature of India, different parts of this plant have been recommended for
various ailments like hypertension, pyrexia and liver disorders. Traditionally, it is used as an antiseptic, aphrodisiac,
astringent, diuretic, refrigerant, emollient, purgative, sedative, stomachic and tonic (3). H. sabdariffa is rich in
phenolic compounds especially anthocyanins (the pigments responsible for the red color of the calyces) and is a rich
source of antioxidants. Numerous studies have shown that anthocyanins and other phenolic compounds are the
major source of antioxidant in Roselle (H. sabdariffa) extract (4). They can scavenge free radical species, partake in
regeneration of other antioxidants and protect cell constituents against oxidative damage (5). Silymarin is derived
from the seeds of Silybum marianum L. It belongs to the family: Asteraceae or compositae. It is commonly called
“milk thistle”. For many centuries, silymarin has been used as a natural remedy for liver and biliary tract diseases.
Its bioactive constituents are flavonolignans which includes silybin, silydianin and silychristin, collectively known
as silymarin (6). Numerous reports have shown that silymarin protects liver cells from a wide variety of toxins,
including acetaminophen, ethanol, carbon tetrachloride (CCl 4) and D-galactosamine (7,8,9,10,11,12) via many
mechanisms such as anti-oxidation, anti-lipid peroxidation, enhanced detoxification and protection against
glutathione depletion (13). The liver is the largest internal organ of the human body and plays a significant role in
food metabolism, detoxification of drugs and foreign compounds, thereby making it one of the most viable target
organs for toxicity (14,15). Thioacetamide (TAA) is an organosulfur, white crystalline compound having formula
CH3CSNH2. It is a hepatotoxicant (16,17) and a carcinogen (Class 2B), designated as a human carcinogen (18).
TAA has been used extensively to induce acute and chronic liver injury in experimental animals (19,20). This is due
to its effect on protein synthesis, RNA, DNA and Gama-glutamyl transpeptidase activity (21). In rat liver,
cytochrome P450 is thought to be responsible for the metabolism of TAA (22). The flavin-containing monooxygenase
(FMOs) systems are also involved in the biotransformation of TAA. The presence of cytochrome P 450 in liver
microsomes converts TAA to its metabolic intermediate, thioacetamide S-oxide (TAASO) and reactive metabolite,
thioacetamide S, S dioxide (TAASO2), which covalently binds to hepatic macromolecules, leading to centrilobular
necrosis and hepatic injury (23). The generation of a large amount of ROS due to TAA can disrupt the antioxidant
defense mechanism (24) and damage biomolecules such as lipids, proteins, and DNA, which in turn can impair
*Corresponding Author’s E-mail: so.ebhohon@mouau.edu.ng
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Ebhohon et al.
cellular structure and function (25). Increased free radical production or reduced free radical scavenging capacity is
responsible for oxidative stress. Oxidative stress is a major imbalance between free radical development and
antioxidant (26,27) which leads to cell death and tissue injuries (28,29).

Chemicals
Thioacetamide (TAA), silymarin and all other chemicals (of analytical grade) used in this study were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Randox (Antrim, U.K) assay kits for alanine aminotransferase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein and bilirubin were used in this study.
Preparation of TAA and Silymarin
Thioacetamide (TAA) was dissolved in normal saline and administered at 300 mg/kg body weight (b.w.t)
intraperitoneally (i.p.) to induce oxidative stress. Silymarin (1g) was dissolved in 100 ml of olive oil and
administered at an oral dose of 100 mg/kg b.w.t.
Plant Collection and Authentication
Dried calyces of Hibiscus sabdariffa were purchased from a local market at Ikwuano Local government area of Abia
State. The plant was authenticated as Hibiscus sabdariffa by Dr Garuba Omosun, a plant taxonomist in the
Department of Plant Science and Biotechnology, Michael Okpara University of Agriculture Umudike, with voucher
specimen number MOUAU/COLNAS/PSB/16/A286. The Calyxes was air dried at room temperature for three
weeks, thereafter, the weight was then measured on a sensitive weighing balance and recorded.
Plant Extraction
The calyces of Hibiscus sabdariffa were pulverized into fine powder and a known quantity (200 g) of the fine
powder was poured into a glass extraction jar. The pulverized calyces were macerated in 500 ml of ethanol for 72
hours, with occasional shaking of the jar to increase extraction. The macerated calyces were strained through a
muslin cloth and then filtered using No 1 Whatman filter paper. The filtrate was collected and stored in a glass
beaker, and then put in a freeze dryer. The filtrate was allowed to dry at ≤ -40ºC for 24 hours. The freeze-dried
extract was stored at 4ºC in an air tight glass container until required for biochemicals assays.
Qualitative Phytochemical Analyses of Hibiscus sabdariffa calyx
Phytochemical screening of Hibiscus sabdariffa L. calyx ethanol extract was carried out to identify secondary
metabolites: flavonoids, saponins, tannins, phenols and glycosides using standard phytochemical methods
(30.31.32,33).
Determination of Lethal Dose (LD50)
Acute toxicity study was carried out on the ethanol extract according to the method described by (34).
Experimental Animals and Design
Healthy adult male Wistar rats weighing 100-120 g were obtained from the animal house unit of the Department of
Biochemistry, University of Nigeria, Nsukka, Nigeria. The animals were kept in wire meshed wooden cages at a
temperature of 25±ºC, in a 12hr light-dark cycle for seven (7) days before the commencement of the experiment.
The animals were maintained under standard housing condition with free access to a standard diet and clean
drinking water ad libitum during the experimental study. The rats were maintained in accordance with the
recommendation of the guide for the care and use of Laboratory animals (35). The animals were randomly assigned
into five (5) groups of five (5) animals each and were treated as follow: Group 1 served as control; group 2 received
thioacetamide (TAA) 300 mg/kg b.w.t intraperitoneally (i.p.), group 3 received TAA (300 mg/kg b.w.t.; i.p.) and
silymarin (100 mg/kg b.w.t) orally, while groups 4 and 5 were given TAA (300 mg/kg b.w.t.; i.p.), and then 250 and
500 mg/kg body weight of the extract respectively via a gavage. All the experimental groups except group 1
received a single dose of TAA on the first day of the experiment. Groups 3-5 received silymarin and extract from
day 2 to day 7 after exposure to TAA. All the animals were sacrificed after day 7 of the experiment.
Ethical Statement
All the experimental handling procedures were performed in strict accordance with protocols approved by the
Animal Care and Ethical Committee of Michael Okpara University of Agriculture, Umudike.
Biochemical Analysis
Blood sample and tissue collection
At the end of the seventh (7th) day, the rats were fasted overnight, anaesthetized by chloroform inhalation in a closed
chamber and thereafter sacrificed. Blood samples were collected via cardiac puncture and put in plain blood sample
bottles. After blood coagulation, the sample tubes were centrifuged at 4000 rpm for 10 minutes to obtain serum
which was used for liver function tests. The liver samples were excised, trimmed of connective tissue and washed
thoroughly in ice cold saline.
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Ebhohon et al.
Preparation of liver homogenates

Ten percent (10%) of tissue homogenates were prepared by homogenizing liver in 0.05 M cold phosphate buffer
(pH 7.4). The homogenates were centrifuged at 4000 rpm for 10 minutes and the supernatant was used for the
estimation of CAT, SOD, GSH, GPx and MDA.
Liver Function Tests
The activities of AST, ALT, and ALP, as well as the concentrations of total bilirubin and total protein (TP) were
determined in serum using their respective Randox kits.
Antioxidant Assays
Estimation of Catalase Activity
Catalase activity in liver homogenate was determined using the modified method described by Cohen et al. (36).
Estimation of SOD Activity
Superoxide dismutase (SOD) activity in liver homogenate was determined using the method described by Misra and
Fridovich (37).
Estimation of GSH Levels
GSH levels in liver homogenate was determined using the method described by Tietze (38).
Estimation of Glutathione Peroxidase (GPx) Activity
Glutathione Peroxidase (GPx) Activity in liver homogenate was determined using the method described by Flohe
and Guùzler (39).
Assessment of Lipid Peroxidation
The concentration of MDA in liver homogenates was determined using the method described by Okhawa et al. (40).
Statistical Analysis
Data are presented as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) using a computer
software, graph pad prism, version 7. Means were compared using Dunnett‟s multiple range tests. Values with
p<0.05 were considered statistically significant.
Results
LD50 of Ethanol Extract of Hibiscus sabdariffa calyx
The LD50 of ethanol extract of Hibiscus sabdariffa calyx was greater than 5000 mg/kg.
Table 1: Phytochemical constituents of Ethanol Extract of Hibiscus sabdariffa calyx

Phytochemical Constituents Hibiscus sabdariffa calyx
Flavonoids +
Saponins +
Tannins +
Phenol +
Glycosides +
Key: + = detected
Effect of Ethanol Extract of Hibiscus Sabdariffa Calyx against TAA induced Changes on Liver Function
Indices
The effect of ethanol extract of Hibiscus sabdariffa calyx on serum ALT, AST, ALP, bilirubin and total protein of
rats exposed to thioacetamide (TAA) are presented in Table 2. The results show significant (p˂0.05) increases in
liver enzyme activities (ALT, AST and ALP) as well as increased level of total bilirubin in serum of group 2 rats,
when compared to control. Administration of extract at varying doses of 250 and 500 mg/kg b.w.t significantly
improved alterations in the activities of serum ALT, AST and ALP. Standard control drug, silymarin given to the
experimental animals at a dose of 100 mg/kg also significantly (p˂0.05) reduced the elevated levels of ALT, AST
and ALP in serum (Table 2). However, there was a significant (p˂0.05) decrease in serum total protein of
thioacetamide (TAA) treated group when compared to control. Administration of extract, as well as silymarin
significantly (p˂0.05) elevated serum level of total protein, when compared to the TAA-treated group. The increase
in serum total protein by the extract, was dose-dependent. In this study, the ameliorative effect of 500 mg/kg b.w.t of
the extract against TAA- induced toxicity was almost comparable to that of silymarin.
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Ebhohon et al.
Effect of Ethanol Extract of Hibiscus Sabdariffa Calyx against TAA induced Changes on Liver Homogenate
Antioxidant Parameters
The effect of thioacetamide (TAA) and ethanol extract of Hibiscus Sabdariffa Calyx on superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GPx) activities, as well as glutathione (GSH) and malondialdehyde
(MDA) levels of liver homogenates are presented in Table 3. The activities of SOD, CAT and GPx were
significantly (p˂0.05) decreased in the TAA treated rats compared to control. Administration of extract (250 and
500 mg/kg) and Silymarin (100 mg/kg) significantly (p˂0.05) increased the activities of SOD, CAT and GPx when
compared to TAA treated group. However, GSH and MDA levels in liver homogenates were significantly (p˂0.05)
decreased and increased respectively in rats treated with TAA when compared to control, while administration of the
varying doses of the extract (250 and 500 mg/kgb.w.t) and 100 mg/kg b.w.t of silymarin significantly increased and
decreased GSH and MDA levels respectively in liver homogenate when compared to the group that was
administered TAA alone.
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Ebhohon et al.
Table 2: Effect of Ethanol Extract of Hibiscus sabdariffa calyx on serum ALT, AST, ALP, bilirubin and total protein of Wistar rats exposed to
thioacetamide (TAA).
Treatment Groups ALT (IU/L) AST (IU/L) ALP (IU/L) Total Bilirubin Total protein (g/dl)
(mg/dl)
Control 22.19 ± 1.98b 18.98 ± 1.97b 50.04 ± 0.99b 0.38 ± 0.01b 5.16 ± 0.26b
Thioacetamide(300mg/kg) only 33.71 ± 1.45 28.74 ± 1.39 58.61 ± 1.42 0.97 ± 0.15 4.90 ± 0.09
Thioacetamide (300 mg/kg 25.68 ± 0.50b 23.83 ± 0.68b 50.50 ± 0.72b 0.45 ± 0.06b 5.47 ± 0.38b
bwt) and Silymarin (100 mg/kg
bwt)
bwt) and Hibiscus sabdariffa
calyx (250 mg/kg bwt)
Values are expressed as mean ± SEM (n =5). Values with superscript „b‟ across a column are statistically significant relative to the untreated thioacetamide group
of rats.
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Ebhohon et al.
Table 3: Effect of Ethanol Extract of Hibiscus sabdariffa calyx on liver homogenate SOD, CAT, GSH, GPx and MDA of Wistar rats exposed to
thioacetamide (TAA).
Treatment Groups SOD (U/mL) CAT (U/mL) GSH (U/mL) GPx (U/mL) MDA (mmole/mL)
Control 14.35 ± 0.16b 15.56 ± 0.71b 25.21 ± 1.47b 10.48 ± 0.01b 3.43 ± 0.21b
Thioacetamide(300mg/kg) only 12.84 ± 0.54 12.34 ± 0.30 14.12 ± 0.03 3.17 ± 0.10 4.94 ± 0.19
bwt) and silymarin (100 mg/kg
bwt)
Values are expressed as mean ± SEM (n =5). Values with superscript „b‟ across a column are statistically significant relative to the untreated thioacetamide group
of rats.
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Ebhohon et al.
Discussion
Phytochemicals are bioactive chemical compounds derived from plants, conjectured to have protective and curative
properties (41). In this study, the phytochemicals present in ethanol extract of Hibiscus sabdariffa calyx were:
flavonoids, saponins, tannins, phenol and glycosides. This finding is similar to a study reported by (42). Marker
enzymes are extensively used to access liver damage in experimental studies. Raised levels of serum enzymes are
suggestive of cellular leakage and loss of functional integrity of cell membrane in liver (43,44). Liver damage is
associated with cellular necrosis, increase in tissue lipid peroxidation and depletion of reduced glutathione levels
(43). In this study, the activities of liver enzymes were elevated in serum of Wistar rats administered thioacetamide
(TAA), indicative of hepatic injury. This may be due to necrosis or membrane damage, which releases the enzyme
into blood circulation, hence it can be measured in serum. Disruption of the membrane‟s well-organized lipid-
bilayer may be due to the presence of reactive oxygen species (ROS) produced due to oxidative stress, which leads
to the escape of detectable amount of these enzymes out of the cell into the extracellular fluid (44). The protective
effect of the extract against TAA- induced hepatic injury was confirmed by reductions in serum levels of ALT, AST
and ALP to near normal levels. Bilirubin is derived from the regular degradation of haemoglobin from the red blood
cells and excreted from the liver in the bile. It is normally present in the blood in small amounts and used by the
liver to produce liver. Increase in bilirubin due to TAA administration may be due to liver cell damage, which result
in the cells not been able to excrete bilirubin in the normal way, thereby causing a build-up of bilirubin in the blood
and extracellular fluid. It may also be due to decreased hepatic clearance (45). Treatment with extract reduced the
concentration of bilirubin in the serum of TAA treated rats, suggestive of the protective effect of the extract and
silymarin. Estimation of total protein in the body helps to evaluate normal liver function from an impaired one. This
is because majority of plasma protein like albumin and globulin are produced in the liver (46). A decrease in total
protein amongst the rats administered TAA, may indicate hepatocellular injury. However, administration of extract
ameliorated total protein in serum. Oxidative stress occurs when there is an imbalance between the production of
reactive oxygen species and the antioxidant defense system. Disruption of the cell‟s normal redox state can cause
lethal effects through the production of peroxides and free radicals that damage all components of the cell, including
proteins, lipids and DNA (47). In this study, thioacetamide induced oxidative damage and hepatotoxicity by
elevating and depleting malondialdehyde (MDA) and glutathione (GSH) levels respectively, while decreases in the
activities of endogenous antioxidant enzymes (SOD, CAT, and GPx) in liver were also observed. Thioacetamide
may have induced oxidative stress in the experimental animals by generating free radicals. Free radicals attack
nucleophilic targets in the cell and membrane phospholipids, thereby leading to disruption of membrane viability
and integrity (48,49,50). Administration of extract to the animals administered thioacetamide significantly
ameliorated the antioxidant status of the liver. The protective property of the extract may be due to the presence of
potent antioxidants: polyphenolic, flavonoids and anthocyanins (4) known to have significantly decreased the
formation of malondialdehyde (MDA) and oxidative damage in the liver (51,52).
Conclusion
This study indicates the hepatoprotective effect of Hibiscus sabdariffa calyx ethanol extract against thioacetamide
(TAA) induced hepatotoxicity and oxidative stress in the liver of male Wistar rats. The ameliorative effect of the
extract is evident by a significant restoration of liver function indices, malondialdehyde (MDA) levels and
antioxidant enzymes activities in TAA-treated rats.
Conflict of interest
There were no conflicts of interest declared by the authors.
Authors’ declaration
The authors thereby declare that the work in this article is their own original work, and that they will be held liable
for any claims connected to the content of this article.
Acknowledgement
The authors appreciate Raymond C. Ibeh, Department of Biochemistry, Federal University of Technology, Owerri,
for his technical assistance.
References
1. Bahaeldeen BM, Adbelatif AS, Adbelhafiz AD. Roselle (Hibiscus Sabdariffa L.) in Sudan, cultivation and
their uses. Bull. Env. Pharmacol. Life Sci 1(6):48-54. 2012.
2. Brown F, González J, Monan, M. Pharmacognostic, Physicochemical and phytochemical analysis of two
cultivars from Hibiscus sabdariffa L. in Cuba. OALibJ 6 (12): 1-8. 2019.
3. Mishra S, Dash DK, Das AK. South Indian leafy vegetable Gongura (Hibiscus sabdariffa L.) as an
important medicinal herb: a review. J. Pharmacogn. Phytochem 7(1S): 2534-2538. 2018.
4. Maciel LG, Vieira do Carmo MA, Azevedo L, Daguer H, Molognoni L, Mendes de Almeida M, Granato D,
31
Ebhohon et al.
Rosso ND. Hibiscus sabdariffa anthocyanins-rich extract: Chemical stability, in vitro antioxidant and
antiproliferative activities. Food Chem. Toxicol 113: 187-197. 2018.
5. Mohamad NR, Abdul Gani SS, Abdul Wahab R, Darham W. Natural antioxidant from Hibiscus Sabdariffa
extract: Assessments on extraction yield, antioxidant capacity and total polyphenol content of different
polarities of solvent extracts. JSST 2(2): 52-60. 2022.
6. Doğan D, Meydan İ, Kömüroğlu AU. Protective effect of silymarin and gallic acid against cisplatin-
induced nephrotoxicity and hepatotoxicity. Int. J. Clin Pract 2022: Article ID 6541026. 2022.
7. Abed NA, Khalaf MM, Alnori MKJ. the potential effect of silymarin against paracetamol-induced
hepatotoxicity in male albino rats. Pharmacogn J 14(5): 558-564.2022.
8. Bektur NE, Sahin E, Baycu C, Unver G. Protective effects of silymarin against acetaminophen-induced
hepatotoxicity and nephrotoxicity in mice. Toxicol. Ind. Health 32(4):589-600. 2016.
9. Talbi A, Khelili K, Remita F, Abdennour C.The benefit of Silybum marianum in ethanol-induced
reprotoxicity of male Wistar rat. Brazilian J. Pharm. Sci 58: 1-10. 2022.
10. Sokar SS, El-Sayad MES, Ghoneim MES, Shebl A.M. Combination of Sitagliptin and Silymarin
Ameliorates Liver Fibrosis Induced by Carbon Tetrachloride in Rats. Biomed. Pharmacother 89:98–
107.2017.
11. Javed S, Ahsan W, Kohli K. Pharmacological influences of natural products as bioenhancers of silymarin
against carbon tetrachloride-induced hepatotoxicity in rats. Clin Phytosci 4(18): 1-9. 2018.
12. Raj PV, Nitesh K, Chandrashekhar HR, Mallikarjuna Rao C, Venkata Rao J, Udupa N. Effect of Lecithin
and silymarin on D-galactosamine induced toxicity in isolated hepatocytes and rats. Indian. J. Clin.
Biochem 25(2):169-74. 2010.
13. Yaman T, Uyar A, Kaya M S, Keles ÖF, Uslu B A, Yener Z. Protective effects of silymarin on
methotrexate-induced damages in rat testes. Braz. J. Pharm. Sci 54(1): e17529. 2018.
14. Mohamed Saleem TS, Madhusudhana Chetty C, Ramkanth S, Rajan VST, Mahesh Kumar K,
Gauthaman.K: Hepatoprotective herbs – A review. Int J Res Pharm Sci 1: 1-5. 228.2010.
15. Katoonizadeh A: Liver regeneration. In: Liver Pathophysiology: therapies and antioxidants. P Muriel (eds.)
pp. 113–123, 2017.
16. Abdel-Rahman RF, Fayed HM, Asaad GF, Ogaly HA, Hessin AF, Salama AAA, Abd El-Rahman SS, Arbid
MS, Mohamed MAE. The involvement of TGF-Β1/FAK/α-SMA pathway in the antifibrotic impact of rice
bran oil on thioacetamide-induced liver fibrosis in rats. PLoS ONE 16: e0260130. 2021.
17. Vokálová L, Lauková L, Čonka J, Melišková V, Borbélyová V, Bábíčková J, Tóthová L, Hodosy J, Vlková
B, Celec P. Deoxyribonuclease partially ameliorates thioacetamide-induced hepatorenal injury. Am. J.
Physiol. Liver Physiol 312: G457–G463. 2017.
18. Low TY, Leow CK, Salto-Tellez M, Chung MC: A proteomic analysis of thioacetamide-
induced hepatotoxicity and cirrhosis in rat livers. Proteomics 4 (12):3960–3974. 2004.
19. Bhakuni GS, Bedi O, Bariwal J, Deshmukh R, Kumar P. Animal models of hepatotoxicity. Inflamm Res 65:
13–24. 2016.
20. Hajovsky H, Hu G, Koen Y, Sarma D, Cui W, Moore DS, Staudinger JL, Hanzlik RP. Metabolism and
toxicity of thioacetamide and thioacetamide S-oxide in rat hepatocytes. Chem. Res. Toxicol 25: 1955–1963.
2012.
21. Zargar S, Wani TA, Alamro AA, Ganaie MA. Amelioration of thioacetamide-induced liver toxicity in
Wistar rats by rutin. Int. J. Immunopathol. Pharm 30: 207–214. 2017.
22. Chandrashekar DV, DuBois BN, Rashid M, Mehvar R. Effects of chronic cirrhosis induced by
intraperitoneal thioacetamide injection on the protein content and Michaelis-Menten kinetics of cytochrome
P450 enzymes in the rat liver microsomes. Basic Clin Pharmacol Toxicol 132(2):197-210. 2023.
23. Ramaiah SK, Apte U, Mehendale HM: Cytochrome P4502E1 induction increases thioacetamide liver injury
in diet-restricted rats. Drug Metab Dispos 29:1088–95. 2001.
24. Zargar S, Wani TA, Alamro AA, Ganaie MA. Amelioration of thioacetamide-induced liver toxicity in
Wistar rats by rutin. Int. J. Immunopathol. Pharmacol 30(3):207-214. 2017.
25. Türkmen NB, Yüce H, Taşlıdere A, Şahin Y, Çiftçi O. The ameliorate effects of nerolidol on
thioacetamide-induced oxidative damage in heart and kidney tissue. Turk. J. Pharm. Sci 19(1):1-8. 2022.
26. Vona, Rosa, Lucia Pallotta, Martina Cappelletti, Carola Severi, Paola Matarrese. "The impact of oxidative
stress in human pathology: Focus on gastrointestinal disorders" Antioxidants 10 (2): 1-26. 2021.
27. Klaunig JE. Oxidative stress and cancer. Curr Pharm Des 4: 4771–4778. 2018.
28. Behl T, Makkar R, Sehgal A, Singh S, Sharma N, Zengin G, Bungau S, Andronie-Cioara FL, Munteanu
MA, Brisc MC, Uivarosan D, Brisc C. Current trends in neurodegeneration: Cross talks between oxidative
32
Ebhohon et al.
stress, cell death, and inflammation. Int. J. Mol. Sci 22(14): 1-19. 2021.
29. He L, He T, Farrar S, Ji L, Liu T, Ma X. Antioxidants maintain cellular redox homeostasis by elimination of
reactive oxygen species. Cell Physiol Biochem 44:532-553. 2017.
30. Harborne JB: Phytochemical Methods. A guide to modern techniques of plant analysis. Chapman & Hall,
London, pp. 40-75, 1973.
31. Harborne JB: Phytochemical methods: a guide to modern techniques of plant analysis. (2nd ed.)
Chapman & Hall, London, 1984.
32. Sofowora A: Medicinal plants and Traditional medicine in Africa. Spectrum Books Ltd, Ibadan, 1993.
33. Trease GE, Evans WC: Pharmacognosy. (15th ed.) London: Saunders Publishers. 2002.
34. Lorke D: A new approach to practical acute toxicity testing. Archives of Toxicology 54: 275-87. 1983.
35. National Institutes of Health Office of Laboratory Animal Welfare Public Health Service policy on the
humane care and use of laboratory animals. Bethesda, MD: NIH, 2002.
36. Cohen G, Dembiec D, Marcus J: Measurement of catalase activity in tissue extracts. Anal Biochem 34: 30-
38. 1970.
37. Misra HP, Fridovich I: The role of superoxide anion in the autooxidation of epinephrine and simple assay
for superoxide dismutase. J. Biol. Chem 247: 3170 - 3175. 1972.
38. Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized
glutathione: applications to mammalian blood and other tissues. Analytical Biochemistry 27: 502 – 522.
1969.
39. Flohe L, Guùzler WA: Assay of glutathione. Methods in enzymology 105: 114-121. 1984.
http://dx.doi.org/10.1016/S0076-6879 (84)05015-1.
40. Okhawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.
Anal Biochem 95: 351 – 358. 1979.
41. Bansal A, Priyadarsini C. Medicinal properties of phytochemicals and their production. natural drugs from
plants. IntechOpen. 2022.
42. Suniarti DF, Suwandi T, Putri SA, Kurnia D. Potential of Hibiscus sabdariffa L. Calyx (Rosella) extract as
antibacterial agent in dental disease: Phytochemical and chemical components profiling. J. Adv. Pharm.
Technol. Res 13(3):202-206. 2022.
43. Erfan OS, Sonpol HM, Abd El-Kader M. Protective effect of rapamycin against acrylamide-induced
hepatotoxicity: The associations between autophagy, apoptosis, and necroptosis. Anat. Rec 304 (9): 1984–
1998. 2021.
44. Rahbardar MG, Farmad HC, Hosseinzadeh H, Mehri S. (2021). Protective effects of selenium on
acrylamide-induced neurotoxicity and hepatotoxicity in rats. Iran. J. Basic. Med. Sci 24 (8): 1041–1049.
2021.
45. Eftekhari A, Ahmadian E, Azarmi Y, Parvizpur A, Fard JK, Eghbal MA. The
effects of cimetidine, N-acetylcysteine, and taurine on thioridazine metabolic
activation and induction of oxidative stress in isolated rat hepatocytes. Pharm. Chem.
J 51 (11): 965–969. 2018.
46. Lala V, Zubair M, Minter DA: Liver Function Tests. In: StatPearls. Treasure Island (FL): StatPearls
Publishing. 2022.
47. Juan CA, Pérez de la Lastra JM, Plou FJ, Pérez-Lebeña E. The chemistry of reactive oxygen species (ROS)
revisited: Outlining their role in biological macromolecules (DNA, lipids and proteins) and induced
pathologies. Int. J. Mol. Sci 22(9):4642. 2021.
48. Bashandy SA, Alaamer A, Moussa SA, Omara E. Role of zinc oxide nanoparticles in alleviating hepatic
fibrosis and nephrotoxicity induced by thioacetamide in rats. Can. J. Physiol. Pharmacol 16: 1–8. 2017.
49. Ganesan K, Sukalingam K, Xu B. Solanum trilobatum L. ameliorate thioacetamide-induced oxidative stress
and hepatic damage in albino rats. Antioxidants 6(3): 68. 2017.
50. Seema Zargar, Mona Alonazi, Humaira Rizwana, Tanveer A. Wani, "Resveratrol reverses thioacetamide-
induced renal assault with respect to oxidative stress, renal function, DNA damage, and cytokine release in
Wistar rats", Oxid. Med. Cell. Longev vol Article ID 17029592019:1-8. 2019.
51. Wang J, Cao X, Jiang H, Qi Y, Chin KL, Yue Y: Antioxidant activity of leaf extracts from different Hibiscus
sabdariffa accessions and simultaneous determination five major antioxidant compounds by LC-Q-TOF-
MS. Molecules 9:21226–21238. 2014.
52. Alalkam E. Hibiscus sabdariffa aqueous extract ameliorates thioacetamide-induced hepatic encephalopathy
in guinea pigs: role of ammonia extraction. Al-Azhar j. pharm. Sci 58(2): 19-36. 2018.
33
Ikponmwosa-Eweka and Omoregie

Comparative Studies on the Phytochemical Content and In Vitro Antioxidant

Capacity of Methanol Extract of Spondias mombin and Chasmanthera
dependens
*Omorede Ikponmwosa-Eweka1 and Ehimwenma Sheena Omoregie2

1. Department of Medical Biochemistry, School of Basic Medical Sciences, University of Benin, Benin City, Edo State,
2. Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City, Edo State, Nigeria
Abstract
Spondias mombin and Chasmanthera dependens are commonly used in traditional medicine. This study investigated
the phytochemical constituents and in vitro antioxidant potential of Spondias mombin stem bark and Chasmanthera
dependens root. The in vitro antioxidant activity study was carried out using ferric reducing antioxidant potential
(FRAP), total antioxidant capacity, diphenyl-2-picryl-hydrazyl (DPPH) scavenging activity, and reducing potential
activity. Qualitative phytochemical screening of the extracts showed that the plants were rich in flavonoids, tannins,
saponins, phenols, terpenoids, and reducing sugar. Both extracts had appreciable quantities of these phytochemicals.
However, S. mombin had a significantly (p ˂ 0.05) higher amount of total flavonoids, total tannins, total phenols, and
proanthocyanidins content than C. dependens. Results from the antioxidant assay conducted revealed that the extracts
had antioxidant potentials. Although, C. dependens had a significantly (p > 0.05) higher FRAP value than S. mombin.
The methanol extract of S. mombin had a better antioxidant effect wherein it could scavenge DPPH radical better,
reduce molybdenum (VI) to molybdenum (V), and its dose-dependent lowering activity. This study revealed that S.
mombin and C. dependens are rich in phytochemicals, which may account for their antioxidant properties. These
findings provide more evidence that these herbs have therapeutic use.
Keywords: Spondias mombin, Chasmanthera dependens, Antioxidant, Phytochemical, Extract
Introduction
Over time, it has become apparent that plants are essential natural reservoirs of secondary metabolites. As a result,
extensive efforts are being directed toward researching and developing phytomedicines as key therapeutic agents for
treating various diseases [1]. This is attributable to their indispensable biological activities, such as detoxifying
compounds, inhibiting cellular damage, and anti-inflammatory effects [2,3]. Several lines of the study indicate that
consuming natural antioxidants can reduce the incidence of various health issues, such as cancer, peptic
ulcers, neurological disorders, and diabetes [4,5]. The beneficial roles of natural antioxidants in maintaining human
health result from their hydrogen-donating propensity in quenching free radicals such as reactive oxygen or nitrogen
species (ROS/RNS), thereby preventing the oxidative damage of cells caused by the action of free radicals [6]. Free
radicals are highly reactive chemical species, most notably hydroxyl radicals and superoxide ions (O 2), which react
with essential biological molecules such as phospholipids, proteins, and nucleic acids, causing oxidative damage in
healthy cells of the body [7]. Recent investigations [8] have established that the protective impact of antioxidants
against oxidative stress is initiated by the binding interaction of essential intracellular signaling proteins with
antioxidants, which regulates their expression and activity. Other methods by which antioxidants combat oxidative
stress include influencing the activities of gut bacteria and epigenetics [9]. Numerous studies have proven that natural
antioxidants in plant extracts prevent the damaging effects of free radicals [10,11]. Due to their lower toxicity than
their chemical equivalents, there is a growing interest in natural therapies, which have diverse applications in the
pharmaceutical, food, and cosmetic industries [12,13].
Chasmanthera dependens is a climbing shrub producing stems 5 meters or more. The plant belongs to the
Menispermaceae family. It is commonly known as Chasmanthera. It is used locally to treat several diseases, including
red-eye infections [14], venereal diseases, and fracture management [15]. On the other hand, Spondias mombin is a
fruiting tree that grows in the African rainforest and along the shore. It belongs to the Anarcardiaceae plant family.
Its common names are yellow mombin, hog plum, and mombin. All plant parts are reported to be medicinally
important in traditional medicine. The antimicrobial, antibacterial, antiviral, and antifungal properties of
Spondias mombin have been reported [16,17,18]. Based on this background, this study sought to compare the
phytochemical constituent and in vitro antioxidant capacity of Spondias mombin stem bark and Chasmanthera
dependens roots.
Materials and Method

Collection and Preparation of Plant Material
The stem bark of Spondias mombin was collected from the Faculty of Engineering, University of Benin, Edo State. At
the same time, the roots of Chasmanthera dependens were gotten from Oba Isin village, Kwara State. The plants were
identified and authenticated in the Department of Plant Biology and Biotechnology, University of Benin, and
*Corresponding Author’s E-mail: Omorede.aguebor@uniben.edu
34
herbarium specimens were assigned voucher numbers UBH 345 and UBH 387, respectively. The plant parts were
S C
cut into pieces to dry under shade and pulverized. The pulverized plants were steeped in methanol for three days. The
contents were stirred many times a day and filtered with Whatman filter paper at the end of the third day. The filtrate
was evaporated to dryness with a rotary evaporator before being frozen with a freeze-dryer. The extracts were
weighed, placed in an airtight container, and refrigerated at 4 oC until use.
Qualitative Phytochemical Screening
Qualitative Phytochemical screening was carried out on the plant samples using established protocols as described by
Harbone [19], Sofowora [20], and Trease and Evans [21]. A stock solution of each extract, with a concentration of 10
mg extract/mL distilled water, was prepared and used for the phytochemical screening.
Quantitative phytochemical screening
Total phenolic and flavonoid contents were determined according to the Folin-Ciocalteu method [22] and Miliauskas
et al. [23], respectively. At the same time, the total proanthocyanidin and total tannin contents of the methanol
extracts of the plant were carried out according to the method of Sun et al. [24] and Polshettiwar et al. [25] with some
modifications.
In vitro antioxidant capacity
Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity was determined by the method described by Jain et
al. [26], while Reducing power was determined by the method described by Lai et al. [27]. Ferric reducing
antioxidant power and total antioxidant capacity was determined by the method described by Benzie and Strain. [28]
and Prieto et al. [29], respectively.
Statistical Analysis
Data were expressed as the mean ± SEM of the 3 measurements using the GraphPad Prism software. Statistical
significance was investigated by one-way analysis of variance. Differences between mean values were investigated by
Duncan multiple range test.
Results
Phytochemical Constituents (Qualitative) of Methanol extracts of S. mombin stem bark and C. dependens root.
Table 1 represents the qualitative phytochemical screening results of methanol extracts of S. mombin stem bark and C.
dependens root. The result revealed the presence of various phytochemicals such as flavonoids, tannins, saponins,
phenols, alkaloids, terpenoids, and reducing sugar in both plant extracts.
Phytochemical Constituents (Quantitative) of Methanol extracts of S. mombin stem bark and C. dependens
roots.
Figure 1 represents the total flavonoids, total phenols, total tannins, and total proanthocyanins of methanol extracts of
S. mombin stem bark and C. dependens roots. The total flavonoids, phenols, tannins, and proanthocyanins content
were significantly (p < 0.05) higher in the stem bark of S. mombin (41.86 mg Quercetin equivalent; 204 mg gallic acid
equivalent; 197.8 mg tannic acid equivalent and 369.1 mg catechin equivalent respectively) when compared with the
extracts from the roots of C. dependens (8.61 mg Quercetin equivalent;199.1 mg gallic acid equivalent; 87.05 mg
tannic acid equivalent and 45.1 mg acid equivalent respectively).
Table 1: Qualitative phytochemical analysis of S. mombin and C. dependens

Phytochemicals Spondias mombin (stem bark) Chasmanthera dependens(root)
Alkaloids + +
Flavonoids + +
Tannins + +
Saponins + +
Terpenoids + +
Phenols + +
Reducing sugar + -
Carbohydrate + +
‘+’ = indicates presence of constituent, ‘–’ = Indicates absence of constituents
35
Figure 1: Quantitative phytochemical constituents of methanol extracts of S. mombin stem bark and C.
dependens root. Total flavonoid is expressed as mg Quercetin Equivalent / g extract; Total phenol is expressed as mg
Gallic Acid Equivalent / g extract, while total tannins and Proanthocyanidin content is expressed as mg Tannic acid
Equivalent / g extract. Values are expressed as mean± SEM, n = 3/group. Different lowercase letters represent a
significant difference between means at P ˂ 0.05.
120
100
80
% Inhibition
60 ascorbic acid
S. mombin
40 C. dependens
20
0
0 50 100 150 200 250
Concentration µg/ml
Figure 2: DPPH’s radical scavenging activity of methanol extracts of S. mombin and C. dependens root. Values
are expressed as mean± SEM, n = 3/group
Table 2: DPPH (IC 50 values), Ferric acid reducing antioxidant potential (FRAP) and Total antioxidant
capacity (TAC) of methanol extracts of S. mombin and C. dependens
Sample DPPH, IC50 FRAP TAC
(µg/ml) (mg Fe (II)/g extract) (mg ascorbic acid Eq/g
extract)
Ascorbic acid 0.02 ± 0.001a 1.9 ± 0.001a
S. mombin 0.15 ± 0.007a 1.91 ± 0.003a 277.0 ± 2.84a

C. dependens 16.59 ± 0.016b 1.334 ± 0.027b 140.93 ± 2.21b
Data represent mean ± SEM of triplicate analysis. Different lowercase letters within the column indicate significant
differences at p ≤ 0.05.
36
In vitro antioxidant capacity of methanol extract of S. mombin stem bark and C. dependens roots
The total antioxidant capacity (TAC), Ferric reducing antioxidant potential (FRAP), 1.1-diphenyl-2-picrylhydrazyl
(DPPH), and reducing power are presented in table 2. The total antioxidant Capacity (TAC) was significantly (p <
0.05) higher in the methanol extract of S. mombin (277 ± 2.84 µg/ml) than extract of C. dependens (140.93 ± 2.21
µg/ml). The ferric reducing antioxidant potential (FRAP) of C. dependens (1.91 ± 0.003 mg/Fe (II)/g) was
significantly (p < 0.05) higher than S. mombin (1.334 ± 0.02667 mg/Fe (II)/g) (Figure 4.3). Also, the FRAP content
of C. dependens was comparable to that of the standard (1.9 ± 0.00 mg/Fe (II)/g).
Figure 2 represents the DPPH radical scavenging activity of methanol extracts of S. mombin and C.
dependens compared to ascorbic acid. S. mombin was able to better scavenge DPPH (2,2-diphenyl-1-picryl-hydrazyl-
hydrate) free radicals than C. dependens. This was evident in the IC50 value comparable to ascorbic acid (standard).
However, the IC50 of C. dependens (16.59 ± 0.016 µg/ml) was significantly different from ascorbic acid. The reducing
power of methanol extracts of S. mombin and C. dependens is represented in Figure 3. The standard and extracts
showed a significant lowering ability, as evidenced by the increased absorbance value. However, when compared
to C. dependens, the lowering power of S. mombin was found to be stronger in a dose-dependent way.
2.0
Absorbance (nm)
1.5 Ascorbic acid
1.0 S. mombin
C. dependens
0.5
0.0
1 2 3 4 5 6
Concentration (µg/ml)
Figure 3: Reducing Potential Activity of S. mombin and C. dependens. Values are expressed as mean ± SEM, n =
3/group.
Discussion
Before the introduction of modern medicine, herbs and preparations were the primary treatment sources worldwide.
Due to their pharmacological, ecological, and other non-hazardous properties, medicinal plants are gaining popularity
as choices for preventing and treating diseases. The therapeutic benefits of plants may be attributed to a single
component or synergy of phytoconstituents [30]. Phytochemicals are essential as protective and disease-fighting
molecules that aid the body in preventing or combating disease and are, therefore, necessary for human survival [31].
Their therapeutic uses in the prevention or treatment of various ailments are the basis for their widespread usage in
traditional or ethnomedicine in various cultures. Ascertaining the phytochemical constituents and antioxidant status of
medicinal plants has become an excellent point to commence research on their therapeutic potential. The qualitative
phytochemical screening of the extracts in this study revealed that both plants were rich in flavonoids, tannins,
saponins, phenols, alkaloids, terpenoids, and reducing sugar. These findings are consistent with those of Maduka et
al. [32] and Enenebeaku et al. [33]. They also discovered the presence of several of these phytochemicals in the
methanol extracts of the stem bark of S. mombin and the roots of C. dependens.
The presence of an appreciable quantity of therapeutically active chemicals, including flavonoids, tannins, phenols,
and proanthocyanin dins, was discovered in methanol extracts of S. mombin stem bark and C. dependens roots.
However, S. mombin extract contained a significantly (p ˂ 0.05) higher concentration of total flavonoids, tannins,
proanthocyanin, and phenol than C. dependens root extract. Flavonoids are the most prevalent group of polyphenolic
substances in the human diet and can be found in abundance in plants. They possess numerous biochemical qualities,
but their capacity to serve as antioxidants is the biochemical trait that is most thoroughly described. Mechanisms of
antioxidant action include suppression of reactive oxygen species (ROS) synthesis by inactivating enzymes or
interfering with the oxidation state of trace elements involved in free radical production, destruction of ROS, and
upregulation or protection of antioxidant defenses [34,35]. Tannins are pervasive and are likely found in all plant
matter. They are polyphenolic compounds with a high molecular weight that are water-soluble and phenolic group-
37
rich. These polyphenolic chemicals are categorized into two primary groups: hydrolyzable and condensed. Due to
their astringent qualities, tannins are utilized as therapeutic substances that promote quick wound healing and the
development of new tissue on inflamed mucosa [36]. Phenols play a significant role in antioxidant activity by
quenching free radicals, singlet oxygen (O2−), or metal ions (Fe2+) due to their lower redox potential. The strong
correlation between the total phenolic contents of a plant and their resultant antioxidant properties has been well
supported by various antioxidant studies conducted on plant extracts [37,38,39.40,41,42]. Diverse modes of action are
responsible for the antioxidant activities of plants. Thus, antioxidant activity cannot be explained adequately using a
single assay paradigm [43]. In this investigation, four complementary in vitro tests were employed to evaluate the
antioxidant activity of S. mombin and C. dependens extracts: the DPPH free radical-scavenging activity, the Ferric
reducing antioxidant potential (FRAP), the reducing power assays, and the phosphomolydenum assay. The ability to
scavenge DPPH radicals is one of the most widely used approaches for determining antioxidative activity. The DPPH
radical is utilized as a substrate for measuring antioxidant activity. The reduction of the DPPH radical is assessed by
the decrease in absorbance caused by the presence of antioxidants in the sample. IC 50 is a significant variable
calculated from the DPPH free radical scavenging test. The IC50 represents the quantity of antioxidants required to
reduce DPPH radical concentration by 50% [44]. The lower IC50 value observed for Spondias mombin extract shows
that it is a more powerful antioxidant than Chasmanthera dependens. Spondias mombin extract demonstrated a
significantly p < 0.05 higher total antioxidant capacity than C. dependens. Using the phosphomolybdate test, the total
antioxidant capacity (TAC) of the extracts was quantified. In the presence of a reducing agent, molybdenum (VI) is
reduced to molybdenum (V), generating a green phosphomolybdate (V) complex, which is the basis of the assay
[45,46]. The electron transfer (ET) process is utilized in this test.
The Ferric reducing potential antioxidant test evaluates the ability of antioxidants present in the test extract to convert
Fe3+ to Fe2+. C. dependens extracts were shown to have a significantly higher (p < 0.05) FRAP value than S. mombin
extract, comparable to the control (ascorbic acid). The findings of the reductive potential test revealed that the extract
of S. mombin had a significantly higher reductive potential than that of C. dependens. Compounds having reducing
power are electron donors and can reduce the oxidized intermediates of lipid peroxidation processes, allowing them to
function as primary and secondary antioxidants [47]. Although C. dependens showed a significantly higher (p > 0.05)
FRAP value than S. mombin, S. mombin possessed a superior antioxidant effect, as it was able to scavenge DPPH
radicals more effectively, convert molybdenum (VI) to molybdenum (V), and exhibit dose-dependent reducing
activity. This could be attributable to phytochemicals such as flavonoids, phenols, and tannins in the extracts. This
confirms the findings of Khan et al. [48], who discovered that phytochemical-rich plants possess a greater antioxidant
capacity.
Conclusion
Our findings suggest that S. mombin and C. dependens extracts could serve as free radical scavengers, acting possibly
as primary antioxidants which could be used in the treatment/management of diseases.
References
1. Gandhi Y, Kumar R, Grewal J, Rawat H, Mishra S K, Kumar V, Acharya, R: Advances in anti-inflammatory
medicinal plants and phytochemicals in the management of arthritis: A comprehensive review. Food chemistry
advances, 100085. 2022.
2. Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB: The relative antioxidant activities of plant-
derived polyphenolic flavonoids. Free radical research, 22(4), pp.375-383. 1995
3. Gengatharan A, Dykes GA, Choo WS: Betalains: Natural plant pigments with potential application in functional
foods. LWT Food Sci. Technol.64:645–649. 2015.
4. Pohl F, Lin-Thoo PK: The potential use of plant natural products and plant extracts with antioxidant properties
for the prevention/treatment of neurodegenerative diseases: In vitro, in vivo and clinical trials. Molecules
23:3283. 2018.
5. Hrelia S, Angeloni C: New Mechanisms of Action of Natural Antioxidants in Health and
Disease. Antioxidants. 9:344. 2020.
6. Williams RJ, Spencer JP, Rice-Evans C: Flavonoids: Antioxidants or signalling molecules? Free Rad. Biol.
Med 36:838–849. 2004.
7. Kumar S: Free Radicals and Antioxidants: Human and Food System. Adv. Appl. Sci. Res. 2:129–135. 2011.
8. Al-Rasheed NM, Fadda LM, Ali HM, Abdel Baky NA, El-Orabi NF, Al-Rasheed NM: New mechanism in the
modulation of carbon tetrachloride hepatotoxicity in rats using different natural antioxidants. Toxicol. Mech.
Meth. 26:243–250. 2016.
9. Majeed M, Pirzadah TB, Mir MA et al: Comparative Study on Phytochemical Profile and Antioxidant Activity
of an Epiphyte, Viscum album L. (White Berry Mistletoe), Derived from Different Host Trees. Plants
(Basel).11;10(6):1191. 2021.
10. Khan MS, Ikram M, Park JS, Park TJ, Kim MO: Gut Microbiota, Its Role in Induction of Alzheimer’s Disease
Pathology, and Possible Therapeutic Interventions: Special Focus on Anthocyanins. Cells. 9:853. 2020.
11. Zengin G, Cakmak YS, Guler GO, Aktumsek A: Antioxidant properties of methanolic extract and fatty acid
composition of Centaurea urvillei DC. subsp. hayekianaWagenitz. Rec. Nat. Prod. 5:123–132
38
12. Naz R, Roberts TH, Bano A, Nosheen A, Yasmin H, Hassan MN, Anwar Z: GC-MS analysis, antimicrobial,
antioxidant, antilipoxygenase and cytotoxic activities of Jacaranda mimosifolia methanol leaf extracts and
fractions. PLoS ONE.15:e0236319. 2020.
13. Wannes WA, Mhamdi B, Sriti J, Jemia MB, Ouchikh O, Hamdaoui G: Antioxidant activities of the essential
oil and methanol extracts from myrtle (Myrtus communis var. Italica L.) leaf, stem and flower. Food Chem.
Toxicol 48:1362–1370. 2010.
14. Ogunlesi M, Okiei W, Ademoye M: Medicinal Plants Used in Treating Eye Infections in Nigeria. In: A
Textbook of Medicinal Plants from Nigeria, Odugbemi, T. (Ed.). University of Lagos Press, Lagos, ISBN: 978-
978-48712-9-7, pp: 299-317. 2008.
15. Odugbemi T: A Textbook of Medicinal Plants from Nigeria. University of Lagos Press, Lagos, Nigeria, ISBN:
978-978-48712-9-7, Pages: 628. 2008
16. Corthout J, Pieters LA, Claeys M, et al: Antiviral; Ellagitannins from Spondias mombin. Phytochemistry,
30:1190. 1991.
17. Abo KA, Ogunleye VO, Ashidi, JS: Antimicrobial potential of Zambesicus and Zygotritonia crocea.
Phytotherapy Research, 13: 494 – 497. 1999.
18. Rodrigues KF, Hasse M: Antimicrobial activities of secondary metabolites produced by endophytic fungi from
Spondias mombin. Journal of Basic Microbiology, 40(4): 261-267. 2000.
19. Harbone JB: Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 3rd Ed. New York:
Chapman and Hall, 49-188. 1998.
20. Sofowora A: Screening Plants for Bioactive Agents. In Medicinal Plants and Traditional Medicine in Africa.
Spectrum Books Ltd. Ibadan. Pp 128-161. 1982.
th
21. Trease GE, Evans WC: A Textbook of Pharmacognosy,13 ed. London: Bailliere-Tyndall Ltd. p 345-356.
1989
22. Cicco N, Lanorte MT, Paraggio M, Viggiano M, Lattanzio V: A reproducible, rapid and inexpensive Folin-
Ciocalteu micro method in determining phenolics of plant methanol extracts. Microchem J,91: 107–110. 2009.
23. Miliauskas G, Vensketonis PR, Beck TA: Screening of radical scavenging of some medicinal and aromatic
plant extracts. Food Chem.,85:231 – 237. 2004.
24. Sun B, Ricardo-da-Silva J.M and Spranger, I: Critical factors of vanillin assay for catechins and
proanthocyanidins. J Agri Food Chem., 46: 4267 - 4274. 1998.
25. Polshettiwar SA, Ganjiwale RO, Wadher SJ, Yeole PG: Spectrophotometric estimation of total tannins in
some ayurvedic eye drops. Indian Journal of Pharmaceutical Sciences, 69(4), p.574.of AOAC (1970). 2007.
26. Jain A, Soni M, Deb L et al: Antioxidant and hepatoprotective activity of ethanolic and aqueous extracts of
Momordica dioica Roxb leaves. Journal of Ethnopharmacology. 115(1): 61-66. 2008.
27. Lai LS, Chou ST, Chao WW: Studies on the antioxidant of Hsian-Tsao (Mesona procumbems Hemel) leaf
gum. Journal of Agricultural and Food Chemistry. 49: 963-968. 2001.
28. Benzie IF, Strain JJ: The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The
FRAP assay. Anal Biochem ,239: 70-76. 1996.
29. Prieto P, Pineda M, Aguila M: Spectrophotometric Quantitative of Antioxidant Capacity through the
Formation of a Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E1.
Analytical Biochemistry, 269: 337. 1999.
30. Sofowora A, Ogunbodede E, Onayade A: The role and place of medicinal plants in the strategies for disease
prevention. African journal of traditional, complementary and alternative medicines, 10(5): 210-229. 2013.
31. Forni C, Facchiano F, Bartoli M: Beneficial role of phytochemicals on oxidative stress and age-related
diseases. BioMed research international, 8:78-82. 2019.
32. Maduka H, Okpogba A, Ugwu C, Dike C, Ogueche P, Onwuzurike D, Ibe D: Phytochemical, antioxidant and
microbial inhibitory effects of Spondias mombin leaf and stem bark extracts.
Journal of Pharmaceutical and Biological Sciences, 9(2):14-17. 2014.
33. Enenebeaku UE, Okotcha EN, Oguoma LMO: Biochemical and haematological enhancement activities of
aqueous and methanol leaves, stem and roots extracts of Chasmanthera dependens (Hochst) and Dictyandra
arborescens (Welw.). Bulletin of the National Research Centre. 45:186. 2021.
34. Halliwell B, Gutteridge JM: Free Radical in Biology and Medicine, Oxford University Press, Oxford, UK.
1998.
35. Mishra A, Sharma AK, Kumar S, Saxena AK, Pandey AK: Bauhinia variegata leaf extracts exhibit
considerable antibacterial, antioxidant, and anticancer activities. BioMed Research International, 10. 2013.
39
36. Chokotho L, Van Hasselt E: The use of tannins in the local treatment of burn wounds-a pilot study. Malawi
Medical Journal, 17(1) :19-20. 2005.
37. Song FL, Gan RY, Zhang Y, Xiao Q, Kuang L, Li H.B: Total phenolic contents and antioxidant capacities of
selected Chinese medicinal plants. Int. J. Mol. Sci.11:2362–2372. 2010.
38. Tosun M, Ercisli S, Sengul M, Ozer H, Polat T, Ozturk E: Antioxidant properties and total phenolic content of
eight Salvia species from Turkey. Biol. Res.42:175–181. 2009.
39. Alali FQ, Tawaha K., El-Elimat T, et al: 3rd, Oberlies N.H. Antioxidant activity and total phenolic content of
aqueous and methanolic extracts of Jordanian plants: An ICBG project. Nat. Prod. Res. 21:1121–1231. 2007.
40. Lin S, Guo H, Gong JDB: Phenolic profiles, β-glucan contents, and antioxidant capacities of colored Qingke
(Tibetan hulless barley) cultivars. J. Cere. Sci. 81:69–75. 2018.
41. Yang XJ, Dang B, Fan MT: Free and bound phenolic compound content and antioxidant activity of different
cultivated blue highland barley varieties from the Qinghai-Tibet plateau. Molecules.23:879. 2018.
42. Ge X, Jing L, Zhao K, Su C, Zhang B, Zhang Q, Li, W: The phenolic compounds profile, quantitative analysis
and antioxidant activity of four naked barley grains with different color. Food Chem. 335:127655. 2021.
43. Prior R, Wu X, Schaich K: "Standardized methods for the determination of antioxidant capacity and phenolics
in foods and dietary supplements". Journal of Agricultural and Food Chemistry, 53 (10): 4290–4302. 2005.
44. Chanda S, Dave R, Kaneria M: In vitro antioxidant property of some Indian medicinal plants. Research
Journal of Medicinal Plants, 5(2) :169-179. 2011.
45. Mashwani Z, Khan MA, Irum S, Ahmad M: Antioxidant potential of root bark of Berberis lycium Royle. from
Galliyat, western Himalaya, Pakistan. Pakistan Journal of Botany, 45(231), p.4. 2013.
46. Jan S, Khan MR, Rashid U, Bokhari J: Assessment of antioxidant potential, total phenolics and flavonoids of
different solvent fractions of Monotheca buxifolia fruit. Osong Public Health and Research
Perspectives, 4(5):246-254. 2013.
47. Ayala A, Muñoz MF, Argüelles S: Lipid peroxidation: production, metabolism, and signaling mechanisms of
malondialdehyde and 4-hydroxy-2-nonenal. Oxidative medicine and cellular longevity, 2014.
48. Khan W, Subhan S, Shams DF, Afridi SG, Ullah R, Shahat AA, Alqahtani A S: Antioxidant potential,
phytochemicals composition, and metal contents of Datura alba. BioMed research international, 5. 2019.
40
Ugbeni, O.C and Uanseoje, S.O

Changes in Some Biochemical Parameters in Red Meat Tendered with

Paracetamol and Extract of Ocimum basilicum.
*
Department of Biochemistry, University of Benin, Benin City, Edo State.
Abstract
Tendering of meat for greater acceptability is paramount to food sellers. The use of paracetamol (PC) for this
purpose is becoming a common practice. We analyzed some biochemical changes associated with beef tendered
with PC and extract of Ocimum basilicum. Fresh beef was divided into 4 groups, A-D. Group A was boiled with
distilled water, group B was boiled with PC (3000 mg/L), group C was boiled with extract of O. basilicum while
group D was boiled with a mixture of PC and extract O. basilicum. Samples were taken for analysis at zero hour
and after every 48 hrs. The remaining samples were stored at -4ºC. pH, Malondialdehyde, Metmyoglobin, Acid
value, total protein and Heme iron were assayed. Our results showed a significant (P< 0.05) increase in Heme iron
content in the samples tendered with PC at the 0 hour and 48 hour when compared to the control. However, there
was no significant difference was in Malondialdehyde content. Total protein content yielded significant (P< 0.05)
decrease at the 0 hour and 96 hour for samples boiled with Ocimum basilicum and that of Ocimum basilicum and
PC when compared to the control. Significant (P<0.05) decrease was also observed in percentage metmyoglobin
content all through the period. pH value showed significant increase for all samples at the 0 hour and at the 144
hour, although it was higher in the sample boiled with PC. Our results are indication that boiling of meat PC is
inimical to the quality of red meat.
Keywords: Red meat, Ocimum basilicum,paracetamol, percentage metmyoglobin, acid value
Introduction
Animal flesh that is consumed as food is called meat [1]. Humans have hunted and killed animals for meat since
prehistoric times. Meat is valued as a rich source of proteineous food containing all the amino acids necessary for
the human body. Protein, water and fat are the major component of meat. It is edible raw, but is normally eaten after
it has been cooked and seasoned or processed in a variety of ways. Unprocessed meat will spoil or rot within hours
or days as a result of infection with and decomposition by bacteria and fungi. Meat is a major component of the
protein food groups and also a major source of important nutrients to the body. Folks consume meat for some
reasons which include: the good taste and a desirable flavor. Furthermore, meats have desirable nutritional benefits
and also enhance social status [2]. Fathoming the best way to process meat for consumption will justify the many
benefits associated with the consumption of meat. The three common nutrients associated with meats are Zinc, Iron
and protein [3]. Meat proteins are considered high quality protein –one that provides essential amino acids- and they
also have high biological values in that a large proportion of proteins from meat are used to synthesize proteins in
the animal‘s body [3]. Ocimum basilicum (Sweet Basil) is one of the most important crops with essential oils as well
as polyphenols, phenolics, flavonoids and phenolic acids .It is part of a group of medicinal plants widely used in
cooking and known for its beneficial health properties, possessing significant antioxidant effects, antinociceptive,
and others [4]. It has been used traditionally for the treatment of anxiety, diabetes, cardiovascular diseases, headaches,
nerve pain, as anti-convulsant and anti-inflammatory, and used in a variety of neurodegenerative disorders [5].
Tenderness is a quality of meat gauging how easily it is chewed or cut. Tenderness is a desirable quality, as tender
meat is softer, easier to chew, and generally more palatable than harder meat. Consequently, tender cuts of meat
typically command higher prices. The use of potash and more recently paracetamol is becoming a common practice
in some parts of African countries including Nigeria. The National Agency for Food and Drug Administration
Control (NAFDAC) has raised serious concern about the safety of consumption of such treated meat [6].We report in
the piece of study scientific data on the biochemical changes that occur in meat tendered with paracetamol and
Ocimum basilicum.
Materials and Method

Preparation of plant Materials: The leaves of Ocimum basilicum were obtained from a local community in Edo
State, South-South Nigeria. It was authenticated in the department of Plant Biology and Biotechnology, University
*Corresponding Author: osezele.ugbeni@uniben.edu.
41
of Benin, Benin City, Nigeria and a voucher number of UBH-0461 was assigned. The leaves were dried under shade
for five days. The dried leaves were ground to fine powder using the mechanical grounding machine. The plant
extract was obtained by soaking 100 g of the ground leaf powder in 1000 ml of distilled water for three days inside a
plastic container and was filtered using a muslin bag and whatman1 filter paper. Water bath was then used to
concentrate the filtrate at a temperature of 40 degrees. The concentrate was freeze dried and the solid sample was
stored in a dry container for use. For further experimental use,100 mg of solid sample was reconstituted in 1 litre of
distilled water.
Experimental design: Paracetamol solution was prepared by dissolving 3000 mg of paracetamol in 1 litre of water
and stored in a plastic container for use. About 2 kg of fresh beef was purchased in an abattoir. The beef was divided
into four different portions labeled A-D. Group 1 was boiled with distilled water, group 2 was boiled with a solution
of paracetamol, group 3 was boiled with a solution of Ocimum basilicum, while group 4 was boiled with a mixture
of potash and Ocimum basilicum in equal proportion, Each set of samples were boiled for 15 mins using a gas
cylinder. The appropriate masses were weighed out for biochemical analysis at intervals of 48 hours starting from
the zero hour to 144 hrs. The rest samples were stored in a freezer at -4ºc till required.
Measurement of pH value: pH denoting ‗potential of hydrogen‘ or ‗power of hydrogen‘ is a scale used to specify the
acidity or alkalinity of an aqueous solution. pH is measured with a pH meter. Meat was boiled for 15 minutes and
allowed to cool. Thereafter an electronic pH meter was placed on the meat to check for the pH of the meat.
Determination of total protein: The assay was carried out using Randox kits. The principle is as described by [7].
Cupric ions, in an alkaline medium, interact with protein peptide bonds resulting in the formation of a coloured
complex whose adsorbance is taken at 546nm.
Determination of percentage metmyoglobin content: The analysis of metmyoglobin content was performed as
described by [8]. Metmyoglobin is the oxidized form of oxygen carrying some protein myoglobin. Metmyoglobin is
the cause of the characteristics brown colouration of meat that occurs due to cooking. The chemistry of beef colour
is due to the presence of the pigment myoglobin. Meat undergoes oxygenation when exposed to air to form
oxymyoglobin. Myoglobin and oxymyoglobin have the capacity to lose an electron which turns the pigment to a
brown colour called metmyoglobin.
Determination of malondialdehyde content of beef: Malondialdehyde content of beef was determined using [9]. This
assay is based on the reaction of malondialdehyde (MDA) with thiobarbituric acid (TBA), forming a MDA-TBA2
adducts that absorbs strongly at 535nm. In the presence of heat and acid, MDA reacts with TBA to produce a
colored end product. The intensity of the colour at 535nm corresponds to the level of lipid peroxidation in the
sample.
Determination of Haem iron contents: Haem iron was determined by means of acidified acetone extraction followed
by spectrophotometry [10]. The minced beef patties sample (2 g) was transferred into a 50 ml centrifuge tube and 9
ml of acid acetone mixture (90% acetone, 8% deionised water, and 2% HCl) was added. The mixture was macerated
using a glass rod and allowed to stand for 1 h at room temperature. The extract was centrifuged at 2200 g for 10
minutes. The supernatant was filtered through Whatman #42 filter paper, and the absorbance was read at 640 nm
against the acid acetone blank. The total pigments were calculated as haematin using the following formula [11]:
Total pigment (mg/kg) = A640 × 680 and haem iron was calculated as follows [10]: Haem iron (mg/kg) = total
pigment (mg/kg) × 8.82/100
Determination of acid value: The analysis of acid value was performed as described by [12]; The acid value is also a
measure of the amount of fatty acids, which have been liberated by hydrolysis from the glycerides. The acid value is
determined by directly titrating the fat in an alcoholic medium against a standard potassium hydroxide/sodium
hydroxide solution.
Statistical analysis: Analysis on the red meat was carried out in triplicates and the results were expressed as mean ±
standard error of mean (SEM). The data was subjected to One Way Analysis of variance (ANOVA) and Turkey‘s
multiple comparisons Test was employed as a post adhoc test. P values of <0.05 were regarded as significant while
p values of >0.05 were regarded as not significant. The lower case alphabet, b, represents ―significant‖ while the
lower case letter a, represents ―not significant‖ as illustrated in the charts below. For simplicity, the following
designations are used: RMPC = Red Meat and Paracetamol, RMS = Red Meat and Spice (Ocimum basilicum),
RMPC = Red Meat and Paracetamol and spice.
Results and Discussion

Treatment of red meat with Paracetamol and Ocimum basilicum, resulted in non-significant difference in groups
containing red meat and Paracetamol, red meat and Ocimum basilicum, and red meat with both Paracetamol and
Ocimum basilicum showed no significance difference when compared to the control (p>0.05).
42
20.00
Malondialdehyde
18.00 a
Malondialdehyde (Mg/g) X 10-4
16.00 a
14.00
12.00 a
10.00 Control
a a a
8.00 RMPC
RMS
6.00 a a
a
a RMPCS
4.00 a
a
2.00
0.00
0hr 48hr 96hr 144hr
Time
Fig 1: Changes in malondialdehyde concentration in red meat tendered with Paracetamol and Ocimum basilicum.
The values are expressed in Mean±SEM. b= represent significant difference against control. a= represent non-
significant difference against control.
14
Heme Iron content a
12
Heme Iron content (mg/kg)
b
10
b
8 a
6 a Control
a a a
b a RMPC
b a
4
RMS
RMPCS
2
0
0hr 48hr 96hr 144hr
Time
Fig 2: Changes in heam iron content in red meat tendered with Paracetamol and Ocimum basilicum. The values are
expressed in Mean±SEM.
43
There was significance difference in heme iron content of the red meat sample boiled with Paracetamol at 0 hour
and 144 hour (P<0.05) when compared to the control. There was however no significant different in the sample
containing Paracetamol at 48 hour and 96 hour (P>0.05) compared to the control. There was no significant
difference in the red meat sample boiled with Ocimum basilicum for all the hours (P>0.05) when compared with the
control. Just like the samples boiled with Paracetamol, there was significant difference for the samples boiled with
Paracetamol and Ocimum basilicum at the 0 hour and 144 hour (P<0.05) but no significant difference at 48 hour and
96 hour (P>0.05) when compared with the control.
Total protein
3.5
a
a a
Total Protein (g/dl)
3
b
2.5 a a
a Control
2
a
1.5 b Rmpc
1 a Rms
a a
0.5 Rmpcs
0
ohr 48hr 96hr 144hr
Time
Fig 3: Changes in total protein concentration in red meat tendered with Paracetamol and Ocimum basilicum. The
values are expressed in Mean±SEM
Apart for the red meat sample boiled with Ocimum basilicum in the 0 hour and 96 hour, and the red meat sample
boiled with Paracetamol and Ocimum basilicum in the 96 hour, there was no other significant difference in the total
protein of red meat in all the samples at all hours when compared to the control.
120 Percentage Metmyoglobin

100
P80 a
a Control
M a a b
C60 RMS
a a
a
a RMPC
b b
(
%40 b
RMPCS
)
20
0
0hr 48hr Time 96hr 144hr
Figure 4: Changes in metmyoglobin iron content in red meat tendered with Paracetamol and Ocimum basilicum.
The values are expressed in Mean±SEM.
44
There was no significant difference in the percentage Metmyoglobin of red meat in all the samples at the 0 hour
when compared to the control (P>0.05) and also for those at 48 hour. There was however significant difference in
the red meat samples boiled with Paracetamol, that boiled with Ocimum basilicum but there was no significant
difference in that boiled with both Paracetamol and Ocimum basilicum at 96 hour. In 144 hour, there was significant
difference in the red meat samples boiled with Ocimum basilicum and that boiled with both Paracetamol and
Ocimum basilicum but not in that boiled with just Paracetamol when compared to the control.
.
7.00
pH
b
6.80 b a
a
b a
6.60 b
a a
6.40 a a
a Control
pH
6.20
Rmpc
6.00 Rms
5.80 Rmpcs
5.60
5.40
0hr 48hr Time 96hr 144hr
Fig 5: Changes in pH in red meat tendered with Paracetamol and Ocimum basilicum .The values are expressed in
Mean±SEM
There was significant difference in all the red meat samples (those boiled with either Paracetamol, Ocimum
basilicum, or both) in the 0 hour when compared to the control (P<0.05). There was no significant difference in all
the samples in the 48 hour when compared to the control, neither was there significant difference in the 96 hour.
There was however significant difference in the red meat sample boiled with Ocimum basilicum in the 144 hour.
Acid value
1.2
Acid Value(mLN/g)
0.8 a
Control
0.6
b Rmpc
a a a a a a
0.4 a a a Rms
a
0.2 Rmpcs
0
0hr 48hr 96hr 144hr
Time
Fig 6 :Changes in acid value content in red meat tendered with Paracetamol and Ocimum basilicum .
The values are expressed in Mean±SEM
45
Significant difference in acid value (P<0.05) was only seen in the sample boiled with Paracetamol and Ocimum
basilicum at the 48 hour when compared to the control.
Nutrition is a key factor in human health. The process of achieving proper nutrition during the processing of meat
has led to many unwholesome practices which may be detrimental to health. Such practices include the use of
paracetamol to tender meat and addition of seasoning agents. Pertaining to meat, time of slaughter is a very
important criterion to take note of as meat tissues starts to lose their antioxidant effect after slaughter and more
complex oxidative processes will emerge. Lipid, Myoglobin and protein oxidation are the major oxidative processes
in uncured meat [2]. Deterioration of lipids and proteins in the meat determines the development of offensive odours,
off-tastes, colour change, and compounds of possible toxicity, which in all, makes the meat undesirable for eating.
Off-odour, off-flavour, rancidity and meat colour change observed in meat are attributable to lipid peroxidation [2].
The statistically insignificant result (except for the samples containing Paracetamol and Ocimum basilicum in the 48
hour) for the acid value of the red meat boiled with our various additives compared to the control shows that the
presence of Paracetamol and Ocimum basilicum in meat does not have high effect on the quality of the meat.
According to [13], the acid value of food is a measure of the quality of food. Also, according to [14], an increase in
acid value, is an indication of high rancidity. We can infer from our work that there is no significant difference in
red meat boiled with either Paracetamol, Ocimum basilicum or with both compared to the control. It is however very
necessary to state that there was an increase in acid value with an increase in time. Thus, storage and temperature
have effect on the acid value of red meat.
The pH of red meat was seen to increase in samples containing Paracetamol, Ocimum basilicum and both
Paracetamol and Ocimum basilicum on the first hour of our analysis (0 hour). This increase was very significant (P<
0.05). However, while the pH of the samples containing Ocimum basilicum continued to increase at the various
intervals of our analysis, there were fluctuations in the pH of the samples containing Paracetamol and those
containing both Paracetamol and Ocimum basilicum. We can therefore deduce that Ocimum basilicum increase the
pH of red meat and so does Paracetamol. However, with time, there is a decline in the pH of samples containing
Paracetamol.
[15]
in his work published in the Czech Journal of Animal Science stated that meat of high quality has ultimate pH at
the range of 5.4-5.6. Decline in meat quality starts to occur at pH>5.8. Meat of high pH have been characterized to
have gummy structure, increase water holding capacity and decreased in specific taste [16]. Thus, it can be concluded
that Ocimum basilicum and Paracetamol decrease the quality of red meat.
Metmyoglobin is the oxidized product of Myoglobin. Oxidation of Myoglobin to Metmyoglobin leads to
discolouratuon of red meat and it is influenced by oxygen concentration, pH, and the presence of aldehyde generated
by lipid oxidation. The colour intensity difference between meat species are primarily caused by differing
concentrations of Myoglobin. Beef has the highest concentrations of myoglobin and is the darkest of meat species.
Following the information provided by the bar chart, the Myoglobin concentration in the control sample could be
seen to increase with increase in hours. However, the presence of either Paracetamol or Ocimum basilicum in the red
meat decreased the concentration of Myoglobin in the red meat. The difference was specifically significant on the
last hour of analysis (144 hour), in the sample containing Ocimum basilicum and Paracetamol (P value is 0.0017).
Myoglobin is the main protein of muscle tissue sarcoplasm, containing centrally located heme, which is a complex
of protoporphyrin. Red meat owes it dark red colour to the presence of large amounts of haem, the content of which
in this meat is 10-fold higher than in white meat [17]. Although meat colour is primarily determined by its content of
myoglobin, hemoglobin and cytochrome c also contributes to it [18]. Haem Iron which is mainly found in myoglobin
in red meat, contributes to the desirable bright red colour and to the most undesirable brown colour of meat. Heme
Iron which main source is red meat is easily absorbed by the body. Iron is a major component of hemoglobin and it
deficiency can lead to aneamia [18].
Taking into cognizance the perspective provided above, we can draw out a tangible statement from the bar chart for
heme iron content (Figure 3.1.2). There was significant difference in the samples containing Paracetamol (P value is
0.0376) and that containing both Paracetamol and Ocimum basilicum (P value is 0.0425). The heme iron content in
these two samples was low compared to the heme iron content of the control. We can also infer that storage plays a
role in reducing the heme iron content of red meat for the control and while there was no other significant difference
with an increase in time, there was however significant difference at the 144 hour for the sample containing
Paracetamol and the sample containing both Paracetamol and Ocimum basilicum, P value (0.0362 and 0.04
respectively).However, there is an increase in the heme iron content in this case. This overboard value for heme iron
content in red meat produce the undesirable dark colour in meat, and excessive consumption of red meat of this kind
can lead to iron toxicity. So, we can draw a conclusive statement that red meat cooked with Paracetamol alter the
heme iron content (either increase or decrease it). This alteration is not favourable (either increase or decrease).
46
Same can‘t be said for the Ocimum basilicum because the samples containing just Ocimum basilicum showed no
significant difference.
For the total protein, there was almost no significant difference but for the samples containing Ocimum basilicum at
the 48 hour and 96 hour P value (0.0239 and 0.0424 respectively). However, the protein content of the red meat was
seen to increase with increase in time.
Malondialdehyde (MDA) is one of the most abundant aldehydes generated during secondary lipid oxidation and also
probably the most commonly used as an oxidation marker [19]. The presence of oxidized lipids in the diet of humans
and animals resulted in an increase of thiobarbituric acid reactive substances (TBARS) in plasma and tissue [20].
MDA is in many instances the most abundant individual aldehyde that results from lipid peroxidation in foods.
Levels of MDA in living organisms have been found to be significantly modified in many pathological situations
(e.g., gastric, lung, or breast cancer, and atherosclerotic or cardiovascular diseases) [21]. Although there was no
significant effect of Paracetamol and Ocimum basilicum on the red meat (P > 0.05), it can however be deduced from
the bar chart that Paracetamol increased Malondialdehyde levels while on an average, Ocimum basilicum decreased
it.
Conclusion
From the results obtained in this study, it was clearly seen that Paracetamol has the ability to alter the amount of
some important parameters in red meat (pH, Myoglobin, Malondialdehyde, acid value and Heme Iron content).
These alterations were in most cases of adverse effects to the meat and invariably will be of adverse effect to
consumers of such meat (red meat). Ocimum basilicum also showed some alterations on the parameters studied, this
was however not to the extent of alterations caused by Paracetamol. Finally, compared to the other samples, the
sample containing Paracetamol and Ocimum basilicum did not show many alterations when compared to the control.
Thus, it can be inferred that Ocimum basilicum acted as a neutralizer to the alteration caused by Paracetamol. It can
therefore be concluded that tendering of meat with Paracetamol (Acetaminophen) will be of a more deteriorating
effect to human health and also, to the quality of red meat.
References
1. Lawrie, RA, Ledward, DA. Lawrie's Meat Science. Seventh English, edition ed. Cambridge England: Woodhead
Publishing Limited. 2006.
2. Osagiede, EC. Some biochemical changes in beef treated with ethanol extract of Piper guineense. A research
project. University of Benin, Benin city, Edo state. Pp. 1-67. 2018.
3. Boler, DD, and Woerner, DR. What is Meat? A perspective from the American Meat science association.Animal
frontiers.7(4): 8-11. 2017.
4. Silva, VA, da Sousa, JP , Pessôa, HF , Freitas, AF, Coutinho, HD, Alves, LN, Lima, EO. Ocimum
basilicum: Antibacterial activity and association study with antibiotics against bacteria of clinical importance,
Pharm Biol, 54(5):863-7. 2016.
5. Bora KS, Arora S, Shri R. Role of Ocimum basilicum L. in prevention of ischemia and reperfusion-induced
cerebral damage, and motor dysfunctions in mice brain. J Ethnopharmacol, 137: 1360-5. 2011.
6. Nigeria Guardian. https://guardian.ng/news/cooking-with-paracetamol-causes-liver-kidney-failure-nafdac-warns/
Feb., 15: 2020.
7. Henry, RJ, Cannon, DC, and Winkelman, JW. "Clinical Chemistry, principles and Techniques", Harper & Row,
2nd Ed. 1974.
8. Krzy-Wick, K. The determination of haem pigments in meat. Meat Science, 7: 29-36. (1982).
9. Buege, JA. and Aust, SD. Microsomal lipid peroxidation. Methods in Enzymology, 53: 302-310. 1978.
10. Clark, EM., Mahoney, AW. and Carpenter, CE. Heme and total iron in ready to eat chickens. Journal of
Agriculture and Food Chemistry, 45, 124-126. 1997.
11. Lee, BJ, Hendricks, DG, Cornforth, DP. A comparison of carnosine and ascorbic acid on colour and lipid
stability in a ground beef pattie model system. Journal of Meat Science, 51, 245–253. 1999.
12. AOACS. AOCS official method, Cd 3d-63: acid value. Champaign, IL(USA): American Chemist‘s Society.
1999.
13. Divine, J, Williams, P. The chemistry and technology of edible oil and fat .1st ed. Pergamon press.London.
1961.
14. Wollat, EE. The manufacture of soap, other detergents and glycerine.2nd ed. Ellis Harwood Pub. England. 1985.
15. Weglarz, A. Meat quality defined based on pH and colour depending on cattle category and slaughter season.
Czech journal of Animal science.55 (12): 548-556. 2010.
47
16. Pipek, P., Harberl, A., and Jelenikova, J. Influence of slaughterhouse handling on the quality of beef carcasses.
Czech journal of Animal Science.39: 371-378. 2003.
17. Bastide, Nadia M., Pierre, Fabrice, HF, Corpet, Denis, E. Heme Iron from Meat andRisk of Colorectal Cancer: A
Meta-analysis and a Review of the Mechanisms Involved. Cancer Prevention Research. 4 (2): 177–184. 2011.
18. Mateusz, B., Słomka, A., and Janicki B. Heme iron in meat as the main source of iron in
the human diet. Journal of Elementology,21(1):303-314. 2016.
19. Barriuso, B.; Astiasarán, I.; Ansorena, D. A review of analytical methods measuring lipid
oxidation status in foods: A challenging task. Eur. Food Res. Technol.1–15. 2013.
20. Ruban, SW. Lipid peroxidation in muscle foods—An overview. Glob. Vet. 3: 509–513. 2009.
21. Czauderna, M, Kowalczyk, J, Marounek, M. The simple and sensitive measurement of malondialdehyde in
selected specimens of biological origin and some feed by reversed phase high performance liquid
chromatography. J.Chromatogr., B. 879:2251–2258. 2011.
48
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desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion
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Volume 22, Numbers 1/2 ISSN 1595-6938

Journal of Society for Experimental

EDITORIAL ADVISORY BOARD

AIM AND SCOPE

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© 2022 Society for Experimental Biology of Nigeria

Proximate and Phytochemical Analyses of Vinca minor L Leaf Extracts 1

Antioxidant Activities of Leaf Extracts of Vinca minor L

Effect of pH Modifications of PCB-Contaminated Soil on Growth and Leaf Characteristics of

Hibiscus Sabdariffa L. Calyx Ethanol Extract Ameliorates Thioacetamide-Induced Hepatic

Comparative Studies on the Phytochemical Content and In Vitro Antioxidant Capacity of 34

NISEB Journal Vol. 22, Nos. 1/2. March/June, 2022 1595-6938/2022

Materials and Methods

Plant materials V. minor

Percentage moisture content is (28.09 %)

Table 2: The yield of crude extract and fractions

The results of the qualitative phytochemical analyses are presented in Table 4

Table 4: Qualitative analysis results

Table 6: Results of Proximate analysis n=2 Composition (g in 100g or %)

Parameter Vinca minor

Table 7 : FTIR results for Vinca minor

Fraction Wavenumber (cm-1) Assignment Likely functional group present

2853 CH3 stretching Aliphatic

1464 CH2 stretching

Likely compound present Steroids

Likely compound present Alkaloids

NISEB Journal Vol. 22, Nos. 1/2. March/June, 2022 1595-6938/2022

Materials and Methods

Corresponding Author’s Email: eosibote@unilag.edu.ng

Conc. (ug/ml) 10 25 50 100 200

Table 2: Nitric oxide free radical scavenging activity (NOSA) of V. minor

Extract Concentration of TAC (mg AAE/g of extract)

NISEB Journal Vol. 22, Nos. 1/2. March/June, 2022 1595-6938/2022

*Corresponding Author’s Email: adeyemi.oyeyemi@fupre.edu.ng

Materials and Methods

Experimental Design and Agronomic Details

Table 1: Some physico-chemical parameters of the experimental soil

[11] Mackova M, Macek T, Ocenaskova J, Burkhard J, Demnerova K, Pazlarova J: Biodegradation of

NISEB Journal Vol. 22, Nos. 1/2. March/June, 2022 1595-6938/2022

*1 Ebhohon, Shirley O., 2Asoya, Ekene V., 2Iyare, Harrison E.

Materials and Methods

Preparation of liver homogenates

Table 1: Phytochemical constituents of Ethanol Extract of Hibiscus sabdariffa calyx

NISEB Journal Vol. 22, Nos. 1/2. March/June, 2022 1595-6938/2022

Comparative Studies on the Phytochemical Content and In Vitro Antioxidant

*Omorede Ikponmwosa-Eweka1 and Ehimwenma Sheena Omoregie2

Materials and Method

Table 1: Qualitative phytochemical analysis of S. mombin and C. dependens

S. mombin 0.15 ± 0.007a 1.91 ± 0.003a 277.0 ± 2.84a

1.5 Ascorbic acid

NISEB Journal Vol. 22, Nos. 1/2. March/June, 2022 1595-6938/2022

Changes in Some Biochemical Parameters in Red Meat Tendered with

Materials and Method

Results and Discussion

120 Percentage Metmyoglobin

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