Biokemistri Volume 32, Number 2

ISSN 0795-8080
BIOKEMISTRI
An International Journal Published by the
Nigerian Society for Experimental Biology
Volume 32, Number 2

June, 2020
BIOKEMISTRI
An International Journal Published by Society for Experimental Biology of Nigeria
Editor-in-Chief
Prof. Clement O. Bewaji, Department of Biochemistry, Faculty of Life Sciences, University of
Ilorin, Ilorin, Nigeria.
Editorial Advisory Board

Dr-Mrs. R. A. Adisa, Department of Prof. Ganiyu Oboh, Department of
Biochemistry, College of Medicine, Biochemistry, Federal University of
University of Lagos, Lagos, Nigeria. Technology, Akure, Nigeria.
Prof. I. S. Afolabi, Department of Prof.-Mrs. Henrietta Oboh, Department of
Biochemistry, Covenant University, Ota, Medical Biochemistry, Faculty of Basic
Nigeria. Medical Sciences, University of Benin,
Benin City, Nigeria.
Dr. R. O. Arise, Department of
Biochemistry, Faculty of Life Sciences, Prof.-Mrs. O. A. Odunola, Department of
University of Ilorin, Ilorin, Nigeria. Biochemistry, Faculty of Basic Medical
Sciences, University of Ibadan, Ibadan,
Prof. Dr. Hatice Demiray, Department of
Nigeria.
Biology, Ege University, Izmir (Bornova),
Turkey. Prof.-Mrs. A. T. Oladiji, Department of
Biochemistry, Faculty of Life Sciences,
Prof. Evans C. Egwin, Department of
University of Ilorin, Ilorin, Nigeria.
Biochemistry, Federal University of
Technology, Minna, Nigeria. Dr. Mehdi Rajabi, Department of Process
Chemistry, Kinentia Bioscience LLC, 7
Prof. O. Farombi, Department of
University Place, Rensselaer, New York
Biochemistry, Faculty of Basic Medical
12144, USA.
Sciences, University of Ibadan, Ibadan,
Nigeria. Dr-Mrs. K. O. Shittu, Department of
Biochemistry, Federal University of
Dr. A. Igunnu, Department of Biochemistry,
Technology, Minna, Nigeria.
Faculty of Life Sciences, University of
Ilorin, Ilorin, Nigeria. Dr. Olutayo Sunday Shokunbi, Department
of Biochemistry, Babcock University, Ilisan
Dr. Olugbenga Morebise, Department of
Remo, Nigeria.
Biochemistry, All Saints University School
of Medicine, Roseau, Dominica. Prof. M. T. Yakubu, Department of
Biochemistry, Faculty of Life Sciences,
University of Ilorin, Ilorin, Nigeria.
BIOKEMISTRI
Volume 32 Number 2 Contents June, 2020
BKR 2020014/32201
Protective activity of root extract of Rhaphiostylis beninensis against carbon
tetrachloride-induced hepatotoxicity in Wistar rats
Uduenevwo Francis Evuen, Augustine Apiamu, Ngozi Paulinus Okolie and Blessing
Ogechukwu Orji .................................................................................................................................. 93
BKR 2020015/32202
Aqueous seed extract of Cola acuminata ameliorated high fat diet-induced
hyperlipidaemia in rats
Musa Oyewole Salawu, Sikirat Abiola Mustapha and Hussein Oyelola Bukoye Oloyede .................. 101
BKR 2020016/32203
Amylase production by Aspergillus flavus immobilized in polysaccharide beads of
Adansonia digitata.
Temitope Temitayo Banjo .................................................................................................................... 115
BKR 2020018/32204
Molecular phylogenetic authentication of the relative evolution of Desplatsia spp. from
Southern Nigeria
Oghale O. Ovuakporie-Uvo, MacDonald Idu, Olumide Afolabi, Michael Kolade Irieabo ................. 125
BKR 2020019/32205
Repression of some cholinergic and monoaminergic enzymes by non-polar solvent
extracts from Rosary Pea (Abrus precatorius)
Bukola C. Adedayo, Sunday I. Oyeleye and Ganiyu Oboh .................................................................. 133
BKR 2020021/32206
Effect of alkaloid-rich extract from eggplant (Solanum kumba) fruit peels on manganese-
induced neurodegeneration in fruit fly (Drosophila melanogaster) model
E. E. Nwanna, S. A. Shodeinde and A. P. Olayemi.............................................................................. 143
BKR 2020023/32207
Effect of bitter leaf (Vernonia amygdalina) aqueous extract on pancreatic α-amylase and
intestinal α-glucosidase repressive potentials of Acarbose
Ayokunle O. Ademosun, Kelvin C. Akuine, Sunday I. Oyeleye, Ganiyu Oboh .................................... 157
iv
U. F. Evuen et al.
0795-8080/2020 $10.00 + 0.00

Biokemistri
Vol. 32, No. 2, June 30, 2020, pages 93-100 © 2020 Nigerian Society for Experimental Biology
Printed in Nigeria http://journals.niseb.org.ng
Also available online at http://www.bioline.org.br/bk
BKR 20200014/32201
Protective activity of root extract of Rhaphiostylis beninensis

against carbon tetrachloride-induced hepatotoxicity in
Wistar rats
Uduenevwo Francis Evuen*1, Augustine Apiamu1, Ngozi Paulinus Okolie2 and Blessing
Ogechukwu Orji3
1
Department of Biochemistry, Western Delta University, P.M.B. 10, Oghara, Delta State
2
Department of Biochemistry, University of Benin, P.M.B. 1154, Benin City, Edo State
3
Department of Biochemistry and Molecular Biology, Federal University Dutsin-ma, Katsina State
*
Correspondence: francdei@yahoo.com; +2348035459896.
(Received January 20, 2020; Accepted March 9, 2020)
ABSTRACT: Liver diseases pose an enormous problem worldwide irrespective of the many strides in modern
medicine. This has necessitated the need to depend on complementary and alternative systems of medicine for liver
ailments. Rhaphiostylis beninensis is a culinary spice with various medicinal applications. The current study examined
the hepatoprotective effects of methanol root extract of Rhaphiostylis beninensis in Wistar rats for a 14-day period.
Twenty experimental rats were randomly divided into four groups labelled A-D comprising five rats each. Rats in
group A were fed with pellets and water ad libitum for 14 days as control. Group B rats received a single intraperitoneal
injection of carbon tetrachloride in a dose of 3 mL/kg of a 50 % (v/v) solution in liquid paraffin only on day 1. Group
C rats were administered the same dose of carbon tetrachloride on day 1 prior to thirteen days administration of the
root extract (500ml/kg body weight). A similar dose of the root extract was given to rats in group D for thirteen days
sequel to Carbon tetrachloride administration on day 14.The substantive elevated (p<0.05) serum levels of liver
biochemical parameters, liver malondialdehyde levels and decreased liver antioxidant enzyme levels (p<0.05) in group
D rats, were restored towards normalcy in rats pre-treated and treated with the spice extract (D and C) as compared
with the control group. The results of this study strongly indicate that the root extract of Rhaphiostylis beninensis
possess hepatoprotective activity against carbon tetrachloride induced hepatotoxicity in Wistar albino rats.
Keywords: Rhaphiostylis beninensis, hepatotoxicity, carbon tetrachloride, malondialdehyde
Introduction
The liver is the chemical plant of the body that is often exposed to a variety of xenobiotics and
therapeutic agents. The body depends on the liver to regulate, synthesize, store and secrete many important
proteins, nutrients, chemicals and to purify and clear toxins or unnecessary substances from the body (Garba
et al, 2009).
Carbon tetrachloride (CCl4) is a well-known liver toxicant and has been frequently used for generating
free radical induced liver injury in rat models (Minami et al, 2005; Junnila et al, 2000; Cui et al, 2009; Kim
et al, 2010). The hepatotoxicity of CCl4 has been reported to be due to its biotransformation by cytochrome
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Biokemistri Volume 32, Number 2 (2020)
P450 system to produce trichloromethyl free radical (CCl3) which readily reacts with molecular oxygen to
form trichloromethyl peroxy radical (Raucy et al, 1993).
Liver disease remains a worldwide health problem in spite of great advancements in modern medicine
(Sowjanya et al., 2013). Different kinds of herbal medicines derived from plant extracts with relatively little
knowledge regarding their modes of action, are being utilized increasingly to treat a wide variety of clinical
diseases (Mathews et al., 1999). Besides, studies have demonstrated that dietary intake of a variety of plant
phytochemicals confer inhibitory effects against oxidative damage (Hollman and Katan, 1999; Yemitan
and Izegbu, 2006). For this reason, a growing interest has been paid to the research of natural antioxidants,
among which spices occupy an important position (Pokorny et al., 2001). Antioxidants retard or inhibit
oxidation of other substances by inhibiting the initiation or propagation of oxidizing chain reactions
(Velioglu et al., 1998). Hence, natural antioxidants can protect the biologically important cellular
components from oxidative processes caused by reactive oxygen species (Su et al., 2007). Included in the
purview of spices with antioxidant potential is Rhaphiostylis beninensis.
Rhaphiostylis beninensis Planch ex Benth (Icacinaceae) is a woody climber with various medicinal
and culinary applications. It is found growing in South-western Nigeria and the West African sub region.
In Nigeria, it has many vernacular names, depending on the location and usage. The plant is called atapata
(Yoruba), osumadin (Benin), kpolokoto (Ibos), umeni (Urhobos) and kumeni (Itsekiris).
The family to which R. beninensis belongs (Icacinaceae) has been reported to contain numerous
bioactive phytochemicals (Nkafaniya et al., 2007; Gandiza et al., 1993). Studies have also revealed various
pharmacological and biological properties of the root bark extracts of the plant. Pharmacological activities
reported for the plant include anti-bacterial, analgesic and anti-inflammatory (Edema et al, 2006; Lasisi et
al., 2010; Ofeimun and Onwukaeme 2006).
In folkloric medicine, the leaf and root are used in the management of arthritis and rheumatism, skin
diseases, mental disorder, convulsion and eye problem (Burkill, 1994; Odugbemi, 2008) while the leaf
decoction is used as a mouth wash and a wash for sores. Oil obtained from the root and various extracts of
the bark and fruit exhibited anti-microbial activity against gram positive and gram-negative bacteria as well
as fungi (Edema et al., 2009; Adebayo-Tayo et al., 2010).
The plant was reported to contain anthraquinones, flavonoids and triterpenes (Ofeimum and
Onwukaeme, 2006). In addition, a thiourea derivative namely, N,N-di (4-methyoxybenzyl) thiourea with
anti-inflammatory activity has also been isolated from the root of the plant (Ofeimum et al., 2014).
Till date, mankind has not been able to find an ideal curative and prophylactic agent for liver disorders.
Thus, the need for an alternative/complementary medicine for liver ailments cannot be overemphasized.
Although several reports abound on some biological and pharmacological properties of Rhaphiostylis
beninensis spice extracts, there is no documentation on the antioxidant ability of the spice in relation to its
hepatoprotective effect on carbon tetrachloride-induced changes in rats. Hence, the present investigation of
the plant.
Materials and Methods
Chemicals
CCl4 was obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and kits
(from Randox laboratories, Crumlin, United Kingdom) used throughout this investigation were of the
highest analytical grade commercially available while the water was glass distilled.
Collection and Authentication of the Plant Material

The roots of Raphiostylis beneninsis were bought from a local market, in Oghara, Delta state. The
roots were identified and validated by Dr. H. A Akinnibosun of the Department of Plant Biology and
Biotechnology, University of Benin. The plant specimen with serial number, UBHp0262 was deposited in
his herbarium.
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U. F. Evuen et al.
Preparation and Extraction of the Plant Material

The plant was washed free of earthy material and the root bark was scrapped off and dried in the
laboratory at room temperature for 1 week and thereafter in an oven maintained at 40 ⁰C for 30 min. The
dried plant material (1500g) was milled and subjected to extraction with 70 % methanol by cold maceration
technique at 20 mg/ml for 4 days. The extract was then concentrated to dryness by a rotary evaporator
(Buchi Rotavator-R, China) to a brownish slurry at 40ºC. This was then dried in an open water bath at 40ºC
and weighed to determine the yield. The concentrated extract was stored in an air-tight container at 4oC
until required.
Phytochemical Screening of the Plant Material

The phytochemical components of Raphiostylis beninensis roots were determined using the methods
of (Sofowora, 1993; Harbone, 1973) with some minor modifications.
Experimental Animals
Twenty (20) male Wistar rats (110-170 g) were selected for the study. The animals were procured
from the animal unit of the Anatomy Department, University of Benin, Benin City and kept in cages for
four (4) weeks.
The animals were allowed to acclimatize to the new environment for two (2) weeks and thereafter,
subjected to two (2) weeks of treatment with free access to pelleted livestock feed, and water ad libitum
with a 12-12 h light/dark cycle respectively.
The handling and treatment of the rats were done based on the approved ethics for the use and care of
experimental animals.
Experimental Design
The animals were randomly divided into four groups labelled A-D comprising five rats each. Rats in
group A were fed with pellets and water ad libitum for 14 days as control. Group B rats received a single
intraperitoneal injection of CCl4 in a dose of 3ml/kg of a 50 % (v/v) solution in liquid paraffin only on day
1.
Group C rats were administered CCl4 (3ml/kg body weight of a 50% (v/v) solution in liquid paraffin
on day 1 prior to thirteen days administration of Methanol root extract of Rhaphiostylis beninensis (MERB)
at a dose of 500ml/kg body weight.
Group D rats were administered 500 mg/kg body weight of MERB for thirteen days sequel to CCl 4
(3ml/kg body weight of a 50% (v/v) solution in liquid paraffin) administration on day 14. On the 15th day,
animals were fasted overnight, sacrificed by cervical dislocation and then dissected.
Preparation of Serum and Liver homogenate

Blood samples were collected by heart puncture using a hypodermal syringe and needle. Blood sample
was collected from the heart vessels into the non-heparinized bottles for serum biochemical analyses, using
standard assay kits (Randox Lab Ltd. UK) and the liver was quickly excised from each rat immediately
after sacrifice. It was rinsed in ice-cold 1.15% potassium chloride, blotted with filter paper and weighed.
Weighed portions were minced with scissors in 4ml of ice-cold 0.1M phosphate buffer, pH 7.4 and
homogenized in a Potter–Elvehjem homogenizer. The homogenates were later centrifuged using
refrigerated centrifuge at 3,000 x g for 15 min at 4ºC to obtain clear supernatants, which were used for
subsequent oxidative stress indicators.
Biochemical Analyses
Biochemical analyses carried out include measurement of the activities of plasma alanine
aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (γ-GT), and
alkaline phosphatase (ALP); plasma total protein and albumin concentrations. The determination of the
95
concentrations of these biochemical parameters was done using commercially available test kits, products
of Randox Laboratories (Crumlin, United Kingdom).
Lipid peroxidation and Antioxidant Biomarkers

Lipid peroxidation was assessed by measuring the formation of malondialdehyde according to the
method described by Iqbal et al. (1996). Catalase (CAT) activity was determined according to the method
of Aebi (1984) and Luck (1974). The level of superoxide dismutase (SOD) activity was measured as
described by Misra and Fridovich (1972).
Statistical Analysis
Data generated in the present study were expressed as Mean ± SEM for the five determinations of each
experimental group. They were statistically analyzed using one-way analysis of variance (ANOVA) and
tukey multiple comparison (TMC) test. The mean results with probability less than 0.05 (p < 0.05) were
considered significant.
Results
Results of the quantitative phytochemical constituents of methanol root extract of R. beninensis spice
(Table 1) indicated that flavonoids and phenols were present in higher concentration in the root extracts
than alkaloids, tannins and saponins.
Table 1: Phytochemical constituents of methanol root extracts of R. beninensis spice
Phytochemical Quantity (%)

Flavonoids 3.72 ± 0.13a
Tannins 0.78 ± 0.04b
Alkaloids 1.74 ± 0.07c
Phenols 2.03 ± 0.07d
Saponins 0.23 ± 0.01e
Values are expressed as mean ± SEM (n=3). Values with different letters are significantly different (p<0.05).
Table 2 reveals relative weight of liver in the control and experimental groups. The relative weight of
Liver for group B animals (carbon tetrachloride administered) were significantly higher (p<0.05) than that
of group A (control) and the other experimental groups (B and C). However, values for groups A, C and D
were significantly similar (p<0.05).
Table 2: Comparison of mean relative weight of the liver
PARAMETER GROUP A GROUP B GROUP C GROUP D

(CONTROL) (CCl4) (MERB + CCl4) (CCl4 + MERB)
Liver weight(g) 4.28 ±0.29a 6.02 ±0.21b 4.32 ±0.13 a 4.14 ±0.15a
Values are expressed as mean ± SEM (n=5). Values with different letter along the same row are significantly different
(p<0.05).
Table 3 shows the effect of methanol root extract of R. beninensis on CCl4 - induced changes in serum
levels of some Liver biochemical parameters. There was significant increase (p<0.05) in the concentrations
of gamma-glutamyl transpeptidase (GGT), aspartate aminotransaminase (AST), alanine aminotransaminase
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U. F. Evuen et al.
(ALT), alkaline phosphatase (ALP) and decreased total protein (TP) and albumin(ALB) levels were
observed following an intraperitoneal administration of CCl4 when compared with normal control.
However, treatment with MERB before and after administration of CCl 4 in groups C and D respectively,
resulted in significant increased level (p<0.05) of total protein and albumin and a concomitant decreased
levels (p<0.05) of other serum biochemical parameters in comparison with the CCl4 treated group. In
general, treatment with MERB extract produced a non-significant difference in the aforementioned
biochemical parameters when compared with the normal control group.
Table 3: Effect of methanol root extract of R. beninensis on CCl4-induced changes in serum levels of
some liver biochemical parameters
Groups GGT(U/L) ALB (g/L) TP(g/L) ALP (U/L) AST (U/L) ALT(U/L)
A 110.10±26.80a 13.80±0.30c 23.69±0.40d 7.10±0.3.10b 12.03±4.30e 60.75±4.01f
B 126.22±0.00b 6.28±0.00d 16.16±0.00e 12.65±0.00c 15.00±1.41f 94.00±0.00h
C 112.67±0.18 a 12.81±0.76c 22.98±1.85d 7.38±1.65b 11.90±0.03e 59.71±5.04f
D 113.99±0.15a 13.23±0.14c 23.38±0.12d 7.46±3.03b 14.84±3.86e 64.08±0.62f
Values are expressed as mean ± SEM (n=5). Values with different letter along the same column are significantly
different (p<0.05).
Table 4 shows the effect of methanol extract of the roots of R. beninensis on CCl4-induced changes in
liver lipid peroxidation product (MDA), Superoxide dismutase (SOD) and Catalase (CAT).
In group B animals, Malondialdehyde (MDA), an index of lipid peroxidation was significantly
increased (p<0.05) by the administration of CCl4 when compared with normal control (A) and the other
experimental groups (B and C).The induced peroxidation due to this increase was attenuated by treatment
with MERB before and after CCl4 administration (groups C and D).
Liver antioxidant marker enzymes (SOD and CAT) activities on administration of CCl 4 on rats were
significantly reduced in group B animals when compared with normal control group (A) and the other
experimental groups (C and D). However, administration of MERB gave a significant increase (p<0.05) in
their activities in groups C and D when compared with the normal control.
Table 4: Effect of methanol root extract of R. beninensis on CCl4-induced changes in levels of catalase,
superoxide dismutase and malondialdehyde in liver of rats
Groups CAT(µmol/g Tissue) MDA(µmol/g Tissue) SOD(µmol/g Tissue)

A 38.73±2.99d 45.40±4.67b 22.55±5.94e
B 32.40±3.24e 73.91±0.00c 16.12±0.00f
C 40.10±0.00d 47.01±2.89b 24.31±1.22e
D 38.92±0.91d 45.10±2.75b 23.20±2.61e
Values are expressed as mean ± SEM (n=5). Values with different letter along the same column are significantly
different (p<0.05).
Discussion
Liver disorders are one of the world’s problems. The conservative treatments of liver problems, such
as acute and chronic liver hepatitis, liver cirrhosis, and fatty liver are often inadequate due to hazardous
effects initiated by hepatotoxic drugs of chemical origin (Weber et al, 2003). The occupational exposure to
chemical compounds such as aliphatic hydrocarbons alters the liver structure and functions (Jaeschke,
97
2008). Moreso, oxidative stress-induced peroxidation is a prominent feature of CCl4- induced liver injury
(Adewale and Orhue, 2015).
Phytochemical analysis of the root extract of R. beninensis reveal that flavonoids and phenols were
present in higher concentration in the root extracts than alkaloids, tannins and saponins. The relative
composition of each phytochemical in the plant is contrary to the analysis of phytochemicals in the stem
extract of R. beninensis from an earlier study (Lasisi et al, 2011).The reason may be due to the differential
quantities of phytochemicals in different parts of a plant (Kouki and Manetas, 2002; Monteiro et al., 2006).
However, the presence of these chemical constituents in the methanol root bark extract of R. beninensis is
an indication that this plant if properly screened would yield drugs of plant origin with pharmacological
significance. This is better supported by the fact that various parts of the plant are known to be involved in
ethnomedicine for the management of numerous ailments.
Alkaloids are made up of heterocyclic nitrogen that has been shown to exhibit antimalarial,
antihypertensive, antiarrhythmic, and anticancer properties (Heikens et al., 1995). Alkaloids have also been
reported to act as CNS stimulant and powerful analgesics (Ashok and Upadhyaya, 2012). Saponins have
been reported to have antimalarial effect (Besong et al., 2016). Flavonoids have been reported to possess
antioxidant, anti-inflammatory, antitumor, antiallergic, and antiplatelet activity (Pal and Verma, 2013).
The presence of these phytochemicals in R. beninensis could be responsible for the various reported
pharmacological activities of the plant (Edema et al, 2006; Lasisi et al., 2010; Ofeimun and Onwukaeme
2006).
In accordance with previous studies (Adewale and Orhue, 2015; Hai et al, 2011; Krishnan and
Muthukrishnan, 2012), administration of CCl4 brought about increased levels of AST, ALT, and ALP, and
GGT in serum of the CCl4 treated group.
This outcome can be attributed to hepatic damage, resulting in an increased rate of synthesis or release
of functional enzymes from bio membranes (Pari and Prasath, 2008). In particular, the relative increase in
plasma levels of ALT is an indicator of hepatocyte damage (Brent and Rumack 1993).
Elevated plasma level of ALP is suggestive of biliary obstruction, such as occur in cholestatic disease
of the liver. Differential diagnosis to ascertain the source of plasma ALP is provided by assay of gamma
glutamyl transferase (GGT).
GGT is a membrane bound enzyme whose activity in plasma increases significantly alongside that of
plasma ALP following biliary obstruction (Zimmerman et al, 1965; Ratanasavanh et al, 1982). Biliary
obstruction, as suggested by the data obtained for both ALP and GGT for the CCl 4- treated group in this
study, implies a failure of biliary secretion.
The significant decrease (p<0.05) in albumin levels in CCl4 treated groups when compared with the
control, suggest liver injury, since these are reliable indices of liver toxicity (Omoniyi and Mathew, 2006).
Lipid peroxidation has been categorized as one of the most important causes of CCl4-induced hepatic injury
(Basu, 2003). Malondialdehyde (MDA) is a cytotoxic reactive aldehyde formed as a byproduct of lipid
peroxidation (Manibusan et al, 2007).
An increase in the hepatic MDA level suggested the enhancement of lipid peroxidation, consequently
leading to hepatic damage as well as the inactivation of the antioxidant defense system in the CCl 4- treated
group.
Administration of methanol root extracts of R. beninensis (MERB) significantly (p<0.05) protected
the rats against CCl4-induced hepatotoxicity as demonstrated by its inhibition of the elevation of serum
AST ALT, ALP, MDA, GGT and Total protein activities in the pre-treated and treated groups. The ability
of MERB to effectively protect the liver against carbon tetrachloride toxicity may be related to the
remarkable antioxidant and anti-inflammatory properties of its chemical constituent, thiourea (Ofeimum et
al, 2014; Kajimoto and Murakami, 1998; Kulkarni et al, 2008).
This reduction in the elevated enzyme levels in the pre-treated and treated groups, indicate that MERB
interferes with the action of CCl3 free radicals produced.
Animals pretreated and treated with MERB showed a significant reduction in the levels of hepatic
peroxidative markers with concomitant improvement in the hepatic antioxidative defense system.
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U. F. Evuen et al.
The suppression of MDA production plausibly promote the activities of SOD and CAT. Therefore, an
increase in antioxidant activity and the inhibition of free radical generation is positively correlated with
hepatic protection.
In conclusion, the results of this study have demonstrated for the first time that, R. beninensis root
extract possess hepatoprotective potential seemingly through enhancing hepatic antioxidant enzyme
activities and inhibition of lipid peroxidation in rats. The hepatoprotective effects of the extract may be
traceable to the presence of potent phytoconstituents of the roots. However, further studies are in progress
to identify the antioxidant ability and characterize the active principles of R. beninensis root extract so as
to propose the fingerprint of the mechanism of action of the plant.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the Laboratory Technologist, Western Delta University for his
technical support and the University’s management for financial support.
References
Adebayo-Tayo, B.C., Adegoke, A.A., Okoh, A.I. and Ajibesin, K.K. (2010). Rationalizing some Medicinal Plants
used in Treatment of Skin Diseases. Afr. J. Microbiol. Res. 4:958-953.
Adewale, O.B., and Orhue, N.E.J. (2015). Aqueous Extract of the Fruits of Xylopia aethiopica
(Dunal) A. Rich. Protects against Carbon Tetrachloride - Induced Hepatotoxicity in Rats. European J. Med. Plants,
9(4): 1-10
Aebi, H. (1984). Catalase, In: Packer L (Eds.). Methods in enzymology.Academic press, Orlando. pp. 121-126.
Ashok, P. K., and Upadhyaya, K. (2012). Tannins are astrigent. J. Pharmacog. Phytochem. 1: 45–50.
Besong, E. E., Balogun, M. E., Djobissie, S. F. A., Mbamalu, O. S., and Obimma, J. N. (2016). A review of Piper
guineense (African Black Pepper). Int. J. Pharm. Pharm. Res. 6: 368–384.
Burkil, H.M. (1999). The useful plants of net. Tropical Africa. 4th Edition Macmillan Press: 1:34-36.
Cui, C.P., Wei, P., Liu, Y., Zhang, D.J., Wang, L.S., and Wu, C.T. (2009). The protective role of hepatopoietin on
liver injury induced by carbon tetrachloride in rats. Hepatol. Res., 39(2):200-206.
Edema, C., Nwodo, O., Eneh, F. and Ogbunugafor, H. (2009b). Investigation of the chemical composition and
biological activity of R. beninensis Dunal (ANNONACAE). Afr. J. Biotechnol., 9(43): 7352-7356.
Edema, M.O., Iyekowa, O., Nnadi, C.F. and Eigbadon, K. (2009a). Chemical characterization and anti-microbial
activity of Raphiostylis beninensis (Planch) root oil. J. Chem. Soc. Nig. 34: 110-112.
Garba, S.H., Sambo, N., and Bala, U. (2009). The effect of the aqueous extract of Kohautia grandiflora on paracetamol
induced liver damage in albino rats. Nig. J. Physiol. Sci., 24(1):17-23
Hai, Z. H., Bing, W., Yong, K. L., Yong, Y. B. and Yan, G. (2011). Hepatoprotective and Antioxidant Effects of
Licorice Extract against CCl4-Induced Oxidative Damage in Rats. Int. J. Mol. Sci. 12: 6529-6543.
Harbone, J.B. (1973). Phytochemical Methods London. Chapman and Hall Ltd. pp49-188.
Heikens, H., Fliers, E., Endert, E., Acknermans, M., and Van Montfrans, G. (1995). Linquorice-induced
hypertension—a new understanding of an old disease. J. Med. Chem. 5: 230–234.
Hollman, P.C.H. and Katan, M.B. (1999). Dietary flavonoids: intake, health effects and bioavailability. Food Chem
Toxicol. 37:937- 942. Int. J. Pharm. Pharm. Sci. 5: 97–106.
Iqbal, M., Sharma, S. D., Zadeh, H. R., Hasan, N., Abdulla, M. and Athar, M. (1996). Glutathione metabolizing
enzymes and oxidative stress in ferric nitrilotriacetate (Fe-NTA) mediated hepatic injury. Redox Report, 2: 385-
391.
Jaeschke,H. (2008): Toxic responses of the liver.In: Klaassen CD (ed) Casarret & Doull’s
Junnila, M., Rahko, T., Sukura, A, and Lindberg, L.A. (2000). Reduction of carbon tetrachloride-induced hepatotoxic
effect by oral administration of betaine in male wistar rats: a morphometric histologic study. Vet. Pathol. 37(3):
231-238.
Kajimoto, G and Murakami, C. (1998). Antioxidant activity of thioureas in Oils. Nippon. Eijo. Shakuryo Gakaishi
51:207-212.
Kim, H.Y., Kim, J.K., Choi, J.H., Jung, J.Y., Oh W.Y., Kim, D.C., Lee, H.S., Kim, Y.S., Kang, S.S., Lee, S.H., and
Lee, S.M. (2010). Hepatoprotective effect of pinoresinol on carbon tetrachloride-induced hepatic damage in mice.
J. Pharmacol. Sci., 112(1): 105-112.
99
Kouki M, and Manetas, Y. (2002). Resource availability affects differentially the levels of gallotannins and condensed
tannins in Ceratonia siliqua. Biochem Syst Ecol. 30(7):631-639.
Krishnan, N. and Muthukrishnan, S. (2012). Effect of Nigella sativa seed extract on carbon tetrachloride-induced
hepatotoxicity in rats. J. Acute Med. 2:107-113/
Kulkarni, R.R., Vikar, A.D and D’mello, P. (2008). Antioxidant and Anti-inflammatory activity of Vitex negundo.
Indian J. Pharm. Sci. 70:838-840.
Lasisi, A.A., Folarin, O.M., Daro, E.O. Akinloye, O.A. and Fasuyi, M.O. (2011). Phytochemical, Antibacterial andv
Cytotoxic evaluation of Raphiostylis beninensis (Hook F. Ex. Planch) Stem bark extract. Int. J. Pharmacol. Biosci.
2:489-495.
Luck, H. (1974).Methods in enzymatic analysis. Academic Press, New York, p. 885.
Matthews, H.B., Lucier, G.W., and Fisher, K.D.(1999). Medicinal herbs in the United States: research needs. Environ
Health Perspect.107:773-778.
Minami, K., Saito, T., Narahara, M., Tomita, H., Kato, H., Sugiyama, H.( 2005). Relationship between hepatic gene
expression profiles and hepatotoxicity in five typical hepatotoxicant administered rats. Toxicol Sci.87:296-305.
Misra, H. P. and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of Epinephrine and a simple
assay for superoxide dismutase. J. Biol. Chem. 247:3170-3175.
Monteiro, J.M., Albuquerque, U.P., Lins Neto, E.M., Araújo, E.L., Albuquerque, M.M., Amorim, E.L. (2006). The
effects of seasonal climate changes in the Caatinga on tannin levels in Myracrodruon urundeuva (Engl.) Fr. All.
and Anadenanthera colubrina (Vell.) Brenan. Rev Bras Farmacogn. 16(3):338-344.
Odugbemi, T. (2008). A Textbook of Medicinal Plants in Nigeria. Tolu Press Lagos, p. 23-97.
Ofeimum, J. O., Onwukaeme, D.N. (2006). Evaluation of Phytochemical Constituent and Pharmalogical activity of
roots of Raphiostylis beninensis(icacinaceae) J. Pharm. Sci. Pharm. Practs. 8:85-90.
Ofeimum, J.O. and Ayinde, B.A. (2017). Preliminary investigation of the aphrodisiac potential of the methanol extract
and fractions of Rhaphiostylis beninensis Planch ex Benth (Icacinaceae) root on male Rats. J. Sci. Pract. Pharm.
4(1):12-188.
Ofeimum, J.O. and Mbionwu, M.I. (2014). Cytotoxic and Growth inhibitory activity of aqueous extracts of root and
leaf of Rhaphiostylis beninensis Planch ex Benth and Pyrenacantha standtii Engl (Icacinaceae). J. Pharm. Biol.
Res. 11(1):8-14.
Ofeimum, J.O., Ayinde, B.A., Igbe, I, Aderogba, M., Adhikari, A., Amjad, H., and Iqbal, M.C. (2014). Anti-
inflammatory Constituent from the Root extract of Rhaphiostylis beninensis (Icacinaceae). Res. J. Phytochem.
8(3):127-132.
Pal, D., and Verma, P. (2013). Flavonoids: a powerful and abundant source of antioxidants. Int. J. Pharm. Pharm Sci.
5(3):95-98
Pari L.,and Prasath A. (2008). Efficacy of caffeic acid in preventing nickel induced oxidative damage in liver of
rats. Chem. Biol. Interact.173:77–83.
Pokorny, J., (2001). Introduction in Antioxidants in Food Practical Applications. (Pokorny, J.,Yanishlieva, N.,
Gordon, M. eds.). CRC Press, Boca Raton, Boston, New York, pp. 1-3
Raucy, J.L., Kraner, J.C., and Lasker, B.(1993). Bioactivation of halogenated hydrocarbons by cytochrome P450. Rev.
Toxicol. 23:1-20.
Sofowora A. (1993). Medicinal Plants and Traditional Medicine in Africa. 2nd Edn. Spectrum Books Limited, Ibadan,
Nigeria, pp. 1-153
Sowjanya, G., Swarnalahtha, D., Shivakala, T. and Mobeena, S.K. (2013). Hepatoprotective Activity –A Review. Int.
J. Phytopharm. 3(2): 37-49.
Srivastava, S.K., Rai, V., Srivastava, M., Rawat, A.K.S. and Mehrotra, S. (2006): Estimation of heavy metals in
different Berberis Species and its Market Samples. Environ. Monit.Assess., 116:315-320.
Su, L., Yin, J.J., Charles, D. Zhou, K. Moore, J. and Yu, L. (2007). Total phenolic contents, chelating capacities, and
radical-scavenging properties of black peppercorn, nutmeg, rosehip, cinnamon and oregano leaf. Food Chem.
100:990-997
Weber, L. W. D. Boll, M. and Stampfl, A. (2003). “Hepatotoxicity and mechanism of action of haloalkanes: carbon
tetrachloride as a toxicological model,” Crit. Rev. Toxicol., 33 (2):105–136.
Yanishlieva, N., Gordon, M.eds). CRC Press, Boca Raton, Boston, New York, pp. 1-3
Yemitan, O.K., Izegbu, M.C. (2006). Protective effects of Zingiber officinale (Zingiberaceae) against carbon
tetrachloride and acetaminophen-induced hepatotoxicity in rats. Phytother Res. 20: 997-1002.
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0795-8080/2020 $10.00 + 0.00

Biokemistri
Vol. 32, No. 2, June 30, 2020, pages 101 – 113 © 2020 Nigerian Society for Experimental Biology
BKR 20200015/32202
Aqueous seed extract of Cola acuminata ameliorated high

fat diet-induced hyperlipidaemia in rats
Musa Oyewole Salawu*1, Sikirat Abiola Mustapha1 and Hussein Oyelola Bukoye
Oloyede1,2
1. Department of Biochemistry, University of Ilorin, Ilorin, Kwara State, Nigeria.

2. Department of Chemical Sciences, Summit University, Offa, Kwara State, Nigeria.
*Corresponding Author: salawu.mo@unilorin.edu.ng Tel: +2348056168553
ABSTRACT: Hyperlipidaemia is a medical condition characterized by an elevation of any or all lipid profiles
and/or lipoproteins in the blood. It has been well established that nutrition plays a vital role in the aetiology of
hyperlipidaemia and cardiovascular diseases. The antihyperlipidaemic potential and toxicological assessment of
aqueous seed extract of Cola acuminata in high-fat diet-induced hyperlipidaemic rats were investigated in this
study. Twenty-four albino rats were grouped into six of four animals per group. Group 1 (control) was fed
formulated feed without high fat. Group 2 was fed a high-fat diet (HFD) but untreated. Group 3 was fed HFD for
eight weeks but was treated with atorvastatin from week 5 to week 8 (RFD). Group 4 was fed HFD for eight
weeks but was treated with extract 10.71 mg/kg bw from week 5 to week 8. Group 5 was fed HFD for eight weeks
but was treated with extract 21.42 mg/kg bw from week 5 to week 8. Group 6 was fed HFD for eight weeks but
was treated with extract 42.84 mg/kg bw. The results showed that rats in group 2 (HFD) presented significantly
(p<0.05) higher levels of blood lipids, Atherogenic Index (AI) and Coronary Disease (CHD) risk ratio
considerably higher than in the healthy control rats (p<0.05). Group 3 (RFD) rats showed significantly (p<0.05)
reduced blood lipid profile compared to the HFD but similar to that of the reasonable control. The AI and CHD
risk ratios were also not significantly different from that of the control. The ALP, ALT activities of RFD Group,
were also not significantly (p<0.05) separate from the control except the AST activity that significantly (p<0.05)
increased. The groups treated with 10.71, 21.42 and 42.84 mg/kg bw extract presented reduced serum level of
lipids and ALP, ALTand AST activities considerably. AI and CHD risk factors were significantly (p<0.05)
decreased, and the reduction was dependent on the dose of the extract. Histopathological assessment of the heart,
kidney and liver tissues of the experimental rats presented no significant changes and no sign of acute or chronic
injury. However, the HFD group showed with overlying pericardial and coronary artery fat. In the liver, there
were mild lymphocytic infiltrations. Therefore, from this study, it is concluded that aqueous extract of Cola
acuminata was able to improve HFD-induced hyperlipidaemia and caused no significant damage to the organs
(heart, kidney, and liver of the rats).
Keywords: Hyperlipidaemia, high-fat diet, Cola acuminata.
Introduction
Hyperlipidaemia is a heterogeneous group of disorders characterized by an excess of lipids in the

bloodstream (Johnson, 2005). This condition is also called hypercholesterolaemia or
hyperlipoproteinaemia (Amit, 2011). The human body is a sophisticated machine for maintaining the
homeostasis of various organs and systems. An unfavourable change often disturbs the balance resulting
101
in the diseased state (Ankur et al., 2012). Excess lipids cause hyperlipidaemia in the diet or by abnormal
fat and lipoprotein metabolism in the body (Harikumar, 2011). Hyperlipidaemia is a significant health
problem in Nigeria and other developing countries (Chukwu, 2011), and it is considered one of the
major risk factors causing cardiovascular diseases (CVDs). CVDs account for one-third of total deaths
around the world and are often thought to be a major cause of death and disability World-wide by the
year 2020 (Ginghina et al., 2011; Jorgenson et al., 2013). Atherosclerosis (a process of arteries
hardening of cholesterol in the arterial wall which causes narrowing of the arteries) and associated
atherosclerosis disorders like coronary, cerebrovascular and peripheral vascular diseases are accelerated
by the presence of hyperlipidaemia (Wells et al., 2007). Prolonged hyperlipidaemia can also lead to
non-alcoholic fatty liver disease-a situation whereby cholesterol accumulates in the liver, causing
congestion (Hall, 2011). The leading cause of hyperlipidaemia are changes in lifestyle habits, a risk
factor being mainly poor diet, i.e. with a fat intake more significant than 40 per cent of total calories,
saturated fat intake greater than 10 per cent of total calories; and cholesterol intake higher than 300
milligrams per day or treatable medical conditions (Durrington,1995). The abnormally high cholesterol
levels are the result of an unhealthy lifestyle, including taking a high-fat diet and other factors like being
overweight, smoking, heavy alcohol use and lack of exercise. Other factors include diabetes, kidney
disease, pregnancy, and an underactive thyroid gland (Kelly, 2010).
Herbal medicines are being used by about 80 per cent of the world population, primarily in
developing countries. They have stood the test of time for their safety, efficacy, cultural acceptability
and low side effects. The chemical constituents present in them are a part of the physiological functions
of living flora, and hence they are believed to have better compatibility with the human body (Kambooj,
2000). More than 70 medicinal plants have been documented to have significant hypolipidaemic action
(Dahlia et al., 2013). During the last decade, an increase in the use of medicinal plants has been observed
in metropolitan areas of developed countries. Medicinal plants play a significant role in the
hypolipidaemic activity. The advantages of herbal medicines reported are effectiveness, safety,
affordability, and acceptability (Dahlia et al., 2013). There are so many medicinal plants extracts that
have recently been reported to have antihyperlipidaemic property. These include Moringa oleifera
(Onwe et al., 2015), extracts of Solanum melongena (Kateregga et al., 2015), Salvadora oleoides
(Deepak, 2012), Brassica oleracea (cabbage) based diet (Oloyede et al., 2015) among others. This
research was carried out to study the hypolipidaemic activity of aqueous extract of Cola acuminata in
high-fat diet-induced hyperlipidaemia.
Thirty-six adult female albino rats with an average weight of 122.56 ± 4.32 g were obtained from
a reputable source. They were housed in plastic cages at room temperature and 12h light-dark cycle.
The animals were randomly divided into six groups (n=6animals per group). The animals were
acclimatized for one week before the commencement of the experiment. They were grouped and fed
with different dietary formula, as shown in Table 1 and given water ad libitum. The extract was gavaged
to experimental groups at three dose levels 10.71, 21.42 and 42.84 mg/kg) equivalent to 13.5, 27 and
54 g kola nut per 70 kg man daily for 30 days. Group 1 served as the control group and was fed with a
control diet (NC). Group 2 was the hyperlipidaemic group (HFD) and was fed a high-fat diet for eight
weeks. Group 3 was fed a high-fat diet for eight weeks but was treated with atorvastatin from week
five. Group 4 was fed a high-fat diet for eight weeks but was treated with extract 10.71 mg/kg b.w from
week five equivalent to the consumption of half kola nut weighing 13.5 g (half standard) by a 70 kg
man per day. Group 5 was fed a high-fat diet for eight weeks but was treated with extract 21.42 mg/kg
b.w from week five equivalent to the consumption of one kola nut weighing 27 g (standard) by a 70 kg
man per day. Group 6 was fed a high-fat diet for eight weeks but was treated with extract 42.84 mg/kg
b.w from week five equivalent to the consumption of 2 kola nuts both weighing 54 g (double standard)
by a 70 kg man per day.
Assay Kits and Reagents

Reagent kits were products of Randox Laboratory Limited. Sucrose solution, formalin solution,
and other reagents were of analytical grade prepared using distilled water.
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M. O. Salawu et al.
Biochemical analysis
The proximate analyses for control and high-fat diet were determined according to the methods of
AOAC (2005). After eight weeks, the animals were sacrificed by jugular puncturing under anaesthesia
using diethyl ether; blood was collected inside centrifuge tubes and centrifuged at 300 rpm for 20 mins.
The clear serum was carefully pipetted into plain sample bottles and kept frozen until needed for
analysis. High-density lipoprotein cholesterol (HDL-c) was determined by the methods of Bachorik
(1996). The low-density lipoprotein cholesterol (LDL-c) was determined using the method of Williams
et al. (1979). The purposes of Tiez (1990) were employed in the determination of Triglycerides. The
cardiovascular disease risk was calculated as described by Mannien et al., (1992) and Atherogenic Index
(AI) was calculated as described by Nwagha et al., (2005). The method described by Reitman and
Frankel (1957) was used in the assay for the activity of alanine aminotransferase and aspartate
aminotransferase. The technique described by Wright et al. (1972) was employed in the test for the
activity of alkaline phosphatase. Bilirubin was determined using the method described by Jendrassik
and Grof (1938). The technique used for the determination of urea in the serum of the animals was as
described by Veniamin and Vakirtzi (1970). Serum uric acid was determined according to the method
described by Tietz (1995). Serum creatinine was measured using the technique described by Bartels
and Bohmer (1972).
Statistical Analysis
Values were expressed as mean ± SEM of 4 replicates (n = 4). Statistical analysis was conducted
using the SPSS software (version 21). Collected data were subjected to one-way Analysis of Variance
(ANOVA), followed by Duncan Multiple Range test for comparisons. The significant differences
between the means were determined at P<0.05 (95 % confidence interval).
Results
Table 1 presents the proximate composition of the control and high-fat diets; from the table, it
could be observed that the crude lipid content of the high-fat diet was significantly (P<0.05) higher than
the control diet. The caloric value of the high-fat diet was also significantly (P<0.05) higher than the
control.
Table 1: Proximate Composition of Control and High Fat Diet
Proximate Composition (%) NC HFD

Moisture 7.32 ± 0.22a 1.81 ± 0.53b
Ash 3.46 ± 0.33a 2.25 ± 0.08a
Carbohydrate 54.47 ± 0.34a 25.23 ± 0.28b
Caloric Value 1344.1 ± 4.23a 2225.4 ± 2.62b
Crude protein 17.14 ± 0.00a 16.28 ± 0.28a
Crude lipids 3.88 ± 0.26a 36.85 ± 0.03b
Crude fibre 5.65 ± 0.06a 5.58 ± 0.08a
Results are mean of 4 determinations ± SEM. Mean along the same column with different superscript
letters are significantly different (p<0.05).
NC = Control, HFD = High-fat diet group, RFD = Reference drug group
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Table 2: Secondary Metabolites in Aqueous Seed Extract of Cola acuminata
Phytochemicals Concentration (mg/100g)
Saponin 0.21 ± 0.00

Phenolics 6.57 ± 0.12
Steroids 51.18 ± 0.17
Flavonoids 60.92 ± 0.16
Anthocyanin 2.00 ± 0.00
Terpenoids 16.43 ± 0.27
Glycosides 8.67 ± 0.04
Triterpenes 74.93 ± 1.75
Alkaloids 10.14 ± 0.70
Table 3 shows the serum and hepatic lipids of rats fed with a high-fat diet and treated with Cola
acuminata aqueous seed extract. The serum and hepatic lipids of rats fed with high-fat diet were
significantly (P<0.05) higher than the control and other groups except for HDL which was significantly
(P<0.05) lower while the hepatic lipids of the reference drug group were not significantly (P<0.05)
different from the treated groups.
Table 3: Serum Lipid Profile and Hepatic Lipids of Rats Fed with High-Fat Diet and Treated with
Aqueous Seed Extract of Cola acuminate
Serum lipid profile(mg/dl) Liver Lipids (mg/dl)

Groups CHOL TRIG HDL LDL CHOL TRIG
NC 81.68 ± 1.60a 25.90 ± 0.44a 75.97 ± 6.44a 2.03 ± 0.05a 146.18 ± 8.56c 88.75 ± 1.77a
HFD 106.91± 1.03c 43.24 ± 0.56c 48.39 ± 1.41b 24.77 ± 2.56c 216.41 ± 4.56b 172.78 ± 3.74c
RFD 82.41 ± 0.64a 24.53 ± 0.09a 82.85 ± 0.61a 2.50 ± 0.04a 154.76 ± 1.34c 102.99 ± 2.99b
10.71mg/kg 88.35 ± 0.67b 32.26 ± 0.59b 68.47 ± 14.93c 7.49 ± 0.13b 171.35 ± 7.26a 103.32 ± 3.83b
extract (half
standard)
21.42mg/kg 86.95 ± 2.54b 29.38 ± 1.44b 75.44 ± 8.34a 4.61 ± 0.27a 174.75 ± 3.45a 101.77 ± 2.21b
extract
(standard)
42.85mg/kg 83.08 ± 0.52a 25.05 ± 0.20a 79.45 ± 7.59a 3.33 ± 0.56a 168.19 ± 3.78a 101.91 ± 3.77b
extract (double
standard)
Results are mean of 4 determinations ± SEM. Mean along the same column with different superscript letters are
significantly different (p<0.05).
NC=control, HFD = High-fat diet group, RFD = Reference drug group
Table 4 shows the Atherogenic Index (AI) and Coronary Heart Disease (CHD) risk ratio of rats
fed with a high-fat diet and treated with aqueous seed extract of Cola acuminata. From the table, it is
observed that the AI and CHD risk ratio of the rats fed on a high-fat diet were significantly (p<0.05)
higher compared with the control and other groups.
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M. O. Salawu et al.
Table 4: Artherogenic Index and Coronary Heart Disease (CHD) Risk Ratio of Rats Fed with High-Fat
Diet and Treated with Aqueous Seed Extract of Cola acuminate
Groups AI Index (x10-1) CHD Risk ratio

NC 0.19 ± 0.01b 1.06 ± 0.01a
HFD 0.36 ± 0.50d 2.23 ± 0.03d
RFD 0.17 ± 0.03a 1.05 ± 0.01a
10.71mg/kg extract 0.20 ± 0.00c 1.12 ± 0.05c
21.42mg/kg extract 0.19 ± 0.08b 1.09 ± 0.03b
42.85mg/kg extract 0.18 ± 0.02b 1.05 ± 0.02b
NC = Control, HFD = High-fat diet group, RFD = Reference drug group, AI = Atherogenic Index, CHD =
Coronary Heart Disease.
Table 5 shows the specific enzyme activities of ALP, ALT, and AST in the serum of animals fed
on High-Fat diet and treated with aqueous seed extract of Cola acuminata. The specific activities of
ALP, ALT, and AST in the serum of animals fed on High-Fat diet (HFD) were significantly (P<0.05)
higher than the control and other groups while the specific activities of ALP, ALT, and AST of the
treated groups were not significantly (P<0.05) different from the control and reference drug group
except for AST which was significantly (P<0.05) higher in the reference group than the control.
Table 5: Specific Enzyme Activity in the Serum of Rats Fed with High-Fat Diet and Treated with
Aqueous Seed Extract of Cola acuminate
Groups Specific Enzyme Activity (nmol min-1 mg protein-1)

ALP ALT AST
NC 85.56 ± 2.88a 25.57 ± 0.98a 62.56 ± 2.33a
HFD 101.70 ± 3.04b 35.85 ± 0.87b 85.41 ± 2.68c
RFD 91.46 ± 4.37a 27.04 ± 0.78a 73.27 ± 0.41b
10.71mg/kg extract 91.35 ± 3.85a 25.67 ± 0.86a 63.70 ± 1.42a
21.42mg/kg extract 87.16 ± 7.03a 24.46 ± 0.86a 60.27 ± 1.97a
42.85mg/kg extract 83.95 ± 3.76a 26.89±1.34 61.30 ± 5.40a
ALP = Alanine aminotransferase, AST = Aspartate aminotransferase, ALP = Alkaline Phosphatase, NC =
Control, HFD = high-fat diet group, RFD = Reference drug group.
Table 6 shows the serum urea, uric acid and creatinine concentrations of rats fed with a high-fat
diet and treated with aqueous seed extract of Cola acuminata. The levels of urea, uric acid and creatinine
were not significantly (p<0.05) different in the experimental and control groups.
105
Table 6: Effect of Aqueous Seed Extract of Cola acuminata on the Kidney Function Indices in the
Serum of Rats Fed with High-Fat Diet
Groups Urea(mg/dl) Uric acid(mg/dl) Creatinine(mg/dl)
NC 12.16 ± 1.71a 1.90 ± 0.09a 2.52 ± 0.32a

HFD 13.04 ± 0.79a 1.69 ± 0.67a 2.60 ± 0.11a
RFD 12.11 ± 0.00a 1.56 ± 0.08a 2.78 ± 0.5b
10.71mg/kg
extract) 12.93 ± 1.03a 1.53 ± 0.09a 2.39 ± 0.27a
21.42mg/kg
extract 13.06 ± 0.04a 1.68 ± 0.04a 2.35 ± 0.54a
42.85mg/kg
extract 12.13 ± 0.56a 1.79 ± 0.02a 2.56 ± 0.53a
Results are mean of 4 determinations ± SEM. Mean along the same column with different superscript
letters are significantly different (p<0.05),
NC = Control, HFD = High-fat diet group, RFD = Reference drug group, C Bil = Conjugated
Bilirubin, T Bil = Total Bilirubin
Table 7 shows the serum total and conjugated bilirubin concentrations of rats fed with a high-fat
diet and treated with aqueous seed extract of Cola acuminata. It shows from the table that the
concentrations of conjugated and total bilirubin of rats fed with a high-fat diet were significantly
(p<0.05) higher than the control and other groups.
Table 7: Effect of Aqueous Seed Extract of Cola acuminata on the Liver Function Indices in the
Serum of Rats Fed with High-Fat Diet
Groups C Bil (mg/dl) T Bil (mg/dl)

NC 25.44 ± 1.12a 65.83 ± 2.54a
HFD 32.90 ± 1.09b 75.86 ± 1.35b
RFD 26.00 ± 0.90a 64.03 ± 2.92a
10.71 mg/kg extract 27.12 ± 2.64a 65.72 ± 2.61a
21.42 mg/kg extract 24.72 ± 2.27a 62.86 ± 2.50a
42.85 mg/kg extract 26.25 ± 2.56a 66.38 ± 2.56a
significantly different (p<0.05),
NC = Control, HFD = High-fat diet group, RFD = Reference drug group, C Bil = Conjugated Bilirubin, T
Bil = Total Bilirubin
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M. O. Salawu et al.
Effect of Aqueous Extract of Cola acuminata on the Heart Histoarchitecture of High- Fat Diet-
Induced Hyperlipidaemic Rats
Results of the histopathology of the heart of both the experimental and control rats show
myocardial tissue composed of a syncytium of cardiac muscle cells and vascular channels (Figs. 1 – 3).
There was no significant hypertrophy, infarction or inflammation seen in the control and other groups
except for HFD which shows myocardial tissue with overlying pericardial and coronary artery fat but
also presented no significant hypertrophy, infarction or inflammation. This is suggestive of the fact that
prolonged consumption of a High-Fat Diet could cause atherosclerosis which is a risk factor for
Coronary Heart Disease.
NC HFD
RFD 10.71 mg/kg b.w.
21.42 mg/kg b.w. 42.84 mg/kg b.w.
Fig. 1: Histoarchitecture of the Hearts of control and experimental rats.
107
Effect of Aqueous Extract of Cola acuminata on the Kidney Histoarchitecture of High- Fat Diet-
Induced Hyperlipidaemic Rats
NC HFD
21.42 mg/kg b.w. 42.84 mg/kg b.w.
Fig. 2: Histoarchitecture of the Kidney of the control and experimental rats.

The histopathological examination of the kidneys of control and experimental rats shows kidney tissues
with preserved architecture comprising normal glomeruli tubules with no features of acute or chronic
damage. There was no significant difference in the kidney histopathology of all groups.
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M. O. Salawu et al.
Effect of Aqueous Extract of Cola acuminata on the Liver Histoarchitecture of High-Fat

Diet-Induced Hyperlipidaemic Rats
NC HFD
21.42 mg/kg b.w. 42.84 mg/kg b.w.
Fig. 3: Histoarchitecture of the Liver of the control and experimental rats
The histopathological examination of the Liver of control and experimental rats shows liver tissue
with preserved architecture, composed of hepatocytes with mild cytoplasmic accumulations (Glycogen
accumulation-normal). The portal tract shows mild lymphocytic infiltration in the High-Fat diet group
which may be due to the membrane leakage, but there are no features of acute/chronic injury.
109
Discussion
Africans, especially Nigerians, eat a lot of fatty meats which expose them to risks associated with
a high-fat diet. Intake of a high amount of saturated and unsaturated fatty acids have been implicated in
the cause of hyperlipidaemia (Varsha et al., 2010) and could be said to be responsible for the induction
of hyperlipidaemia in rats (Harikumar et al., 2013).
A high-fat diet prepared with animal fat was rich in saturated and unsaturated fatty acids (Table
5). The crude fat content of the high-fat diet was significantly (P<0.05) higher than the control diet
(Table 5). Intake of a High-fat diet has been known to induce hyperlipidaemia in previous studies
(Monike et al., 2011; Oloyede et al., 2015; Karam et al., 2018). Administration of high-fat diet
demonstrated a significant increase in total cholesterol, low- density lipoprotein and triglycerides with
a decrease in high-density lipoprotein cholesterol when compared with the control (Table 7).
Kola nuts have been reported to have lipolytic (fat-burning) properties (Arun, 2012; Salawu et al.,
unpublished 2018). Thus the use of Cola acuminata extract at varying concentrations was carried out
in this study of the effectiveness of kola nut, in ameliorating high-fat diet-induced hyperlipidaemia. The
serum and hepatic lipids of the untreated high-fat diet group were found to be significantly (P<0.05)
higher than the control and other groups. The serum lipid profile of the extract-treated groups was found
not to be significantly (P<0.05) different from the control and RFD group, except the 42.84mg/kg bw
extract group presented the least values for lipid profile parameters thus, could be assumed to be the
most effective dose in ameliorating high-fat diet-induced hyperlipidaemia in the rats. This is consistence
with a recent study by Nku et al., (2014) that high consumption of kola nut does reduce the risk of
coronary heart disease and another previous research has also shown that chronic administration of kola
nut significantly decreased body weight (Hwu and Lin 2010).
Hyperlipidaemia is associated with heart diseases. Coronary heart disease is the leading cause of
death in developed countries. This alarming statistic is partly attributable to lifestyle, and partly due to
the genetic factors that make humans highly susceptible to atherosclerotic vascular disease. The
principal metabolic causes of atherosclerosis include hyperlipidaemia, hypertension, obesity, and
diabetes mellitus (Dhandapani, 2007).
The atherogenic index is a direct pointer to exposure to atherosclerosis which is a precursor of all
cardiovascular diseases. The high atherogenic index may imply that atherosclerotic plaques have been
deposited on the walls of the arteries (Kaushik et al., 2014). These plaques are caused primarily by
deposition of low-density lipoproteins (LDL) on the walls of the arteries. They may lead to partial or
complete blockage of the flow of blood in the arteries (Harikumar et al., 2013). The result of this study
showed that the atherogenic index (AI) of the HFD untreated group was significantly (P<0.05) higher
than the control and other groups which means that there is high probability that atherosclerotic plaque
has started forming in the arterial walls of the rats of which prolonged exposure to the high-fat diet may
later result to atherosclerosis. In contrast, the AI of the extract-treated groups reduced but not to the
level of the reference drug (atorvastatin) (Table 8). However, long term treatment with aqueous extract
of Cola acuminata might give a better result.
Cardiovascular disease (CVD) risk ratio is a reliable marker for predicting the risk of coronary
heart diseases like heart attack, myocardial infarction, atherosclerosis, etc. CVDs account for one-third
of total deaths around the world, and it is believed that it will turn out to be the leading cause of death
and disability World-wide by the year 2020 (Ginghina et al., 2011; Jorgenson et al., 2013). The CVD
risk ratio of the HFD untreated group was significantly (P<0.05) higher than the control and other
groups, suggesting that the chance of developing CVDs is high in the HFD untreated group, agreeing
with the report by Wells et al. (2007) that the presence of hyperlipidaemia accelerates atherosclerosis
and other CVDs. On the other hand, the CVD risk ratio of the extract-treated groups significantly
(P<0.05) reduced but not to the level of the reference drug. This also implies that long term treatment
with the extract may give a better result.
Results from the study indicate the presence of saponins, phenolics, terpenoids, flavonoids, and
alkaloid in the aqueous extract seed extract of Cola acuminata, which is in agreement with a report by
Dewole et al., (2013). The concentration of phenolics and flavonoids might have contributed to the
reduction of oxidative stress and hence improving the hyperlipidemic activity. Phenolics and
flavonoids, as reported by El-Tantawy et al., (205) have the potential for lowering the hyperlipidaemia.
110
M. O. Salawu et al.
Alaninet aminotransferase and aspartate aminotransferase (ALT and AST) are two closely related
enzymes of clinical importance (Huncrantz et al., 1986), especially in the assessment of liver and kidney
functions. Both enzymes increase in many disorders related to liver damage; hence they have been
proven to be sensitive indicators of liver-cell injury (Pratt and Kaplan 2000). ALT is more elevated than
AST in various necro-inflammatory conditions of the liver, reflecting its greater efficiency as a liver
disease marker (Rosenthal and Haight 1989). The activities of ALT and AST in the serum of the
untreated animals (HFD) were found to increase significantly (P<0.05) and this may be due to damage
to the membranes of the liver leading to leakage of the enzymes out of the liver, and this is consistent
with the finding of Oloyede et al., (2015) that high-fat diet formulated with goat fat caused damage to
the membranes of heart and liver while the increase in the activity of AST in the reference drug group
(RFD) suggests localized system of toxicity of the reference drug (Table 9).
There was also a significant (P<0.05) increase in the serum activity of ALP in the untreated group
(Table 5), too suggestive of damage to the membranes in the organs of the rats. ALP is a biomarker of
the plasma membrane and is widely used in the assessment of liver injury, an increase in serum activity
of ALP indicates altered membrane integrity (Saukkonen et al., 2006).
Urea is the end product of protein catabolism. Amino acid deamination takes place in the liver
which is also the site of the urea cycle where ammonia is converted into urea that is excreted through
the urine (Amadi et al., 2012). Urea varies directly with protein intake and inversely with the rate of
excretion(Adebayo et al., 2003). The functional capacity of the kidney can be assessed by determining
the serum electrolyte, urea, uric acid, and creatinine concentrations. In this study, the serum urea, uric
acid and creatinine concentrations of the experimental rats were not significantly (P<0.05) different
from that of the control. This indicated that the administration of aqueous extract of Cola acuminata
did not alter glomeruli and tubular functions of the experimental rats. Biu et al. reported a similar result.,
(2009) who said that Cola nitida extract caused no significant changes in the plasma creatinine level
and also in consistence with Salawu et al., unpublished (2018), that the administration of aqueous
extract of the two varieties of Cola nitida did not cause any significant (P<0.05) changes in the
creatinine and urea levels in the serum.
The level of conjugated and total bilirubin can be used to monitor the excretory function of the
liver (Yakubu et al., 2003). Increase (P<0.05) in total and direct bilirubin concentrations of the untreated
group as compared to other groups might be a result of haemolysis of red blood cells. It has been
reported that there is a relationship between serum lipids and erythrocyte membrane fragility (Cooper,
1977) and that increasing hyperlipidaemia (Particularly hypertriglyceridaemia) is associated with
increased haemolysis. It is, therefore, possible, that increased lipid concentrations alter the composition
of the erythrocyte membrane leading to increased fragility of the membrane and subsequent leakage of
cellular content such as haemoglobin. It is the breakdown of haemoglobin that produces bilirubin.
Results of the histopathology of the heart of both the experimental and control rats show
myocardial tissue composed of a syncytium of cardiac muscle cells and vascular channels. There was
no significant hypertrophy, infarction or inflammation seen in the control and other groups except for
HFD which shows myocardial tissue with overlying pericardial and coronary artery fat but also
presented no significant hypertrophy, infarction or inflammation.
This is suggestive of the fact that prolonged consumption of a High-Fat Diet could cause
atherosclerosis which is a risk factor for Coronary Heart Disease.
The histopathological examination of the kidneys of control and experimental rats shows kidney
tissues with preserved architecture comprising normal glomeruli tubules with no features of acute or
chronic damage. There was no significant difference in the kidney histopathological examination of all
groups.
The histopathological examination of the Liver of control and experimental rats shows liver tissue
with preserved architecture, composed of hepatocytes with mild cytoplasmic accumulations (Glycogen
accumulation-normal). The portal tract shows mild lymphocytic infiltration in the High-Fat diet group,
but there are no features of acute/chronic injury. There was no significant difference in the kidney
histopathological examination of all groups.
Conclusion
The result of this study shows that a high-fat diet formulated with goat fat was able to induce
hyperlipidaemia and caused membrane lipid damage to the liver. Cola acuminata extract of varying
111
concentrations was able to reduce blood lipids to almost normal levels. The histopathological
assessment of the heart, kidney, and liver of the experimental and control animals presented no
significant changes in the biochemistry of the rats.
References
Amit G, Vandana S, Sidharth M (2011). Hyperlipidaemia: An Updated Review. Inter J of Biopharma &Toxicol
Res; 1:81-89.
Ankurrohilla, NidhiDagar, SeemaRohilla, AmarjeetDahiya, Ashok Kushnoor (2012). Hyperlipidaemia- a Deadly
Pathological Condition. Inter J Curr Pharma Res; 4:15-18
AOAC (2002). Official Methods of Analysis. Association of Analytical Chemists. 18th edn, Washington DC.
Chukwu, O. (2011). Heart disease, stroke cost Nigerian $800m yearly. Pmnews Sept. 21. Available at
http://www.pmnewsnigeria .com/2011/09/21/heart-disease-
Cooper R. A. Abnormalities of cell-membrane fluidity in the pathogenesis of disease. N Engl J Med 1977;
297:371-7.
Dahlia Salam. A, Surya A. S, Dawn V Tomy, Dr. Betty Carla, Dr.Arun Kumar*, Dr. C. Sunil*A review of
hyperlipidaemia and medicinal plants. Int.J.A.PS.BMS, oct-Dec.2013, Vol.2. (4) ,219-237.
Dewole E.A, Dewumi D.F.A, Alabi J.Y.T, Adegoke A (2013). Proximate and phytochemical of Cola nitida and
Cola acuminata. Pakistan journal of biological sciences 16(22):1593-6.
Dhandapani R. Hypolipidemic activity of Ecliptaprostrata(L.) L. leaf extract in atherogenic diet induced
hyperlipidemic rats. Indian J Exp Biol. 2007; 45:617–9.
El-Tantawy Walid Hamdy, Abeer Temraz, Hoda E. Hozaien, Omayma D. El-Gindi and Kamilia F. Taha (2015).
Anti-hyperlipidemic activity of an extract from roots and rhizomes of Panicum repens L. on high cholesterol
diet-induced hyperlipidaemia in rat DOI 10.1515/znc-2014-4147.
Ginghina, C., Bejan, I., Ceck, C. D. Modern risk stratification in coronary heart disease.J. Med. Life. 4(4): 377-
86 (2011).
Ginghina, C., Bejan, I., Ceck, C. D. Modern risk stratification in coronary heart disease.J. Med. Life. 4(4): 377-
86 (2011).
Hall, J. E. (2011). Guyton and Hall textbook of medical physiology (12th edition). Philadelphia, P.A. Saunders
Elsedvier. P. 157.
Harikumar, K., AbdulAlthaf, S., Kishorekumar, B., Ramunaik, M., and Suvarna, C. H. (3013). A Review on
hyperlipidaemia. International Journal of novel Trends on pharmaceuticals sciences (3): 4. 59-71.
Huncrantz R. Galuman H. Lindberg G. Nilsson LH. (1986) Liver investigation in 14 asymptomatic patients with
moderately elevated activities of serum aminotransferases. Scand J Gastroentrol 21: 109-113.\
Hwu CM, Lin KH. Uric acid and the development of hypertension. Med SciMonit. 2010; 16(10): RA224-RA230.
Johnson MC (2005) Hyperlipidaemia disorders in dogs. Compendium 27: 361-70.
Jorgensen, T., Capewell, S., Prescott, E., Allender, S., Sans, S., Zdrojewski, T. Population-level changes to
promote cardiovascular health. Eur. J. Prev. Cardiol., 20(3):409-21 (2013).
Karam I, Ma N, Yang Y-J, Li J-Y (2018) Induce Hyperlipidaemia in Rats Using High Fat Diet Investigating Blood
Lipid and Histopathology. J Hematol Blood Disord 4(1):104
Karam I, Ma N, Yang Y-J, Li J-Y (2018) Induce Hyperlipidaemia in Rats Using High Fat Diet Investigating Blood
Lipid and Histopathology. J Hematol Blood Disord 4(1):104
Kaushik, V., Shivali, A. and Vipin, S. (2014). Hyperlipidaemia: Its management and induction. International
Journal of pharmaceutical sciences and Research. 5(8):3152-3158
Kelly RB. Diet and exercise in the management of hyperlipidaemia. Am Fam Physician. 2010;81: 1097-102.
Manninen V, Tenkanen L, Koskinen P, et al. Joint effects of serum triglyceride and LDL cholesterol and HDL
cholesterol concentrations on coronary heart disease risk in the Helsinki Heart Study: implications for
treatment. Circulation. 1992; 85: 37–45.
Monike, G.P., Andresa, M. M., Marina, R. B., Camila, P. D., Maria, A. G., Guilherme, V. P. and Alceu
A. J. (2011). A high-fat diet as a model of fatty liver disease in rats. Acta cir. Bras. Vol.26 1678-2674.
Nku CO, Ikpi DE, Nna VU, Agiande GU. Altered serum lipid profile in albino Wistar rats following the
consumption of cola nitidarubra (kola nut). Australian Journal of Basic and Applied Sciences 2014; 8(13): 82-
89.
Nwagha IU, Igweh JC, Okaro JM. The Effects of Menopause on the Serum lipid profile of Normal Females of
South-East Nigeria. Nigerian Journal of Physiological Sciences. 2005;20(1-2):48–53 ISSN 1684-5315.
Oloyede, H.O.B., Aina G.O., Salawu, M.O., Ajiboye T.O., Oladiji, A.T., Yakubu M.T., Nafiu., M.O. Brassica
oleracea (cabbage) based diet ameliorates high fat diet-induced hyperlipidaemia in rats. Centerpoint journal
(Science Edition) vol. 21, No 1, Pages 183-203, 2015.
112
M. O. Salawu et al.
Reitman, S. And Frankel, S. (1957). A colorimetric method for determination of serum glutamate-oxaloacetate
and pyruvate transaminase. Am. J. Clin path. 28. 56-59.
Rosenthal P, Haight M. (1989) Aminotransferase as a prognostic index in infants with liver disease. Clin Chem.
36: 346 -348.
Salawu et al., 2018 (unpublished) ‘Composition and effects of administration of aqueous extract of cola nitida
seeds on Wistar rats’. Department of biochemistry university of Ilorin.
Saukkonen, J.J, Cohn, D.L., Jasmer, R.M., Schenker, S., Jereb, J.A. (2006). An Official ATS Statement:
Hepatotoxicity of antituberculosis therapy. Am J RespirCrit Care Med 174: 935-952.
Tietz, N.W. (1990). Clinical guide to laboratory test (2nd ed) W.B. saunders company Philadelphia, USA. 554-
556.
Varsha, D., Shubhangi, S., Mangesh, P.N. and Naikwade, S. (2010). Antihyperlipidemic activity of
Cinnamomumtamalanees on high cholesterol diet induced rats. International Journal of Pharm Tech
Research. 2(4): 2517-2521.
Wells, G. B., Dipiro, J., Schwinghammer, T., Hamilton, C. Pharmacotherapy Handbook 7thedn, USA, McGraw
Hill Companies, 2007; pp98-108.
Wells, G. B., Dipiro, J., Schwinghammer, T., Hamilton, C. Pharmacotherapy Handbook 7thedn, USA, McGraw
Hill Companies, 2007; pp98-108.
Williams, P. (1979). High-density lipoprotein and coronary risk factor, Lancet 1: 72.
Yakubu, M. T., Bilbis, L. S., Lawal, M., and Akanji, M. A. (2003). Evaluation of selected parameters of rat liver
and kidney function following repeated administration of yohimbine. Biokemistri 15: 50-56.
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T. T. Banjo
0795-8080/2020 $10.00 + 0.00

Biokemistri
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Amylase production by Aspergillus flavus immobilized in

polysaccharide beads of Adansonia digitata.
Temitope Temitayo Banjo
Department of Biological Sciences, Crawford University, PMB 2001, Igbesa, Ogun State, Nigeria.
Corresponding author: E-mail: topebanjo4rever@gmail.com

Telephone: +2347030121326, +2348066373050
ABSTRACT: A novel matrix for the immobilization of amylase produced by Aspergillus flavus was exploited in
this study. Spores of A. flavus were immobilized on Adansonia digitata matrix cross-linked with glutaraldehyde
(2.5 %) and the effects of gel concentration (9 – 13 %), spore load (100 – 500 mg), bead size (2 - 7 mm) and
bead number (2 - 10) on amylase activity were determined. Optimum amylase activity of 280 U/ml was
obtained under batch fermentation at 10 % gel concentration, 280 mg spore load with 9 beads of 3.0 mm bead
size at 96 h of fermentation compared to 190 U/ml by the free cells. The immobilized A. flavus retained amylase
activity of 250 U/ml after four repeated cycle and also exhibited increased activities over the free cells. This
study shows the potential of Adansonia digitata as a novel matrix for increased amylase production.
Keywords Amylase; Immobilization; Adansonia digitata; Aspergillus flavus.
Introduction
Recent discoveries on the potentials of using microorganisms as biotechnological source of

industrially relevant enzymes have led to increased search for microorganisms with enzymes of
industrial relevance (1). Amylases are hydrolytic enzymes employed in the processing industries for
the hydrolysis of starch into simpler sugar constituents (2). This group of enzymes has a wide
application in the brewing, textile, detergent and pharmaceutical industries. In recent times, the
application of α-amylase in the field of laundry and dish washing detergents is on the increase (3).
Several attempts have been made to optimize the culture conditions of different strains of fungi
because the amylase of fungal origin was found to be more stable than the bacterial enzymes on a
commercial scale (4). Moreover, many fungi had been found to be good sources of amylolytic
enzymes. Studies have been reported on the production of amylase by Rhizopus sp. and Aspergillus
niger (5). Also, a high amylolytic activity in biomass production has been reported by Ikenebomeh
and Chikwendu (6).
Microbial cells and enzymes immobilized by entrapment in polymeric matrices are currently
receiving attention as industrial biocatalysts. The immobilized form of enzyme offers several
advantages, including repeated use of the enzyme, ease of product separation, improvement of
enzyme stability, and continuous operation in packed-bed reactors (7).The most frequently used
matrices include agar, agarose, alginate, carrageenan and polyacrylamide (8). Some of these matrices
are expensive and also have weak mechanical strength (9). However, in Nigeria, there are some
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underutilized natural polymers such as Adansonia digitata with hydrocolloid properties comparable
with conventional entrapment agents.
Adansonia digitata belongs to the family Bombacaceae and is predominant in the semi-arid and
arid zones of Africa. The seed is underutilized, non-toxic, cheap, readily available and biodegradable
key economic species used daily in the diet of rural communities in West Africa with various
important medicinal and food uses (10). Whole cell and enzyme immobilization with Afzelia africana,
Detarium microcarpum and Irvingia gabonensis had been reported (11-13). However, there are no
known reports on immobilization of amylase with A. digitata, hence, the need for this study. The aim
of this study therefore is to use a novel matrix, Adansonia digitata for the immobilization of amylase
produced by Aspergillus flavus.
Fungal source
Pure strains of Aspergillus flavus were obtained from the Culture Collection Center of the
Federal University of Agriculture, Abeokuta, Nigeria. The strains were sub cultured on Sabouraud
Dextrose Agar to revive the cultures.
Pretreatment of Adansonia digitata seeds.
Dehulling of A. digitata seeds

The seeds of A. africana were removed from the coats mechanically by the use of sharp objects.
The seeds were later washed with water and sun dried.
Milling of A. digitata seeds

The sun dried seeds were grounded with laboratory mortar and pestle after which it was milled
into powdery form using a local milling machine. This was sieved through 1.0 mm sieve to obtain a
fine powder which was kept in an airtight container until further use.
Removal of Lipids from A. digitata seeds

The powder sample of A. digitata was defatted as described by Shivani et al. (14) using a soxhlet
apparatus. The extraction was carried out using hexane as the solvent for 6 h. The defatted sample of
the A. digitata was removed and oven dried at 105 °C for 1 hour. The difference between the initial
and final weight of the sample was taken as the lipid content of the sample as shown below;
Percentage of lipid = W1 - W2/ W1 = W3/ W1 ×100/1 %
Where W1 = Initial weight of the sample

W2 = Final weight of the sample
W3 = Difference between the initial and final weight
Screening of amylase positive molds

This was carried out by using the method of Kareem et al. (15). The plate medium is made up of
nutrient agar containing 1 % Starch (Starch-Agar). The medium was autoclaved at 121°C for 15mins
at pH 4.5. The plates were inoculated with pure culture of Aspergillus flavus and incubated at 30° C
for 72 h. The plates were flooded with grams iodine solution to confirm the formation of clear zones
around the mould.
Amylase production
This was carried out using the medium of Osho et al. (2001). The production medium was
adjusted to pH 4.5 before autoclaving at 121°C for 15 min. Spores of Aspergillus flavus (2 × 109
spore/ml) were inoculated on the sterilized medium and incubated at 30 °C for 120 h.
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T. T. Banjo
Determination of amylase activity

Crude amylase extract (0.5 ml) from Aspergillus flavus was added to 1.5 ml of the 2%
gelatinized starch and incubated at 65°C for 10 min. The reaction was stopped with 0.5 ml of NaOH
and HCl. The Solution was made up to 10ml out of which 0.5 ml of aliquot was added to 0.5 ml of
iodine solution. This was also made up to 10 ml and the absorbance was measured at 470 nm
colorimetrically using the method of Harahito et al. (16)
Immobilization of Aspergillus flavus and Aspergillus tamarii on A. digitata matrix

This was carried out as described by Kareem et al. (13). Defatted powder of A. digitata was
cross-linked using glutaraldehyde (2.5 %) v/v. Spores of Aspergillus flavus were mixed with 100 mls
of cross-linked A. digitata slurry at 35°C under vigorous stirring. The slurry was made into spherical
beads by dropping through a syringe into ethanolic formaldehyde for 24 h.
Properties of free and immobilized amylase produced
Effect of fermentation time on amylase production.

The effect incubation time on amylase production was studied by assaying for the amount of
amylase produced at 24, 48, 72, 96 and 120 h of incubation respectively as previously described.
Effect of temperature on amylase production

The effect of temperature on the quantity of amylase produced was studied at different
temperatures (30, 35, 40 and 45 °C). The amylase activity was determined as previously described.
Effect of pH on amylase production

The effect of pH on amylase production was studied at pH range 4.0 – 8.0 (pH 4.0, 5.0, 6.0, 7.0
and 8.0) and incubated at the optimum temperature. The amylase activity was determined as
previously described.
Optimization of Immobilization of Aspergillus flavus on A. digitata matrix
Effect of gel concentration on amylase production by immobilized cells

The effect of gel concentration on amylase production was investigated. Various gel
concentrations (9, 10, 11, 12 and 13 %) were used and the amylase produced determined at 96 h of
fermentation.
Effect of spore load on amylase production by immobilized cells

The effect of spore load on amylase production was studied at optimum gel concentration by
weighing spores in the range 100 – 500 mg. The spores were mixed with defatted powder activated
with 2.5 % (v/v) glutaraldehyde solution. Beads were formed and the amylase activity determined at
96 h of fermentation.
Effect of bead size on amylase production by immobilized cells

At optimum gel concentration and spore load, the effect of bead sizes on amylase production was
studied by varying the bead sizes in the range 2–7 mm. This was achieved by dropping gel through
laboratory dropper of various diameter sizes and amylase activity determined at 96 h of fermentation.
Effect of number of beads on amylase production by immobilized cells.

The effect of the number of beads on amylase production was investigated by varying the
number of beads in the range 2–10. This was carried out at optimum gel concentration and bead sizes.
The amylase activity was determined at 96 h of fermentation.
Effect of reusability on amylase production by immobilized cells.

The effect of reusability of the immobilized cells of Aspergillus flavus on amylase production
was investigated. At the end of each batch, the beads were washed in phosphate buffer (pH 7.0),
117
dried, and used for the next batch. The amylase activity at the end of each batch was determined at 96
h of fermentation.
Comparative yield of amylase produced by free and immobilized cells of Aspergillus flavus in
Adansonia digitata matrix
A comparative study of amylase produced by the free and immobilized cells of Aspergillus flavus
in A. digitata was carried out. The amylase activity by the free and immobilized cells was determined
and compared at 96 h of fermentation.
Results and Discussion
Screening of amylase positive molds

The zone of clearance exhibited around colonies of the Aspergillus flavus (A) showed their
amylolytic potentials. The non-amylase producing mould (B) gave no zone of clearance around its
colony because amylase was not produced (Plate 1). The clear zones around colony of the mould on
Starch-Agar showed that it is an amylase producer able to degrade the starch in the medium around.
This showed the mould to be an amylolytic microorganism. The zone of clearance increases as a
function of increased amylase production by the mould.
A B
A = Amylase positive mould B= Non-amylase producing mould
Plate 1: Amylase and Non-amylase producing moulds
Effect of incubation time on amylase production by Aspergillus flavus

Studies on the effect of incubation time on amylase production showed that amylase yield peaked
at 96 h of fermentation. Amylase yields of 170 U/ml and 180 U/ml were produced by the free and
immobilized cells of Aspergillus flavus respectively. Thus, 96 h was adopted as the optimum
incubation time for further studies. Amylase production reduced with increase in incubation time for
the free and immobilized cells (Table 1). This is in agreement with the works of Osho et al. (2001)
who reported an optimum incubation time of 96 h for amylase production by Aspergillus oryzae
immobilized in gelatin matrix. Fadahunsi and Garuba (17) reported that the decrease in amylase
production beyond the optimum incubation time could be attributed to catabolite repression caused by
glucose released into the medium. It could also be as a result of the secretion of proteolytic proteins
which are known to cause the denaturation of proteins (18).
Effect of pH on amylase production

The effect of pH on amylase production showed that optimum amylase yields of 170 U/ml and
175 U/ml were produced at pH 6 and 4 by the free and immobilized cells of A. flavus respectively.
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T. T. Banjo
Thereafter, an increase in pH led to a decrease amylase production (Fig. 1). This variation in amylase
production as pH of the medium varies could be as a result of the various morphological changes
induced in microbes due to the changing pH. The optimum pH of the free amylase was pH 6, while
that of immobilized enzyme tends towards acidic medium with optimum pH 4 (Figure 1). This may be
a result of the micro environmental pH of the polysaccharide hydrogel support. This result correlates
with the findings of Fadahunsi and Garuba (17) who reported an optimum pH of 6 for amylase
production by Aspergillus flavus. Similarly, Sasi et al. (19) reported that the maximum amylase
production by a strain of Aspergillus spp was at pH 6.
Table 1: Effects of incubation time on amylase production by Aspergillus flavus.
Fermentation time (Hours)

Isolates
12 24 36 48 60 72 84 96 108 120
Free cells 0 40 60 90 100 120 150 170 140 100
(U/ml)
Immobilized 0 60 90 110 125 140 165 180 160 110
cells (U/ml)
Effect of temperature on amylase production

The effect of temperature on amylase production showed that optimal amylase activities of 180
U/ml and 190 U/ml were produced by the free and immobilized cells of Aspergillus flavus
respectively at 40 ºC (Fig. 2). However, as the temperature was increased to 40 ºC, amylase activities
by the free and immobilized cells reduced to 140 U/ml and 160 U/ml respectively. Reduced amylase
activity at higher temperature above the optimum may be as a result of the inactivation of amylase
enzyme or suppression of cell viability. However, low amylase activity observed at low temperature
values may be as a result of reduction in the metabolic activities of the microorganism and
consequently, the amylase production (20)
Effect of Gel concentration on amylase production

Optimum amylase production of 240 U/ml was achieved with gel concentration of 12 % as
shown in Figure 3. Further increase in gel concentration resulted in decrease in amylase production.
Increase in gel concentration reduces the pore size of the bead which interfered with the entry of
substrate into the bead, thus leading to decreased amylase production. Whereas at lower gel
concentration, the beads were unstable and fragile which resulted in poor immobilization and reduced
amylase production. This may be due to larger pore size of the beads and consequently leakage of
enzyme from the beads will increase (21).
200
activity (U/ml)
150
Amylase
free cells
100
immobilized cells
50
0
4 5 6 7 8
pH
Fig 1: Effect of pH on amylase production by free cells of Aspergillus flavus
119
Fig 2: Effect of temperature on amylase production by free cells of Aspergillus flavus
Fig 3: Effect of gel concentration on amylase production by Aspergillus flavus
Effect of spore load on amylase production by immobilized cells

The influence of spore load on amylase production was investigated and presented in figure 4.
Amylase activity of 250 U/ml was produced at optimum spore load of 300 mg. However, higher
concentration of spores did not lead to increased amylase production. At spore load of 500 mg,
amylase activity reduced to 170 U/ml. It has been reported that the mechanical strength of gel
particles decreases with increasing cell loading and may be accountable for this decrease in amylase
production (22).
Effect of Bead Size on ascorbic acid production

The significance of bead size in amylase production was also studied as shown in Figure 5.
Optimum amylase activity of 260 U/ml was produced at bead size of 3 mm. An Increase in the bead
size to 7.0 mm resulted in amylase activity of 90 U/ml. This is due to the fact that at lower bead size,
the gel matrix is thinner, which makes the fungal mycelia in the gel cavities more accessible to
substrate than at higher bead size (8).
Effect of Number of Beads on amylase production

The effect of the number of beads on amylase production showed that amylase production
increased with increase in the number of beads (Fig. 5). Optimum amylase activity of 280 U/ml was
achieved with 9 beads. However, further increase in bead number did not result into increased
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T. T. Banjo
amylase activity. Amylase activity became asymptotic above the optimum number of beads. This
agrees with Kareem et al. (12) who reported that as reaction rate was maximum at 15 beads, further
increase in bead number will achieve the same rate.
Fig 4: Effect of spore load on amylase production by Aspergillus flavus
Fig 5: Effect of bead size on amylase production by Aspergillus flavus
Effect of Beads Reusability on amylase production

One of the main advantages of immobilization is the ease of separation and reusability. The
effect of reusability of beads on amylase activity showed that 250 U/ml of enzyme activity was
produced after 4 repeated uses (Fig. 7). However, there was a decline in amylase activity with further
use of the immobilized cells. The decrease in enzyme activity could be due to the leakage of enzyme
from the beads, occurring due to the weakening of the binding strength between the beads and the
enzyme (23). Similarly, Osho et al. (8) reported that the low yield of amylase in sequential batch
fermentations could be due to low stability of the gel beads.
Comparative amylase production by free and immobilized cells of Aspergillus flavus in

Comparative studies on amylase production by the free and immobilized cells of Aspergillus
flavus revealed that immobilized cells of A. flavus gave an improved amylase activity of 280 U/ml
while the free cells gave a lower activity of 190 U/ml (Fig. 8). When free cells are compared with
immobilized cells, the productivity obtained in the latter is considerably higher, due to high cell
density and immobilization-induced cellular or genetic modifications (24).
121
Fig 6: Effect of bead number on amylase production by Aspergillus flavus
Fig 7: Effect of bead reusability on amylase production by Aspergillus flavus
Fig. 8: Comparative amylase production by free and immobilized cells of Aspergillus flavus in
122
T. T. Banjo
Conclusion
The present study showed that immobilized cells of Aspergillus flavus produced a higher yield of
amylase compared to the free cells. Furthermore, the immobilized A. flavus retained 250 U/ml of
enzyme activity after 4 repeated uses. This shows the potential of Adansonia digitata as matrix for
enhanced amylase production.
References
1. Akpan I, Bankole MO, Adesermowo AM, Lantunde – Dada GO: Production of α- amylase by Aspergillus
niger in a cheap solid medium using rice bran and agricultural material. Tropical Science 1999; 39: 77-79.
2. Okolo BN, Ezeogwu LI, Mba CN: Production of raw starch digesting amylase by grown on native Starch
sources. Journal of Science Food and Agriculture 2006; 69: 109-115.
3. Van der Maarel MJEC, Van der Veen B, Vitdehaag JCM, Leemhuis H, Dijkhuizen, L: Properties and
application of starch converting enzymes of α-amylase family. J. Biotechnol. 2002; 49: 137- 55.
4. Abu EA, Ado SA, James DB: Raw starch degrading amylase production of mixed culture of Aspergillus
niger and Saccharomyces cerevisiae grown on Sorghum pomace. Afr. J. Biotechnol. 2005; 4: 785-790.
5. Abe J, Bergman FW, Obeta K, Hizukuri S: Production of the raw starch degrading amylase of Aspergillus
sp. K-27. Appl. Microbiol. Biotechnol. 1988; 27:447-450.
6. Ikenebomeh MJ, Chikwendu AE: Aspergillus niger biomass production in cassava whey medium. Nig. J.
Microbiol. 1997; 11: 52-63.
7. Abdel-Naby MA, Sherif AA, El-Tanash AB, Mankarios AT: Immobilization of Aspergillus oryzae
tannase and properties of the immobilized enzyme. Journal of Applied Microbiology 1999; 87(1): 108–
114.
8. Osho MB, Akpan I, Kareem SO: Production of amylase by Aspergillus oryzae immobilized in a gelatin
matrix. Nigerian Journal of Microbiology 2001; 15 (2):87-91.
9. Park JK, Chang HN: Citric and gluconic acid production from fig by Aspergillus niger using solid state
fermentation. Journal of Microbiology and Biotechnology 2000; 25:298-304.
10. Assogbadjo AE, Gle’ le KR, Edon S, Kyndt T, Sinsin B: Natural variation in fruit characteristics, seed
germination and seedling growth of Adansonia digitata L. in Benin. Springer science 2010; 41:113-125.
11. Banjo TT, Kareem SO, Popoola TO, Akinloye O: Biosynthesis of ascorbic acid by Aspergillus flavus and
Aspergillus tamarii immobilized in Afzelia africana matrix. Food and Applied Bioscience Journal 2018; 6
1): 39-52.
12. Kareem SO, Oladipupo IO, Omemu AM, Babajide JM: Production of Citric acid by Aspergillus niger
immobilized in Detarium microcarpum matrix. Malaysian Journal of Microbiology 2012; 9(2):161-165.
13. Kareem SO, Adio OQ, Osho MB: Immobilization of Aspergillus niger F7-02 Lipase in Polysaccharide
Hydrogel Beads of Irvingia gabonensis matrix. Enzyme Research 2014:1-7.
14. Shivani P, Khushbu P, Faldu N, Thakkar V, Shubramanian RB: Extraction and analysis of Jatropha
curcas L. seed oil. Afr. J. Biotechnol. 2011; 10 (79): 8210-18213.
15. Kareem SO, Akpan I, Oduntan SB: Cow Pea waste: A novel substrate for solid State production of
amylase by Aspergillus oryzae. African Journal of Microbiological Research 2009; 3 (12):974–977.
16. Harahito T, Eizo T, Naoko O, Akiko NG: Screening of alpha-amylase suitable for evaluating the degree of
starch retrogration. Starch/Starke 1992; 21: 29-32.
17. Fadahunsi IF, Garuba OG: Amylase production by Aspergillus flavus associated with the biodeterioration
of starch based fermented foods. New York Science Journal 2012; 5 (1):13-18.
18. Gupta A, Gupta VK, Modi DR, Yadava LP: Production and characterization of αamylase from Aspergillus
niger. Biotechnology 2008; 7: 551-556.
19. Sasi A, Kani M, Panneerselvam A, Jegadeesh G, Muthu K, Kumar MR: Optimizing the conditions of α-
amylase by an Estuarian strain of Aspergillus sp. African Journal of Microbiology Research 2009;
4(8): 581-586.
20. Mazutti M, Bender JP, Treichel H, Di-Luccio M: Optimization of Inulinase production by solid-state
fermentation using sugarcane bagasse as substrate. Enzyme Microbial and Technology 2006; 39: 56–59.
21. Talekar S, Chavare S: Optimization of immobilization of α-amylase in alginate gel and its comparative
biochemical studies with free α-amylase. Rec. Res. Sci. Technol. 2012; 4: 1–5.
22. Dong HL, Cheol HP, Jong MY, Seung WK: Lipase immobilization on silica gel using a cross-linking
method. Journal of Industrial and Chemical Engineering 2006; 12:777-782.
23. Jaiswal N, Prakash O: Immobilization of soybean 𝛼-amylase on gelatin and its application as a detergent
additive. Asian Journal of Biochemistry 2011; 6 (4): 337–346.
123
24. Ivanova V, Petrova P, Hristov JM: Application in the ethanol fermentation of immobilized yeast cells in
matrix of alginate/magnetic nanoparticles, on chitosan-magnetite microparticles and Cellulose-coated
Magnetic nanoparticles. Int. Rev. Chem. Eng. 2011; 3:289 – 299.
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O. O. Ovuakporie-Uvo et al.
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BKR 20200018/32204
Molecular phylogenetic authentication of the relative

evolution of Desplatsia spp. from Southern Nigeria
Oghale O. Ovuakporie-Uvo*1, MacDonald Idu1, Olumide Afolabi2, Michael Kolade
Irieabo3
1
Department of Biological Sciences (Plant Science and Biotechnology Unit), University of Medical Sciences,
PMB 536, Ondo State, Nigeria.
2
Phytomedicine Research Unit; Department of Plant Biology and Biotechnology, University of Benin, PMB 1154,
Benin City, Edo State, Nigeria.
3
African Biosciences Ltd, Ibadan Expressway, Nigeria
4
Department of Biological Sciences, University of Abuja, PMB 127, FCT, Abuja, Nigeria.
For correspondence: ooghale@unimed.edu.ng
ABSTRACT: Desplatsia spp. Is among threatened species on the list of the International Union for Conservation
of Nature (IUCN). On the taxonomic classification of the species, they are reported as belonging to Tiliaceae by
some authorities while others report as Malvaceae family. In this study, Random Amplified Polymorphic DNA
markers (RAPD) was used to evaluate the genetic relatedness of Desplatsia subericarpa and Desplatsia dewevrei
collected from Southern Nigeria with the aim of ascertaining their exact plant family. Fresh leaves samples of test
plants were collected alongside already established plant members of Tiliaceae (Corchorus olitorius) and
Mavaceae (Abelmoschus esculentus). The genomic DNA was extracted from leaves using Bioline isolate II plant
Genomic DNA kit. Three-cluster analysis was conducted based on Nei’s genetic distance matrices. Results
showed clear RAPD bonding patterns. The combination of 10 random primers generated 84 bands all of which
were polymorphic (100%). Findings suggest that though closely related to the family Tiliaceae, both species of
Desplatsia are neither relatives of Tiliaceae nor Malvaceae families. Further and advanced study is recommended
to appropriately classify Desplatsia spp. into a plant family to avoid the disparity in their present taxonomic
classification.
Keywords: Desplatsia spp.; Southern-Nigeria; Phylogeny; Tiliaceae; Malvaceae; RAPD
Introduction
Random Amplified Polymorphic DNA (RAPD) analysis is a multi-locus arbitrary finger-printing

technique useful in determining genetic relationships of various species (1-3). RAPD analyses are
efficient, economical and tend to produce genetic markers suited to the assessment of population, race
and species-specific genetic variation (4). Genetic variations between plant materials may result from
variations in DNA sequences and ecological effects. “The assessment and maintenance of genetic
variation, which involves the use of biochemical and molecular markers, is crucial for providing a fount
of adaptability to environmental stress (5)”. Several efficient genetic markers are used to reveal genetic
variability within and among the same set of plant samples, including random amplified polymorphic
DNA (RAPD)-based polymerase chain reaction (PCR), a DNA marker, and isozymes, protein markers.
These markers differ from each other with respect to genomic abundance, level of polymorphism
detected, locus specificity, reproducibility, technical requirements, cost, and the type of data generated
(6, 7). RAPD has been used for the assessment of genetic relationships and variation in Paspalum
vaginatum (8), variation in populations of Ranunculus reptans (9) and Changeful smyrnioides (10).
125
RAPD was the first PCR- based molecular markers to be employed in genetic variation analysis (11,
12).
Desplatsia (or Desplatzia) is a genus of small trees native to tropical Africa formerly classified as
Tiliaceae. It is distributed across West African countries; Ivory Coast, West Cameroons, across the
Congo Basin to Rwanda, Ghana and the Southern Nigeria (13, 14). The genus was initiated by
Bocquillon in 1867, with a single species (Desplatsia subericarpa) though, the genus contains a few
more species (15, 16). In addition to Desplatsia subericarpa, the other more recognized species are
Desplatsia chrysochlamys, Desplatsia lutea and Desplatsia dewevrei with Desplatsia caudata,
Desplatsia chrysophylla, Desplatsia floribunda, Desplatsia klainii, Desplatsia mildbraedii and
Desplatsia trillesiana as other recorded names (13). According to Burkil (17), Desplatsia (subericarpa)
belongs to the plant family Tiliaceae while Desplatsia dewevrei belongs to the family Malvaceae
according to Ken Fern (18) and Hassler (14). The experimental question; Is it possible for two species
of the same genera to belong to different plant families? It is certain that phenotypic traits can be reliable
measures of genetic differences (19). Phenotypic variation is positively associated with genetic
diversity, but it is dependent on environmental factors as well as on the interaction between genotypes
(20). Morphological characters may be unstable due to environmental influences; so that methods to
assess and detect genetic diversity have extended from analysis of discrete morphological traits to
biochemical and molecular traits (21). Therefore, morphological characterization which allows analysis
of discrete morphological traits to biochemical and molecular traits (21) in the presence of
environmental variation (5) is necessary. This research is aimed at defining the genetic relatedness or
differences amongst Desplatsia spp in order to verify if the Desplatsia spp are of the family Tiliaceae
or Malvaceae,
DNA Extraction:
Fresh leaf samples of Desplatsia subericarpa, Desplatsia dewevrei, Corchorus olitorius, and
Abelmoschus esculentus were dried and preserved in silica gel until need for DNA extraction. Total
genomic DNA was extracted from these leaf samples using Bioline Isolate II Plant Genomic DNA kit
according to the manufacturer’s protocol. The DNA obtained was quantified using Nanodrop
Spectrophotometer and the integrity was verified on 1% agarose gel at African Biosciences Ltd Ibadan,
Nigeria.
PCR Amplification and RAPD analysis:

The PCR amplification was performed using Solis Biodyne FirePol Ready-To-Load PCR Master mix
and 10 random decamers. A 10 µl reaction was prepared for each sample per primer. Each 10 µl
reaction contains; 2 µl master mix (5x), 1 µl primer (10 µM), 2 µl template DNA (10 ng/µl) and 5 µl
nuclease-free water. The PCR program includes an initial denaturation at 95oC for 3 min, denaturation
at at 94oC for 30 secs, annealing at 37oC for 1 min, extension at 72oC for 30 sec and final extension at
72oC for 10 min. The denaturation, annealing and extension steps were allowed to run for 40 cycles.
The fragment analysis was performed on 2% agarose gel in 1x TBE buffer at 80v for 50 mins. The gel
was stained precast with ethidium bromide to a concentration of 0.5 µg/ml. The list of primers used in
the study and their sequences are presented thus;
Primers Sequence (5’ → 3’)
OPA 03 AGTCAGCCAC
OPA 13 CAGCACCCAC
OPA 15 TTCCGAACCC
OPA 17 GACCGCTTGT
OPA 19 CAAACGTCGG
OPAB 02 TGATCCCTGG
OPAB 06 TGCTCTGCCC
OPAB 08 GTCCACACGG
OPAB 11 GTAGACCCGT
OPAB 14 TCCGCTCTGG
126
Data Analysis:
The gel bands were scored into a binary matrix using Pyelp 1.4 (22). The estimation of Nei’s genetic
distances and construction of the phylogenetic tree (UPGMA algorithm) were on Genalex 6.502 (23)
and MEGA 7 (24) respectively.
Genomic DNA isolated from fresh leaves of Desplatsia spp; Desplatsia subericarpa and
Desplatsia dewevrei alongside Abelmoschus esculentus and Corchorus olitorius which served as
outgroups were investigated in this study. Figure 1 shows the gel images from the 10 primers used.
Presence of a band was scored as 1 and its absence as 0. The binary matrix was manually edited and
missing values were represented as -1.
Figure 1: RAPD Fingerprints for the 10 primers used in this study.
Key: Order of samples on gel images: M 1 2 3 4 5 6 7 8 9 10 11 12 M

M- DNA Ladder (100bp)
Desplatsia subericarpa: 1 2 3 4 7 8
Desplatsia dewevrei: 3 5 6 9 10
Abelmoschus esculentus: 11
Corchorus olitorius: 12
The combination of the 10 random primers generated 84 bands, all of which were polymorphic
(100%). The 84 polymorphic bands contained 20 unique and 64 non-unique bands (Table 1). Desplatsia
subericarpa and Desplatsia dewevrei had 58 detectable bands out of which bands 10 and 12 were
private bands unique to each species. Abelmoschus esculentus had 30 while Corchorus olitorius had 23
detectable bands out of which 2 and 3 were private (Figure 2).
127
Table 1: Bands distribution and polymorphism revealed by the primers
Primers Total No of Monomorphic Polymorphic Bands Percentage

Bands (Common) Bands Unique Non-unique Polymorphism
OPA 03 4 0 0 4 100
OPA 13 11 0 0 11 100
OPA 15 6 0 3 3 100
OPA 17 11 0 4 7 100
OPA 19 12 0 4 8 100
OPAB 02 5 0 3 2 100
OPAB 06 9 0 1 8 100
OPAB 08 9 0 2 7 100
OPAB 11 10 0 2 8 100
OPAB 14 7 0 1 6 100
Total 84 0 20 64 100
Figure 2: Band patterns across populations
The Nei’s genetic distance matrix revealed that A. esculentus and C. olitorius are genetically farther
apart from each other than each is from the Desplatsia spp (Table 2). This portrays a triangular
relationship which can be easily visualised in the Principal Coordinate Analysis PCoA (Figure 3). The
Principal Coordinate Analysis (PCoA) based on Nei’s genetic distances with data standardization
showed that the first, second and third coordinates accounted for 63.24, 34.21 and 2.55% of the observed
variations respectively (Table 3). The triangular relationship demonstrates that the genus Desplatsia is
closer to A. esculentus for some genetic characters, but closer to C. olitorius for other genetic characters.
For characters captured by the first coordinate (which covers most of the variations observed), the
Desplatsia spp are closer to C. olitorius but closer to A. esculentus on the fewer variations captured by
the second coordinate (Table 3 and Figure 6).
128
Table 2: Nei’s pairwise genetic distance matrix
D. subericarpa D. dewevrei A. esculentus C. olitorius

Desplatsia subericarpa 0.000
Desplatsia dewevrei 0.096 0.000
Abelmoschus esculentus 0.354 0.367 0.000
Corchorus olitorius 0.293 0.311 0.461 0.000
Figure 3: Principal Coordinate Analysis (PCoA) of A. esculentus, C. olitorius and Desplatsia spp.
The phylogenetic tree in Figure 4 revealed 3 main clusters (A, B and C), one of which belongs to
the Abelmoschus esculentus and Corchorus olitorius (Cluster C). The Desplatsia spp formed two close
and chimeric clusters. Figure 5 is a condensed phylogenetic tree constructed from the Nei’s genetic
distance (Table 3) between the different populations.
Table 3: Eigen values and proportions by axis and sample Eigen vectors
Axis No. 1 2 3
% 63.24 34.21 2.55
EigenValue 0.037 0.020 0.002
Desplatsia subericarpa 0.041 -0.074 -0.029
Desplatsia dewevrei 0.042 -0.084 0.026
Abelmoschus esculentus -0.150 0.019 0.000
Corchorus olitorius 0.108 0.086 0.001
129
Figure 4: Sample phylogenetic tree – Unweigted Paired Group Method with Averages (UPGMA)
Figure 5: Population phylogenetic tree (UPGMA)
The population phylogenetic tree revealed a closer relationship between Desplatsia spp and
Corchorus olitorius than between the former and Abelmoschus esculentus. Though the core families
that make up Malvales (Bombaceae, Malvaceae, Sterculiaceae and Tiliaceae) form a well-supported
monophyletic group within Malvales, the only one of the four core families that represent a
monophyletic group is Malvaceae (15, 25). This may, therefore, suggests why Desplatsia spp share
some genetic similarities with the family Tiliaceae and Malvaceae. However, Desplatsia spp did not
closely cluster with any of the two type species and in fact, the cluster pattern observed from the
130
phylogenetic trees and PCoA in this study suggests that Desplatsia subericarpa and Desplatsia
dewevrei belong to an entirely different family that is neither Tiliaceae nor Malvaceae. Perhaps they
belong to Grewioideae as suggested by hinsley (13) and seconded in studies carried out by Brunken and
Muellner (26). Further/ advance research involving more samples of the suspected plant family
members (Tiliaceae, Malvaceae, Grewioideae) is recommended.
Conclusion
The present study is the first report on the genetic diversity of Desplatsia spp using RAPD method.
It is recommended that more studies including other related and well-established family type species be
carried out with the aim of verifying if Desplatsia spp truly belongs to other families than Malvaceae
and Tiliaceae.
References
1. Ramshini H, Naghavi MR, Alizadeh H. Comparison of genetic diversity based on total and sharp bands of
RAPD data in wheat. Asian J. Plant Sci. 2005; 4(2):123-127.
2. Sadder MT, Ateyyeh AF. Molecular assessment of polymorphism among local Jordanian genotypes of the
common fig (Ficus carica L.). Sci. Hort. 2006; 107: 347-351.
3. Shinde VM, Dhaliwal K, Mahadik KR, Joshi KS, Patwardhan BK. RAPD analysis for determination of
components in herbal medicine. Evid. Based Complement Alternat. Med. 2007; 4: 21-23.
4. Aagaard JE, Krutoviskii KV, Strauss SH. RAPDs and allozymes exhibit similar levels of diversity and
differentiation among population and races of Douglas fir. Heredity, 1998; 81: 69-78.
5. Mondini L, Noorani A, Pagnotta MA. Assessing plant genetic diversity by molecular tools. Diver. 2009; 1:
19-35.
6. Goncaves MB, Suetterlin P, Yip P, et al. Neurogenesis in an age-dependent manner. Mol. Cell. Neurosci.
2008; 38: 526-536.
7. Kumar RV, Tripathi YK, Shukla P, Ahlawat S. Genetic diversity and relationships among germplasm of
Jatropha curcas L. revealed by RAPDs. Trees- Struct. Funct. 2009; 23: 1075- 1079.
8. Lin ZW, Jarret RL, Duncan RR et al. Genetic relationships and variation among ecotype of seashore
paspalum (Paspalum vaginatum) determined by random amplified polymorphic DNA markers. Genome
1994; 37: 1011–1017.
9. Fischer M, Husi R, Daniel P, et al RAPD variation among and within small and large populations of the
rare clonal plant Rananculus reptans (Rananculaceae). Am. J. Bot. 2000; 87(8): 1128- 1137.
10. Fu C, Qiu Y, Kong H. RAPD analysis of genetic diversity in Chagium smyrnioides (Apiaceae), an
endangered plant. Bot. Bull. Acad. Sin. 2003; 44: 13-18.
11. Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acid Res. 1990;
18: 7213-7218.
12. Schierwater B, Ender A. Different thermostable DNA polymerase may apply to different RAPD products.
Nucleic Acids Res. 1993; 21: 4647-4648.
13. Hinsley SR. Classification of Malvaceae: Overview, composition; position; division, Malvaceae Info
(Home), (2006). Accessed on 20 December 2019, @
http://www.malvaceae.info/Classification/overview.html.
14. Hassler M. World Plants: Synonymic Checklists of the Vascular Plants of the World. In: Species 2000 &
ITIS Catalogue of Life, 2016 Annual Checklist (Roskov Y., Abucay L., Orrell T., Nicolson D., Flann C.,
Bailly N., Kirk P., Bourgoin T., DeWalt R.E., Decock W., De Wever A., eds). Digital resource at
www.catalogueoflife.org/annual-checklist/2016. Species 2000: Naturalis, Leiden, the Netherlands.
15. Kubitzki K, Chase MW. Introduction to Malvales. In: Kubitzki, K., Bayer, C. (Eds.). The Families and
Genera of Vascular Plants, vol. V, Flowering Plants, Dicotyledons: Expanded Caryophyllales, Capparales
and Malvales. Springer, Berlin, 2003; 12–16.
16. Hutchinson J. The Genera of Flowering Plants (Angiospermae).Clarendon Press, Oxford. 1967; 2: 536-567.
17. Burkill HM. The useful plants of West Tropical Africa.2nd Edition.Volume 1, Families A–D. Royal Botanic
Gardens, Kew, United Kingdom. 1985.
18. Ken F. Useful Tropical Plants Database. Available online @
<tropical.theferns.info/viewtropical.php?id=Desplatsia+subericarpa>. 2014
19. Singh SK, Singh CM, Lal GM. Assessment of genetic variability for yield and its component characters in
rice (Oryza sativa L.). Res. Plant Biol. 2011; 1: 73-76.
20. Moose SP, Mumm RH. Molecular plant breeding as the foundation for 21st-century crop improvement. Plant
Physiol. 2008; 147: 969- 977.
131
21. Muthusamy S, Kanagarajan S, Ponnusamy S. Efficiency of RAPD and ISSR markers system in accessing
genetic variation of rice bean (Vigna umbellata) landraces. Electronic J. Biotech. 2008; 11(3): 1-9.
22. Pavel AB, Vasile CL. PyElph- A Software Tool for Gel Images and Pylogenetics. BMC Bioinfo. 2012;
13(9).
23. Peakall R, Smouse PE. GENALEX 6: Genetic Analysis in Excel. Population Genetic Software for Teaching
and Research – An Update. Bioinfo. 2006; 28: 2537- 2539.
24. Kumar S, Stecher G, Koichiro T. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0. Mol.
Biol. Evol. 2015.
25. Nyfeller R, Bayer C, Alverson WS, et al. Phylogenetic analysis of the Malvadendrina clade (Malvaceae
s.I.) based on plastid DNA sequence. Org. Div. Evol. 2005; 5: 109-123
26. Brunken U, Muellner AN. A New Tribal Classification of Grewioideae (Malvaceae) Based on
Morphological and Molecular Phylogenetic Evidence. Syst. Bot. 2012; 37 (3): 699-711.
132
B. C. Adedayo et al.
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Repression of some cholinergic and monoaminergic enzymes by

non-polar solvent extracts from Rosary Pea (Abrus precatorius)
Bukola C. Adedayo1*, Sunday I. Oyeleye2 and Ganiyu Oboh1
1
Functional Foods, Nutraceuticals and Phytomedicine Unit, Biochemistry Department, Federal University of
Technology, P.M.B. 704 Akure, Nigeria
2
Department of Biomedical Technology, Federal University of Technology, P.M.B. 704 Akure, Nigeria
*Corresponding author: email address chrisadeb2013@yahoo.com
ABSTRACT: This study focused on the effects of four different solvents (ethanol, acetone, dichloromethane (DCM)
and N-hexane) extractions of Abrus precatorius leaves on neurological enzymes [acetylcholinesterase (AChE),
butyrylcholinesterase (BChE) and monoamine oxidase (MAO)], which are markers of Alzheimer’s disease (AD), as
well as the antioxidant [1,1-diphenyl-2 picrylhdrazyl] (DPPH) radical scavenging and ferric reducing antioxidant
property (FRAP) potentials. The results revealed that acetone and N-hexane extracts had the highest AChE
inhibitory effect having an EC50 value of 0.28 and 0.27 mg/ml, respectively, with no significant difference, while
ethanol (EC50 = 0.61 mg/ml) extract had the least on BChE activity. Also, on MAO activity, acetone (0.24 mg/ml)
and N-hexane (0.24 mg/ml) extracts had the highest inhibitory effect with no significant difference. The total phenol
and flavonoid contents of the extracts ranged between 4.51 - 9.47 mg GAE/100 g and 0.03 - 0.41 mg QE/100 g
respectively. All the extracts scavenged DPPH radical and possessed Fe reducing power. The inhibition of
cholinesterases and MAO activities as well antioxidative potential of A. precatorius could be some of its possible
mechanisms explore in folklore for the management of neurodegenerative diseases. However, in vivo and clinical
studies should be carried out to ascertain these claims.
Keywords: Abrus precatorius, cholinesterases, neurodegeneration, antioxidants, solvent
Introduction
In recent years, plants are commonly used as an alternative source to combat health problems in
developing countries, because of the several side effects exhibited by synthetic drugs (Costa et al., 2015).
Abrus precatorius is a leguminous crop of the plant family Fabacea. It is a slender perennial climber that
twines around trees, shrubs and hedges with no special organ of attachment. Leaves of A. precatorius are
glabrous with long internodes, having a slender branches with cylindrical wrinkled stem (Hara and
Williams, 1979; Fernando, 1988). A. precatorius is also known as rosary pea, precatory pea or bean,
jequirity, crab's eye, John Crow bead, Indian licorice, Akar Saga, gideegidee or Jumbie bead in Trinidad
and Tobago (Wagstaff, 2008). This plant is well known for its seeds, which are found in a variety of
colours such as red, which is the most common variety with a glossy appearance with the black band at
133
the end that attaches to the plant and also black and orange seeds (Fernando, 2001). The seed is used as
beads and in percussion instruments, and is known for its toxic nature due to the presence of the toxic
principle, abrin (Bisby, 1994).
Management of neurodegenerative conditions, especially Alzheimer’s disease (AD) has been with
the use of drugs, such as cholinesterase inhibitors, consequently increasing brain’s acetylcholine. This is
because in AD conditions, there are elevated cholinesterase activities (Vladimir-Kneevic et al., 2014),
which function in degrading the neurotransmitters acetylcholine and butyrylcholine thereby giving rise to
the symptoms observed in AD. Nevertheless, due to the apparent toxicity and cost encountered with the
use of synthetic cholinesterase inhibitors, the focus is now on the use of natural sources of cholinesterase
inhibitors as a remedy for this disease (Vladimir-Kneevic et al., 2014). Monoamine oxidase (MAO)
inhibitors have also been employed in the therapeutic measure towards alleviating Parkinson disease
(Riederer et al., 1989). Studies on some sea weeds (Yoon et al., 2008), vegetables and herbs (Ferreira et
al., 2006; Mukherjee et al., 2007) have shown the great therapeutic importance in cholinesterase
inhibition. Oral consumption of water extract of dried A precatorius leaves and roots in Brazil has been
reportedly served as a nerve tonic (Elisabetsky et al., 1992). According to Prashant et al. (2011), method
of extraction, solvent type, polarity as well as the concentration of the solvent, among others, does affect
the extraction of phytochemical compounds. Hence, this study aims to determine the effects of different
solvent extractions on the cholinesterases [acetylcholinesterase (AChE), butrylcholinesterase (BChE)],
monoamine oxidase (MAO) and antioxidant properties of rosary pea (Abrus precatorius) leaves.
Sample Collection
Abrus precatorius leaves were collected from Ilogbo Ekiti, Southwest, Nigeria. The authentication of
the plant was done in the Department of Crop and Pest Management, Federal University of Technology,
Akure, Nigeria. The leaves were air dried for 7 days and ground into powder.
Preparation of extracts
The air-dried leaves of the plant were powdered with a laboratory grinder to obtain a powder, which
was then subjected to extraction using different solvents (N-hexane, acetone, ethanol and
dichoromethane) followed by shaking in an orbital shaker for 4 h at room temperature. The extracts were
subjected to rotary evaporator to obtain a powder form which was reconstituted and centrifuged at 4000
RPM to obtain clear supernatant, which were then stored for subsequent analysis.
Chemicals and reagents

All chemicals used were purchased from Sigma Co. (St. Louis, MO). Unless stated otherwise, all
chemicals used were of analytical grade and the water was glass distilled.
Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) inhibition assay

Inhibition of AChE was assessed by a modified colorimetric method of Perry et al., 2000. The AChE
activity was determined in a reaction mixture containing 200 µL of a solution of AChE (0.415 U/ml in 0.1
M phosphate buffer, pH 8.0), 100 µl of 5, 5’-dithiobis (2-nitrobenzoic) acid solution (3.3 mM in 0.1 M
phosphate-buffered solution, pH 7.0) containing NaHCO3 (6 mM), the extracts (0 - 100 µl), and 50 µl
phosphate buffer, pH 8.0. After incubation for 20 min at 25ºC, 100 µl of 0.05 mM acetylthiocholine
iodide solution was added as the substrate, and AChE activity was determined as changes in absorbance
reading at 412 nm for 3 min at 25°C using a spectrophotometer. 100 µl of butrylthiocholine iodide was
used as a substrate to assay butyrylcholinesterase activity, while all other reagents and conditions were the
same. The AChE and BChE inhibitory activities were expressed as percentage inhibition.
134
Monoamine oxidase (MAO) inhibition assay

The MAO inhibitory activity of the extracts was carried out according to Green and Haughton,
(1961) and Turski et al. (1973) with slight modification. Briefly, the reaction mixture contained 25 mM
phosphate buffer (pH 7), 12.5 mM semicarbazide, 10 mM benzylamine (pH adjusted to 7), tissue
homogenate and appropriate dilutions of extracts in a total reaction volume of 2 ml. After 30 min, 1 ml of
acetic acid was added and boiled for 3 min in boiling water bath followed by centrifugation. The resulting
supernatant (1 ml) was mixed with equal volume of 0.05% of 2, 4-DNPH and 2.5 ml of benzene was
added after 10 min incubation at room temperature. After separating the benzene layer it was mixed with
equal volume of 0.1 M NaOH. Alkaline layer was decanted and heated at 80°C for 10 min. The orange-
yellow colour developed was measured at 450 nm. The MAO inhibitory activity was expressed as
percentage inhibition.
Determination of total phenol content

The total phenol content was determined according to the method of Singleton et al., (1999). Briefly,
appropriate dilutions of the extracts (200 µl) were oxidized with 2.5 ml 10% Folin-Ciocalteau’s reagent
(v/v) and neutralized by the addition of 2.0 ml of 7.5 % sodium carbonate. The reaction mixture was
incubated for 40 min at 45oC and the absorbance was measured at 765 nm in the spectrophotometer. The
total phenol content was subsequently calculated as gallic acid equivalent.
Determination of total flavonoid content

The total flavonoid content was determined using a slightly modified method reported by Meda et
al., (2005). Briefly 0.5 ml of appropriately diluted sample was mixed with 0.5 ml methanol, 50 µl 10%
A1C13, 50 µ1 of 1M potassium acetate and 1.4 ml distilled water, and allowed to incubate at room
temperature for 30min. The absorbance of the reaction mixture was subsequently measured at 415 nm; the
total flavonoid content was subsequently calculated.
1,1-diphenyl-2 picrylhydrazyl (DPPH) free radical scavenging ability

The free radical scavenging ability of the extracts against DPPH (1,1-diphenyl-2 picrylhdrazyl) free
radical was evaluated as described by Gyamfi et al. (1999). Briefly, appropriate dilution of the extracts (0
– 500 µl) was mixed with 1 ml, 0.4 mM methanolic solution containing DPPH radicals, the mixture was
left in the dark for 30min and the absorbance was taken at 516 nm. The DPPH free radical scavenging
ability was subsequently calculated.
Determination of ferric reducing antioxidant power (FRAP)

The reducing power of the extracts was determined by assessing the ability of the extract to reduce
FeCl3 solution as described by Oyaizu (1986). A 500 µl aliquot of the extracts was mixed with 2.5 ml of
200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was
incubated at 50ºC for 20 min and then 2.5 ml of 10% trichloroacetic acid was added. This mixture was
centrifuged at 801 × g for 10 minutes. 5 ml of the supernatant was mixed with an equal volume of water
and 1 ml of 0.1% ferric chloride. The absorbance was measured at 700 nm and ferric reducing power was
subsequently calculated using ascorbic acid equivalent.
Data analysis
The results of the three replicates were pooled and expressed as a mean±standard deviation (S.D.). A
Student’s t-test, one-way analysis of variance (ANOVA) and least significance difference (LSD) were
carried out. Significance was accepted at p < 0.05. EC50 was determined using linear regression analysis.
135
Results
The effect of different solvent extraction of A. precatorius on the activities of acetylcholinesterase

(AChE), butyrylcholinesterase (BChE) and monoamine oxidase (MAO) in vitro are presented in Figures
1, 2 and 3 respectively with their EC50 values in Table 1. The result revealed that all the extracts inhibited
AChE in a dose dependent manner (0 - 0.58 mg/ml); however, N-hexane extract (IC50= 0.27 mg/ml) had
the significantly (p < 0.05) highest inhibition of AChE activity than other extracts while ethanol extract
(IC50= 0.45 mg/ml) had the least inhibition of AChE activity in vitro.
Figure 1. Butyrylcholinesterase inhibitory activity of different solvent extraction of Rosary pea leaf
Values represent mean ± standard deviation (n=3).
136
Figure 2. Acetylcholinesterase inhibitory activity of different solvent extraction of Rosary pea leaf
137
Figure 3. Monoamine Oxidase inhibitory activity of different solvent extraction of Rosary pea leaf
Table 1: IC50 of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), Monoamine oxidase (MAO)

and DPPH inhibitory activities of Abrus precatorius extracts.
Sample Extract IC50 (mg/ml)

AChE BChE MAO DPPH
Ethanol 0.45  0.0.03a 0.61  0.0.02a 0.35  0.0.05a 0.85  0.0.01a
DCM 0.28  0.0.01b 0.65  0.0.01a 0.38  0.0.03a 0.81  0.0.01a
Acetone 0.28  0.0.03b 0.54  0.0.02 b
0.24  0.0.02b 0.82  0.0.02a
N-Hexane 0.27  0.0.02b 0.36  0.0.02c 0.29  0.0.02ab 0.76  0.0.01b
Values represent mean ± standard deviation (n=3). Values with the same superscript along the same column are not
138
The result also revealed that all the extracts inhibited BChE in a dose dependent manner (0 - 0.58
mg/ml); however, N-hexane extract (IC50= 0.36 mg/ml) had the significantly (p<0.05) highest inhibition
of BChE activity than other extracts while DCM extract (IC50= 0.65 mg/ml) had the least inhibition of
BChE activity in vitro. The result of MAO inhibition revealed that all the extracts inhibited MAO in a
dose dependent manner (0 - 0.5 mg/ml); however, N-hexane extract (IC50= 0.29 mg/ml) had the
significantly (p<0.05) highest inhibition of MAO activity than other extracts while DCM extract (IC 50 =
0.38 mg/ml) had the least inhibition of MAO activity in vitro. Furthermore, the DPPH* radical
scavenging ability of A. precatorius extracts revealed that all the solvent extraction of Abrus precatorius
scavenged DPPH* radical in a dose-dependent manner (0-1.67 mg/ml) with N-Hexane extract (IC50 =
0.76 mg/ml) having the highest significantly (p<0.05) DPPH* scavenging ability and ethanol extract (IC50
= 0.85 mg/ml) having the least scavenging ability.
The results of the total phenol, total flavonoid and ferric reducing antioxidant property (FRAP) of the
extracts of Abrus precatorius are shown in Table 1 respectively. The result revealed that acetone extract
(9.47 mg GAE/100g) had significantly (p<0.05) higher total phenol content than other solvent extracts
with ethanol extract (4.51 mg GAE/100g) having the least total phenol content. The result also revealed
that acetone extract (0.4 mg QE/100g) had significantly (p<0.05) higher total flavonoid content than other
extracts with ethanol extract (0.03 mgQE/100 g) having the least total flavonoid content. However, the
FRAP result revealed that the extracts were able to reduce Fe2+ with the DCM extract (9.8 mg AAE/g)
having higher FRAP than other extracts with the least observed with the ethanol extract (7.4 mg AAE/g).
Figure 4. DPPH Radical Scavenging activity of different solvent extraction of Rosary pea leaf
139
Table 2: The total phenol (mg GAE/100g), total flavonoid (mg QE /100g) and Ferric reducing antioxidant
property (FRAP) (mg AEE/g) of Abrus precatorius extracts.
Samples Total phenol Total flavonoid Reducing power

(mg GAE/100g) mg QE/100g) (mg AEE/g)
Ethanol 4.51  0.02c 0.03  0.01c 7.42  0.03b
DCM 7.62  0.06b 0.23  0.01b 9.81  0.03a
Acetone 9.47  0.01a 0.41  0.02a 7.81  0.02b
N-Hexane 5.25  0.02c 0.04  0.01c 8.33  0.01ab
Values represent mean ± standard deviation (n=3). Values with the same superscript along the same column are not
Discussion
Different clinical reports have demonstrated the chemopreventive ability of plant-derived phenolic
compounds to be inexpensive, accessible, readily available and acceptable approach to different disease
control and management (Huang et al., 2010). The inhibitory effect of different solvent extraction of A.
precatorius leaves on some key enzymes linked to neurodegeneration [acetylcholinesterase (AChE),
butyrylcholinesterase (BChE) and monoamine oxidase (MAO)] in vitro used in this study suggests their
potential usefulness in the management of neurodegenerative conditions. The result revealed that all the
extracts inhibited AChE, BChE and MAO activities in a dose dependent manner. However, the N-hexane
extract of the A. precatorius leaf had the highest inhibitory effects on AChE and BChE activities while
acetone extract had the highest on MAO activity. The inhibition of AChE and BChE activities of the
extracts agreed with earlier reports on several plants showing cholinesterase inhibitory activity, thereby
making them relevant in the treatment of neurodegenerative disorders (Das et al., 2002; Perry et al.,
2001). Likewise, the observed inhibition of MAO activity by various extracts of A. precatorius leaves is
in accordance with earlier reports on MAO inhibitors being shown to be effective in the management
and/or treatment of neurological disorders (Youdim et al., 2006)
Several studies reported that AD brain is under intense oxidative stress (Ademosun and Oboh, 2014)
and decrease in the cholinergic neurons has been shown to promote the amyloid protein deposition in the
AD brain, which in turn favour amyloid protein-associated oxidative stress and neurotoxicity (Butterfield
and Lauderback, 2002). The inhibition of AChE, BChE and MAO by the extracts of A. precatorius leaf is
suggestive of their neuroprotective potentials. Medicinal plants such as Abrus precatorius have been used
in folklore medicine for the management of neurodegenerative diseases, though there is little or no
information on its possible therapeutic mechanisms. The result of this study revealed that all the extracts
had a considerable amount of total phenolic content, however the acetone leaf extract had the significantly
(p<0.05) highest total phenol content than other extracts with same trend noticed for the total flavonoid
content. Phenolic compounds can protect the human body from free radicals, whose formation is
associated with the normal natural metabolism of aerobic cells. The antiradical activity of flavonoids and
phenols is principally based on the structural relationship between different parts of their chemical
structure (Rice-Evans et al., 1996). Natural polyphenols are capable of removing free radicals, chelate
metal catalysts, activating antioxidant enzymes, reducing α- tocopherol radicals, and inhibiting oxidases
(Amic et al. 2003; Alia et al. 2003).
Oxidative stress has been a major factor linked with the pathogenesis and progression of
neurodegenerative diseases such as AD. The reducing powers of the extracts [ethanol, dicholoromethane
(DCM), Acetone and N-hexane) of A. precatorius were assessed based on their ability to reduce Fe3+ to
Fe2+ and the results are presented in Table 4.1 as ascorbic acid equivalents. As revealed by the results, all
the extracts were able to reduce Fe3+ to Fe2+, however, DCM extract (9.81 mg AEE/g) had the
significantly (p<0.05) highest reducing property than other extracts. Reducing power is a novel
140
antioxidant defence mechanism; the two mechanisms available to affect this property are by electron
transfer and by hydrogen atom transfer (Dastmalchi et al., 2007). This is because the ferric-to-ferrous iron
reduction occurs rapidly with all registrants, with half reaction reduction potentials above that of
Fe3+/Fe2+, the values in the ferric reducing antioxidant property (FRAP) assay will express the
corresponding concentration of electron-donating antioxidants (Halvorsen et al., 2002).
Antioxidant capacities of the various solvent extracts of A. precatorius as typified by their DPPH*
radical scavenging and Fe2+ chelating abilities were assessed and as revealed by the results, all the
extracts scavenged DPPH* radical dose dependently. However, the N-Hexane extract exhibited the
strongest DPPH* radical scavenging ability than other extracts. An important mechanism of antioxidant
activity is the ability to collate and deactivate the transition metals such as iron, which is capable of
generating free radicals from Fenton reactions. The antiradical activity of flavonoids and phenols is
principally based on the structural relationship between different parts of their chemical structure (Rice-
Evans et al., 1996). DPPH radical is used as a stable free radical donor to determine the antioxidant
activity of natural compounds and the scavenging of stable radical (DPPH) is considered a valid and easy
assay to evaluate scavenging activity of antioxidants (Suhaj, 2006; Ozturk et al., 2007; Maizura et al.,
2011).
Conclusion
The inhibition of AChE, BChE and MAO with their antioxidant properties of different solvent
extractions of Abrus precatorius make them a good source for the management of neurodegenerative
conditions. Nevertheless, in vivo experiments and clinical trials are recommended.
References
Ademosun AO, Oboh G: Comparison of the inhibition of monoamine oxidase and butyrylcholinesterase activities by
infusions from green tea and some citrus peels. International Journal of Alzheimer’s Disease. 2014.
Alia M, Horcajo C, Bravo L, Goya L: Effect of grape antioxidant dietary fiber on the total antioxidant capacity and
the activity of liver antioxidant enzymes in rats. Nutritional Research. 2003; 23: 1251–1267.
Amic D, Davidovic-Amic D, Beslo D, Trinajstic N: Structure radical scavenging activity relationship of flavonoids.
Croatia Chemica Acta. 2003; 76:55- 61.
Bisby F: Phytochemical Dictionary of the Leguminosae, Volume 1 CRC Press. 1994.
Butterfield DA, Lauderback CM: Lipid peroxidation and protein oxidation in Alzheimer’s disease brain: Potential
causes and consequences involving amyloid β-peptide-associated free radical oxidative stress. Free Radical
Biology and Medicine. 2002; 32:1050–1060.
Costa RMPB, Vaz AFM, Xavier HS, Correia MTS, Carneiro-da-Cunha MG: Phytochemical screening of Phthirusa
pyrifolia leaf extracts. Free-radical scavenging activities and environmental toxicity. South African Journal of
Botany. 2015; 99:132–137.
Das A, Shanker G, Nath C, Pal R, Singh S, Singh HK: A comparative study in rodents of standardized extracts of
Bacopa monniera and Ginkgo biloba anticholinesterase and cognitive enhancing activities. Pharmacology
Biochemistry and Behavior. 2002; 73: 893-900.
Dastmalchi K, Dorman HD, Koşar M, Hiltunen R: Chemical composition and in vitro antioxidant evaluation of a
water-soluble Moldavian balm (Dracocephalum moldavica L.) extract. LWT-Food Science and Technology.
2007; 40(2): 239-248.
Elisabetsky E, Figueiredo W, Oliveria G: Traditional Amazonian Nerve Tonics as Antidepressant Agent:
Chaunochiton kappleri: A Case Study. Journal of Herbs, Spices and Medicinal Plants. 1992;1(1-2): 125-162.
Fernando R: Poisonous plants: Abrus precatorius L. National Poison Information Centre, Colombo. 1988
Fernando C: Poisoning due to Abrus precatorius (jequirity bean). Anaesthesia. 2001; 56: 1178-1180.
Ferreira A, Proença C, Serralheiro MLM, Araújo MEM: The in vitro screening for acetylcholinesterase inhibition
and antioxidant activity of medicinal plants from Portugal. Journal of Epidemiology. 2006; 108: 31-37.
Green AL, Haughton TM: A colorimetric method for the estimation of monoamine oxidase Biochemical Journal.
1961; 78: 172–176.
141
Gyamfi MA, Yonamine M, Aniya Y: Free-radical scavenging action of medicinal herbs from Ghana: Thonningia
sanguinea on experimentally induced liver injuries. General Pharmacology. 1999; 32: 661– 667.
Halvorsen BL, Holte K, Myhrstad MCW, Brikmo I, Hvattum E, Remberg SE, Wold
AB, Haffner K, Baugerod H, Andersen LM, Moskaug JO, Jacobs DR, Blomhoff RA: Systematic screen of total
antioxidant in dietary plants. Journal of Nutrition. 2002; 132: 461 – 471.
Hara H, Stearn WT, Williams LHJ: An enumeration of the flowering plants of Nepal. Volume I. British Museum
Natural History, London. 1978.
Huang WY, Cai YZ, Zhang Y: Natural phenolic compounds from medicinal herbs and dietary plants: potential use
for cancer prevention. Nutrition and Cancer. 2010; 62: 1–20.
Maizura M, Aminah A, Wan Aida WM: Total phenolics content and antioxidant activity of kesum, ginger (Zingiber
officinale) and turmeric (Curcuma longa) extract. International Food Research Journal. 2011; 18: 529 – 534.
Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG: Determination of the total phenolic, flavonoid and
praline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chemistry. 2005;
91:571 – 577.
Mukherjee PK, Kumar V, Mal M, Houghton PJ: Acetylcholinesterase inhibitors from plants. Phytomedicine. 2007;
14: 289 – 300.
Oyaizu M: Studies on products of browning reaction: antioxidative activity of products of browning reaction
prepared from glucosamine. Japanese Journal of Nutrition. 1986; 44: 307–315.
Ozturk M, Aydogmus-ozturk F, Emin-duru M: Antioxidant activity of stem and root extracts of Rhubarb (Rheum
ribes): An edible medicinal plant. Food Chemistry. 2007; 103: 623–630.
Perry NSL, Houghton PJ, Theolad AE, Jenner P, Perry EK: In vitro inhibition of human erythrocyte
acetylcholinesterase by Salvia lavandulaefolia essential oil and constituent terpenes. Journal of Pharmacy and
Pharmacology. 2000; 52: 895 – 902.
Prashant T, Bimlesh K, Mandeep K, Gurpreet K, Kaur H: Phytochemical screening and extraction: a review.
Internationale Pharmaceutica Sciencia. 2011; 1: 98 – 106.
Rice-Evans C, Miller NJ, Paganga G: Structure-antioxidant activity relationships
of flavonoids and phenolic acids. Free Radicals in Biology and Medicine. 1996; 20: 933–956.
Riederer P, Sofic E, Rausch WD: Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains
Journal of Neurochemistry. 1989; 52: 515–520.
Singleton VL, Orthofer R, Lamuela-Raventos RM: Analysis of total phenols and other oxidation substrates and
antioxidants by means of Folin–Ciocalteau’s reagent. Methods in Enzymology. 1999; 299: 152–178.
Suhaj, M: Spice antioxidants isolation and their antiradical activity: a review, Journal of Food Composition and
Analysis. 2006; 19: 531–537.
Turski W, Turska E, Gross Bellard M: Modification of the spectrophotometric method of the determination of
monoamine oxidase Enzyme. 1973; 14: 211– 220.
Vladimir-Kneevic S, Blaekovic B, Kindl M, Vladic J, Lower-Nedza AD, Brantner AH: Acetylcholinesterase
inhibitory, antioxidant and phytochemical properties of selected medicinal plants of the Lamiaceae family.
Molecules. 2014; 19: 767–782.
Wagstaff DJ: International poisonous plants checklist: an evidence-based reference. CRC Press. 2008
Yoon NY, Chung HY, Kim HR, Choi JS: Acetyl and butyrylcholinesterase inhibitory activities of sterols and
phlorotannins from Ecklonia stolonifera. Fish Science. 2008; 74: 200 – 207.
Youdim MBH, Edmondson D, Tipton KF: The therapeutic potential of monoamine oxidase inhibitors. Nature
Reviews Neuroscience. 2006; 7: 295–309.
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Effect of alkaloid-rich extract from Eggplant (Solanum kumba) fruit

peels on manganese-induced neurodegeneration in fruit fly
(Drosophila melanogaster) Model
E. E. Nwanna*1, S. A. Shodehinde 2 and P.O. Aro1
1
Functional Foods and Nutraceutical Unit, Department of Biochemistry, Federal University of Technology, P.M.B
704 Akure, Ondo State, Nigeria.
2
Biochemistry Department, Adekunle Ajasin University Akungba Akoko Ondo State, Nigeria
*Corresponding Author: eenwanna@futa.edu.ng; esthernwanna@gmail.com
ABSTRACT: Eggplant fruits commonly called garden egg in Nigeria have been found to be rich in phytochemicals
with various therapeutic properties, however little is known about the alkaloid-rich constitutes. This study sought to
identify and investigate the effect of alkaloid-rich extract obtained from eggplant fruit peels and its biological
activities on manganese-induced (MgCl2) toxicity in fruit fly (Drosophila melanogaster). Fruit flies were induced
with (MgCl2) and were subsequently treated, homogenized and the acetylcholinesterase (AChE) enzyme activity, in-
vivo reactive oxygen species and in-vitro antioxidants such as ferric reducing antioxidant property (FRAP) and 2,2’-
azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTs*) were determined, while the gas chromatography coupled
with mass spectra (GC-MS) was used to analyse the alkaloid constitutes in the peels. The results reveal that the
alkaloid-rich extract (0.05-0.10mg/ml) diet had high antioxidant property which reduced the reactive oxygen species
level generated in the fruit flies and also increased the life span of the fruit flies with 50% reduction in mortality rate
with an increased locomotors performance. The alkaloid-rich diet also modulated the activity of AChE enzyme in all
the treated flies. The GC-MS showed alkaloids with anti-oxidative, anti-inflammatory and cholinergic inhibitor
activities. Conclusively, this study suggests that the eggplant fruit peels diet could reduce neuronal stress.
Keywords: Eggplant fruit peels, alkaloid, Drosophila melanogaster, acetylcholinesterase, GC-MS chromatography
(Received February 2, 2020; Accepted March 17, 2020)
Introduction
Neurodegenerative diseases (NDDs) are traditionally defined as disorders with selective loss of
neurons and distinct involvement of functional systems defining clinical presentation. Neurodegenerative
diseases affect the central nervous system causing progressive nervous system dysfunction. These
debilitating and incurable conditions are characterized by loss of neuronal cell functions and are often
associated with atrophy of the affected nervous system structures. An important subset of
neurodegenerative disease concerns dementias associated with aging. [1]. Alzheimer's disease (AD) is the
most common clinically recognized dementia in aging populations, and 43% of people 85 or older are
thought to suffer from Alzheimer's in the United States [1] Parkinson's disease (PD), another common
nervous system disorder associated with the elderly, affects 1-3% of the population over 60. United
Nations population projections estimate a world population of 400 million people 80 years of age or older
143
by the year 2050 [2]. Given the financial, societal and personal impact of the burden of these
neurodegenerative diseases, determining the cause, prevention and treatment have become a major focus
for basic and clinical research.
Management of neurodegenerative conditions, especially Alzheimer’s like disease (AD) has been
with the use of drugs such as tacrine, donepezil and rivastigmine which are cholinesterase inhibitors
consequently increasing the brain’s acetylcholine. This is because in AD conditions there are elevated
cholinesterase activity [3]. Several studies have shown that monoamine oxidase (MAO) inhibitors could
countervail some of these processes through various neuroprotective mechanisms, such as interaction with
the mitochondrial outer membrane, as well as the up-regulation of anti-apoptotic proteins and neurotropic
factors [4, 5].
In recent years the use of these synthetic drugs have declined drastically due to their adverse side
effects and much prevalence/importance have been given to the traditional ways of treating these
neurodegenerative conditions. Study has shown that plant-based products could be used in the
management of neurodegenerative diseases [6] Thus, recent efforts are focused on plant products.
Phytochemicals have been reportedly serves as natural cholinesterase inhibitors with little or no side
effects, found useful as a dietary intervention in the management of neurodegenerative disorders [7].
Human beings have used plants for the treatment of diverse ailments for thousands of years [8, 9). Rural
areas of many developing countries still rely on traditional medicine for their primary health care needs
and have found a place in day-to-day lives. These medicines are relatively safer and cheaper than synthetic
or modern medicine [10]. Thus, the quest for natural plant products rich in antioxidant is gaining much
importance in neurotoxicity management and in the treatment. However, one of the major concerns of
researchers is in the reduction in the number of higher laboratory animals for research and testing due to
ethical issues. Therefore, the fruit fly Drosophila melanogaster, has been recommended by the European
Centre for the Validation of Alternative Methods (ECVAM) for promoting the 3Rs (reduction, refinement
and replacement) of laboratory animal usage in toxicity studies [11]. Drosophila possesses systems which
control nutrient uptake, storage and metabolism and these systems have been reported to be analogous to
those of humans [12, 13].
Drosophila melanogaster is known for its high sensitivity to toxic substances and is being considered
as a useful model for toxicity studies as well as evaluating the biological action of pharmacological agents
[14] Drosophila has been previously demonstrated to be a useful model for elucidating the mechanisms
underlying manganese-induced neurotoxicity [15]. Alkaloids are plant-originated nitrogen-containing
compounds [16]. Some cholinesterase inhibitors (galantamine and rivastigmine) commonly used to treat
neurodegenerative-related diseases are alkaloid origin [17] highlighting the importance of alkaloid-rich
plants as an alternative source for the management of neurodegenerative diseases. Eggplant (Solanum spp)
is an edible fruits vegetable with different species that include wild relatives as well as primitive cultivars
and landraces of the important vegetable crop [18]. The African eggplant fruit play a central role in the
tradition and culture of people in sub-Saharan Africa [19]. It is offered as a gift in traditional ceremonies,
such as marriage, child naming and other social occasions as a sign of blessing and fruitfulness [19]. It is
an important to fruit-crop found useful in traditional folklore medicine as it is used to treat certain
ailments. It has proven beneficial to patients suffering from anemia because of its rich iron content. Rich
meal of garden eggs is used to induce lactation in freshly delivered women [20]. Frequent consumption of
garden eggs reduces intraocular pressure (glaucoma) and convergence insufficiency [21]. In addition, it
prevents heart diseases and blood pressure [22].
Phytochemical screening of the whole eggplant fruit and relatives of this genus have revealed the
presence of steroidal glycoalkaloids, tannins, sesquiterpenoids and other essential bioactive compounds as
highlighted in [19, 22-24] studies, but there is little or no information on the indigenous eggplant fruit
peels activities in biological systems as literature reported that the colour part of a fruit has the highest
phytochemicals, therefore for those who only consume the outer part known as the flesh or peel there is
still need to investigate further if it can still be beneficial to them because previous studies on eggplant
fruits have shown that high consumption of the fruit due to its rich dietary fiber might have induced
constipation in some subject [18,19] as such it could be the reason why some avoid the consumption of
144
E. E. Nwanna et al.
the whole eggplant fruit. Furthermore, recent study on indigenous eggplant whole fruit polyphenol extract
and diet have revealed that it could manage neuronal issue in diabetic model. So, this study was designed
to investigate the effect of alkaloid-rich extract from eggplant fruit peels (Solanum kumba) on
acetylcholinesterase enzyme linked to Alzheimer’s disease (AD) like symptoms using fruit fly Drosophila
melanogaster as a model.
Sample collection and identification

Eggplant fruits were obtained from a local farm in Akure, Ondo State, Nigeria during the month of
April, 2018. Authentication of the sample was carried out at the Department of Forestry and Wood
Technology Federal University of Technology, Akure, Nigeria with a Voucher number IFE-17718 while
the green skin was peeled from the whole fruit for the experiment.
Drosophilia melanogaster Stock Culture
Wild type D. melanogaster (Oregon strain) stock culture was obtained from Department of
Biochemistry, University of Ibadan, Oyo State. The flies were maintained and reared on normal diet made
up of corn meal medium containing 1% w/v brewer's yeast and 0.08% v/w nipagin at constant temperature
and humidity (25 ± 1oC; 60% relative humidity respectively) under 12 h dark/light cycle conditions. All
the experiments were carried out with the same D. melanogaster strain.
Reagents
Chemical reagents such as acetylthiocholine iodide, sulphanilamide, reduced glutathione, n-n-diethyl-
para-phenylenediamine (DEPPD), ferrous sulphate, semicarbazide were procured from Sigma Al-drich
Co. (St Louis, Missouri, USA). Trichloroacetic acid (TCA) and sodium acetate was sourced from Sigma
Al-drich, Chemie GmbH (Steinheim, Germany), hydrogen peroxide, methanol, acetic acid, hydrochloric
acid, aluminium chloride, potassium acetate, sodium dodecyl sulphate, Iron (II) sulphate, potassium
ferrycyanide and ferric chloride were sourced from BDH Chemicals Ltd., (Poole, England). Ascorbic acid
and starch were products of Merck (Darmstadt, Germany). Except stated otherwise, all other chemicals
and reagents were of analytical grades and the water was glass distilled.
Instruments
Test tubes, beaker, water bath, centrifuge machine, weighing balance, spectrophotometer, cuvettes,
conical flask, measuring cylinder, standard flask, micro pipette, amber bottles, centrifuge tubes, universal
bottles, microplates, microplate readers.
Methods
Extraction of alkaloid
The plant material was air-dried for one week; ground mechanically using an electric blender to
powder which was then used for analysis. The alkaloid constituent was then determined using the method
of [25].
Stock solution preparation
From 50g of the sample 4.58g of the alkaloid extract was gotten. This alkaloid extract was dissolved
with 3ml Dimethyl Sulphoxide (DMSO) after which 60mls of water was added. Thereafter, the solution
was poured into a stock bottle and stored in the refrigerator for subsequent analysis.
Preparation of Drosophila food using Agar method
The diet preparation was done for toxicological studies on D. melanogaster according to the method
of [26].
Experimental Design
The flies (both gender, 3–5 days old) were divided into 7 groups containing 60 flies each. Group I
was placed on normal diet alone while group II – VII, were placed on basal diet containing; Solanum
145
kumba (SM) alkaloid extract of 0.1% and 0.05% inclusion in the diet (equivalent weight replacement) as
shown.
Groups:
I Basal Diet
II Basal Diet + manganese chloride (Mgcl2)
III Basal Diet + Manganese chloride (Mgcl2) + 0.1% SM
IV Basal Diet + Manganese chloride (Mgcl2) + 0.05% SM
The flies were exposed to these treatments for 5 days and the vials containing flies were maintained
at room temperature. All experiments were carried out in triplicate (each experimental group was carried
out in five independent vials) [27].
Survival Study
A study was conducted to assess the effect of alkaloid extract inclusion on survival rate of flies after
five days of exposure. Flies both genders, of 3–5 days old were divided into four groups containing 60
flies each. Each group was exposed to different alkaloid extract inclusion (0.1 and 0.05%). The flies were
observed daily for the incidence of mortality and the survival rate were determined by counting the
number of dead flies for the first five days. The data were subsequently analysed and plotted as
cumulative mortality and percentage survival after the treatment period [27, 28].
Measurement of Locomotors Performance (Negative Geotaxis)

The negative geotaxis assay was used to evaluate the locomotors performance of flies [29]. In brief,
after the treatment period of five days, the flies from each group were briefly immobilized in ice and
transferred into a clean tube (11 cm in length 3.5 cm in diameter) labelled accordingly. The flies were
initially allowed to recover from immobilization for 10 mins and thereafter were tapped at the bottom of
the tubes. Observations were made for total number flies that crossed the 6 cm line within a period of 6 s
and recorded. The results are expressed as percentage of flies that escaped beyond a minimum distance of
6 cm in 6 s during three independent experiments.
Preparation of Tissue Homogenate

The flies were immobilized in ice and homogenized in 0.1 M phosphate buffer, pH 7.4. The resulting
homogenates were centrifuged at 10,000 X g, at 40C for 10 mins in a Kenxin refrigerated centrifuge
Model KX3400C (KENXIN Intl. Co., Hong Kong). Subsequently, the supernatant was separated from the
pellet into labelled Eppendorf tubes and used for the various biochemical assays.
In-vitro biochemical assays

2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) radical scavenging ability
The 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS*) scavenging ability of
the fruits was determined according to the method described by [30]. The Trolox equivalent antioxidant
capacity (TEAC) was subsequently calculated using trolox as the standard.
Determination of reducing property (FRAP)

The reducing property of the extracts was determined by assessing the ability of the extract to reduce
FeCl3 solution as described by [31]. The absorbance was measured at 700 nm in the JENWAY UV-
Visible spectrophotometer. Then, the ferric reducing antioxidant property was subsequently calculated as
ascorbic acid equivalent.
Lipid Peroxidation and thiobarbibutric Acid reactions
The lipid peroxidation assay was carried out using the modified method of [32]. TBARS
(thiobarbituric acid reactive species) produced was measured at 532 nm using spectrophotometer.
Malondialdehyde (MDA) produced was calculated and reported as percentage of control.
146
E. E. Nwanna et al.
In-vivo biochemical assays

Determination of Total Protein
Total Protein content of fly homogenates were measured by the Coomassie blue method according to
[33] using bovine serum albumin (BSA) as standard.
Reactive Oxygen Species (ROS) level

ROS level in the whole fly tissue homogenates was estimated as H2O2 equivalent according to by the
method of [34] with slight modifications. The absorbance was measured at 505 nm using a
spectrophotometer. ROS levels was estimated from an H2O2 standard calibration curve and expressed as
Unit/mg protein, where 1 unit = 1 mg H202/L.
Acetylcholinesterase (AChE) Activity Assay

Acetylcholinesterase activity was assayed according to the method of [35]. The AChE activity was
thereafter calculated and expressed as mmolAChE/min/mg protein.
Data Analysis
The results of replicate readings will be pooled and expressed as mean ± SEM values. One-way
Analysis of Variance (ANOVA) will be used to analyze the results followed by Turkey column
comparison test, with levels of significance accepted at p<0.05, p<0.01 and p<0.001. All statistical
analysis was carried out using the software Graph pad PRISM (V.5.0) [36].
Results
Table 1 reveals the in vitro antioxidant property of alkaloid extract of eggplant (Solanum kumba)
using radical species 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) radical scavenging ability
(ABTS), also ferric reducing antioxidant property (FRAP).
Fig 1: Solanum kumba fruits.
147
Table 1: Antioxidant activity of alkaloid-rich extract from eggplant fruit peels (Solanum kumba)
Sample ABTS FRAP

(mmolTEAC/µM) (µgAAE/g)
Eggplant peel 0.65±0.06 32.51±2.53
Values represent means of triplicate reading.
Fig 2: Effect of dietary inclusion of alkaloid-rich extract from eggplant fruit peels on the MDA produced
(% control) in D. melanogaster at different concentration. Bars represent mean ± SEM. Bars with
different letter shows significantly different (P<0.05).
KEYS
GROUPS
I Basal
II Induced
III Basal Diet + Eggplant 0.5mg/ml +MnCl2
IV Basal Diet + Eggplant 1mg/ml+ MnCl2
148
E. E. Nwanna et al.
FIG 3: Day 7 Survival Rate (%) of D. melanogaster fed with supplemented alkaloid-rich extract of eggplant fruit
peels. Bars represent mean ± SEM. Bar with the same letter are not significantly different (P < 0.05).
KEYS
GROUPS
I Basal Diet
II Basal Diet + MnCl2
III Basal Diet + Eggplant 0.5mg/ml + MnCl2
FIG 4: Effect of dietary inclusion of alkaloid-rich extract from eggplant fruit peels on the percentage climbing
activity in 6cm in 6 seconds in D. melanogaster. Bars represent mean ± SEM. Bar with the same letter are not
significantly different (P < 0.05).
KEYS
GROUPS
I Basal Diet
149
FIG 5: Effect of dietary inclusion of alkaloid-rich extract of eggplant fruit peels on the ROS activity in D.
melanogaster. Bars represent mean ± SEM. Bars with different significantly different (P<0.05).
KEYS
GROUPS
I Basal Diet
AChE
0 .1 0
A C h E (u n it/m g p ro te in )
0 .0 8
0 .0 6
0 .0 4
0 .0 2
0 .0 0
I
I
II
IV
II
G ro u p s
FIG 6: Effect of dietary inclusion of alkaloid-rich extract of eggplant fruit peels on the Acetylcholinesterase activity
(µmolAcsch/h/mg/protein) in D. melanogaster. Bars represent mean ± SEM with significantly difference (p<0.05)
KEYS
GROUPS
I Basal Diet
150
E. E. Nwanna et al.
Table 2. Alkaloid identify in the eggplant peel using GC-MS chromatography
Constituents Retention Time %

1,2-Ethanediamine, N,N'-dimethyl- 8.20 0.43
1,3-Bis(2-chloroethyl)urea 11.96 4.53
1-Pentanol,4-amino 12.93 4.68
1,2,5-Oxadiazol-3-carboxamide 24.05 2.72
Octadenamide-9- (Oleamide) 32.23 2.22
% Total 14.58
Fig 7. Chromatogram of the alkaloid characterized from the eggplant fruit
Table 1 reveals the antioxidant property of the eggplant fruit peels which was able to reduced radical
species formed. Figure 2 shows the effect of dietary inclusion of alkaloid-rich eggplant fruit peels extract
on the lipid peroxidation activity in D. melanogaster. It was observed that there was no significant
difference (P<0.05) in the treated groups when compared with the basal group however the induced group
with pro-oxidant sodium-nitroprusside (SNP) have an increased lipid peroxides generated when
compared to the basal group and the eggplant fruit peels treated groups. Figure 3 shows the effect of
alkaloid-rich extract of eggplant fruit peels on the life span of D. melanogaster. Dietary inclusion of
MnCl2 reduced the life span of Drosophila melanogaster significantly (p>0.05) when we compared
control flies and the treated groups with eggplant fruit peels diet checking by the number of days required
to reach 50% mortality which clearly revealed that the alkaloid-rich extract might have attenuated
manganese induced neurotoxicity which could have led to neurodegenerative state, however on day 7
survival of D. melanogaster revealed significantly (p<0.05) difference in the percentage survival of the
flies induced with MnCl2 and induced with eggplant peels extract inclusion. While Fig 4 shows the effect
of dietary inclusion of eggplant fruit peels extract on locomotors (climbing movement) known as negative
geotaxis in D. melanogaster, the flies fed with diet induced with MnCl2 had the same climbing rate in
151
comparison to the control group but significantly low in the induced group without any treatment thus, the
treatment enhanced the locomotors activity. Fig 5 shows the effect of dietary inclusion of the eggplant
fruit peels extract on the Reactive Oxygen Species (ROS) level. This revealed that flies fed with eggplant
fruit peels extract diet had a reduced Reactive Oxygen Species (ROS) level (P< 0.05) when compared with
the untreated groups. Fig 6 shows the effect of dietary inclusion of eggplant fruit peels extract on
acetylcholinesterase (AChE) enzyme in D. melanogaster, flies induced with manganese chloride had a
significantly (p<0.05) increased in acetylcholinesterase enzyme activity when compared with the control
and the eggplant fruit peels treated groups, it was also observed that the diet significantly (p<0.05)
decreased the AChE level to the positive control level. While Table 2 and Fig 7 show the quantified
alkaloid presence in the eggplant fruit and their peaks in the chromatogram which reveals that
Octadenamide-9- (Oleamide) was the most abundant alkaloid which have been found to various
therapeutic ability including cholinergic inhibitor.
Discussion
Diet has been implicated to play a vital role in ageing processes [37]. Dietary phenolic such as
phenolic acids and flavonoids are beneficial for longevity by reducing oxidative stress, regulating signal
transduction and gene expression [37]. Several reports have confirmed that these phytochemicals are more
abundant in the skin, peel of fruits and vegetables because these ‘chemicals” have been found to serve as
protection and self-defense from insect and other predators. [39]. Drosophila melanogaster (fruit fly) has
been recently used as an alternative model for screening of therapeutic agents for the treatment of
degenerative diseases using Manganese-induced toxicity as a model for induced neurodegeneration [15,
38, 28] pathology. The positive health effects of phenolic phytochemicals are linked to their ability to
counter the negative effects of reactive oxygen species generated during cellular energy metabolism [39].
Flavonoids are also known as a class of widely distributed phytochemicals with antioxidant activities.
Phytochemical investigation of eggplant (Solanum kumba) and relatives of this genus [18] has revealed
presence of high levels of steroidal glycoalkaloids, tannins, sesquiterpenoids and other essential bioactive
compounds as stated[19, 23, 24]. The results of this study reveals that eggplant fruit peels have anti-
oxidative property from the in vitro antioxidant indices, the presence of the phytochemicals in it could
have mopped up the peroxides form during neurodegenerative state. A growing body of evidence suggests
that the D. melanogaster exposure to Manganese (Mn) might led to locomotors dysfunction, oxidative
stress and mortality [40, 41]. Although little is known about the precise mechanism of action of Mn in D.
melanogaster but, its involvement in dopaminergic (Daergic) neurodegeneration and oxidative stress is
well supported by previous studies [40, 41] which reported that Mn can induce the loss of Daergic
neurons, increase reactive species, increase MDA, nitric oxide, and decrease the activity of antioxidant
enzymes [40,41]. Also, the notion that Mn might led to both the oxidation of dopamine and also the
mitochondrial dysfunction are the two potential mechanisms by which Mn-induced oxidative stress was
documented [42, 43,44].
This present study demonstrated that dietary exposure to manganese at 30 mmol per kg caused a
significantly (p<0.05) increase in the cumulative number of dead flies and consequently, a significant
reduction in the percentage of life flies following 7 days of the treatment regimen. This observation could
be attributed to the cytotoxic effect of manganese (Mn) which has been previously reported. However,
dietary supplementation of eggplant (Solanum kumba) fruit peels significantly prevented the manganese
mediated toxicity by decreasing the mortality and consequently increased the (%) of the life flies to which
the result followed the same trend with the flies locomotor ability as well as increase in their climbing
performance. Acetylcholinesterase (AChE) is a serine protease that hydrolyses acetylcholine, a
neurotransmitter which regulates motor function and locomotion [45] an increase in its activity in the flies
could have led to neurodegeneration in the fly which invariably resulted to dead flies. Dietary
supplementation of eggplant fruit peels alkaloid–rich extract could have been associated with the
decreased in AChE enzyme activity and also an increased climbing performance in flies, thus confirmed
the protective role of eggplant fruit diet in Drosophila melanogaster induced manganese neurotoxicity.
152
E. E. Nwanna et al.
Plant-based alkaloid extract from eggplant fruit decreases the level of acetylcholine in the
acetylcholinergic receptors in the brain of the flies which could have prevented the flies in developing the
neurodegenerative disorders as a result of the toxicant. The observed activity is basically due to the anti-
oxidant, anti-amyloid, anticholinergic agent, adenosine receptors agonists, cholinesterase inhibitor, MAO
inhibitor, α-synuclein aggregation inhibitor, nicotine and dopaminergic agonist properties in the eggplant
fruit peels [46]
Conclusion
This study shows that alkaloid-rich eggplant fruit peels could protect and/or prevent manganese
induced oxidative stress. More so, supplementation of the indigenous eggplant fruit diet could be
associated with neuroprotection as observed with AChE enzyme activity. Therefore eggplant fruit whole
and its peels could be a promising functional food candidate against neuronal stress and disorders thus, the
need for its regular consumption as an alternative to synthesized drugs.
References
1. Wright WA. Geographic and ethnic variation in Parkinson disease: a population-based study of US Medicare
beneficiaries. Neuroepidemiology. 2010. 24, 143-151.
2. Alzheimer's Disease Fact and Figures, Alzheimer's Association. Facts and Figures. 2008
3. Rao AV and Agarwal S. Role of Antioxidant Lycopene in Cancer and Heart Disease. Journal of the
American College of Nutrition 2000.19, 563-569.
4. Youdim MB Edmondson D and Tipton KF. The therapeutic potential of monoamine oxidase inhibitors.
NatrevNeurosci 2006. 7(4):295.doi:10.1038/nrn1883.PMID:16552415.
5. Carradori S, Secci D, Bolasco A, Chimenti P, and D’Ascenzio M. Patent-related survey on new monoamine
oxidase inhibitors and their therapeutic potential. Expert Opin Ther Pat 2012. 22(7):759–801.
doi:10.1517/13543776.2012.698613.
6. Ferreira A, Proeca C., Serralheiro MIM and Aradjo MEM. The in vitro screening for acetylcholinesterase
inhibition and antioxidant activity of medicnal plants from Portugal. Journal of Ethno-Pharmacology.2006. 108,
31-37
7. Orhan I, Sener B, Choudhary MI and Khalid A. Acetylcholinesterase and butyrylcholinesterase inhibitory
activity of some Turkish medicinal plants. Journal Ethnopharmacol.2004. 91, 57-60.
8. Sofowora A. Medicinal plants and Traditional Medicinal in Africa. New York: John Wiley and Sons. 1982
9. Hill AF. Economic Botany: A text book of usefu plants and plant products. 2 nd Edn. New York: McGraw Hill
book company, Inc. 1989
10. Iwu MW, Duncan AR and Okunji CO. New antimicrobial of plant origin. In: Janick J (editor). Perspectives on
new crops and new uses. ASHS Press, Alexandria, 1999. pp, 457-462.
11. Benford DJ, Hanley AB, Bottrill K, Oehlschlager S, Balls M, Brance F, Castegnara JJ, Descotes J, Hemminiky
K, Lindsay D and Schilter B. Biomarkers as predictive tools in toxicity testing. The report and
recommendations of ECVAM workshop 40.Alt Lab Anim 2000. 28: 119–131.
12. Baker KD and Thummel CS. Diabetic larvae and obese flies-emerging studies of metabolismin Drosophila.
Cell Metab 2007. 6:257–266.
13. Jafari M. Drosophila melanogaster as a model system for the evaluation of anti-aging compounds. Fly (Austin)
2010. 4:253–257.
14. Adedara IA, Rosemberg DB, Souza DO, Kamdem JP, Farombi EO, Aschner M, and Rocha JBT. Biochemical
and behavioural deficits in lobster cockroach Nauphoeta cinerea model of methylmercury exposure. Toxicol
Res 2015. 4:442–451.
15. Mora M, Bonilla E, Medina-Leendertz S, Bravo Y, Arcaya JL. Minocycline increases the activity of superoxide
dismutase and reduces the concentration of nitric oxide, hydrogen peroxide and mitochondrial malondialdehyde
in manganese treated Drosophila melanogaster. Neurochem Res 2014. 39: 1270–1278.
16. Meyers RA. Encyclopedia of Physical Science and Technology Alkaloids (3 rd Ed.). Academic Press 2002.
17. Konrath EL, Passos COS, Klein LC and Henriques AT. Alkaloids as a source of potential anti cholinesterase
inhibitors for the treatment of Alzheimer’s disease. J Pharm Pharmacol. 2013. 65(12):1701–1725.
doi:10.1111/jphp.12090.
153
18. Nwanna EE, Ibukun EO and Oboh G. Nutritional contents and antioxidant activities of african garden egg
(Solanum aethiopicum) cultivars. Advances in Food Sciences. 2013. 35 (1): 30-35.
19. Chinedu SN, Olasumbo AC, Eboji OK, Emiloju OC, Arinola OK and Dania DI. Proximate and Phytochemical
Analyses of Solanium aethiopicum L. and Solanium macrocarpon L. Fruits. Research Journal of Chemical
Sciences. 20111(3) 63-71.
20. Blay E. Garden eggs and egg plant production in Ghana. The Legion Agricultural Research and Extension
Journal. 1991. 3: 97-100.
21. Igwe SA, Akunyili DN and Ogbogu C. Effects of Solanum melongena on some visual functions of visually
active Igbos of Nigeria. Journal of Ethnopharmacology 2003. 86 (2-3): 135-138.
22. Okon UE, Aniete AA and Bassey NE. Technical efficiency and its determinants in Garden Egg (Solanum spp.)
production in Uyo Metropolis, Akwa Ibom State, Nigeria. Field Action Science Report, Special Issue (1).2010.
23. Cipollini ML and Levey DJ. Antifungal activity of Solanum fruit glycoalkaloids. Implications for frugivory and
seed dispersal. Ecology 1997.78:799-809.
24. Shamin S, Ahmed SW and Azhar I. Antifungal activity of Allium, Aloe and Solanum species. Pharmaceutical
Biology. 2004. 42,491-498.
25. Harbone JB. Phytochemical methods: A guide to modern techniques of plant analysis. Chapmam and Hall Ltd,
London: 1973 Pp 279.
26. The Bloomington Drosophila stock centre, Indiana University. Drosophila media recipes.
(2002).htpp://flystocks.bio.indiana.edu/media-recipes.htm
27. Abolaji OA, Kamdem JP, Lugokenski TH, Nascimento TK, Waczuk EP, Farombi EO, Loreto EL et al
.Involvement of oxidative stress in 4-vinyl cyclohexene-induced toxicity in Drosophila melanogaster. Free
Radic. Biol. Med 2014. 71: 99–108
28. Adedara IA, Abolaji AO, Rocha JB, Farombi EO. Diphenyl diselenide protects against mortality, locomotor
deficits and oxidative stress in Drosophila melanogaster model of manganese-induced neurotoxicity.
Neurochem Res. 2016.
29. Le Bourg E and Lints FA. Hypergravity and aging in Drosophila melanogaster climbing activity. Gerontology
1992. 38(1-2):59-64.
30. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C Antioxidant activity applying an
improved ABTS radical cation decolorisation assay. Free Rome, Italy 1999.
31. Oyaizu M. Studies on products of browning reactions: antioxidative activities of products of browning reaction
prepared from glucosamine. Japanese Journal of Nutrition 1996. 44, 307-315.
32. Ohkawa H, Ohishi N, and Yagi K. Assay for lipid peroxide in animal tissues by thiobarbituric acid reaction.
Analytical Biochemistry, 1979 95(2):351-8. DOI: 10.1016/0003-2697(79)90738-3.
33. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem. 1976. 72:248-54.
34. Hayashi K, Chuva de Sousa lopes SM, Surani MA. Germ cell specifications in mice.Science 2007. 316
(5823):394-6.
35. Ellman GL and Fiches FT. Quantitative determination of peptides by sulfhydryl groups Arch, Biochem
Biophys; 1959. 82: 70-72.
36. Zar, JH. Biostatistical Analysis. New Jersey: Prentice-Hall, 1986.
37. Rossi L, Mazzitelli S, Arciello M, Capo CR, Rotilio G. Benefits from dietary polyphenols for brain aging and
Alzheimer's disease. Neurochem Res 2008. 33(12):2390–2400.
38. Bonilla E, Contreras R, Medina-Leendertz S, Mora M, Villalobos V, Bravo Y. Minocycline increases the life
span and motor activity and decreases lipid peroxidation in manganese treated Drosophila melanogaster.
Toxicology 2012. 294:50–53.
39. Urquiaga I and Leighton F. Plant polyphenol antioxidants and oxidative stress. Bio. Res; 2000. 33:55–64.
40. Ternes AP, Zemolin AP, da Cruz LC, da Silva GF, Saidelles AP, de Paula MT, Wagner C, et al. Drosophila
melanogaster an embryonic model for studying behavioral and biochemical effects of manganese exposure.
Excli Journal 2004. 13:1239–1253.
41. Bonilla-Ramirez L, Jimenez-Del-Rio M and Velez-Pardo C. Acute and chronic metal exposure impairs
locomotion activity in Drosophila melanogaster: a model to study Parkinsonism. Biology 2011.
42. Aschner M, Guilarte TR, Schneider JS and Zheng W. Manganese: recent advances in understanding its
transport and neurotoxicity. Toxicol Appl Pharmacol 2007. 221:131–147
43. Avila DS, Puntel RL and Aschner M Manganese in health and disease. Met Ions Life Sci 2013. 13:199–227.
44. Farina M, Avila DS, da Rocha JB and Aschner M. Metals, oxidative stress and neurodegeneration: a focus on
iron, manganese and mercury. Neurochem Int 2013. 62:575–594
154
E. E. Nwanna et al.
45. Day J, Damsma G, Fibiger HC. Cholinergic activity in the Rat hippocampus, cortex and striatum correlates
with locomotor activity: an in vivo micro dialysis study. 38: 723–729
46. Hussain G, Rasul A, Anwar H, Aziz N, Razzaq A. et al (2018). Role of plant derived alkaloids and their
mechanism in neurodegeneration disorders. Inter. Journal of Biological Sciences, 14(3), 341-357.
48 Nwanna EE Ibukun EO Oboh G. Eggplant (Solanum spp) supplemented fruits diet modulated the activities of
ectonucleoside triphosphate diphosphohydrolase (ENTPdase), monoamine oxidase (MAO), and
cholinesterases (AChE/BChE) in the brain of diabetic Wistar male rats. Journal of Food Biochemistry 2019. 1-
11.
155
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0795-8080/2020 $10.00 + 0.00

Biokemistri
BKR 20200023/32207
Effect of bitter leaf (Vernonia amygdalina) aqueous extract

on pancreatic α-amylase and intestinal α-glucosidase
repressive potentials of Acarbose
Ayokunle O. Ademosun1, Kelvin C. Akuine1, Sunday I. Oyeleye1,2, Ganiyu Oboh1*
1
Functional foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology, Akure,
PMB 704 Akure, Ondo State, Nigeria
2
Department of Biomedical Technology, Federal University of Technology, Akure, PMB 704 Akure, Ondo State,
Nigeria
*Correspondence e-mail: goboh@futa.edu.ng

Telephone no.: +2347031388644
ABSTRACT: There is a continuous search for antidiabetic drugs. The available synthetic drugs, including Acarbose
could only provide temporary relief with some negative effects. This has resulted in the co-administration of
Acarbose with plants possessing antidiabetic potential such as bitter leaf. In Nigeria, co-administration of bitter leaf
(BL) vegetable with acarbose (a synthetic inhibitor of α-amylase and α-glucosidase) in the management of Type-2
diabetes mellitus is on the increase, basically to potentiate the therapeutic value and amelioration of side effects of
Acarbose. However, there is no information on the influence of BL on the effectiveness of Acarbose. This study
determined the influence of BL on the α-amylase and α-glucosidase inhibitory properties of Acarbose. BL extract,
acarbose and their combinations (75:25, 50:50, and 25:75) were prepared and their effect on α-amylase and α-
glucosidase, and the production of Fe2+-induced thiobabituric reactive acid species (TBARS) in pancreas
homogenate were determined. Results revealed that all the combinations exhibited the antagonistic effect except
50:50 combined ratios, which exhibited additive effects. All the samples inhibited TBARS production except 100%
Acarbose. This study shows that the use of bitter leaf with Acarbose could reduce the potency of Acarbose or the
bitter leaf.
Keywords: Acarbose, α-amylase, α-glucosidase; bitter leaf; food-drug interaction
(Received February 8, 2020; Accepted March 17, 2020)
Introduction
Diabetes mellitus is among the world's leading metabolic diseases, affecting millions of people
across the globe (Maiti et al., 2004). The growing incidence of diabetes and unfavourable side effects of
controlling synthetic drugs has geared researchers to search for natural products with minimal side effect,
and herbal materials rich in phenolic compounds have demonstrated equal if not the same potential with
synthetic drugs (Adefegha et al., 2016; Erasto et al., 2009; Oboh et al., 2015). An example of such plants
157
is bitter leaf (Vernonia amygdalina Del. Family: Astercieace), locally known as “Ewuro” by Yoruba
speaking people of Nigeria. BL is widely consumed as a vegetable and for the management of Type-2
diabetes, having α-amylase and α-glucosidase inhibitory properties (Atangwho et al., 2013; Leonard et
al., 2002; Oboh et al., 2016). As a result, it is often consumed locally with Acarbose to potentiate the
therapeutic value and reduce the side effects of Acarbose without considering possible food-drug
interaction.
Acarbose is an effective synthetic antidiabetic drug (Ademiluyi et al., 2016; Oboh et al., 2016b). It
acts by inhibiting the activities of α-amylase and α-glucosidase, hence, controlling the release of blood
glucose level (Adefegha et al., 2016). However, this drug could only provide temporary relief with
attendance abdominal discomfort, flatulence among other side effects (Ademiluyi et al., 2016; Adefegha
et al., 2016). In this study, the effects of phenolic-rich extract from bitter leaf (BLE), acarbose and their
various combinations on α-amylase and α-glucosidase activities, and on the production of Fe2+-induced
thiobarbituric reactive acid species (TBARS) in pancreas homogenate. Phenolic constituents of bitter leaf
were also determined using HPLC-DAD.
Chemical and reagent

Acarbose was procured from Glenmark Generics Limited, Middlesex, UK. Porcine α-amylase and
pancreatic α-glucosidase were procured from Sigma Aldrich Co. Other chemicals and reagents were of
analytical grade. The water used was glass distilled.
Collection of Bitter leaf and samples preparation

Bitter leaf was freshly harvested from a farmland, and authenticated by A. A. Sorungbe (voucher
numbers FUTA/BIO/201), Department of Biology, Federal University of Technology, Akure. The leaf
was air-dried and blended into powdery form using laboratory blender. Thereafter, the phenolic-rich
extract and 25 mM of acarbose were prepared in accordance with the report of Adefegha et al., (2016).
Acarbose (25 mM) and bitter leaf extract (BLE, 0.1 mg/ml) were prepared with distilled water.
Thereafter, sample mixtures were prepared as follows: 100% ACA; 75% ACA: 25% BLE; 50% ACA:
50% BLE; 25% ACA: 75% BLE; 100% BLE. All samples were stored at 4°C for subsequent analysis.
α-Amylase and α-glucosidase activities assays

The effects of the samples on α-amylase and α-glucosidase activities were carried out using the
methods described in the study of Ademiluyi et al., (2016). The enzyme inhibitory effects of the samples
were expressed as percentage inhibition.
Handling of experimental animals

Wistar strain male albino rats (weighing between 180 and 210g) were purchased from the Animal
Breeding Colony of Animal Health and Production (APH) Department, Federal University of
Technology, Akure for this study, and their handling were in accordance to the regulation published by
the National Institutes of Health (NIH, 2011) for the care and Use of Laboratory Animals, and Ethics
Committee of the Federal University of Technology, Akure, Nigeria. They had access to the feed and
water ad libitum for 14 days of acclimatization period.
Preparation of pancreas homogenate and lipid peroxidation assay

The rats’ pancreas tissue was isolated and placed on ice, weighed and rinsed in cold 0.9% normal
saline (1:3, w/v), subsequently homogenized in phosphate buffer (pH 7.4). The homogenate was
centrifuged at 5,000 × g. The clear supernatant obtained was used for thiobarbituric acid reactive species
(TBARS) assay according to the method described by Shodehinde et al. (2017). The TBARs produced
was measured at 532 nm and subsequently calculated.
158
Determination of reducing power, Fe2+ chelating and hydroxyl radical scavenging abilities of the
samples
The reducing power of the samples was determined by assessing the ability of the samples to reduce
FeCl3 solution as described by Pulido et al., (2000), and was subsequently calculated and expressed as
ascorbic acid equivalent (AAE). The ability of the samples to chelate Fe2+ was carried out using the
method described by Shodehinde et al. (2017). The method of Halliwell and Gutteridge, (1981) was used
to determine the scavenging ability of the hydroxyl (OH) radical produced from Fe 2+/H2O2 induced
decomposition of deoxyribose.
High Performance Liquid Chromatography Analysis (HPLC-DAD)

The quantification of phenolic compounds in the extract was carried out according to the method
described by Ademosun et al. (2015) using high performance liquid chromatography coupled with Diode-
Array Detector (HPLC-DAD). The chromatography peaks were confirmed by comparing its retention
time with those of reference standards and by DAD spectra (200 to 600 nm). Chromatography operations
were carried out in triplicates under ambient condition. Limit of detection (LOD) and limit of
quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using
three independent analytical curves, as defined by Ademosun et al. (2017).
Data Analysis
The results of triplicate experiments were pooled and expressed as mean ± standard deviation (SD).
Means were compared by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range
test. Significant difference was accepted at p ≤ .05. GraphPad Prism version 5.00 for Windows was used
for statistical analysis.
Regulation of blood glucose level is one of the therapeutic points in the management of T2DM.
Acarbose, an α-amylase and α-glucosidase inhibitor is being used to control blood glucose level
(Ademiluyi et al., 2016; Adefegha et al., 2016). Although, about 2% of acarbose could be absorbed, after
oral dosing (Oboh et al., 2016b), however, according to Boath et al. (2012), co-administration with some
berries exhibited synergistic effect on α-glucosidase activities in vitro, and possibly reduced its side
effects. In this study, Acarbose, BLE and their various combinations inhibited α-amylase and α-
glucosidase activities (Fig. 1). The various combinations exhibited reduced α-amylase inhibitory effect
compared to 100% ACA, but higher than that of 100% BLE (Fig. 1A). The combinations also exhibited
lower α-glucosidase inhibitory effect when compared to 100% ACA (Fig. 1B). Interestingly, 100% BLE
had high α-glucosidase (71.76 ± 1.20%) inhibition than α-amylase (48.95 ± 2.01%). The effect of the
BLE on α-amylase and α-glucosidase agree with earlier reports on the mild inhibition of α-amylase
activity, but stronger α-glucosidase inhibitory properties of plant materials (Ademiluyi et al., 2016),
which could be an added advantage over acarbose that has stronger α-amylase inhibitory properties
(Adefegha et al., 2015). Chlorogenic and caffeic acids, rutin and quercetin, which were among the
phenolic identified in bitter leaf, have been reported to inhibit α-amylase and α-glucosidase activities
(Adefegha et al., 2015). Therefore, we could affirm that the enzymes inhibitory effect of the extract could
be linked with the phenolic. However, contrary to the reports of Oboh et al. (2016b), Adefegha et al.
(2016) and Boath et al. (2012), that phenolic content of the plant materials may potentiate the therapeutic
value of Acarbose in the management of T2D, this study shows that there could be a conflicting effect
when bitter leaf is consumed with acarbose
The antioxidative properties of the samples were also determined, via four antioxidant assays
method: inhibition of Fe2+-induced TBARs production, ferric reducing antioxidant power (FRAP), Fe2+
chelation and OH radical scavenging abilities. The incubation of rat's pancreatic homogenate with 250
159
mM FeSO4 leads to significant (p < 0.05) increase in TBARs (173.2 ± 3.17%) content relative to the basal
(100%) (Fig 2A). But, the addition of the samples reversed the TBARs levels while the combinations and
100% BLE had a stronger inhibitory effect on TBARs production compared to 100% ACA. The result of
the reducing power (FRAP) of the samples shows that the 100% BLE had the highest (p < 0.05) reducing
property compared to the combinations, while 100% ACA had the least (Fig 2B). Figure 2C revealed that
the combinations and 100% BLE had higher Fe2+ chelating abilities compared to 100% ACA. The result
of the hydroxyl radical scavenging ability (Fig.2D) revealed that the samples scavenged OH radical, and
100% BLE had the highest, while 100% ACA had the least.
Figure 1: α-Amylase (A) and α-glucosidase (B) inhibitory abilities of various combinations of acarbose
(ACA) and bitter leaf extract (BLE) (*p < 0.05 vs. 100% ACA).
160
Figure 2: A-Inhibition of TBARS, B-Ferric reducing antioxidant power (FRAP), C-Fe 2+ chelating and
D-hydroxyl radical scavenging abilities of various combinations of acarbose (ACA) and bitter leaf extract
(BLE) (*p < 0.05 vs. 100% ACA, **p < 0.05 vs. 100% BLE).
Report has shown that pancreatic β-cells are prone to oxidative damage, probably due to limited
antioxidant defense systems and attack from reactive species (Ademiluyi et al., 2016). Although Fe2+ is
very important and its quantity is required in a small amount for proper metabolic function of the
biological system, however, its free form in the circulation can initiate production of reactive species and
ultimately oxidative stress; a major culprit in the pathophysiology DM (Swaminathan et al., 2007). This is
confirmed by this study as incubation of 250 μM Fe2+ solution with the pancreas homogenate caused a
significant (p < .05) increase in TBARS level. However, the addition of the samples caused reduction of
TBARS level, which could either be as a result of ferric-to-ferrous reducing ability of the samples (Fig
2B) or their Fe2+ chelating and hydroxyl radical scavenging abilities; an indication of their corresponding
concentration of electron donating antioxidants molecules of the samples (Halvorsen et al., 2002).
Furthermore, the high antioxidant activity of 100% BLE compared to 100% ACA and the combinations
could be due to its phenolic composition (Table 1).
Polyphenols are considered to be strong antioxidant molecules capable of preventing oxidative
damage to pancreatic β-cells, owning to their abilities to prevent TBARS production, reduction of ferric-
to-ferrous, chelate transition metals and scavenging of radicals (Oboh et al., 2015: Adefegha et al., 2015),
161
due to its redox properties of their numerous hydroxyl groups (Adefegha et al., 2015). Table 1 and Fig 3
represent the HPLC-DAD phenolic analysis of BLE, which revealed 9 phenolic compounds, four of
which were phenolic acids, while the remaining five were flavonoids. Quercitrin (6.37 mg/g), luteolin
(4.15 mg/g), p-coumaric acid (4.11 mg/g), rutin (4.11 mg), quercetin (3.72 mg/g) caffeic (2.05 mg/g),
orientin (2.01 mg/g), chlorogenic acid (1.79 mg/g) were detected (Table 1) alongside with some trace
phenolics (Fig 3). Reports have shown that the correlations between the antioxidant capacities of plant
extracts and their phenolic constituents are statistically significant (Dudonné et al., 2009; Katalinic et al.,
2006). Therefore, in this study, the observed varieties of phenolic acids and flavonoids, found in the BLE
extract could be responsible for its biological activities, as well as the enhanced antioxidant activities of
ACA as observed in the various combinations tested.
Figure 3 – Representative high performance liquid chromatography profile of bitter leaf extract. Catechin
(peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), p-coumaric acid (peak 4), rutin (peak 5),
orientin (peak 6), quercitrin (peak 7), quercetin (peak 8) and luteolin (peak 9)
Conclusion
This study revealed that the phenolic-rich extract from bitter leaf and Acarbose, and their various
combinations exhibited α-amylase and α-glucosidase inhibitory and antioxidant effects in vitro. The
combinations exhibited reduced α-amylase and α-glucosidase inhibition compared to 100% ACA and
BLE, while the extract appeared to enhance antioxidative effects of ACA. This study shows that the co-
administration of bitter leaf and Acarbose could reduce the α-amylase and α-glucosidase inhibitory effect
of Acarbose. However, BLE enhanced antioxidative potential of Acarbose.
162
References
Adefegha SA, Oboh G, Ejakpovi II, Oyeleye SI (2015) Antioxidant and antidiabetic effects of gallic and
protocatechuic acids: a structure–function perspective. Comp Clin Pathol 24: 1579-1585.
Adefegha SA, Oboh G, Omojokun OS, Jimoh TO, Oyeleye SI (2016) In vitro antioxidant activities of African birch
(Anogeissus leiocarpus) leaf and its effect on the α-amylase and α-glucosidase inhibitory properties of acarbose.
J Taibah Univ Med Sci 11: 236-242.
Adefegha SA, Oboh G, Oyeleye SI, Ejakpovi I (2016) Erectogenic, antihypertensive, antidiabetic, anti‐oxidative
properties and phenolic compositions of Almond Fruit (Terminalia catappa L.) Parts (Hull and Drupe)–in vitro.
J Food Biochem 41: 12309.
Ademiluyi AO, Oyeleye SI, Oboh G (2016) Biological activities, antioxidant properties and phytoconstituents of
essential oil from sweet basil (Ocimum basilicum L.) leaves. Comp Clin Pathol 25: 169-176.
Ademosun AO, Oboh G, Passamonti S, Tramer F, Ziberna L, et al. (2017) Modulation of HMG-CoA reductase and
glutathione-linked enzymes and protection against pro-oxidant induced oxidative damage in colon (Caco-2) cells
and rat colon homogenates by phenolic extracts from Shaddock (Citrus maxima) peels. J Appl Biomed 15: 1-8.
Atangwho IJ, Egbung GE, Ahmad M, Yam MF, Asmawi MZ (2013) Antioxidant versus anti-diabetic properties of
leaves from Vernonia amygdalina Del. growing in Malaysia. Food Chem 141: 3428-3434.
Boath AS, Stewart D, McDougall GJ (2012) Berry components inhibit α-glucosidase in vitro: Synergies between
acarbose and polyphenols from black currant and rowanberry. Food Chem 135: 929-936.
Dudonné S, Vitrac X, Coutiere P, Woillez, M, Mérillon, J.M (2009) Comparative study of antioxidant properties and
total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC
assays. J Agric Food Chem 57: 1768-1774.
Erasto P, Van de venter M, Roux S, Grierson DS, Afolayan AJ (2009) Effect of leaf extracts of Vernonia
amygdalina on glucose utilization in chang liver, C2C12 muscle and 3T3-L1 cells. Pharma Biol 47: 175–181.
Halliwell B, Gutteridge JMC (1981) Formation of thiobarbituric acid reactive substance from deoxyribose in the
presence of iron salts. FEBS Letter 128: 347-352.
Halvorsen BL, Holte K, Myhrstad MCW, Barikmo I, Hvattum E, et al. (2002) Systematic screening of total
antioxidants in dietary plants. J Nutr 132: 461–471.
Katalinic V, Milos M, Kulisic T, Jukic M (2006). Screening of 70 medicinal plant extracts for antioxidant capacity
and total phenols. Food Chem 94: 550-557.
Leonard BS, Karen LL, Bruce B, Thomas FK, Jay HH (2002) Complementary and alternative medicine in chronic
liver disease. Hepatol 34: 595–603.
Maiti R, Jana D, DAS, UK, Ghosh, D (2004) Antidiabetic effect of aqueous extract of seed of Tamarindus indica in
streptozotocin-induced diabetic rats. J Ethnopharmacol 92: 85–91.
National Institute of Health (NIH) (2011) Guide for the care and use of laboratory animals. US. Department of
Health Education and Welfare. USA: NIH Publication
Oboh G, Ademosun AO, Ayeni PO, Omojokun OS, Bello F (2015) Comparative effect of quercetin and rutin on α-
amylase, α-glucosidase, and some pro-oxidant-induced lipid peroxidation in rat pancreas. Comp Clin Pathol 24:
1103-1110.
Oboh G, Akinyemi AJ, Adeleye B, Oyeleye SI, Ogunsuyi OB, et al., (2016) Polyphenolic compositions and in vitro
angiotensin-I-converting enzyme inhibitory properties of common green leafy vegetables: A comparative study.
Food Sci Biotech 25: 1243-1249.
Oboh G, Ogunsuyi OB, Ogunbadejo MD, Adefegha SA (2016b) Influence of gallic acid on α-amylase and α-
glucosidase inhibitory properties of acarbose. J Food Drug Anal 24: 627-634.
Pulido R, Bravo L, Saura-Calixto F (2000) Antioxidant activity of dietary polyphenols as determined by a modified
ferric reducing/antioxidant power assay. J Agric Food Chem 48: 3396–3402.
Shodehinde SA, Oyeleye S.I., Olasehinde TA, Adebayo AA, Oboh G et al (2017). Lasianthera Africana leaves
inhibits α-amylase α-glucosidase, angiotensin-I converting enzyme activities and Fe2+-induced oxidative damage
in pancreas and kidney homogenates. Oriental Pharma Exp Med 17: 41-49.
Swaminathan S, Fonseca VA, Alam MG, Shah SV (2007) The role of iron in diabetes and its complications.
Diabetes Care 30: 1926-1933.
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ISSN 0795-8080

Volume 32, Number 2

Editorial Advisory Board

0795-8080/2020 $10.00 + 0.00

Protective activity of root extract of Rhaphiostylis beninensis

(Received January 20, 2020; Accepted March 9, 2020)

Keywords: Rhaphiostylis beninensis, hepatotoxicity, carbon tetrachloride, malondialdehyde

Materials and Methods

Collection and Authentication of the Plant Material

Preparation and Extraction of the Plant Material

Phytochemical Screening of the Plant Material

Preparation of Serum and Liver homogenate

Lipid peroxidation and Antioxidant Biomarkers

Table 1: Phytochemical constituents of methanol root extracts of R. beninensis spice

Phytochemical Quantity (%)

Table 2: Comparison of mean relative weight of the liver

PARAMETER GROUP A GROUP B GROUP C GROUP D

Groups CAT(µmol/g Tissue) MDA(µmol/g Tissue) SOD(µmol/g Tissue)

0795-8080/2020 $10.00 + 0.00

Aqueous seed extract of Cola acuminata ameliorated high

1. Department of Biochemistry, University of Ilorin, Ilorin, Kwara State, Nigeria.

(Received January 21, 2020; Accepted March 9, 2020)

Keywords: Hyperlipidaemia, high-fat diet, Cola acuminata.

Hyperlipidaemia is a heterogeneous group of disorders characterized by an excess of lipids in the

Materials and Methods

Assay Kits and Reagents

Table 1: Proximate Composition of Control and High Fat Diet

Proximate Composition (%) NC HFD

Table 2: Secondary Metabolites in Aqueous Seed Extract of Cola acuminata

Phytochemicals Concentration (mg/100g)

Saponin 0.21 ± 0.00

Serum lipid profile(mg/dl) Liver Lipids (mg/dl)

Groups AI Index (x10-1) CHD Risk ratio

Groups Specific Enzyme Activity (nmol min-1 mg protein-1)

21.42mg/kg extract 87.16 ± 7.03a 24.46 ± 0.86a 60.27 ± 1.97a

42.85mg/kg extract 83.95 ± 3.76a 26.89±1.34 61.30 ± 5.40a

Groups Urea(mg/dl) Uric acid(mg/dl) Creatinine(mg/dl)

NC 12.16 ± 1.71a 1.90 ± 0.09a 2.52 ± 0.32a

Groups C Bil (mg/dl) T Bil (mg/dl)

21.42 mg/kg extract 24.72 ± 2.27a 62.86 ± 2.50a

42.85 mg/kg extract 26.25 ± 2.56a 66.38 ± 2.56a

RFD 10.71 mg/kg b.w.

21.42 mg/kg b.w. 42.84 mg/kg b.w.

Fig. 1: Histoarchitecture of the Hearts of control and experimental rats.

RFD 10.71 mg/kg b.w.

21.42 mg/kg b.w. 42.84 mg/kg b.w.

Fig. 2: Histoarchitecture of the Kidney of the control and experimental rats.

Effect of Aqueous Extract of Cola acuminata on the Liver Histoarchitecture of High-Fat

RFD 10.71 mg/kg b.w.

21.42 mg/kg b.w. 42.84 mg/kg b.w.

Fig. 3: Histoarchitecture of the Liver of the control and experimental rats

0795-8080/2020 $10.00 + 0.00

Amylase production by Aspergillus flavus immobilized in

Corresponding author: E-mail: topebanjo4rever@gmail.com

Keywords Amylase; Immobilization; Adansonia digitata; Aspergillus flavus.

(Received January 22, 2020; Accepted March 9, 2020)

Recent discoveries on the potentials of using microorganisms as biotechnological source of

Materials and Methods

Pretreatment of Adansonia digitata seeds.