ISSN 0795-8080

BIOKEMISTRI
An International Journal Published by the
Nigerian Society for Experimental Biology

Volume 32, Number 1

March, 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 1 Contents March, 2020

BKR 2020003/32101
Heavy metals correlate with cellular adenosine triphosphate and fructose levels in
municipal dumpsite exposure
Oluwakemi T. Oyelowo, Oju E. Omamogho, Ikechukwu F. Ezenwajiaku, Olajumoke A.
Morenikeji and Adekunle Mofolorunso ............................................................................................... 1

BKR 2020004/32102
Effects of ethanol leaf extract of Vernonia amygdalina on some indices of liver function,
oxidative stress and lipid profile in aluminium chloride intoxicated male Wistar rats
Edet Okon Akpanyung, Utibe Evans Bassey, Utonne Texubong Noah and Grace Sylvester
Effiong ................................................................................................................................................. 11

BKR 2020005/32103
Amelioration of paracetamol-induced nephrotoxicity in mice by aqueous extract from the
calyx of Hibiscus sabdariffa Linn.
Blessing O. Orji, Frederick O. Obi, Emmanuel U, Modo, Martin Osibemhe, Catherine A.
Otitolaiye ............................................................................................................................................. 23

BKR 2020006/32104
Gastroprotective potentials of aqueous extract of Persea americana seed against Aspirin-
induced ulcer in Wistar rats
M. O. Salawu, A. Shittu, M. O. Nafiu and H. O. B. Oloyede ............................................................... 35

BKR 2020010/32105
Acute toxicity study of crude methanol leaf extract of Ficus exasperata Vahl on male
Wistar albino rats
Shemishere B. Ufuoma, Anyebe A. Daniel, Tajudeen Yahaya, Liman U. Umar and Ahmad
Bello .................................................................................................................................................... 47

BKR 2020011/32106
Starch and triglycerides in the root of maize (Zea mays) and cowpea (Vigna unguiculata)
grown in crude oil polluted soil treated with ash from palm bunch
Stella Oghomwen Olubodun, George E. Eriyamremu and Chinedu Eze ............................................ 55

BKR 2020013/32107
Inhibition of some enzymes implicated in diabetes mellitus by raw and blanched extracts
of African Lettuce (Launaea Taraxacifolia)
Bukola C. Adedayo, Sunday I. Oyeleye, Ganiyu Oboh ....................................................................... 69

BKR 2020031/32108
Features, Evaluation and Treatment of Coronavirus Disease 2019 (COVID-19)
Marco Cascella; Michael Rajnik; Arturo Cuomo; Scott C. Dulebohn; Raffaela Di Napoli ............... 77
iv
 O. T. Oyelowo et al.

0795-8080/2020 $10.00 + 0.00

Biokemistri
Vol. 32, No. 1, March 31, 2020, pages 1 - 10 © 2020 Nigerian Society for Experimental Biology
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BKR 2020003/32101

Heavy metals correlate with cellular adenosine

triphosphate and fructose levels in municipal dumpsite
exposure
Oluwakemi T. Oyelowo1*, Oju E. Omamogho1, Ikechukwu F. Ezenwajiaku2, Olajumoke
A. Morenikeji 2, Adekunle Mofolorunso1
1
Department of Physiology, University of Lagos, Lagos, Nigeria
2
Department of Zoology, University of Ibadan, Ibadan, Nigeria

*Author for Correspondence: Oluwakemi T. Oyelowo ([email protected]) Tel:

+23480367333891

(Received January 16, 2020; Accepted March 9, 2020)

ABSTRACT: This study assessed the influence of direct exposure of rodents to Awotan Solid Waste Dumpsite
(ASWD) in Ibadan, Nigeria. Reproductive energy content was evaluated by assessing testicular ATP activity,
testicular polychlorinated biphenyls (PCBs), testicular heavy metal and fructose levels in seminal vesicle and
coagulating glands. The exposure of the male rats was from postnatal day 22 and for a period of 10 weeks. The
direct municipal dumpsite exposure resulted in testicular heavy metal accumulation viz: mercury > iron > zinc >
nickel > cadmium > arsenic. There was a decrease in serum lactate dehydrogenase (LDH) concentration, an
increase in testicular LDH concentration (markers of cellular ATP) and a decrease in the fructose level in the
seminal vesicle of the dumpsite exposed (DSE) animals compared with the control. The PCBs were not detected
in the testis of DSE animals but there was significant correlation between testicular LDH and some metals. It is
concluded that some possible mechanisms by which direct exposure to a dumpsite elicits energy depletion in the
male rat testes could be through the inhibition of fructose level and LDH activity.

Keywords: Heavy metal, ATP, Fructose, Dumpsite, Polychlorinated biphenyls

Introduction

Reproductive disorders, high infertility rate, increased birth defects, repressed immunological
function, and increased frequency of cancers have been linked to exposure to landfill sites (1,2). Landfill
which refers to the deposition of waste in an area specially designed for such is a major method in waste
management hierarchy in developing countries (1). Landfills may contain household or municipal
wastes, industrial, biomedical or infectious wastes (3). The landfills thus can contain toxic substances
such as heavy metals, dissolved organic matter, polychlorinated biphenyls and inorganic macro
compounds. Some of these xenobiotics have been classified by The United States Environmental
Protection Agency to be of concern to the male reproductive health (4).
Heavy metal environmental pollution even at low levels and their subsequent long-term collective
health effects are among the leading causes of health concerns the world over (5). There have been
studies suggesting that heavy metals are a major source of oxidative stress in the cell and that they play
an important role in the aetiology of diverse human pathologies like carcinogenesis (6). Research has
also revealed that heavy metals directly modify and /or damage the DNA structure (7). There have also
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been adverse effects of these metals on male reproduction (8). The adverse effects include altering the
morphology and functional systems, fluctuating secretory functions as well as altering energy balance
resulting in infertility or impotence (9). Exposure to cadmium for instance has been reported to affect
the prostate function and serum testosterone levels (10). It was reported that workers exposed to
chromium (VI) had significantly higher serum follicle stimulating hormone (FSH) concentration and
lower seminal plasma zinc levels, sperm concentration, motility and lactate dehydrogenase (LDH)
levels (11) and significantly higher percentage of abnormal sperm than control workers (12).
Lactate dehydrogenase is involved in metabolic processes of sperm in the seminal plasma and is
closely related to reproductive performance of the males (13). Lactate dehydrogenase (LDH-C4) is
known as a major isoenzyme of sperm synthesized in the testes during spermatogenesis. Lactate
dehydrogenase and its isoenzyme fulfils a specialized function for the metabolic development
requirements of adult male (13) and it is related to metabolic processes by which the stem cells obtain
their energy (14). Lactate dehydrogenase and its isoenzyme are important markers of seminiferous
epithelium activity in the diagnostics and treatment of male infertility (15). An in vitro study revealed
that LDH-C4 is also involved in sperm capacitation (16). Lactate dehydrogenase activity is a marker of
cellular adenosine triphosphate (ATP) thus the level of cellular ATP during anaerobic conditions has
been widely assessed using LDH activity. The role of adenosine triphosphate (ATP) as the energy
metabolism of the mitochondria is a major factor supporting multiple functions of the sperm. They
harbour significant metabolic pathways during germ cell development and fertilization, energy
maintenance and fertility (17). Testicular energy metabolism is desired to maintain spermatogenesis and
research reveals that the germ cells depend on lactate for energy. Lactate dehydrogenase is associated
with survival and maturation of germ cells and adenosine triphosphate production.
The occurrence of health symptoms with solid waste management workers, as well as people living
near municipal dumpsites is increasing (18,19). Previous studies have linked municipal dumpsites to
the adverse effects of leachates on sperm and testicular activities (20-24). It remains to be determined
if there is a link between the direct exposure to municipal landfill and its effects on the male reproductive
activity.
The fact that the Awotan Solid Waste Dumpsite is located not too far from a residential settlement
and the possibility of using the solid waste dumpsite mixture for agricultural soil enrichment, considered
this study of importance, to evaluate the reproductive energy content in the male rats. The energy
content was evaluated by assessing adenosine triphosphate energy production, testicular
polychlorinated biphenyl content, metal content on testicular function. Also, fructose levels in the
seminal vesicle and coagulating glands, of directly exposed rats to the municipal dumpsite were
assessed.

Materials and Methods

Study area
The study area, Awotan Solid Waste Dumpsite (ASWD) is one of the three government approved
open dumpsites in the city of Ibadan, Nigeria. It is located within 07’27.719’-07’27.811’ North and
003’51.003’- 003’50.999’East, Ibadan. The ASWD receives 36000 tonnes of municipal wastes in an
area of 14 hectares and it has been active since 1998 (25). Residential settlements, block industries,
markets, schools and churches are located within thirty meters radius to the dumpsite.

Animal Model and Experimental Protocol

All procedures of animal handlings and experimental protocols conformed with the
internationally accepted guideline for laboratory animal use and care (NIH publication No.85-
23, revised 1985), as approved by the Institutional Animal Care and Use Ethics Committee of
the College of Medicine, University of Lagos. Brown rats (Rattus norvegicus) of 22 days old
were housed in the vicinity of the Awotan Solid Waste Dumpsite for 10 weeks and they fed on
dumpsite waste. The rats were exposed to the municipal waste according to Luvizutto et al.,
(26) with slight modifications. The municipal solid waste which is a mixture of complex
materials (food wastes, market wastes, microorganisms, plants, chemical contaminants and

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 O. T. Oyelowo et al.

minerals) was given to the rats at 5000ppm ad libitum. This served as the dumpsite exposed
(DSE) group (n=12). Daily monitoring of the rats was done to observe any clinical signs. The
laboratory rats (Rattus norvegicus domesticus) of 22 days old were bred in the Institution’s
laboratory animal facility and they served as the control (n=12). The rats were provided with
rat pellets and water ad libitum. All animals were subjected to a natural 12:12h light-to- dark
photoperiod. Rats were maintained under the controlled conditions of temperature (35 ± 2˚C)
and humidity (50 ± 5%). At the end of the 10 weeks’ period, DSE animals were returned to the
laboratory animal facility. Twenty-four hours after the DSE animals were brought to the
laboratory facility, the blood from the animals was drawn from retro-orbital venous plexus for
serum lactate dehydrogenase (LDH) assay. The animals were then killed by cervical
dislocation. The testes, epididymis, seminal vesicles/coagulating glands were removed rapidly
and weighed. The testes, seminal vesicle and coagulating glands were processed for further
assays.

Lactate Dehydrogenase (LDH) Assay

The serum and testicular homogenates were assayed for LDH activity using a commercially
available kit (Randox Laboratories, UK). The LDH assay was performed and according to the
manufacturer’s instructions.

Fructose Assay
Fructose level was estimated from the seminal vesicles and coagulating gland homogenate by
means of Fructose test kit (FertiPro N.V. Industriepak Noord 32, 8730 Bernem, Belgium). The assays
were carried out according to the manufacturer’s instructions. The seminal fructose was done
photometrically.

Testicular Heavy Metal Analysis

The right testis of animals from each group were washed in ice-cold 1.15% KCl solution, then
blotted and weighed. They were subsequently homogenized in 4 volumes of homogenizing buffer
(50mM Tris-HCL mixed with 1.15% KCL [pH adjusted to 7.4]) using a homogenizer. The testes
supernatants were used to determine heavy metal levels (27). The levels of lead, copper, nickel, zinc,
iron, cadmium, arsenic, and mercury in the filtrates of the digested samples were determined using an
atomic absorption spectrophotometer (Perkin Elmer A Analyzer 200, USA).

Polychlorinated biphenyls (PCBs) concentration determination

The testis sample (2g) each, was weighed into 250ml conical flask and 10ml of dichloromethane
was added to the sample. The mixture was shaken vigorously on the shaker for about 45minutes. It was
then transferred to the ultrasonic bath for ultrasonic extraction for 2hours. The isolation of
Polychlorinated biphenyls (PCBs) from the lipid matrix was done by solid phase extraction in a normal
phase mode. Activated silica gel was loaded onto a glass chromatographic column (i.d 20 mm, height
400 mm) and conditioned with dichloromethane. The effluents were then concentrated using a rotary
evaporator and the samples were thereafter dissolved in 1 ml acetone and transferred to viral bottles for
Gas Chromatography Mass Spectroscopy (GCMS) analysis. The analyses were performed with Agilent
Tech model 7890A gas chromatograph system coupled with an MS Agilent Tech 5975C VL MSD
(USA). The identification of the found components was confirmed by comparison of the retention
indices with those of authentic compounds and with the NIST 02 library.

Statistical Analysis
The results were presented as mean ± standard error of mean (SEM) and were subjected to post
hoc test using Duncan’s multiple range tests using GraphPad Prism-5 statistical software (San Diego,
California, U.S.A.). The differences were considered significant at p< 0.05.

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Results

Body Weight and Absolute Reproductive Organ Weights

There was no significant difference in the final body weights of the control and the DSE animals.
However, the absolute weights of the epididymis, testis and seminal vesicle and coagulating glands
were significantly increased in the DSE animals compared with the control group (Table 1).

Table 1: Body weight and Reproductive organ weights after DSE for 10 weeks in rats

Variables Control DSE

Body weight initial (g) 40.01 ± 0.01 40.01 ± 0.01

Body weight final (g) 202.40 ± 4.17 185.30 ± 7.65
Epidydimal weight (g) 0.39 ± 0.01 0.58 ± 0.06*
Testicular weight (g) 0.48 ± 0.01 0.72 ± 0.03*
Seminal vesicle and CG
weight (g) 0.39 ± 0.01 2.25 ± 0.07*

Data are expressed as mean ± SEM for 12 rats/ group. *p <0.05 compared with the control

Testicular Heavy Metal Concentrations

The exposure of the rats to the dumpsite resulted in a significant increase in the levels of nickel,
zinc, iron, cadmium, arsenic and mercury accumulation in the testis of the animals compared with the
control (Figs 1 and 2). However, there was a significant decrease in the level of lead in the DSE animals
compared with the control (Fig 2).

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O. T. Oyelowo et al.

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Seminal vesicle and coagulating gland Fructose level

There was a significant decrease in the level of fructose found in the seminal vesicle and
coagulating glands compared with the control (Fig 3).

Activity of Serum and Testicular Lactate Dehydrogenase (LDH) (Functional marker of ATP).
The activity of the serum LDH was significantly inhibited in the rats from the dumpsite compared
with the control rats however, the dumpsite exposure was associated with a significant increase in the
testicular activities of LDH compared with the control (Fig 4). Testicular LDH significantly correlated
with mercury and copper (Table 2).

Polychlorinated biphenyls (PCBs) concentration

Polychlorinated biphenyls (PCBs) were absent in the testis of the dumpsite-exposed animals.

Table 2: Correlation matrix between Heavy metals and Testicular LDH, Serum LDH, Fructose in control
animals

Cu Ni As Zn Hg Fe Cd Pb
Testicular LDH 0.66* 0.30 0.47 -0.36 0.70 0.67 0.72 0.72
Control Serum LDH -0.1 0.10 0.01 -0.31 0.18 0.47 -0.36 -0.36
Fructose -0.43 0.06 0.02 -0.58 -0.24 -0.49 -0.95 -0.95**

*Correlation is significant at the 0.05 level. **Correlation is significant at the 0.01 level.

Table 3: Correlation matrix between Heavy metals and Testicular LDH, Serum LDH, Fructose in
dumpsite exposed (DSE) animals.

Cu Ni As Zn Hg Fe Cd Pb
Testicular LDH 0.77* -0.19 0.04 -0.60 0.83* 0.12 -0.23 0.54
DSE Serum LDH -0.10 -0.08 0.38 -0.22 -0.14 0.27 0.27 0.41
Fructose -0.55 0.01 -0.04 0.30 -0.62 -0.62 -0.62 -0.03
*Correlation is significant at the 0.05 level. **Correlation is significant at the 0.01 level.

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 O. T. Oyelowo et al.

Discussion

There was an accumulation of mercury, arsenic, cadmium, iron, zinc and nickel in the testes of the
dumpsite exposed (DSE) animals. A previous study from our laboratory (28) reported that heavy metal
contents found in the soil of Awotan Solid Waste Dumpsite (ASWD) which is the study area for this
research were above the USEPA permissible limits which would have resulted in the accumulation of
these toxic heavy metals signifying their roles in testicular toxicity observed in the DSE animals.
Although the levels of lead and copper were above the USEPA permissible limits in the soil of ASWD,
they were not accumulated in the testis of the animals exposed to the dumpsite. A probable reason is
that metals interact additively, synergistically as well as antagonistically and affect each other’s
absorption, distribution and excretion. This interference with metabolism can reduce the concentration
in the organism or decrease the bioavailability. The competition between lead and/or cadmium and zinc
for the same binding sites in enzymes, proteins, and transporters can affect structure and/or function of
cell membranes, change enzyme activity, induce oxidative stress and apoptosis which all have grave
consequences on cell growth, development, and differentiation. Thus, these interactions contribute to
interindividual differences in susceptibility to adverse effects of metals in men (29).
According to a study by Sharpe et al. (30) sertoli cells proliferate during the foetal, neonatal, and
pre-pubertal periods and each of these periods is particularly sensitive to the adverse effects of metals.
Testicular toxicity in rats has also been shown as a good predictor in human subjects (31). Arsenic and
cadmium which are classified as the first and seventh most hazardous substances (32) were significantly
increased in the DSE group. Arsenic and its compounds have been reported to disrupt ATP production
when exposed to animals by way of water drinking (33). The observation in this study corroborates
other studies that reported that cells exposed to arsenic showed a considerable depletion in ATP and
glycogen levels in the liver and other tissues. Cadmium has been reported to cause severe damage to
spermatogenic epithelium in an animal model (34). Other reports have shown that at concentrations
greater than (0.003 mg/L), cadmium had direct injury on the testes by damaging germ cells and Sertoli
cells and subsequently reducing sperm quality (34). The increase in cadmium level in the dumpsite-
exposed animals also confirms the fact that cadmium accumulates in the male reproductive organs,
especially the testis of both humans and animals (35). Cadmium has been shown to negatively affect
accessory sex organ functions (10).
The increase in testicular and epididymal weights (36) has been reported in cadmium exposure
which corroborates the findings of this study. The increase in the organ weights generally indicates an
excessive accumulation of interstitial fluid and maybe a sensitive indicator of decreased sperm
production (36). Some studies have earlier reported that cadmium mediated histological changes in the
testes, epididymis and accessory sex organs (37). Moreover, exposure to cadmium, lead and arsenic
may contribute to prostate cancer development and some reports have associated their exposure with
increased serum prostate specific antigen (PSA) (10). Metals can affect the male reproductive system
directly when they target specific reproductive organs. When there is an accumulation of metals in the
testis, epididymis, prostate, seminal vesicle, they impair progressive sperm motility (38). Metals affect
the secretory function of the seminal vesicles leading to the loss of fertility, libido or impotence (39).
The seminal plasma is a mixture of secretions derived principally from the major accessory sex
glands such as the seminal vesicle, prostate, Cowper’s glands with minor contributions from the
epididymis, ampulla gland etc. Since neither the testis nor epididymal sperm contains fructose, the
immotile spermatozoa encounter fructose only after it has been intermixed with seminal plasma during
ejaculation. This occurs at a crucial moment when the sperm requires a high degree of motility requiring
a source of quick available energy. This energy is provided by the metabolic process of fructolysis
during which lactic acid is formed. Since growth and secretory function of the accessory sex glands are
under the control of the testis, the level of fructose in seminal plasma appears to be a precise indicator
of the function of the Leydig cell system of the testis. The decreased fructose level as well as an
increased accumulation of heavy metals in the testis observed in this study is consistent with the earlier
report. Scientific evidence suggests that hypofunction of these accessory sex organs affects sperm
motility and sperm chromatin stability which may produce infertility (41). The seminal fructose
concentration is an assessment of the seminal vesicle and could also give useful indications of the male
reproductive function (42).
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The level of cellular adenosine triphosphate (ATP) during anaerobic conditions had been widely
assessed using LDH activity because it is a stable enzyme. LDH is an oxidoreductase enzyme that
catalysis the interconversion of pyruvate and lactate. Cells release LDH into the bloodstream after tissue
injury. Animals exposed to the dumpsite had a significant depletion of LDH activity in the serum.
Inhibition of LDH which is a key enzyme of glycolytic pathway caused by the dumpsite exposure in
the absence of oxygen which is needed for ATP production would slow down the metabolic pathway in
charge of energy production. This outcome supports (43,44) who had previously reported that patients
with abnormal spermatogenesis had low levels of ATP. Additionally, ATP is necessary for the synthesis
of cholesterol, ketone bodies and fatty acids. In aerobic conditions, pyruvate is converted to acetyl CoA
catalyzed by pyruvate dehydrogenase which may be oxidized in the tricarboxylic acid (TCA) cycle to
produce ATP. The findings in this study thus suggest that heavy metals in the dumpsite could disrupt
ATP production by inhibiting pyruvate dehydrogenase when competing with the phosphate group thus
uncoupling oxidative phosphorylation. This inhibits energy-linked decrease of NAD+, mitochondrial
respiration, and ATP synthesis in the serum. It is interesting to note that a different interplay of events
occurred in the testis of the DSE animals, as there was an increase in the level of LDH activity in the
testes. Lactate dehydrogenase is the functional marker of ATP and it is required in its production. LDH
is also connected with existence and development of germ cells. The increase in testicular LDH activity
thus observed in this study may indicate an adaptive mechanism by the testes to assuage germ cell death
and azoospermia in DSE rats.
The significant correlation between the testicular LDH, mercury and copper, as well as fructose
and nickel and lead in the DSE animals further buttress the fact that the heavy metal bioaccumulation
in the testis affected the energy supply to the testis thus the reason for the adaptive mechanism.
Polychlorinated biphenyls (PCBs) are one of the groups of persistent organic pollutants and they have
been linked to reproductive difficulties (45). In this study PCBs were absent in the testes of the DSE
rats. According to a study (46), the PCB levels in the AWSD topsoil were below detection limit which
would have been the reason it was not detected in the testis of the DSE rats. This however does not rule
out the possibility of the presence of organic pollutants at the dumpsite as there was an accumulation of
polyaromatic hydrocarbons (PAHs) according to their study.

Conclusions
The direct exposure to a municipal dumpsite elicits detrimental effects on the energy supplies
needed for male reproductive activity. The exposure compromised testicular LDH and decreased the
LDH activity in the serum which are markers of cellular ATP. The inhibition of LDH activity and a
decrease in fructose levels observed could be possible mechanisms by which direct dumpsite exposure
elicited energy depletion and toxicity in the male rat. PCBs were absent thus the biotoxic effects and
energy depletion could be linked to heavy metal interactions which was additive, synergistic,
antagonistic and individual in nature. Indeed, an alteration in LDH and fructose levels would be
expected to compromise the energy supplies to germ cells. The human male has relatively low fertility
potential compared with other mammals and is much more susceptible to metal toxicity, there is
therefore the need for a critical assessment and discouragement of siting residential areas near
dumpsites.

Conflicts of interest
The authors declare they do not have any conflicts of interest.

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19. Bortey-Sam N, Nakayama SMM, Ikenaka Y, Akoto O, Baidoo E, Mizukawa H, Ishizuka M. Heavy metals
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20. Adedara IA, Oyebiyi OO, Lawal TA, Adesina AA, Farombi EO. Involvement of oxidative stress in municipal
landfill leachate-induced toxicity in boar sperm. Environ. Toxicol. Pharmacol. 2013; 36: 972-978.
21. Adedara IA, Lawal TA, Adesina AA, Oyebiyi OO, Ebokaiwe AP, Farombi EO. Sperm functional parameters
and erythrocytes oxidant-antioxidant imbalance during municipal landfill leachate treatment withdrawal in
rats. Environ. Toxicol. Pharmacol. 2014; 37:460-467.
22. Adedara IA, Awogbindin IO, Adesina AA, Oyebiyi OO, Lawal TA, Farombi EO. Municipal landfill leachate-
induced testicular oxidative damage is associated with biometal accumulation and endocrine disruption in
rats. Arch. Environ. Contam. Toxicol. 2015; 68:74-82.
23. Akintunde JK, Oboh G, Akindahunsi AA. Testicular membrane lipid damage by complex mixture of leachate
from municipal battery recycling site as indication of idiopathic male infertility in rat. J. Interdiscip.
Toxicol.2013a; 6(4): 192-197.
24. Akintunde JK, Oboh G, Akindahunsi AA. Inhibition of key markers linked with spermatogenesis and cellular
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101007/s00244-014-0068-9.
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26. Luvizutto JFL, Solano M De LM, Martinez MF, Fernandez CD, Umbuzeiro G de A, Camargo JLde. Potential
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27. Farombi EO Adewolo OA& Ajimoko YR Biomarkers of oxidative stress and heavy metal levels as indicators
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28. Adeyemo A, Morenikeji OA. Helminths and heavy metals in soils from a dumpsite in Ibadan city. Nigeria.
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29. Telišman S. Interactions of essential and/or toxic metals and metalloid regarding interindividual differences
in susceptibility to various toxicants and chronic diseases in men. Arh. Hig. Rada. Toksikol. 1995; 46:459-
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30. Sharpe RM, McKinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of Sertoli cells and
their relevance to disorders of testis function in adulthood. Reproduction 2003; 125:769-784.
31. Rosner M, Dolznig H, Schipany K, Mikula M, Brandau O. Human amniotic fluid stem cells as a model for
functional studies of genes involved in human genetic diseases or oncogenesis. Oncotarget. 2010; 2:705–
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32. ATSDR. Agency for Toxic Substances and Disease Registry Minimum risk levels (MRLs). 2013. Available
online at http://wwwatsdrcdcgov/mrls/pdfs/atsdr_mrls_february_2012pdf
33. Klaassen C, Watkins J. In: Casarett and Doull’s essentials of toxicology. McGraw-Hill. pp.512,2003.
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J. Exp. Pathol. 2010; 91 (2):125–131.
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epididymal toxicity. Toxicol. Pathol. 2002; 30(4): 507–520.
37. Ohtani K, Yanagiba Y, Ashimori A, et al. Influence of injection timing on severity of cadmium-induced
testicular toxicity in mice. J. Toxicol. Sci. 2013; 38(1):145–150.
38. Hess RA. Effects of environmental toxicants on the efferent ducts epididymis and fertility. J. Reprod. Fertil.
1998; 53: 247-259.
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40. Martinez CS, Torres JG, Peçanha FM et al. 60-Day Chronic Exposure to Low Concentrations of
HgCl2Impairs Sperm Quality: Hormonal Imbalance and Oxidative Stress as Potential Routes for
Reproductive Dysfunction in Rats. PLoS One 2014; 4: 9(11).
41. Gonzales GF, Villena A. Influence of low corrected seminal fructose levels on sperm chromatin stability in
the semen from men attending an infertility service. Fertil. Steril. 1997; 67:763.
42. Trang NT, Sang TT, Hoang N, Khanh NTG, Duc TT. Assessment of the level of seminal zinc and fructose
concentration in seminal plasma of Vietnamese infertile men. MOJ Biorg. Org. Chem. 2018; 2(4):185-190.
43. Parlakpinar H, Tasdemir S, Polat A, et al. Protective effect of chelerythrine on gentamicin-induced
nephrotoxicity. Cell Biochem. Funct. 2006; 24:41–48.
44. Walker PDY, Barri SV. Oxidant mechanisms in gentamicin nephrotoxicity. J. Ren. Fail. 1999; 21:433.
45. USEPA. Ambient water quality criteria for chloride. Washington DC: US Environmental Protection Agency
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46. Oketola AA, Akpotu SO. Assessment of solid waste and dumpsite leachate and topsoil. Chem. Ecol. 2014;
31(2):134-146.

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BKR 2020004/32102

Effects of ethanol leaf extract of Vernonia amygdalina on

some indices of liver function, oxidative stress and lipid
profile in aluminium chloride intoxicated male Wistar rats
Edet Okon Akpanyung1, Utibe Evans Bassey2, Utonne Texubong Noah1, and Grace Sylvester
Effiong1
1
Department of Biochemistry, University of Uyo, Uyo, Akwa Ibom State, Nigeria
2
Department of Biochemistry, Obong University, Etim Ekpo, Akwa Ibom State, Nigeria

Author for Correspondence: [email protected]; +234(0)7037917688

(Received January 16, 2020; Accepted March 9, 2020)

ABSTRACT: Aluminium is a widely distributed element with established toxicity to the liver. The present study
evaluated the protective potential of ethanol leaf extract of Vernonia amygdalina against the toxic effects of aluminium
chloride in male Wistar rats by measurement of some indices of liver function, oxidative stress and lipid profile.
Twenty-five (25) male Wistar rats weighing 180 – 220g were randomly divided into five groups of five animals per
group. Group 1 served as control and was given normal drinking water. Group 2 received 100 mg/kg body weight of
aluminium chloride while Group 3 was administered 400 mg/kg body weight of ethanol leaf extract of Vernonia
amygdalina for 14 days each. Group 4 received 400 mg/kg body weight of ethanol leaf extract of Vernonia amygdalina
and 100 mg/kg body weight of aluminium chloride concomitantly for 14 days while Group 5 received 100 mg/kg
body weight of aluminium chloride for 14 days and was allowed another period of 14 days without treatment. Assay
for the activity of some diagnostic enzymes in serum and histological assessment of the liver were carried out.
Parameters of oxidative stress and lipid profile were also assayed. Aluminium chloride significantly (p < 0.05)
increased the activities of ALT, AST, and ALP, concentrations of MDA, total cholesterol, triglyceride and LDL-
cholesterol compared to control. However, the leaf extract effectively suppressed the AlCl3-induced increase in
enzyme activities, increased the activities of SOD and catalase, reduced the concentrations of total cholesterol and
triglyceride as well as MDA in Group 4. Histological assessment of the hepatocytes corroborated the observed changes
in enzyme activities. It is therefore concluded that ethanol leaf extract of Vernonia amydalina has protective effect
against aluminium chloride induced hepatotoxicity, oxidative stress and alterations in lipid profile in male Wistar rats.

Keywords: Aluminium Chloride, Liver Function, Lipid Profile, Oxidative Stress, Vernonia amygdalina

Introduction

Aluminium is the third most abundant element in the earth’s crust and constitutes about 8% of the total
mineral components [1] It is widely used in the manufacture of cosmetics, cookware, food additives and
toothpaste [2]. It serves as a component of pharmaceuticals such as antacids, buffered aspirin, vaccines and
injectable allergens [3-5]. Aluminium is also used in the purification of drinking water [6, 7]. Industrial
waste and particulate matter generated by cement producing factories contain high amount of aluminium

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and individuals who reside around the vicinity are exposed to high levels of this metal [8]. Food sources of
aluminium include corn, yellow cheese, salt, herbs, spices and tea [9].
Human exposure to aluminium is inevitable because of its presence food, pharmaceuticals and drinking
water [10, 11]. The average normal daily intake of aluminium for adults is 1-10 mg [12]. It has been reported
that aluminium is poorly absorbed after oral intake and in plasma 80-90% of this element is transported
bound to transferrin [13]. Aluminium is usually excreted from the body through urine [14, 15].
Al-Kahtani, [16] reported that the ingestion of excessive amounts of aluminium may cause damage to
various organs such as liver, kidneys, bone, lungs and heart. Aluminium has been implicated in the
pathogenesis of Alzheimer’s and Parkinson’s diseases [17]. The toxicity of aluminium was observed to be
mediated by the generation of free radicals hence various antioxidant compounds and plant extracts are
reported to play a role in ameliorating the toxic effects of this element [13, 18-21].
Vernonia amygdalina Del. (commonly known as bitter leaf) is a shrub 2 – 5 m tall that grows
predominantly in tropical Africa [22]. The leaves of this plant are green in color, elliptical, 6 mm in length,
with characteristic odor and taste [23]. They are rich in nutrients and are used as vegetable in the preparation
of soups [24-26]. Bitter leaves extracts have for long been used in ethnomedicine as antimalarial,
antimicrobial, anthelmintic, antidiabetic and anticancerous medications [27-31]. The free radical
scavenging activities of the leaf extracts of Vernonia amygdalina have also been reported by various authors
[32-36]
In view of the reported involvement of free radicals in the toxicity of aluminium, the present study
investigated for the first time, the protective potential of ethanol leaf extract of Vernonia amygdalina against
the toxic effects of aluminium chloride in male Wistar rats by measurement of some indices of liver
function, oxidative stress and lipid profile.

Materials and Methods

Plant Materials and Aluminium chloride

Vernonia amygdalina leaves were obtained from Atan Ikot Ekpene village in Ikot Ekpene Local
Government Area, Akwa Ibom State, Nigeria. The leaves were identified and authenticated by the curator
in the Department of Botany and Ecological Studies, Faculty of Science, University of Uyo, Uyo, Akwa
Ibom State, Nigeria. Pure crystalline aluminium chloride was obtained from Guangdong Guanghua Sci-
Tech Company Limited, Shantou, Guandong, China.

Preparation of Extract
The leaves of Vernonia amygdalina were washed, air dried at room temperature for two weeks and
ground into powder using a manual grinder. The powdered sample (200 g) was macerated in 1L of 80%
ethanol for 48 hours at room temperature and filtered through a Whatman No.1 filter paper. The filtrate was
concentrated in a water bath at 40oC and stored in a refrigerator at 4oC until required for the experiment.

Experimental Design
Twenty-five (25) male Wistar rats, weighing 180 – 220 g were obtained from the Animal House,
Faculty of Basic Medical Sciences, University of Uyo, Uyo. They were fed standard laboratory diet, housed
in wire meshed cages, maintained under standard environmental conditions of temperature, 23 ± 2 0C,
relative humidity, 60% and 12 hour light/dark cycles. The rats were allowed free access to drinking water
and rat chow. After an acclimatization period of seven days, the animals were randomly divided into five
(5) groups with 5 rats in each group. Group 1 served as control. Group 2 was administered 100 mg/kg of
aluminum chloride daily for 14 days. Group 3 received 400 mg/kg of ethanol leaf extract of Vernonia
amygdalina for 14 days. Group 4 received 100 mg/kg of aluminum chloride and 400 mg/kg of ethanol leaf
extract of Vernonia amygdalina simultaneously. Group 5 received aluminum chloride for 14 days and was
allowed a wash out period of 14 days before sacrifice. Administration of aluminium chloride and extract
was carried out by oral gavage between the hours of 8 and 10 am daily. The study was carried out in

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 E. O. Akpanyung et al.

accordance with the “Guide for the Care and Use of Laboratory Animals” [37]. Institutional approval for
the conduct of this study was obtained from the Postgraduate School, University of Uyo, Nigeria.

Collection of Serum Samples

At the end of the experimental period, the animals were allowed to fast overnight and then sacrificed
using ketamine as anaesthesia. Blood samples were collected by cardiac puncture into sterile plain bottles
and allowed 30 minutes to clot. Serum was separated by centrifugation at 3000 g for 15 minutes using a
Bench Top Centrifuge (MSE minor, England). The serum samples were stored frozen until required for
analysis.

Biochemical Assay
The serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and
alkaline phosphatase (ALP) were assayed using the Fortress Diagnostic Reagent Kit.
MDA was determined by the measurement of thiobarbituric acid reactive substances in serum [38].
SOD was assayed by the ability of the enzyme to inhibit the autoxidation of pyrogallol [39]. The assay for
catalase was carried out by measurement of the rate of decomposition of hydrogen peroxide [40]. GSH was
determined by measuring the reduction of Ellman’s reagent at 412 nm [41]. Glutathione peroxidase was
determined with the Fortress Reagent kit using the manufacturer’s guidelines.
Total cholesterol, triglycerides and HDL-cholesterol were determined using diagnostic kits supplied
by Randox Laboratories, England. LDL-cholesterol was estimated based on the principles of Friedewald et
al., [42].

Histopathological Studies
Sections of the liver were preserved in buffered formaldehyde and fixed in 10% formalin for
histological examination as described [43]. The tissues were processed into 5 µm thick sections, stained
with hematoxylin-eosin and then observed under a photomicroscope (Olympus BX 41).

Results

The Effect of Ethanol Leaf Extract of Vernonia amygdalina on the Activity of Some Serum Enzymes
in Male Wistar Rats Intoxicated with Aluminium Chloride
The effect of ethanol leaf extract of Vernonia amygdalina on the serum enzyme activity of male Wistar
rats treated with aluminium chloride is presented in Table 1.0. Significantly elevated activities of ALT,
AST and ALP (p ˂ 0.05) were observed following administration of aluminium chloride (Group 2) when
compared to the control. Administration of Vernonia amygdalina leaf extract along with aluminium
chloride modulated the increase in the activities of ALT, AST, and ALP. The activities of the liver enzymes
in this group were significantly reduced compared to Group 2 and tended towards the values obtained for
the control. After allowing a wash out period of 14 days (Group 5), enzyme activities remained elevated
compared to the control.

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Table 1: Effect of ethanol leaf extract of Vernonia amygdalina on the activity of some serum enzymes in
male Wistar rats intoxicated with aluminium chloride

GROUPS – TREATMENT ALT (U/L) AST (U/L) ALP (U/L)

GROUP 1 – Control 14.89 ± 1.17 38.53 ± 3.91 77.03 ± 2.59
GROUP 2 - Aluminum Chloride 31.10 ± 3.54a 57.12 ± 4.58a 92.36 ± 3.67a
GROUP 3 – Vernonia amygdalina 19.71 ± 2.36b 44.88 ± 0.60a,b 72.95 ± 0.49b
GROUP 4 - Aluminum Chloride and
22.04 ± 2.82a,b 39.07 ± 1.68b,c 78.61 ± 3.72b
Vernonia amygdalina
GROUP 5 - Aluminum Chloride then 14 Days
24.77 ± 1.27a,b 52.38 ± 2.04a,d 87.66 ± 5.99a,b,d
without Treatment

Data presented as Mean ± Standard Error of Mean (SEM). Values are considered statistically different at P<0.05. a =
significantly different when compared to Group 1; b = significantly different when compared to Group 2; c =
significantly different when compared to Group 3; d = significantly different when compared to Group 4.

Effect of Ethanol Leaf Extract of Vernonia aamygdalina on Aluminium Chloride Induced Alterations
in Some Indices of Oxidative Stress in male Wistar Rats
The effect of ethanol leaf extract of Vernonia amygdalina on aluminium chloride induced changes in
some indices of oxidative stress is presented in Table 2.0. This table indicates that oral administration of
AlCl3 resulted in a significant decrease (p ˂ 0.05) in the activity of GPX and a significant increase (p ˂
0.05) in the concentration of MDA when compared to the control group. However, the decrease was
significant (p < 0.05) in only in GPx and MDA activities. Treatment with ethanol leaf extract of Vernonia
amygdalina alone showed a significant decrease in SOD activity and MDA concentration when compared
to the control. Simultaneous administration of ethanol leaf extract of Vernonia amygdalina and aluminium
chloride induced a significant increase in the activities of SOD, CAT and GPx with a significant decrease
in concentration of MDA. The administration of AlCl3 for 14 days followed by a wash out period of 14
days resulted in a significant increase in the activities of CAT and GPx as well as MDA concentrations
when compared to the AlCl3 treated group.

Table 2: Effect of ethanol leaf extract of Vernonia amygdalina on aluminium chloride induced alterations
in some indices of oxidative stress in male Wistar rats.

Groups SOD CAT GSH GPX MDA

(U/ml) (μm/min/ml) (U/ml) (mmol/min/ml) (μM)

1 0.76±0.02 8.33±0.68 0.29±0.01 3.83±0.50 3.23±0.27

2 0.58±0.06 7.49±0.24 0.37±0.04 1.39±0.12a 18.51±0.92a

3 0.37±0.11a 8.38±0.76 0.27±0.01b 2.75±0.28b 1.76±0.13a,b

4 0.94±0.12b,c 12.25±0.26a,b,c 0.30±0.03 4.95±0.52b,c 1.10±0.09a,b

5 0.51±0.15d 8.00±0.24b,d 0.29±0.03 4.31±0.35b 11.04±0.22a,b,d

Data presented as Mean ± Standard Error of Mean (SEM). a = significantly different when compared to Group 1; b =
significantly different when compared to Group 2; c = significantly different when compared to Group 3; d =
significantly different when compared to Group 4.

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 E. O. Akpanyung et al.

Effect of Ethanol Leaf Extract of Vernonia amygdalina On AlCl3 Induced Changes in Lipid Profile of
Male Wistar Rats.
The effect of ethanol leaf extract of Vernonia amygdalina on AlCl3 induced changes in lipid profile is
presented in Table 3.0. Oral administration of AlCl3 induced a significant increase (p < 0.05) in the serum
concentrations of total cholesterol (TCHOL), triacylglycerol (TAG), low density lipoprotein cholesterol
(LDL-C) and very low density lipoprotein cholesterol (VLDL-C) while high density lipoprotein cholesterol
(HDL-C) level decreased significantly when compared to the control. There was no significant difference
(p > 0.05) in the parameters of lipid profile when the animals were treated with the extract alone.
Administration of ethanol leaf extract of Vernonia amygdalina and AlCl3 resulted in a significant decrease
in the lipid profile indices when compared to the control and AlCl3 treated group. Administration of AlCl3
for 14 days followed by a wash out period of 14 days also instigated a significant decrease in the lipid
profile parameters when compared to the AlCl3 treated group.

Table 3: Effect of ethanol leaf extract of Vernonia amygdalina on aluminium chloride-induced changes in
lipid profile of male Wistar rats

Groups TCHOL TAG (mmol/L) HDL-C LDL-C

(mmol/L) (mmol/L) (mmol/L)

1 126.93±1.93 82.43±1.18 28.84±0.26 81.61±1.92

2 143.07±2.43a 88.25±1.92a 28.53±0.58 96.89±2.23a

3 122.16±0.55b 80.08±1.63b 28.19±0.59 77.95±0.58b

4 101.64±0.90a,b.c 70.02±0.96a,b,c 22.86±1.30a,b,c 64.78±1.67a,b,c

5 125.64±2.08b,d 81.02±1.87b,d 25.54±0.70a,b 83.90±2.13b,d

Data presented as Mean ± Standard Error of Mean (SEM). a = significantly different when compared to
Group 1; b = significantly different when compared to Group 2; c = significantly different when compared
to Group 3; d = significantly different when compared to Group 4.

The effects of AlCl3 and ethanol leaf extract of Vernonia amygdalina on the histology of the liver are
shown in Figure 1 (L1 – L5). Liver of the control and Vernonia amygdalina group (L1 and L3) showed
normal lobular architecture with normal hepatic cell, central vein and sinusoid. Administration of AlCl 3
caused dilated, congested, widely infiltrated sinusoid, a mild micro vesicular steatosis and foci of
degenerative changes (L2) which were reversed by co-administration of ethanol leaf extract and AlCl3 (L4).
In group 5, the toxic effects of AlCl3 were observed to persist after a wash out period of 14 days (L5).

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Effect of Ethanol Leaf Extract of Vernonia amygdalina on AlCl3 Induced Histopathology of the liver
in Male Wistar Rats.

L1 L2

CV
$

L3 L4
$
CV CV

L5 Figure 1.0: Photomicrograph of liver section of albino rats in Group 1

(Control) Showed lobular architecture of the liver with normal hepatic cell
(arrow), central vein (CV) and sinusoid ($); Group 2 (administered
aluminum chloride) with normal hepatic cell (arrow), central vein (CV) and
CV dilated, congested and widely infiltrated sinusoid ($). Also seen is a mild
microvesicular steatosis (arrow head) and foci of degenerative changes
(asterisk); Group 3 (administered Vernonia amygdalina) Showed lobular
architecture of the liver with normal hepatic cell (arrow), central vein (CV)
$ and sinusoid ($); Group 4 (concomitantly administered aluminum chloride
and Vernonia amygdalina) with normal arrays of hepatic cells (short arrow),
central vein (CV). Also seen is a widely diffused microvesicular and
macrovesicular steatosis (arrow head); Group 5 (administered aluminum
chloride and allowed for 14 days without treatment) with normal hepatic
cell (arrow), central vein (CV) and dilated, congested sinusoid ($). Also seen
is a mild microvesicular and macrovesicular steatosis (arrow head).

Figure 1: Photomicrograph of liver section of aluminium chloride intoxicated male albino Wistar rats
treated with ethanol leaf extract of Vernonia amygdalina.

Discussion

The leaf extract of Vernonia amygdalina is reported to be hepatoprotective and to possess antioxidant
properties [32, 44, 45]. It has also been observed that the hepatotoxic effect of aluminium chloride is
mediated by the generation of free radicals via oxidative stress [46, 47]. Hence, this study evaluated the
protective potentials of ethanol leaf extract of Vernonia amygdalina against aluminium chloride induced
toxicity in male Wistar rats.
The aminotransferase enzymes (AST, ALT) as well as alkaline phosphatase (ALP) are biomarkers of
toxic injury to the liver [48]. In the present study, it was observed that the administration of AlCl 3 caused a
significant increase in the activities of these enzymes indicating hepatotoxicity. This is in line with the
report of other authors [46, 49-51]. Ethanol leaf extract of Vernonia amygdalina significantly reduced the

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 E. O. Akpanyung et al.

activities of these marker enzymes when compared to the aluminum chloride treated group. This is an
indication of the protective effect of the extract against the hepatotoxic effect of aluminium chloride. It is
noteworthy that administration of ethanol leaf extract of Vernonia amygdalina alone induced a significant
increase in the activity of AST when compared to control. Such increase in enzyme activity was earlier
observed by Ojiako and Nwanjo [52] who attributed the elevated enzyme activity to some unspecified extra
hepatic sources. Enzyme activities remained elevated after discontinuation of treatment with AlCl3
indicating that the hepatocytes would require a longer period of time for self-recovery from AlCl3 induced
damage. The protective effect of the leaf extract of Vernonia amygdalina against chemically induced
hepatic damage has been attributed to its rich content of natural antioxidants which confer stability on the
cell membrane and protect the tissues from free radical damage thereby preventing further leakage of
marker enzymes into circulation [53].
The histological reports of the liver tissues in the present study confirmed the observations from
biochemical parameters measured. The liver of the control group revealed normal cyto-architecture of
hepatocytes with normal central veins and sinusoids. However, degenerative changes were observed in the
photomicrograph of the aluminium chloride intoxicated group with other features such as steatosis and
dilated sinusoids indicating toxicity resulting from the administration of aluminium chloride. Similar
reports on toxicity of aluminium chloride on the liver of albino rats have been documented by other authors
[19]. Normal cellular architecture of the liver was observed in the group co-administered with AlCl3 and
ethanol extract of Vernonia amygdalina. This was indicative of the protective potential of the extract of
Vernonia amygdalina against the toxic effect of AlCl3. Hepatoprotective effect of ethanol extract of
Vernonia amygdalina has been reported [54] and the present study corroborates same. Mild degenerative
features were still observed in Group 5 with washout period of 14 days implying that longer period would
be required for the toxic effect of AlCl3 to wear off naturally.
The assessment of lipid profile is a vital diagnostic procedure because of the fact that dyslipidemia
plays an important role in the pathogenesis and progression of vascular diseases [55]. Various authors have
reported adverse effects of aluminium chloride on lipid profile [13, 56, 57]. These authors observed that
intoxication with aluminium chloride produced dyslipidemia characterized by increase in total cholesterol,
triacylglycerol, low density lipoprotein cholesterol and decreased concentrations of high density lipoprotein
cholesterol. In the present study, administration of aluminium chloride precipitated a significant increase in
all lipid fractions except HDL-cholesterol. Hyperlipidemia as observed in this study is a risk factor for the
development of cardiovascular diseases [55]. However, the altered lipid profile induced by aluminium
chloride was reversed in animals treated with ethanol leaf extract of Vernonia amygdalina. The
hypolipidemic effect of Vernonia amygdalina has also been reported by other authors [58-60]. The lipid
fractions were also restored to normal values after a wash out period of 14 days (Group 5). This would
imply that the animals have the potential for self-recovery from AlCl3 induced dyslipidemia. The lipid
lowering effect of Vernonia amygdalina leaf extract has been attributed to its phytochemical constituents
such as flavonoids, tannins and saponins [33, 60-62].
Oxidative stress has been defined as disturbance in the balance between free radical generation and
antioxidative processes in favor of radical production [63]. Lipid peroxidation is one of the hallmarks of
oxidative stress. It is a free radical mediated chain reaction which results in oxidative deterioration of
polyunsaturated lipids with malondialdehyde as one of its toxic products. Hence, malondialdehyde is used
as a biomarker of oxidative stress induced lipid peroxidation [64]. In the present study, administration of
AlCl3 induced a significant increase in serum concentrations of MDA. This is an indication of increased
lipid peroxidation precipitated by AlCl3 which is in line with the observation of other authors [13, 46, 47].
Ethanol leaf extract of Vernonia amygdalina attenuated the AlCl3 induced increase in serum concentrations
of MDA due to the antioxidant potential of this extract [32, 37, 59, 63, 65].
There are various defense mechanisms that prevent, limit or ameliorate the harmful effects of free
radicals. These include the antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase)
as well as the non-enzyme antioxidants (reduced glutathione) [66]. Reduced glutathione (GSH) plays an
important role in scavenging ROS and detoxification of xenobiotics. It participates in non-enzymatic
conjugation and becomes depleted during exposure to toxicants [67]. Superoxide dismutase catalyzes the

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dismutation of the highly reactive superoxide anion to molecular oxygen and hydrogen peroxide [40].
Catalase is involved in the conversion of hydrogen peroxide to water and oxygen [41]. Glutathione
peroxidase catalyzes the reduction of hydrogen peroxide and hydroperoxides formed from fatty acids thus
effectively removing toxic peroxides from living cells [67].
There are indications that decreased antioxidant levels are the yardstick of oxidative stress in biological
systems [68]. In the present study, administration of AlCl3 caused a non-significant decrease in the
concentration of GSH as well as decreased activities of superoxide dismutase and catalase. However, the
decrease in the activity of glutathione peroxidase was significant compared to control. Other authors had
reported a significant decline in all antioxidant parameters following the administration of AlCl3 [13, 46,
69]. The Agency for Toxic Substances and Disease Registry [70] reported that aluminium accumulates
mainly in the liver, testes, kidneys and brain. Thus, the manifestations of aluminium toxicity are likely to
be more severe at organ/tissue level compared to extracellular fluid. The decrease in antioxidant capacity
has been attributed to reduced synthesis of GSH, SOD, Catalase and GPx caused by higher intracellular
concentration of aluminium and/or the accumulation of free radicals [46, 71] as well as inhibition of the
expression of endogenous antioxidants [72]. Ethanol leaf extract of Vernonia amygdalina caused a
significant increase in all the antioxidant parameters (SOD, GPx, Catalase, GSH). This is consistent with
earlier reports [13, 46, 69]. The beneficial effect of the extract could originate from a host of phytochemical
constituents such as polyphenols which have been reported to act as inducers of these antioxidant enzymes
[73].

Conclusion
It can therefore concluded that ethanol leaf extract of Vernonia amydalina has protective effect against
aluminium chloride induced hepatotoxicity, oxidative stress and alterations in lipid profile in male Wistar
rats.

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Amelioration of paracetamol-induced nephrotoxicity in mice

by aqueous extract from the calyx of Hibiscus sabdariffa
Linn.
Blessing O. Orji1*, Frederick O. Obi2, Emmanuel U. Modo3, Martin Osibemhe1 and
Catherine A. Otitolaiye4
1
Department of Biochemistry and Molecular Biology, Federal University Dutsin-ma, Katsina State, Nigeria.
2
Department of Biochemistry, University of Benin, Benin City, Edo State, Nigeria.
3
Department of Biochemistry, Madonna University Nigeria, Elele Campus, Rivers State, Nigeria.
4
Department of Biochemistry, Sokoto State University, Sokoto, Nigeria.

*Corresponding author E-mail: [email protected], Tel: +2348022226832

(Received January 16, 2020; Revised version received March 12, 2020; Accepted March 28, 2020)

ABSTRACT: The impact of co-administration of paracetamol and aqueous extract of Hibiscus sabdariffa Linn
calyx on nephrotoxicity prompted by paracetamol, under acute and sub-acute treatments was assessed. Four groups,
each of them having 5 mice were involved in this study: control, extract-treated, paracetamol-treated and co-
treatment with extract and drug. The extract and drug were given by oral route (250mg/kg and 500mg/kg
respectively) to the animals. Treatment with paracetamol significantly increased (P≤0.05) creatinine and urea, and
reduced bicarbonate and sodium levels when compared to the control group. Also significantly reduced (P≤0.05)
were superoxide dismutase and catalase activities alongside the level of reduced glutathione, while the value of
malondialdehyde was significantly increased (P≤0.05). Co-administration was seen to attenuate the changes brought
about by paracetamol in the parameters studied. Assessment of histopathology of kidney segments indicated that
treatment with the drug caused acute tubular necrosis and necrotizing pyelitis while co-treatment of drug with
extract provided protection. Co- administration of paracetamol and extract was shown to enhance kidney function in
response to toxicity caused by paracetamol in mice. The source of this amelioration may be due to the antioxidant
components of the extract which have been widely reported.

Keywords: Acute; co-administration; Hibiscus sabdariffa Linn; nephrotoxicity; paracetamol; sub-acute.

Introduction

Paracetamol, also called acetaminophen, is a drug commonly used as painkiller and to manage fever
(Aghababian, 2010). It is usually found in medications for flu (Hanna and Zylicz, 2013). Since the drug
is easily obtainable in most drug outlets, abuse is prevalent and has been found to cause hepatic and renal
damages (Gunnel et al., 2000). Andersson et al., (2011) gave a clue about how paracetamol manages
pain. They discovered that N – acetyl – p - benzoquinoneimine (NAPQI) (paracetamol metabolite),

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prevails on transient receptor potential ankyrin subtype 1 (TRPA1) in the spinal cord to subdue the
sensory information coming from the surface of the dorsal horn, to relieve ache.
Metabolism of paracetamol following ingestion takes place largely in the liver by glucuronidation
(approximately 63%) and sulphation (34%). At therapeutic doses of paracetamol, NAPQI is cleared by
glutathione (Dahlin et al., 1984). NAPQI has been held accountable for harmful effects of paracetamol
(Waring, 2012). When the drug is taken in excess, intracellular glutathione levels are diminished and
NAPQI increases leading to death of tissue (Bessems and Vermeulen, 2001). This also leads to
proliferation of reactive species. The harmful effects of Paracetamol include severe tubular necrosis,
which is the key source of kidney failure (Blantz, 1996).

There has been growing interest in the intake of plants and plant products due to their health benefits
and lower adverse reactions when compared to orthodox treatments (Hu et al., 2003). Hibiscus
sabdariffa Linn is one of the plants which have attracted interest due to the bioactive agents found in it.
Hibiscus sabdariffa Linn is called Roselle and well known for a drink Nigerians call Zobo. Its calyces
have been found to contain nutrients such as protein, fat, carbohydrates, fiber, vitamin C, β-carotene,
calcium and iron (Ismail et al., 2008). Also reportedly found in the calyces are antioxidants which include
anthocyanin, quercetin and protocatechuic acid (Hirunpanich et al., 2005).
This study aimed to elucidate the effects of acute and sub-acute co-treatment of paracetamol and
Hibiscus sabdariffa Linn calyx extract on nephrotoxicity brought about by treatment with paracetamol,
using the mice as experimental model.

Materials and Methods

Materials
Reagents and chemicals used in this study were of analytical grade.

Plant
Hibiscus sabdariffa Linn calyces were obtained from Karu market, Abuja, FCT. Identification of the
plant was carried out at the Department of Plant Biology and Biotechnology, University of Benin, Benin
City by Mr. Joseph Erhabor. Thereafter, a sample (identification number UBHm 0261) was placed at the
Herbarium, University of Benin, Benin City.

Animals
Forty mice weighing between 27 and 32g, gotten from a breeder in Benin City, were used for this
study. They were kept in cages made of wood in the Department of Biochemistry, University of Benin
animal house. They were allowed two weeks for adaptation before the start of study. Also, unrestricted
access to tap water and food (Growers mash, Bendel Feeds and Flour Mills Ltd, Ewu, Edo State) was
granted to them.

Ethical Approval
This study was done in line with the conventional procedures recognized by National Institute of
Health Guide for the Care and Use of Laboratory Animals and approved by Ethic Committee of the
Faculty of Pharmacy, University of Benin, Benin city, Nigeria.

Plant extract preparation

Dry calyces of Hibiscus sabdariffa Linn were crushed into suitable particles, which was left to soak
in distilled water (1:3w/v) for 24 hours at 4°C (Dafalla and Mustafa, 1996). The solution was sifted and
doses equivalent to 250 mg/kg (Dahiru et al., 2003) were prepared.

Preparation of paracetamol

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 B. O. Orji et al.

Paracetamol base powder (Huang Gang Yin He Aati Pharmaceutical Co. Ltd. China) was made
available by Late by Dr. G. C. Josephs (Department of Pharmaceutical Microbiology and Biotechnology,
University of Benin). The powder was dissolved completely in dimethyl sulfoxide (DMSO) (2.5%
aqueous solution of DMSO), after which distilled water was added to make up the necessary measure.

Experimental design and treatment schedule

This work comprised of two (2) categories: acute (contact for not more than 24 hours) and sub-acute
(contact for 4 weeks). Each category was made up of four (4) groups: control, extract only, paracetamol
only and concurrent administration of paracetamol and extract. There were 5 mice in each group.
Paracetamol was administered orally (500 mg/kg body weight); Hibiscus sabdariffa Linn calyx extract
(HSCE) was also administered orally at 250 mg/kg.

Acute Study
Twenty mice which were distributed into four groups were used in this study. Group 1, the control
was administered aqueous DMSO. Animals in the 2nd group were given extract only (zero time and 8h
later). The 3rd group received paracetamol only (zero time and 8h later), and in the 4th group, there was
concurrent administration of drug and extract (zero time and 8h later). At the end of the treatments, the
mice were sacrificed in 24 hours.

Sub-acute Study
Twenty mice, grouped into four were also used in this study. The control, group 1 was given only
aqueous DMSO. Group 2 was given extract only once a day, group 3 paracetamol only once a day and
group 4 received concurrent administration of paracetamol and extract once a day. The treatment lasted
for 4 weeks after which all the animals were sacrificed.

Collection and preparation of samples for analyses

After sedating the mice with chloroform, blood was taken by heart puncture and placed in plain
sample bottles. Clotted blood samples were centrifuged at 4,000 rpm for 10 minutes in order to obtain
sera which were stored at -20⁰. The kidney was also removed and rinsed in very cold saline. Kidneys from
mice in applicable groups were fixed for histopathological investigations. Also for biochemical analyses,
a measured weight of the tissue was homogenized in phosphate buffered saline (PBS) 50mM pH 7.4,
centrifuged at 3500rpm for 15 minutes to obtain the supernatant which was employed.

Biochemical analyses
Analyses conducted on serum include creatinine (Bartels et al., 1972), urea (Weatherburn, 1967),
bicarbonate (Tietz et al., 1986), sodium ion (Maruna, 1958; Trinder, 1951), chloride (Skeggs and
Hochstrasser, 1964) and potassium (Terri and Sesin, 1958). Reduced glutathione (GSH) (Tietz, 1969),
malondialdehyde (MDA) (Buege and Aust, 1978), superoxide dismutase (SOD) (Misra and Fridovich,
1972) and catalase (Cohen et al., 1970) were carried out on kidney homogenate supernatant.

Statistical analysis
Data obtained were presented as mean ± S.E.M. Analysis for significance was done by one way
ANOVA and mean values that differed significantly were identified using the Duncan’s multiple range
test. P ≤ 0.05 was considered significant.

Results

Effects of HSCE on acute paracetamol exposure

Kidney function parameters

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Table 1 shows results for tests conducted on serum to ascertain kidney function. Creatinine and urea
were significantly increased (P ≤ 0.05) while bicarbonate and sodium were significantly decreased (P ≤
0.05) in the groups administered only paracetamol, relative to control. Co – administration of extract and
paracetamol significantly reduced (P ≤ 0.05) creatinine and urea while bicarbonate was significantly
increased (P ≤ 0.05), relative to paracetamol only group.

Table 1: Effects of aqueous HSCE on kidney function parameters in serum of mice on acute
paracetamol exposure

Biochemical Control Extract Paracetamol (zero Concurrent

parameter (serum) (zero time time and 8h later) administration
and 8h (zero time and 8h
later) later)

Creatinine (mg/dL) 9.10±0.00b 9.36±0.26b 10.92±0.32a 9.62±0.32b

Urea (mmol/L) 5.96±0.26b 6.16±0.20b 9.59±0.30a 6.14±0.20b
Bicarbonate 44.26±0.14a 44.13±0.17a 41.83±0.17b 44.13±0.17a
(mmol/L)
Sodium (mEq/L) 182.93±0.39a 182.93±0.39a 174.41±0.49c 180.55±0.35b
Chloride (mEq/L) 92.44±0.16a 92.50±0.22a 93.08±0.22a 92.57±0.12a
Potassium (mEq/L) 3.90±0.06a 3.87±0.05a 4.00±0.06a 3.88±0.07a
Values are Mean ± SEM (n=5). Values with different letters within a row differ significantly from each other (P ≤
0.05). Values with same letters within a row do not differ significantly from each other (P ≥ 0.05).

Antioxidants and lipid peroxidation in the kidney

Table 2 shows results for antioxidant and lipid peroxidation assays carried out in the kidney.
Relative to control, MDA was significantly increased (P ≤ 0.05) in the paracetamol treated group. SOD
was significantly reduced (P ≤ 0.05) in the paracetamol only while co – administration significantly
increased (P ≤ 0.05) SOD, relative to paracetamol only group. Paracetamol also significantly (P ≤ 0.05)
reduced GSH concentration relative to control, while co – administration significantly increased (P ≤
0.05) GSH concentration relative to paracetamol only group.

Table 2: Effects of aqueous HSCE on antioxidants and lipid peroxidation in the kidney of mice on
acute paracetamol exposure

Biochemical Control Extract Paracetamol Concurrent

parameter (zero time (zero time and 8h administration
(kidney) and 8h later) (zero time and 8h
later) later)

MDA (mol/g 0.07±0.00b 0.07±0.00b 0.09±0.00a 0.07±0.00b

tissue)
SOD (Units/mg 0.06±0.00a 0.06±0.00a 0.05±0.00b 0.06±0.00a
tissue)
CAT (Units/g 7.31±0.00a 7.44±0.00a 5.85±0.00c 6.79±0.00b
tissue)
GSH (mmol/L) 0.07±0.00a 0.07±0.00a 0.06±0.00b 0.07±0.00a
Values are Mean ± SEM (n=5). Values with different letters within a row differ significantly from each other (P ≤
0.05).

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 B. O. Orji et al.

Kidney ultrastructure of mice exposed to concurrent administration of paracetamol and extract at

the acute phase.

The control mouse showed normal architecture of the kidney, composed of glomerulus (A) and
tubules (B), separated by interstitial space (C) (plate 1). The mouse that received extract only at zero time
and 8h later showed mild interstitial congestion (A) (plate 2). The mouse that received paracetamol only
at same time interval showed focal cloudy swelling of tubular epithelial cells (A) and narrowing of lumen
(B) (plate 3), while the mouse that received extract and paracetamol simultaneously (co-administration) at
same time interval showed patent tubular lumen (A) (plate 4).

Plate 1: Photomicrograph of control mouse’ kidney (H & E, x400).

Plate 2: Photomicrograph of kidney from mouse given extract at zero time and 8h later (H & E,
x400).

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Plate 3: Photomicrograph of kidney from mouse given paracetamol at zero time and 8h later (H
& E, x400).

Plate 4: Photomicrograph of kidney from mouse co-administered paracetamol and extract at zero
time and 8h later (H & E, x400).

Effects of HSCE on sub-acute paracetamol exposure

Kidney function parameters

Table 3 shows results for tests conducted in serum to assess kidney function. Paracetamol
significantly increased (P ≤ 0.05) urea, creatinine and chloride levels while bicarbonate and sodium were
reduced, relative to control. Co - administration of extract and paracetamol significantly reduced (P ≤
0.05) creatinine and chloride while bicarbonate and sodium were increased, relative to paracetamol - only
group.

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 B. O. Orji et al.

Table 3: Effects of aqueous HSCE on kidney function parameters in serum of mice on sub – acute
paracetamol exposure

Biochemical Control Extract only Paracetamol only Co-administration

parameter (serum) of paracetamol and
extract

Creatinine (mg/dL) 9.07±0.23b 9.07±0.23b 11.16±0.19a 9.64±0.19b

Urea (mmol/L) 5.76±0.03c 5.73±0.04c 9.42±0.23a 6.16±0.05b
Bicarbonate 43.54±0.17a 43.71±0.00a 41.31±0.17b 43.85±0.14a
(mmol/L)
Sodium (mEq/L) 185.07±0.48a 184.68±0.48a 155.93±0.25b 185.46±0.39a
Chloride (mEq/L) 92.50±0.16b 92.69±0.06b 93.40±0.22a 92.76±0.08b
Potassium (mEq/L) 3.90±0.05a 3.90±0.04a 4.01±0.06a 3.90±0.03a
Values are Mean ± SEM (n=5). Values with different letters within a row differ significantly from each other (P ≤
0.05). Values with same letters within a row do not differ significantly from each other (P ≥ 0.05).

Antioxidants and lipid peroxidation in the kidney

Table 4 shows results for antioxidants and lipid peroxidation assays carried out in the kidney.
Paracetamol significantly increased (P ≤ 0.05) MDA while SOD and catalase activities, and GSH level
were reduced, relative to control. Co – administration of paracetamol and extract significantly increased
(P ≤ 0.05) catalase activity and GSH level, relative to paracetamol – only group. Also, co-administration
significantly decreased MDA relative to paracetamol only group, though still significantly higher than the
control. For catalase, co-administration returned values to control while treatment with extract caused a
significant increase.

Table 4: Effects of aqueous HSCE on antioxidants and lipid peroxidation in the kidney of mice on
sub - acute paracetamol exposure:

Biochemical Control Extract only Paracetamol only Co-administration

parameter (serum) of paracetamol and
extract
MDA (mol/g tissue) 0.07±0.00c 0.06±0.00c 0.12±0.00a 0.08±0.00b
SOD (Units/mg
0.06±0.00a 0.06±0.00a 0.04±0.00c 0.05±0.00b
tissue)
CATALASE Units/g
tissue) 7.25±0.04b 7.73±0.01a 5.21±0.03c 7.15±0.04b

REDUCED
GLUTATHIONE 0.07±0.00a 0.07±0.00a 0.05±0.00b 0.07±0.00a
(mmol/L)
Values are Mean ± SEM (n=5)
Values with different letters within a row differ significantly from each other (P ≤ 0.05).

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 Biokemistri Volume 32, Number 1 (2020)

Kidney ultrastructure of mice exposed to concurrent administration of paracetamol and extract at

the sub-acute phase.
The control mouse showed normal architecture of the kidney, composed of glomerulus (A) and
tubules (B), separated by interstitial space (C) (plate 5). The mouse that received extract only showed
unremarkable glomerulus (A) and tubules (B) (plate 6). The mouse that received paracetamol only
showed patchy tubular cloudy swelling (A) and focal infiltrates of inflammatory cells (B) (necrotizing
pyelitis) (plate 7) while the mouse that received extract and paracetamol simultaneously (co-
administration) showed patchy tubular cell cloudy swelling (A) and interstitial congestion (B) (plate 8).

Plate 5: Photomicrograph of control mouse’ kidney (H & E, x400).

Plate 6: Photomicrograph of kidney from mouse given extract only for 4 weeks (H & E, x400).

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 B. O. Orji et al.

Plate 7: Photomicrograph of kidney from mouse given paracetamol only for 4 weeks (H & E,
x400).

Plate 8: Photomicrograph of kidney from mouse co-administered paracetamol and extract for 4
weeks (H & E, x400).

Discussion

Paracetamol, also called acetaminophen, belongs to a subgroup of analgesics called aniline

derivatives and is very effective in the relief of mild fever and pains. It is understandably very safe when
given in the correct therapeutic dose but its overdose remains the major cause of liver injury and even
death in many parts of the world among all drug toxicities (Linda et al., 2009; Yayla et al., 2014).
Paracetamol undergoes metabolic activation by hepatic microsomal cytochrome P450 mixed function
oxidase system (mainly the enzyme CYP2E1) to N‐acetyl‐P‐benzoquinone imine (NAPQI). The active
metabolite (being highly electrophilic) binds quickly to intracellular proteins, leading to both structural
and functional changes (Monira et al., 2012). NAPQI is the active metabolite at the center of virtually all
the metabolic disorders experienced when an overdose of acetaminophen occurs. The mechanism by
which paracetamol ingestion causes nephrotoxicity is less described unlike its induction of hepatotoxicity.
With reference to information from clinical and animal studies, some of the potential mechanisms of
nephrotoxicity are the cytochrome P-450 pathway, prostaglandin synthesis and N-deacetylase enzymes

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 Biokemistri Volume 32, Number 1 (2020)

(Bessems and Vermeulen, 2001). These enzymes are very sensitive markers employed in the diagnosis of
kidney diseases.
In the assessment of kidney injury, levels of urea and creatinine in serum should be first determined.
From the results shown in Table 1 and Table 3 on the effects of HSCE on acute and sub-acute
paracetamol exposure in kidney function parameters, Creatinine and urea were significantly increased (P
≤ 0.05) on both acute and sub-acute paracetamol exposure. This finding was in tandem with the work of
Dogukan et al., (2016) who reported a significant increase in the urea and creatinine levels of rats
exposed to paracetamol. These increased levels of urea and creatinine may be indicators of acute tubular
necrosis (Adebayo et al., 2003; Yakubu et al., 2003). Bicarbonate and sodium were significantly
decreased (P ≤ 0.05) in the groups administered paracetamol only, relative to control in the acute and sub-
acute paracetamol exposure. This was also similar to the work of Pakravan et al., (2015) who reported an
alteration in the electrolyte levels of rats exposed to acetaminophen at toxic levels. Co – administration of
extract and paracetamol led to opposite effects for urea, creatinine and electrolytes as observed when only
paracetamol was administered. These obvious differences could be as a result of the actions of the
phytochemical constituents of the extract such as flavonoids in inhibiting the actions of the toxic
metabolite NAPQI and also stabilizing the cell membranes of the intracellular proteins and other
compounds (Parker et al., 2017).
From the results shown in Table 2 and Table 4 on the effects of HSCE on acute and sub-acute
paracetamol exposure in antioxidant and lipid peroxidation parameters, interesting observations were
made. The generation of hydroxyl radical and other powerful radicals can initiate a chain reaction of lipid
peroxidation in which polyunsaturated fatty acids are converted into lipid peroxides. Malondialdehyde
(MDA) is a major indicator of lipid peroxidation (Nielsen et al., 1997). In the present study, MDA was
significantly increased (P ≤ 0.05) compared to control in the paracetamol treated group for the acute and
sub-acute paracetamol exposed group. This agreed with the work of Zoubair et al., (2013) who reported
increased MDA levels in oxidative stressed mice exposed to paracetamol and hydrogen peroxide. Increase
in the levels of MDA could be as a result of cellular membrane damage initially caused by an increase in
radical formation (Niedernhofer et al., 2003), in this case, by the actions of the toxic metabolite NAPQI.
However, treatment with H. sabdariffa aqueous extract caused a significant decrease (p<0.05) in the
MDA concentration of the treatment group.
Likewise, SOD, catalase and GSH were significantly reduced (P ≤ 0.05) in the paracetamol only
group for the both the acute and sub-acute exposure. Co – administration significantly increased (P ≤
0.05) SOD, catalase and GSH relative to paracetamol only group. This result is consistent with the finding
of Parker et al., (2017) who observed an increase in the above mentioned parameters following treatment
with aqueous extracts of H. sabdariffa after acetaminophen-induction. The protective effect observed
appears to be due to the antioxidant properties of this plant (Liu et al., 2006). It has been recorded that the
aqueous extract of Hibiscus sabdariffa is enriched in high antioxidant constituents, mainly flavonoids and
vitamin C (Hirunpanich et al., 2006), which serves as an antioxidant and a reductant (Wang et al., 2000).
The histological examination on the kidney tissue supported our results obtained.
In conclusion, administration of paracetamol at toxic levels has been shown to have deleterious
effects on the kidney of mice. However, its co-administration with aqueous extract of H. sabdariffa has
shown that the extract contains some compounds that are effective against the reactive oxygen species
generated by paracetamol toxicity, thereby restoring the nephrotic structure and integration close to its
original state.

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Gastroprotective potentials of aqueous extract of Persea

americana seed against Aspirin-induced ulcer in Wistar rats
M. O. Salawu*, A. Shittu, M. O. Nafiu and H. O. B. Oloyede

Department of Biochemistry, Faculty of Life Science, University of Ilorin, PMB 1515, Ilorin, Kwara State, Nigeria.

*Corresponding Author: [email protected]

Tel:- +2348056168553

(Received January 16, 2020; Revised version received March 10, 2020; Accepted March 28, 2020)

ABSTRACT: The study was designed to evaluate the gastro-protective potential of aqueous seed extract of Persea
americana on aspirin-induced gastric ulcer in rats. Thirty adult rats were randomly divided into six groups of five.
Group 1 was not induced with aspirin while groups 2-6 received a single-dose of aspirin of 400 mg/kg bw to induce
gastric ulcer. Groups 2 and 3 received water and omeprazole respectively; while groups 4, 5 and 6 received the aqueous
extract (110, 220 and 440 mg.kg bw respectively) for 14 days. Ulcer index, gastric volume, gastric pH, total acidity,
total carbohydrate, total protein, pepsin of gastric juice and antioxidant activity of the plant extracts were determined.
The extract showed a significant (p<0.05) decrease in ulcer index and total acidity in the extract-treated and the
omeprazole-treated groups when compared to the control group. There was no significant difference in the gastric
juice volume and total protein of the gastric juice of all the extract-treated groups when compared to the control. There
was a significant (P<0.05) decrease in malondialdehyde level in the gastric tissues of the extract-treated and an
increase in the reduced Glutathione, catalase, superoxide dismutase, activities compared to the control. The histology
of the stomach tissues in the extract-treated groups showed slight and moderate ulceration when compared to the
normal control group. The extract therefore has protective activity on aspirin-induced gastric ulceration in rats.

Keywords: Persea americana, avocado, aspirin, gastric ulcer, ulcer index, antioxidant.

Introduction

Gastric ulcers are frequent and severe diseases, which have been a significant cause of morbidity and
mortality for more than a century (Hoogerwer and Pasricha, 2006). The pathophysiology of gastric ulcer
disease is based on an imbalance between aggressive and protective factors in the stomach (Vell, 2005).
Gastric ulcers are caused by psychological and physiological stress, excessive acid, free radicals, alcohol
use, the side effect of non-steroidal anti-inflammatory drugs (NSAIDs), Helicobacter infection or free
radicals or a combination of two or more of these causes (Harbison and Dempsey, 2005).
Currently, the non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin are
preferred drugs for various diseases like arthritis, inflammation, and cardiovascular protection. However,
they cause gastrointestinal complications such as ulcers and erosions.
Non-steroidal anti-inflammatory drugs NSAIDs also generates oxygen free radicals that are known to
play a role in the pathogenesis of mucosal injury (Biswas et al., 2003). Aspirin exerts its effect through

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inhibition of cyclooxygenase the enzyme responsible for the synthesis of prostaglandin. The most adverse
effect of aspirin is irritation of the gastric mucosa. Various synthetic anti-ulcer drugs are presently available,
and some of these like misoprostol esomeprazole, omeprazole, lansoprazole, pantoprazole is specifically
used to cure the NSAID induced gastric ulcer. However, each of these drugs confers simpler to severe side
effects, warranting a search for non-toxic and inexpensive antiulcer medication (Miederer, 1986, Yesilada
and Gurbuz, 2003).
Plants are essential in our everyday existence. They provide our foods, produce the oxygen we breathe,
and serve as raw materials for many industrial products such as clothes; foot wears and so many others.
Plants also provide raw materials for our buildings and in the manufacture of biofuels, dyes, perfumes,
pesticides, adsorbents and drugs. The plant kingdom has proven to be the most useful in the treatment of
diseases, and they provide an essential source of all the world's pharmaceuticals. The most important of
these bioactive constituents of plants are steroids, terpenoids, carotenoids, flavonoids, alkaloids, tannins
and glycosides. Plants in all facet of life have served a valuable starting material for drug development
(Ajibesin, 2011). Antibiotics or antimicrobial substances like saponins, glycosides, flavonoids and alkaloids
etc. are found to be distributed in plants. Yet, these compounds were not well established due to the lack of
knowledge and techniques. The phytoconstituents which are phenols, anthraquinones, alkaloids,
glycosides, flavonoids and saponins are antibiotic principles of plants.
Plants are now occupying a prominent position in allopathic medicine, herbal medicine, homoeopathy
and aromatherapy. Medicinal plants are the sources of many essential drugs of the modern world. Many of
these indigenous medicinal plants are used as spices and food plants; they are also sometimes added to
foods meant for pregnant mothers for medicinal purposes (Akinpela and Onakoya, 2006). Many plants are
cheaper and more accessible to most people, especially in the developing countries than orthodox medicine,
and there is a lower incidence of adverse effects after use. These reasons might account for their worldwide
attention and use. The medicinal properties of some plants have been documented by some researchers
(Akinpelu and Onukoya, 2006). Medicinal plants are of great importance to the health of individuals and
communities. It was the advent of antibiotics in the 1950s that led to the decline of the use of plant
derivatives as antimicrobials. Medicinal plants contain physiologically active components which, over the
years, have been exploited in the traditional medical practices for the treatment of various ailments
(Ajibesin, 2011). A relatively small percentage of less than 10% of all the plants on earth is believed to
serve as sources of medicine.
Plants have been used for medicine from time immemorial because they have fitted the immediate
personal need, are easily accessible and inexpensive. In the recent past, there has been a tremendous
increase in the use of plant-based health products in developing as well as developed countries resulting in
exponential growth of herbal products globally. Herbal medicines have a strong traditional, or conceptual
base and the potential to be useful as drugs in terms of safety and effectiveness leads to treating different
diseases. No studies have been reported for its antiulcer activity. Therefore, an attempt has been made to
evaluate the antiulcer potential of aqueous extract of Persea americana seed (Avocado) due to its potential
in antiulcer traditionally in some part of Ilorin
Persea americana (avocados) is one of the 150 varieties of avocado pear. The tree is widely cultivated
in tropical and subtropical areas (Lu et al., 2005). The seed of Persea americana (avocado seed) has diverse
application in ethnomedicine, ranging from treatment for diarrhoea, dysentery, toothache, intestinal
parasites, skin treatment and beautification. The avocado seed oil has much health benefits, e.g. for
controlling human weight (mainly used for obese for weight loss) (Lopez et al., 1996; Roger, 1999). Persea
americana leaves have been reported to have or possess anti-inflammatory and analgesic activities
(Adeyemi et al., 2002). Antioxidant activity and phenolic content of seeds of avocado pear were found to
be higher than 70% (Song and Barlow, 2004). The edible part (fruit) is trendy in vegetarian cuisine, making
a substitute for meat in sandwiches and salads, because of its high-fat content and high in valuable, health-
promoting fats (Lu et al., 2005). The fruit is not sweet but fatty, almost distinctly, yet subtly flavoured, and
of smooth, almost creamy texture. Avocado fruits in many countries such as Mexico, Brazil, South Africa
and India are frequently used for milkshakes and occasionally added to ice-cream (Zeldes, 2010).

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 M. O. Salawu et al.

Materials and Methods

Equipment:
The major equipment used for the study were 1ml insulin springe, oral cannula, oven, water bath,
dissecting set, electrical weighing balance, pH meter, centrifuge, spectrophotometer and chemical are of
analytical grade.

Plant sample collection and identification:

The plant fruit was collected from Ilorin, Kwara State and authenticated at the Herbarium unit in the
Department of Plant Biology, University of Ilorin, Ilorin. Where voucher number UILH/002/748 was
obtained.

Methodology

Preparation of plant sample

The fruit of Persea americana was obtained from Ipata market in Ilorin, and the seed was removed,
the seed of Persea americana samples were rinsed in clean water, cut into small piece and oven-dried 45oC
temperature. The dried seed samples were pulverized into powder using mortar and pestle, and the powder
obtained was used to prepare the extracts.

Aqueous extract preparation:

Five hundred grams of powdered seed, 2.5L portion of distilled water was added and stored at room
temperature for 48 hours and shaken at intervals. At the end of the extraction, the crude extract was filtered
using a muslin cloth and Whatman filter paper No.1. The aqueous extract obtained was concentrated to
dryness at 45ᵒC using a water bath. The dried extract was stored in an airtight sample bottle until required
for analysis.

Proximate Analysis:
This refers to the determination of the major constituents of the food sample, and it is used to assess if
a sample is within its normal compositional parameters or has somehow been contaminated. This method
partitioned nutrients in food samples into six components: water, ash, crude protein, fat, crude fibre and
moisture

Qualitative phytochemical screening

Phytochemical analysis extract was carried out using the method described by Sofowora (1993) for
the detection of saponins, tannins, phenolics, alkaloids, steroids, triterpenes, phlobatannins, glycosides and
flavonoids.

Experimental animals:
A total of 30 healthy Wistar rats weighing between 150-200g (6-8 weeks) old were obtained and kept
in well-aerated laboratory cages in the animal house, Department of Biochemistry, University of Ilorin,
Ilorin, Nigeria. They were allowed to adjust to the laboratory environment for two weeks before the
commencement of the experiment. The animals were fed with growers mash and water was provided ad
libitum during the stabilization period. The animals were divided into extract treatment groups and the
control groups.

Animal Grouping
The rats were randomly divided into six groups, with five animals per group. The extracts were
reconstituted in distilled water and administered orally daily for 14 days.

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Group 1: Normal control was fed standard growers mash and water ad libitum.
Group 2: Animals were administered a single-dose of aspirin orally (400 mg/kg).
Group 3: Animals were administered a single-dose of aspirin 400mg/kg and treated with omeprazole
40mg/kg for 14 days
Group 4: Animals were administered single-dose of aspirin 400mg/kg and treated with aqueous extract of
Persea americana 110mg/kg for 14days
Group 5: Animals were administered a single-dose of aspirin 400mg/kg and treated with aqueous extract
of Persea americana 220mg/kg for 14 days
Group 6: animals were administered single of aspirin 400mg/kg and treated with aqueous extract of Persea
americana 440mg/kg

Histopathological study
The stomachs were washed thoroughly with saline before tissue samples were collected and stored in
10% formalin solution. Biopsies were obtained from these samples. Sections (5 μm) were taken from the
biopsies and stained with hematoxylin and eosin (H & E) before visual inspection for damage under a light
microscope (100×) (Oloyede et al., 2015).

Statistical analysis
The values are reported as means ± S.E.M. Statistical difference was determined using ANOVA and
differences in the ways were tested by GraphPad prism 6.

Results

Qualitative phytochemical screening of aqueous extract of Persea Americana seed

Medicinal plants are the backbone of the traditional system of medicine and are enriched in
phytochemical constituents which serve as lead compounds in drug discovery and design. In the current
study, the aqueous extract of Persea Americana was prepared, and phytochemical analysis was performed
that confirms the presence of saponin, alkaloids, glycosides, anthocyanin, fixed oil.

Table 1: Qualitative and quantitative phytochemical screening of aqueous extract of Persea americana
seed

Phytochemical parameters Qualitative Quantitative

Saponin + 0.17 ± 0.00 (mg/100g)
Tannin -
Phenolics -
Phlobatanin -
Steroids + 17.10 ± 1.75 (µg/100g)
Flavonoids -
Anthocyanin + 11.56 ± 0.05 (µg/100g)
Terpenoids -
Glycosides + 1.86 ± 0.07 (mg/100g)
Triterpenes -
Alkaloids + 17.23 ± 0.05 (mg/100g)
Coumarin -
Fixed oil +
Key:- (+) indicates the presence of an insufficient phytochemical amount, while (-) indicates the absence of a
phytochemical and results are mean ± S.E.M. (n = 3). P<0.05 were considered as statistically.

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 M. O. Salawu et al.

Proximate analysis result

Table 2: Proximate Composition of aqueous extract of Avocado seed (Persea americana)

Sample Concentration %
Crude protein 5.79
Crude lipid 2.55
Total ash 22.50
Moisture content 7.18
Crude fibre 0.25
Carbohydrate 61.82

Determination of Gastric Ulcer Parameters

Biochemical Analysis

Table 3: Effect of Persea americana aqueous seed extract on gastric pH, total acidity gastric juice volume
and ulcer index in the stomach of rats

Treatment (n = 5) pH Total acidity Gastric volume Ulcer index

Normal control group 6.35±0.23a 10.0±0.57a 2.56±0.18a -
300 mg/kg Aspirin-induced 1.60±0.21c 2.05±0.28c 3.18±0.13a 11.15±0.12c
untreated
40 mg/kg Omeprazole 5.35±0.49a 7.67±0.67b 2.60±0.15a 3.54±0.19a
110 mg/kg P. americana 2.05±0.06b 4.50±0.64c 2.90±0.175a 8.91±0.05b
220 mg/kg P. Americana 4.28±0.15a 9.0±0.57a 2.75±1.11a 4.92±0.05a
440 mg/kg P. Americana 3.45±0.26b 6.75±0.75b 2.66±0.24a 6.67±0.24b
Results are mean ± S.E.M. (n = 3). P<0.05, were considered statistically significant when compared to uninduced
untreated (control) group.

Specific activities of P. Americana on biochemical parameters

Table 4: Effect of aqueous seed extract Persea americana on pepsin activity, total protein and total
carbohydrate level in gastric juice of rats stomach

Treatment (n = 5) Total CHO g/100g Total protein mg/dl Pepsin activity

Normal control group 22.50±8.85a 4.189±1.05a 25.50±0.09a
300 mg/kg Aspirin-induced 29.6±3.20a 5.63±0.50a 16.93±0.62b
untreated
40 mg/kg Omeprazole 18.0±1.00b 3.82±0.50a 24.49±0.06a
110 mg/kg P. americana 24.6±4.80a 3.45±0.24a 20.96±1.48b
220 mg/kg P. Americana 26.4±3.60a 3.57±1.28a 22.84±0.28a
440 mg/kg P. Americana 26.05±10.05a 4.24±0.99a 25.75±0.59a
Results are mean ± S.E.M. (n = 3). P<0.05, were considered statistically significant when compared to uninduced
untreated (control) group.

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 Biokemistri Volume 32, Number 1 (2020)

Antioxidant potential of P. americana on gastric tissue of Wistar rat

g p r o t e in )
3 100
g p r o t e in ) c n o rm a l c o n tr o l g ro u p
a B n o rm a l c o n tr o l g ro u p

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n o r m a l c o n tr o l g r o u p

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10 n o rm a l c o n tr o l g ro u p
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b 100
4 0 m g / k g b . w o f o m e p r a z o le a 4 0 m g /k g b .w o f o m e p r a z o le
6 b
a c t v it y

b b 1 1 0 m g /k g b .w p . a m e r ic a n a
c 1 1 0 m g / k g b .w p . a m e r ic a n a
4 c 2 2 0 m g /k g b .w p . a m e r ic a n a
2 2 0 m g / k g b .w p . a m e r ic a n a 50
p r o t e in

4 4 0 m g /k g b .w p . a m e r ic a n a
2 4 4 0 m g / k g b .w p . a m e r ic a n a
C A T

0 0

Figure 1: (A)Specific activities Malondialdehyde (MDA) on gastric tissue treated with Persea americana
and omeprazole (B) Specific activities reduced glutathione (GSH-Red) on gastric tissue treated with Persea
americana and omeprazole (C) Specific activities Superoxide Dismutase (SOD) on gastric tissue treated
with Persea americana and omeprazole (D) Specific activities Catalase (CAT) on gastric tissue treated with
Persea americana and omeprazole (E) Protein level of gastric tissue treated with aqueous extract of Persea
americana seed omeprazole
Results are mean ± S.E.M. (n = 3). p<0.05, were considered statistically significant when compared to
uninduced untreated (control) group.

40
M. O. Salawu et al.

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 Biokemistri Volume 32, Number 1 (2020)

Discussion

Persea americana fruits are rich sources of bioactive phytochemicals (Ding et al., 2007) The medicinal
values of a plant depends on the phytochemicals such as alkaloids, steroid, saponin, glycoside, anthocyanins
and other nutrients like as amino acid, proteins, which produce definite physiological actions on the human
body (Abhishek and Avinash, 2013).
The observed protective effect of aqueous seed extract and ability to reduce ulcer index groups treated
with the Persea americana is an indication of its vasoconstricting impact due to some phytochemicals
(saponin) present in the plants. These results compared favourably with the gastric ulcer lowering effects
of Exoecaria aggallocha (Thirunavukkarasu and Ramanathan, 2009) and the astringent action of saponin
(Enechi and Nwodo, 2014). The free radical scavenging ability of anthocyanin has been reported to protect
the gastrointestinal tract from ulcerative and erosion lesion. Also, the reduction in the ulcer index in the

42
 M. O. Salawu et al.

extracts pretreated group could be due to the antioxidant activity of the plant; this is in agreement with the
report of (Havsteen, 2002; Repetto and Llesuy, 2002).
These results show that moisture content is low, as revealed. The moisture content of any food is an
index of its water activity (Frazier and Westoff, 1978). It is used as a measure of stability and the
susceptibility to microbial contamination, implying that aqueous seed extract of Persea americana will be
very likely to have a long shelf life because of its low moisture content.
The protein content of aqueous seed extract Persea americana is not appreciably high to meet the
required daily protein of 23-55 g (Chaney, 2006). The use of aqueous seed extract of Persea americana as
a protein source is therefore not encouraged, however, in extreme conditions of protein deficiency; aqueous
seed extract of Persea Americana may be used as a protein source.
Ash content of 22.5% for aqueous seed extract of Persea americana suggests the right level. Ash
content of aqueous seed extract of Persea americana indicates it contains the right level of mineral materials
because low ash content suggests high mineral composition (Egharevba and Kunle, 2010).
Carbohydrate level of 61.8% for aqueous seed extract of Persea americana indicates that aqueous seed
extract of Persea americana is a rich carbohydrate source and has potentials to provide fuel and energy for
daily activities (Yisa et al., 2010).
The volume of acid present in the gastric secretion, which encompasses HCl, pepsinogen, mucus,
bicarbonates, intrinsic factor and protein reflects acid volume. Exposure of unprotected lumen of the
stomach to accumulating acid could facilitate ulceration (Olsen, 1988). Another major aggressive factor
responsible for ulcers is the content of acid present in the gastric juice. Over secretion of histamine
contributes to an increased flow of gastric juice (Grossman, 1978). When the concentration of hydrogen
ions in gastric juice decreases, it is reflective of high pH. The genesis of ulcer and gastric damage is
facilitated by hydrogen ions which serve as another driving factor (Lullmann et al., 2000). Decreased
prostaglandin level impairs almost all aspects of gastroprotection and increases acid secretions which, in
turn, aggravate the ulcer (Miller, 1983). Histamine (H2) receptor activity stimulates adenylate cyclase
system and in turn causes increases in calcium ion concentrations (Enechi and Nwodo., 2014), which
ultimately leads to activation of proton pump and consequently leads to hyperacidity and ulcer (Al-Mofleh
et al., 2006).
The carbohydrate content and the total protein content in the gastric secretion were reduced, and the
pepsin activity was increased in the animals treated with Persea americana extract, showing the active
mucous formation of the tissue. The antioxidant enzyme system plays a vital role in defence of cells against
oxidative damage. It has been reported that antioxidant properties of anthocyanins from several plant
extracts possess stimulatory action and exert a stimulatory effect on transcription and gene expression of
certain antioxidant enzymes (Sreelatha et al., 2009)
Lipid peroxidation can be used as an index for measuring the damage that occurs in membranes of
tissue as a result of free radical generation, leading to aggravated tissue damage during stomach ulceration
(El-Missiry et al., 2001). These present studies are in line with these previous data. Enhanced lipid
peroxidation (LPO) is a measure of membrane damage as well as the alteration in the structure and function
of cellular membranes (Halliwell, 1995). In this present study, treatment with aqueous seed extract of
Persea americana significantly reversed the aspirin-induced changes in Malondialdehyde (MDA), these
significant reductions in MDA level suggest decreased lipid peroxidation which might be due to the
antioxidant properties of the plant against free radical generation and thus its anti-ulcerogenic activity. A
similar observation was reported by (Sathish et al., 2011)
This study shows that all treatments with omeprazole and aqueous seed extract of Persea americana
increased the GSH content significantly (p<0.05). This affected the antioxidant defence system positively
and reduced the gastric damage considerably. GSH protects gastrointestinal tissue lipids from oxidative
damage. As reported in a previous study, in gastric tissue damaged by aspirin, glutathione level is lowered
after induction (Albayrak et al., 2015). glutathione detoxifies hydrogen peroxide and organic acids
chemically; hydrogen peroxide accumulates in the absence of GSH (Dalle-Donne et al., 2003).
In the present study, SOD activity decreased significantly in the untreated aspirin group of animals,
which may be due to the excessive formation of superoxide anions. These excessive superoxide anions

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 Biokemistri Volume 32, Number 1 (2020)

might inactivate SOD and decrease its activity. The activities of the H2O2 scavenging enzymes CAT also
reduced significantly in the stress-induced group of animals. SOD is a crucial defence enzyme that catalyzes
the dismutation of superoxide anions into O2 and H2O2 (Manneersk, 1987).
Catalase traps the harmful hydrogen peroxide and converts into water and oxygen. Catalase is a
haemoprotein containing four heme groups, that catalyses the decomposition of H2O2 to water and O2 and
thus, protects the cell from oxidative damage by H2O2 and OH (Gupta et al., 2004). The activity of CAT
was found to be decreased in aspirin untreated rats. The inhibition of CAT activity during aspirin-induced
ulcer may be due to the increased generation of reactive free radicals, which can create oxidative stress in
the cells. There was a significant difference between the aqueous seed extract of Persea americana
pretreated group and the untreated aspirin group. The administration of aqueous seed extract of Persea
americana increased CAT activity showing excellent antioxidant properties when compared with the
standard drug omeprazole.
Histopathological studies on the gastric mucosa revealed that aspirin administration induced mucosal
ulceration associated with a significant increase in lipid peroxidation. This was manifested as mucosa
epithelial necrosis, and leukocytic infiltration. This effect on mucosal oxidative stress and histological
derangement was following the reports of (Valcheva-Kuzmanova et al. 2007 and El-Moselhy et al. 2009).
the aqueous seed extract of Persea americana had some protective effect against aspirin-induced
inflammatory infiltration and congestion at the ulcer sites this may be due to its anthocyanin content.
anthocyanin could scavenge free radicals, inhibit lipid peroxidation, and increase prostaglandins and
mucosal content of the gastric mucosa; showing cytoprotective effects (Alanko et al., 1999),
The decrease in the protein content of the gastric mucosa and increase the in gastric juice of the
ulcerogenic group may be due to damage in the gastric mucosa which results, in the leakage of protein into
the gastric juice. Treatment with aqueous seed extracts of Persea americana increased the mucosal protein
which indicates its ability to enhance cell proliferation and stimulates the growth of the gastric mucosa
(Sathish et al., 2011)

Conclusion
The aqueous seed extract of Persea americana offered some protection against aspirin-induced gastric
mucosal damage. The antioxidant compounds present in Persea americana seed extract play a protective
role against the production of reactive oxygen species and lipid peroxidation. The present study revealed
that Persea americana extract has promising phytochemicals for the development of alternative treatment
against gastric ulcer.

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Acute toxicity study of crude methanol leaf extract of

Ficus exasperata Vahl on male Wistar albino rats
Ufuoma B. Shemishere*1 Daniel A. Anyebe1, Yahaya Tajudeen2, Umar U.
Liman1 and Ahmad Bello1
1
Department of Biochemistry and Molecular Biology, Federal University, Birnin Kebbi, Kebbi State,
Nigeria.
2
Department of Biology, Federal University Birnin, Kebbi, Kebbi state, Nigeria.
*Corresponding Author: [email protected]; +2348065712129

(Received January 18, 2020; Accepted March 9, 2020)

ABSTRACT: There is an increase in demand for the use of traditional folk medicine globally due to
their low cost, efficacy and easy accessibility especially for people living in developing countries. This
study was carried out to assess the acute toxicity as well as the LD 50 of Ficus exasperata Vahl in male
Wistar albino rats. Lorke’s method was adopted for the determination of the LD 50 of the crude
methanol leaf extract of plant. The extract administration was in two phases. In phase one, doses of 10,
100 and 1000 mg/kg body weight of rats was administered while in phase two, 1500, 3000 and
5000mg/kg body weight of rats was orally administered. Behavioral changes, signs of toxicity and
mortality were observed from the point of administration to 24 hours in the first phase only and 14 days
in the second phase included. The qualitative phytochemicals screening revealed the presence of
alkaloids, tannins, flavonoids, cardiac glycosides, saponins and steroids. Signs of toxicity observed in
the rats administered with 1000mg/kg body weight of rats were uneasiness, sluggishness and dizziness
after 24 hours of observation. However death was not recorded in all the groups after 14 days of
administration. There was dose dependent increase in the level of the selected liver enzymes assayed
(AST and ALT) across all the groups. Liver malondialdehyde concentration in the rats administered
higher doses differed significantly (p < 0.05) when compared with the control group. From our study,
crude methanol leaf extract of Ficus exasperata vahl possess medicinally important phytochemicals
and the acute toxicity studies revealed that the extracts has LD 50 above 5000mg/kg because no death
was recorded. However, the results of the liver enzymes and lipid peroxidation suggested that
prolonged use of the extract may cause damage to the liver and some vital organs. It is recommended
that a long-term study such as sub-chronic toxicity studies should be conducted to know the long term
effect of the leaf extract of Ficus exasperata Vahl.

Keywords: Phytochemicals screening, Acute Toxicity Studies, Ficus Exasperata, Malondialdehyde,

Liver enzymes

Introduction

Ficus exasperata Vahl (Moraceae), with its various species, is widely used in traditional
medicine. In tropical African countries like Nigeria, the plant is used locally for the treatment
of a variety of diseases/disorders such as coughs, hemorrhoids anxiety disorders, epilepsy,
high blood pressure, rheumatism, arthritis, cancer, intestinal pains, colics, bleeding and

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wounds (Cousins et al., 2002). The presence of important phytochemicals with antioxidant
and antimicrobial activities has been well reported (Awala et al., 2017; Pallav et al., 2014).
Recent toxicity studies in rats involving crude aqueous and ethanol extracts of Ficus
exasperata roots have shown potential hepatic and renal toxicity as reflected by significantly
increased serum transaminases and bilirubin (Ahmed et al., 2012). The screening of plant
extracts for their activities against diseases should be combined with evaluation of the
toxicities of such plant extracts with traditionally acclaimed therapeutic properties (Efosa and
Ngozi 2018). This study was carried out to determine the phytochemical constituents and
acute toxicity studies of crude methanol leaf extract of Ficus exasperata Vahl in male Wistar
rats.

Materials and Methods

Sample Collection
The Ficus exasperata plant leaf sample was collected in March, 2019 at Unguwar Jeji
Village, Kalgo Local Government, Kebbi State, Nigeria. They were identified by a plant
taxonomist in the Department of Biology, Federal University Birnin Kebbi were a herbarium
specimen with voucher number BIOHB/0034 was deposited. The leaves were washed with
clean water and air dried at room temperature. After drying, the leaves were ground with an
electric blender and the obtained powder form were stored in air tight containers till needed
for further analysis.

Sample Extraction
An amount of 1000g powder of Ficus exasperata Vahl leaf was soaked into 2000ml of
99.8% methanol for 72 hours (3 days). The sample was then filtered using muslin cloth after
three days soaking. The filtrates were concentrated in a vacuum rotary evaporator at 50℃,
after which the concentrated crude extracts were exposed to allow the remaining methanol to
evaporate. The percentage yield was calculated after which the solid extract was stored in the
refrigerator until further use

Experimental Animals
Adult male Wistar rats weighing 150- 250g, bred in Biology Department Animal house,
Federal University Birnin Kebbi, were used in this study. They were kept in clean plastic
cages, fed with rat pellets and watered ad libitum and left to acclimatize for two weeks. A
standard protocol was observed in accordance with the Good Laboratory Practice (GLP)
Regulations of the WHO (1998).

Phytochemical Screening
Preliminary phytochemical screening was carried out using standard procedure as
described by Sofowora (1993), Trease and Evans (1989) and Harborne (1973).

Acute Toxicity Studies

Standard method was used as described by Lorke (1983). The experiment was conducted
in two phases. In the initial phase, rats randomly divided into 4 groups of 3 animals each.
Groups 1, 2 and 3 were orally administered 10, 100 and 1000 mg/kg body weight crude
methanol leaf extract respectively. The fourth group, which was the control, was administered
distilled water orally. Behavioral observation and body weight changes were monitored
during the 14 days period of study. Prior to sacrifice all animals fasted for 12 hours and
were weighed and euthanize in a chloroform chamber.
In the second phase, twelve (12) Male Wistar were divided into 4 groups of three
animals per group. Groups 1, 2 and 3 were orally administered 1500, 3000 and 5000 mg/kg
body weight crude methanol leaf extract. The fourth group which was the control was
administered distilled water. Behavioral observation, body weight changes were carried out
during the 14 days period of study. Death was monitored over a period of 24 hours and the

48
 S. B. Ufuoma et al.

serum liver function enzymes as well as lipid peroxidation parameters in the liver was
analysed at the end of 14 days period.

Liver Enzymes Level Estimation

Serum ALT Estimations: Serum alanine aminotransferase was assayed by the method
described by Raitman et al. (1975) and Schmidt et al. (1963). The assay is based on the
reaction between α-ketoglutarate and L-alanine catalyse by alanine aminotransferase to give
L-glutamate and pyruvate, the pyruvate hydrazone complex formed between pyruvate and 2,
4-dinitrophenylhydrazine was measured at 546nm.

Serum AST Estimation: Serum aspartate aminotransferase was measured according to

the methods of Raitman et al. (1975) and Schmidt et al.(1963). The assay is based on the
reaction between α-ketoglutarate and L-aspartate catalysed by aspartate aminotransferase to
give L-glutamate and oxaloacetate, the oxaloacetate-hydrazone complex formed between
oxaloacetate and 2,4-dinitrophenylhydrazine was measured at 546nm.

Lipid Peroxidation Estimation

Thiobarbituric acid assay (TBARS) method was used for malondialdehyde concentration
determination as described by Janero (1990). The assay is based on the reaction between the
malondialdehyde in the sample and thiobarbituric acid in the reagent to give a pink coloured
complex measured at 535nm.
After dissection, the liver was isolated, weighed and chilled in ice-cold 0.9％ NaCl.
After washing the liver homogenate was prepared in a ratio of 1g of wet liver tissue to 9ml of
1.15% KCl using Teflon homogenizer.

Calculation:

The malondialdehyde concentration of sample can be calculated using extinction

co-efficient of 1.56 x 105m-1cm-1

TBARS activity = O.D x V x 1000

Ax v x 1 x Y

Where O.D = absorbance of sample test at 535nm

V=Total volume of the reaction = 3ml
A = molar estimation co-efficient of product = 1.56 x 105m -1cm-1
1 = Light path = 1cm
v = volume of tissue extract used = 1ml
Y = mg of tissue in the volume of sample used.

Statistical Analysis
All analyses were carried out in triplicate and, where applicable, results are expressed as
mean ± SEM. The data were subjected to one-way analysis of variance (ANOVA) and Tukey
Post-hoc test was used for the multiple comparison. P values less than 0.05 (p < 0.05) were
regarded as statistically significant.

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Results

The extraction process yielded 12.64 % (w/w) at room temperature. The obtained results
of the qualitative phytochemicals screening of crude methanol extract of Ficus exasperata
leaf extract revealed the presence of alkaloid, tannin, flavonoids,, saponin and steroids.
However, cardiac glycoside was not detected (Table 1). Signs of toxicity observed were
unease, sluggishness and dizziness three hours after administration of extract as from the
dosage of 1000mg/kg body and above. Changes in the weight in both treated groups and
control groups were also observed (Table 2).
The results revealed variations in the level of both ALT and AST at lower doses but
slight increase was recorded at higher doses. The results also revealed that malondialdehyde
concentration obtained from those with lower doses (10mg-1500mg) did not show significant
difference from control. However, those groups that were administered 3000 mg/kg and 5000
mg/kg of the extract showed significant difference from control (Table 5).

Table 1: Phytochemical screening of crude methanol extract of Ficus exasperata vahl

leaf

Phytochemicals Inference
Alkaloids +
Cardiac glycoside -
Flavonoid +++
Phenol +++
Saponin +
Steroid +
Tannin ++
Terpenoid ++
Keys: +: Present in a trace concentration; ++: Present in a medium concentration; +++: Present in a
high concentration; -: Absent or in negligible amount.

Table 2: Effect of administering different doses of crude methanol extract of Ficus

exasperata leaf on body weight of albino rat over a period of two weeks

Dose (mg/kg b.wt) Day 0 Day 7 Day 14

Control 203.86d±2.40 207.69d±1.56 212.42d±1.99
10 206.48a±7.96 203.84a±7.05 198.56a±9.40
100 218.76b±2.06 214.16b±3.63 209.43b±1.99
1000 232.09c±9.46 227.53c±6.75 223.68c±5.75
1500 201.26e ± 2.35 203.62e ± 3.66 202.70e ± 4.32
3000 224.81f ± 4.21 220.82f ± 6.32 217.62f ± 5.78
5000 238.10g ± 4.32 236.17g ± 6.67 231.28g ± 3.29
Data expressed as mean ± standard deviation; mg/kgbw: Milligram Per Kilogram Body Weight. Same
super script means there is no significance difference while different super script means there is
significance difference (p<0.05). ANOVA was used for the multiple comparism (Turkey).

50
 S. B. Ufuoma et al.

Table 3: Effect of administering different doses of crude methanol extract of Ficus

exasperata leaf on the organ weight of male Wistar rats

Dose(mg/kg Liver Kidney Lungs Heart Spleen

Control 6.49a±1.30 1.26b±0.10 1.15a±0.10 0.65a±0.08 0.48a±0.55
10mg 5.88a±0.41 1.39b±0.02 1.45b±0.49 0.75b±0.10 0.63b±0.14
100mg 9.05c±1.27 1.85c±0.22 1.84c±0.09 0.87c±0.09 0.77c±0.15
1000mg 8.52b±1.11 2.15d±0.19 1.94±0.22 1.13d±0.17 0.76c±0.12
1500mg 8.72b ± 1.02 1.14a ± 0.04 1.39b ± 0.04 0.80c ± 0.21 0.72c ± 0.06
3000mg 9.34c ± 0.93 2.16d ± 0.23 1.98d ± 0.06 1.20e ± 0.01 0.84d ± 0.03
5000mg 10.04d ±1.11 2.18d ± 0.21 1.48b ± 0.03 1.36f ± 0.06 0.87d ± 0.02
Data expressed as mean ± standard deviation; mg/kg bw: Milligram Per Kilogram Body Weight. Same
super script means there is no significance difference while different super script means there is
significance difference (p<0.05). ANOVA was used for the multiple comparison (Tukey).

Table 4: Effect of administering different doses of crude methanol extract Ficus

exasperata leaf on the major serum liver marker enzymes (ALT and AST) of male
Wistar rats

Experiments Dose(mg/kg b.wt) ALT (U/L) AST (U/L)

Control 0 5.60a±0.80 7.93a±0.53
Phase 1 10 9.60b±1.60 8.87a±0.81
100 12.27b±0.92 16.80b±2.45
1000 15.57c±1.76 22.05c±0.35
Phase 2 1500 19.68d±0.08 27.30d±0.15
3000 21.76d±0.06 47.25e±0.25
5000 24.00e±0.05 53.90f±1.10
Data expressed as mean ± standard deviation; mg/kgbw: Milligram Per Kilogram Body Weight. Same
super script means there is no significance difference while different super script means there is
significance difference (p <0.05).

Table 5: Effect of administering different doses of Ficus exasperata methanol leaf

extract on the lipid peroxidation using liver tissue of male Wistar rats

Experiment Dose(mg/kg b.wt) MDA conc.in (moleMDA/g

wet tissue × 10 -5)
Control 0.865a±0.002
Phase 1 10 0.764b±0.139
100 0.866a±0.051
1000 0.942c±0.019
Phase 2 1500 1.250 d ± 0.12
3000 1.370 d ± 0.04
5000 1.670e ± 0.06
Data expressed as mean ± standard deviation; mg/kg bw: Milligram Per Kilogram Body Weight. Same
superscript means there is no significant difference while different super script means there is
significance difference (p<0.05).Student T-test was used for single comparison.
MDA=Malondialdehyde

Discussion

Studies have demonstrated that phytochemicals possessed various pharmacological

properties. It is obvious that most therapeutic effects of medicinal plants are a function of
their phytochemical constituents and, in recent years, secondary plant metabolites

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(phytochemicals) have been investigated extensively as sources of medicinal agents

(Krishnaraju, 2005). The presence of alkaloid, tannin, flavonoids, cardiac glycosides, saponin
and steroids in Ficus exasperata crude leaf extract detected in this study was also reported by
Awala et al. (2017), who worked on the same crude methanol extract of Ficus exasperata
vahl leaf. More so, Ugwah-Oguejiofor et al. (2011), also reported the present of tannin,
saponin, flavonoid, volatile oils, steroids and glycosides at various concentrations.
Phytochemicals are thought to have a positive and negative effect on animals upon
administration. For instance tannins and anthraquinones are thought to have both proxidant
and antioxidant effects on the body. While the antioxidant protects the tissues and organs, the
proxidant damages the tissues and organs.
The weight changes of the animals during the period of observation which was more
visible at higher doses, suggest the presence of tannins and other phenolics which are thought
to interfere with absorption of nutrients making them unavailable and thereby reducing feed
intake (Kumar and Singh, 1984). The organ weights were not significantly different form
the control. Generally, the reduction in body weight is a simple and sensitive index of toxicity
after exposure to toxic substances. Body weight changes are indicators of the adverse effects
of drugs and chemicals and it would be regarded as significant if the body weight loss is more
than 10% from the initial body weight (Teo, 2002).
From the results of ALT and AST, it can be suggested that since ALT and AST are
mostly released as a result of liver injury. As such elevation of the level of these enzymes can
be an indication of cellular damage, leakage and loss of functional integrity of hepatic cell
membrane due effects of the plant extract. But this is not always true as other organs and
physiological conditions can result to the release AST in the serum. The results obtained in
these studies revealed that malondialdehyde concentration obtained of those with lower doses
(10mg-1500mg) did not showed significant difference with control. More so, the groups that
were administered 3000mg/kg and 500mgkg of the extract showed (MDA concentration) of
more than 1.00 mole MDA/g tissue wet ×10-5. It can suggested that increased in doses might
be a reason for the increase of malondialdehyde which serves as a primary index of lipid
peroxidation.

Conclusion
This study has established that crude methanol extract of Ficus exasperata leaf obtained
from the northern part of Nigeria, particularly (Kebbi State), is rich in phytochemicals. The
LD50 appears to be more than 5000mg/kg body weight because no death of the rats was
recorded throughout the period of the study. However, the methanol extract of Ficus
exasperata leaf manifested slight signs of toxicity at higher doses as the level of the liver
enzymes increased as well as malondialdehyde concentration (MDA). In a nutshell, prolonged
use of specific plant extract may lead to toxicity irrespective of its medicinal importance.
We suggest future research on the effect of the leaf extracts of this plant on
histopathological and hematological parameters. Furthermore, it is recommended that a
long-term study such as sub-chronic toxicity and chronic studies to be conducted to know the
actual LD50 of the leaf extract of Ficus exasperata Vahl. Finally, it is suggested that further
studies should focus on the isolation, purification and characterization of the active
constituents of crude methanol extract of Ficus exasperata leaf in order to add to our
knowledge of its medicinal profile.

Conflicts of Interest: No Conflicts of Interest

References

Awala, S.I., Ajayi, O.E., Alabi, O.A Ajayi O. and Olalekan, O.T. (2017). Exploration of the
antimicrobial properties of Ficus exasperata leaves from Akure metropolis. Journal of Advances in
Research 9(5):1-6.

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Cousins, O.N and Micheal A.H. (2002). Medicinal properties in the diet of Gorillas. An
ethno-pharmacological evaluation. African Journal of Studies Monograph, 23(2): 65-89.
Efosa Godwin Ewere and Ngozi paulinius okolie (2018).’Phyochemicals, Antioxidant Activity and
acute toxicity of stem Banc Extract of Irvingia gabonensis O’Ronee Bail”.EC Pharmacology
and Toxicology 6.6; 390-399
Harborne, J.B. (1973). Phytochemical methods. London: Chapman and Hall. l.t.d Pp. 52-114
Heo, S.J. (2005) Antioxidant Activities of Enzymatic Extracts from Brown Seaweeds. Journal of
Bioresource Technology, (96)14:1613-1623.
Janero, D.R. (1990). Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid
peroxidation and peroxidative tissue injury. Free Radical. Journal of Biological Medicine , 9, 515–
540.
Krishnaraju, A.V. (2005). Assessment of Bioactivity of Indian Medicinal Plants using Brine Shrimp
(Artemiasalina) lethality assay. International Journal of Applied Science and Engineering, 3(2):
125-134.
Kumar, R. and Singh, M. (1984). Tannins: their adverse role in ruminant Nutrition. Journal of
Agriculture and Food Chemistry, 32:447-453.
Lorke, D. (1983). A new approach to practical acute toxicity testing. Arch. Toxicology. 54: 275-287
Nagalapar,S,K and paramjyothi. S, (2010). In vitro antioxidant activity of Launaea pinntitida Cass
leaves, The Bioscan 5:105-108.
Pallav, K.D., Ragini, G. and Anupam, K.P. (2014). Phytochemical analysis and evaluation of
antimalarial activity of Azadiracchta indica. The Journal Pharmacological Innovation , 3(9):12-16.
Reitman. S., and Frankel. S. (1957). A colorimetric method for determination of serum glutamate
oxaloacetate and alanine oxaloacetate. American Journal of Biomedical Research 28; 56-58.
Schmidt, E. and Schmidt, F.W. (1963). A colorimetric method for determination of serum glutamate
oxaloacetate and alanine oxaloacetate. Journal of Enzyme, Biological clinic, 3;1-5
Sofowora, A. (1993). Medicinal Plants and Traditional Medicine in Africa. Ibadan Nigeria: Spectrum
Books, Pp.191-289.
Teo, S. (2002). A 90 day oral gavage toxicity study of D-methylphenidate and D, L methylphenidate
in sprague -dawley rats. Journal of Toxicology, 179: 183-196.
Trease, G. and Evans, W. (1989).Pharmacognosy (11th ed.,) Pp. 45-50.
Ugwah-Oguejiofor, O., Chinenye, J., Bello, S., Emmanuel, U., Etuk, Vincent U. Igbokwe, Oguejiofor
M. U. and Raymond, U.O. (2011). Preliminary toxicity and phytochemical studies of the aqueous
extract of Ficus platyphylla in female albino rats. International Research Journal of Pharmacy and
Pharmacology 1(5):086-092.

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Starch and triglycerides in the root of maize (Zea mays) and

cowpea (Vigna unguiculata) grown in crude oil polluted soil
treated with ash from palm bunch
Stella Oghomwen Olubodun1, George E. Eriyamremu2 and Chinedu Eze2
1
Department of Medical Biochemistry, School of Basic Medical Sciences, University of Benin, Benin City, Nigeria.
2
Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City, Nigeria.
Emails: [email protected], [email protected], [email protected]
1
Corresponding author: [email protected]; +2348023411948

(Received January 18, 2020; Accepted March 9, 2020)

ABSTRACT: The ash from burnt oil palm fruit bunch has been commercially known and used as natural fertilizer for
neutralizing peaty and acidic soils. This study was conducted to evaluate the use of ash from palm bunch (APB) as a
source of biological fertilizer and soil amendment to neutralize the acidic pH created by crude oil contamination on
the starch and triglycerides contents in the roots of maize and cowpea seedlings. The physicochemical characteristics
of the APB reveals it may be a good source of nutrients to improve soils to be used for agriculture. The results shows
that application of APB significantly (P<0.05) increased soil pH, soil sodium, potassium, calcium and magnesium.
The plant height were significantly increased and comparable to that of the control. This shows that APB contains
nutrients important for plant growth and may contribute to bioremediation. There were significant changes in the
starch and triglyceride contents of the maize and cowpea seedlings in contaminated soils when compared with control.
The study reveals alterations comparable to control with the application of APB, however, the degree of response of
the two plants to APB differed. The study suggests that APB could be used as fertiliser to increase the pH and the
nutrient contents of acidic soils and thus improve the soil quality. Further studies are needed to elucidate the
mechanism of alteration of starch and triglyceride contents in the plant species.

Keywords: APB, Cowpea, Crude Oil, Maize, Polluted Soil, Starch, Triglycerides

Introduction

Maize (Zea mays) belongs to the family Poaceae. Its cultivation started as a subsistence crop in the
Nigerian diet but has gradually become a more important crop. Maize has risen to a commercial crop on
which many agro-based industries depend as raw materials. It is a cereal crop of temperate and subtropical
zones. It grows in most agro ecological areas especially in the Niger Delta region where crude oil industrial
activities are predominant (1). Maize has also been shown to tolerate a variety of stressful conditions and
environmental extremities ranging from drought to heavy metals contamination (2).

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Cowpea (Vigna unguiculata L.) is a dicotyledonous crop in the subfamily Faboideae (3). It has been
said to originate in southern Africa. It is well adapted to conditions in many parts of the world. The seeds
are most often harvested and dried for storage and consumption either after cooking whole or milled as a
flour product and used in various recipes, providing a major source of dietary protein that nutritionally
complements low-protein cereal and tuber crop staples (3).
Soil contamination by crude oil is increasingly becoming a global menace not only to plants and
animals but to the ecosystem in general. The dependence on crude oil and its refined products as major
sources of energy continues to make it a “necessary evil”. Increase in population, rapid industrialization
and complete disregard for environmental health has continued to have impact on the soil. Crude oil spill
due to human activities or through accident is the main cause of water and soil pollution. Crude oil
constituents have been shown to belong to the family of carcinogens and neurotoxic organic pollutants (4).
The soils contaminated by crude oil have been reported to have moderately acidic pH and seeds planted
have reduced percentage germination, reduced growth and development (5, 6). In severe conditions, the
plant roots may die, and this would prevent uptake of water and other nutrients. It can also disrupt plant and
water relationship in soil (7).
Since crude oil pollution is a threat to the environment, the remediation of oil-contaminated soils is a
major challenge for environmental research. The use of plants to remove pollutants from the environment
or to render them harmless (bio-remediation) becomes necessary because other chemical methods are
cumbersome, produce toxic by-products and are very expensive (8, 9). Bio-remediation offers a cost
effective remediation technique, compared to other remediation methods, because it is a natural process
and does not usually produce toxic by-products. It also provides a permanent solution as a result of complete
mineralization of the contaminants in the environment. The advantages of bio-remediation include: i)
destruction rather than transfer of the contaminants to another medium, ii) minimal exposure of workers to
the contaminants, iii) long-term protection of public health and possible reduction in the duration of the
remediation process (4 , 10). Various studies have shown the effectiveness of organic fertilizers in
bioremediation (6, 11). Although different materials such as poultry waste, cow dung, etc, have been used
for remediation of soil contaminated with crude oil, the use of palm bunch ash is relatively new.
Oil palm is very important in Nigeria, for its economic value. When the oil in the fruit is extracted
from the nuts, the empty bunches are thrown away, constituting nuisance in the environment. Palm bunch
is the solid waste that remains after processing of oil palm fruits. Palm fruit bunches are removed after
ripening and the fruits processed to express edible industrial mesocarp and seed oils. Palm bunch ash is
obtained by burning the solid waste (palm bunch), generated during the processing of oil (7). In its natural
state, plant ash has been applied as an amendment to soils and as a substitute for fertilizer. Palm bunch ash
is reported to be alkaline (pH > 10) in nature and contains relatively high potassium and sodium contents.
Palm bunch ash is an effective fertilizer and liming material for increasing soil fertility, pH and nutrient
uptake because of its rich content in nitrogen, phosphorus, potassium, calcium and magnesium. Since palm
bunch ash is a good source of sodium and potassium (12, 13), it can be exploited in remediation of
contaminated soil (14). It has been shown that the effect of palm bunch ash on crops is due to its possession
of vital mineral elements needed for growth and development. Palm bunch ash contributes varying amount
of calcium, phosphorus, potassium and magnesium which affect the yield of crop (7, 15).
In recent years studies have shown the usefulness of ash from palm fruit bunch in soil fertility
restoration by providing essential constituents needed for plants growth and protection (4, 16). The aim of
this study was to assess the qualities of ash from palm bunches in protecting plants against crude oil
pollution. The main objectives were to analyse the composition of ash made from oil palm bunches, it’s
effects in soil exposed to crude oil and its effects on the starch and triglyceride contents of growing cowpea
and maize seedlings.

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Materials and Methods

The study was carried out at the University of Benin, Benin City, Edo State, Nigeria. The soil was
collected from an uncultivated land with no history of crude oil contamination in Edo State, Nigeria. Top
soil samples were collected from an uncultivated land with no history of crude oil contamination by digging
holes with a plastic spade at five different locations within the land to a depth of about 15cm. The soil
samples was collected into polythene bags and taken to the laboratory. All the soil samples were made into
a composite soil by mixing equal amounts from each location, thoroughly. Four hundred grams (400g) of
the composite soil was weighed into 120 polythene bags. The soils were air-dried at room temperature (28-
31oC), crushed in a porcelain mortar and sieved through a 2mm sieve. The air-dried < 2mm samples were
stored in polythene bags and labeled. Ash from oil palm bunch was applied by incorporating appropriate
quantities into the soils and properly mixed to ensure even distribution within the soil. All treatments were
transferred into evenly perforated planting bags and incubated.
Maize (Zea mays) and cowpea (Vigna unguiculata L.) seeds were bought from a local market in Benin
City, Edo State, Nigeria and identified, in the Department of Crop Science, University of Benin, Benin
City, Nigeria. Seed viability was assessed by floatation method. The seeds were placed in a beaker
containing tap water and stirred. The seeds that did not float were regarded as viable seeds.
Bonny Light Crude Oil was obtained from Warri Refinery and Petrochemical Company Delta State,
Nigeria. A portion of the crude oil was fractionated by a modified method of (17) into water soluble fraction
(WSF) and water insoluble fraction (WIF). For the fractions, a 1:1 dilution of 100 ml of crude oil was put
in a 1 litre conical flask and constantly stirred with a magnetic stirrer for 48h. The WSF then separated from
the WIF in a separating funnel.
A pilot study was conducted in an earlier experiment with 0.1%, 0.2% and 0.3% crude oil. In the
experiment, 0.3% crude oil contamination was found to have the highest stress level and adverse effect on
the plants. The composite soils were treated with distilled water (control) and 0.3% whole crude (WC), or
with the water insoluble fractions (WIF) of the crude oil in the laboratory. The soil in the bags contaminated
with whole crude (WC) and water insoluble fractions (WIF) were mixed thoroughly in their respective
polythene bags containing 400g top soil with the aid of a plastic spade.
The seeds were planted by a modified version of (18). Three viable maize or cowpea seeds were sown
in 500g sandy loam soil with a depth of about 1 cm and watered daily with distilled water. The time and
number of seeds that sprouted from each bag were noted and the germination percentage seedling in each
treatment was calculated using the formula:

Germination Percentage = Number of seeds that sprouted x 100

Number of seeds planted 1

In each bag, three (3) viable cowpea and maize seeds were planted. Equal amounts of seeds that
sprouted were harvested on the 7th day and Starch and Triglycerides contents were assessed in the roots
after thorough washing with tap water.
The physicochemical analysis of the soil and APB were assayed according to the methods described
by A.O.A.C, (19) using Atomic Absorption Spectrophotometer (AAS), Bulk Scientific. Potassium (K) was
determined by aspirating directly into flame photometer (PFP7) while calcium (Ca) and Sodium in the
extract was determined using the Atomic Absorption Spectrometer (AAS). The pH, Particle Size Analysis,
Organic Carbon, Organic matter, Potassium, Sodium and Calcium were assayed and the parameters
determined were expressed in percentages and centimole per kilogram (Cmol/kg).
The roots were recovered for the analysis. Weighed quantity of the roots was oven dried at 60 oC for
48h to constant weight. After drying, the tissue was immediately placed in a desiccator before the final
weighing. A portion of the root tissue was crushed and boiled in a few ml of isopropanol to activate
phospholipases, then homogenized in 20 volumes (w/v) of chloroform and methanol (2:1 v/v) and stored at

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9-4oC until ready for use. A portion of the tissue was put into 80% ethanol and stored. The extraction of
lipids was done by the methods of Folch et al (20). A suitable aliquot of lipid extract was evapourated to
dryness to determine the lipid content by weighing. The methods used for the estimation of different lipid
classes were according to Munshi et al (21). The extraction and estimation of starch, total soluble sugars
and reducing sugars were done by earlier described methods (21).
The data collected were subjected to analysis of variance (ANOVA), using Instat-Graphpad statistical
package (version 6), and where significant differences were observed, Duncan’s multiple comparisons test
at 5% probability level was used to compare the treatment means. The results of the study were expressed
as mean ± standard error of mean (SEM).

Results and Discussion

The results of the physicochemical properties of the soil and ash from palm bunch (APB) are shown
in Table 1. The results obtained using the universal soil classification method show that the soil used in the
experiment were sandy loam soil. The results obtained for total organic carbon and total organic matter
were significantly higher (P < 0.05) in the crude oil contaminated soil when compared to control soil but
higher in the APB. The total organic carbon (TOC) and Total Organic Matter (TOM) values obtained in the
results are comparable to those earlier reported (6, 22). The levels of organic matter in soils affect the soil
chemical and physical processes and acts as an indicator of the soils ability to hold plants (22).

Table 1: Physicochemical characteristics of the soils and ash from palm bunch (dry weight)

Parameter/Sample Control 0.3% WC APB

pH (H2O) 6.28±0.03a 5.94±0.03a 8.50
Total Organic Carbon (%) 2.60±0.01a 3.90±0.01b 6.82
Total Organic Matter (%) 2.34±0.02a 4.68±0.02b 8.26
Mg2+ (Cmol/kg) 2.50±0.02a 2.90±0.02a 18.98
K+ (Cmol/kg) 0.42±0.02a 0.20±0.02b 421.28
Na+ (Cmol/kg) 0.50±0.02a 0.39±0.04a 19.42
Ca2+ (Cmol/kg) 3.00±0.02a 4.14±0.05b 73.48
Clay (%) 15.34 16.28
Silt (%) 10.30 10.70
Sand (%) 74.36 73.02
Values are mean of three (n=3) replicates ± standard error of mean, Cmol/kg (centimoles of charge per kg soil =
meq/100 g). Means of the same row carrying different notations are statistically different at P<0.05

However, pH was significantly lower (P < 0.05) in the crude oil contaminated soil when compared to
control soil but higher in APB. The low pH indicates that the control and crude oil contaminated soils were
slightly acidic. Similar pH values have been reported for soils with cassava processing effluents (23), soils
in the Niger Delta and some soils in other parts of Nigeria (6). However, it is at variance with pH values
reported for dumpsites (24) which is similar to the pH observed in APB. The observed pH values in the
soils may have altered the physicochemical compositions of the soil as well as the chemical fractionation
(22). The soil pH which was originally acidic (pH: 6.28) and made more acidic by 0.3% crude oil
contamination (pH: 5.94) may have increased to near/above neutral by addition of the ash from palm bunch
whose pH (8.50) is alkaline (14). The increase in pH may have been involved in the remediation process of
the soil by the ash from palm bunch which may have led to the increase in plant height.
Similarly, the exchangeable potassium (K) and sodium (Na) of the soil which were reduced
respectively after contamination, may have been remediated by addition of the APB with high nutrient
contents (K and Na). Treatment with APB may have increased the original Ca and Mg concentration which
may have led to the increase in nutrient contents in the soil. Since K, Na, Mg and Ca are essential plant

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 S. O. Olubodun et al.

nutrients, the use of APB for soil remediation may have resulted in the enrichment of the soil with vital
nutrients.
The high total organic carbon (TOC) content of APB may also increase the control and 0.3%
contaminated soil because Adjei and Boahen (25) reported that TOC of soils increased with the application
of APB. Similar increase may be observed for the soil total organic matter (TOM), and other nutrients an
indication of soil fertility improvement by applying the APB (4, 14).
The effects of different crude oil fractions and APB on germination percentage, starch and triglyceride
contents in maize is shown in Table 2. The result shows that treatment of crude oil polluted soils with ash
from oil palm bunch resulted in remediation of the soil leading to enhanced sprouting of maize seeds (Table
2 and Figure 7). Several researches have observed that the presence of crude oil in soils hinders seed
germination and attributed these situation to the hydrophobic nature of the soils which leads to
unavailability of water and oxygen essential for seed germination (6, 15 & 26). In this study APB enhanced
germination percentage. This indicates that the hydrophobic nature of the soil may have been reversed by
the APB which may have resulted to water and oxygen being available for germination enhancement thus
improving the soil fertility. This result agrees with the study of other researchers for enhancement of soil
fertility with palm fruit ash (4, 7, 14 &15).

Table 2: Ash from Palm bunch on the effect of 0.3% contamination of various fractions of crude oil on
percentage germination, total protein, starch and triglyceride contents in the root of maize 7 days post
germination

Maize 7 days Germination % Total protein Starch Triglyceride

Control 100 0.337 ± 0.043a 0.078 ± 0.006a 0.640 ± 0.033a
RMD 100 0.371 ± 0.021b 0.173 ± 0.004b 0.680±0.122ab
WC 70 0.054 ± 0.025c 0.200 ± 0.004bc 0.579±0.035ac
WC+R 94 0.138 ± 0.050d 0.201 ± 0.005bcd 0.722 ± 0.143d
WIF 56 0.039 ± 0.026e 0.069 ± 0.008ae 0.593±0.029ace
WIF+R 86 0.083 ± 0.002f 0.070 ± 0.006aef 0.621 ± 0.054acef
Values are represented as mean ± SEM (n=3). Total protein unit is in mg/ml, Starch content is in mg g -1, while
triglyceride unit is in g 100 g-1

The results also show significantly higher concentrations of total protein (P<0.05) in the treated groups
against that of control, but WIR was slightly lower. The various remediated groups shows an improvement
in the total protein concentrations as against their respective non-remediated groups. That is, remediated
group only was higher than control group in protein content; while WC+R showed higher protein content
than WC. Similarly, WIF+R showed higher protein content than WIF (Figure 1).
The starch content was highest in WC group than in control and lowest on WIF (P<0.05). Their various
remediated groups showed no significant changes in starch content (Figure 3).
Triglyceride was significantly higher in WC than control and WIF and lowest in WIF. The remediated
only was higher than control in triglyceride content and the remediation of the treated groups was also in
triglyceride content than the untreated groups (Figure 5).
The mobilization of complex polymers such as starch, proteins and lipids from storage tissues such as
cotyledons is one of the most studied processes on seedling development. These polymers or seed reserves,
are used as energy sources and building blocks for seedling growth during germination (27). The reduction
in starch and triglyceride contents in the maize root observed in the study are consistent with earlier reports
that a reduction in oxygen supply may lead to reduction in adenosine triphosphate (ATP) production which
invariably leads to accelerated sugar metabolism and glycolysis (28, 29 & 30). The decrease in starch and

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 Biokemistri Volume 32, Number 1 (2020)

lipid contents of the cowpea root observed in the study compares favourably with increase in glucose
contents previously reported (31). This may indicate that starch and triglycerides were the main source of
energy in the plant during crude oil exposure. Thus starch and lipids can be converted into glucose under
stress condition as starch and lipids (storage carbohydrate) are dissociated as primary metabolites in stress
condition to overcome the energy demand by the plant for growth and to overcome oxygen deprivation.
The decrease in starch content may be related to the toxicity of crude oil because starch and/or lipids helps
the plant in providing energy in time of emergency for survival. The results suggests that starch and/or
lipids could be used by the growing plants to produce glucose as energy source to sustain the metabolic
activities occurring in the plant (5).

Table 3: Ash from Palm bunch on the effect of 0.3% contamination of various fractions of crude oil on
percentage germination, total protein, starch and triglyceride contents in the root of cowpea 7 days post
germination

Cowpea 7 days Germination% Total protein Starch Triglyceride

Control 100 0.642 ± 0.01c 0.077±0.004a 0.667 ± 0.015a
Remediated 100 0.876 ± 0.02b 0.095±0.002ab 0.690 ± 0.023b
WC 60 0.851 ± 0.02a 0.103±0.004ac 0.251 ± 0.012ac
WC+R 90 0.763 ± 0.02abc 0.133±0.009abc 0.507 ± 0.020bd
WIF 52 0.644 ± 0.03abc 0.098±0.003ade 0.387 ± 0.041ef
WIF+R 86 0.706 ± 0.01abc 0.099±0.004bcd 0.585 ± 0.028abc
Values are represented as mean ± SEM (n=3). Total protein unit is in mg/ml, Starch content is in mg g -1 and
Triglyceride unit is in g 100 g-1.

0 .5
C o n tro l

0 .4 R
WC
0 .3
m g /m l

W C+R
W IF
0 .2
W IF + R

0 .1

0 .0

T o ta l P ro te in

F ig 1 : E f f e c t o f C r u d e o il a n d p a lm f r u it b u n c h a s h o n T o t a l P r o t e in o f m a iz e s e e d lin g s

60
 S. O. Olubodun et al.

1 .0
C o n tro l

0 .8 R
WC
0 .6
m g /m l
W C+R
W IF
0 .4
W IF + R

0 .2

0 .0

T o ta l P ro te in

F ig 2 : E ffe c t o f C ru d e o il a n d p a lm fru it b u n c h a s h o n b e a n s e e d lin g s

The increased starch content observed in the WC fraction when compared to control maize may be a
switch to triglyceride degradation as lipids are those compounds which dissociated as primary metabolites
in stress condition to overcome the energy demand by the plant for growth (32). While the increased starch
content observed in the remediated fraction may be a compensatory rise by the nutrients present in APB.
The results suggests that crude oil may have inhibited the growth of the plant possibly because of the
decrease in starch and triglyceride content (5). However, the compensatory non-significant increase in
starch content in the APB added group, may be attributed to the favorable soil condition established by the
APB (7).
The effects of different crude oil fractions and APB on germination percentage, starch and triglyceride
contents in cowpea is shown in Table 3. The result shows that treatment of crude oil polluted soils with ash
from oil palm bunch resulted in remediation of the soil leading to enhanced germination percentage of
cowpea seeds (Table 3 and Figure 8). Several researches have observed that the presence of crude oil in
soils hinders germination of seeds and attributed this condition to the hydrophobic nature of the soils which
leads to unavailability of water and oxygen essential for seed germination (6, 15 & 33). In this study APB
enhanced germination percentage. This indicates that the hydrophobic nature of the soil may have been
reversed by the APB which may have resulted to water and oxygen being available for germination
enhancement thus improving the soil fertility. This result agrees with the study of other researchers for
enhancement of soil fertility with palm fruit ash (4, 7, 14 & 15).
The results shows that total protein content of cowpea 7 days post germination was significantly
improved in WC when compared with control and was considerably reduced in WIF than in the control.
The remediation of the various treated soils as well as the remediation of the control was progressively
increased compared to their non-remediated groups (P<0.05) (Figure 2).
The starch content in the treated groups WC and WIF were increased significantly as against the
control. The remediation of WC and WIF was able to increase significantly their starch content (P>0.05)
as well as the remediation only also increasing the starch level of the control (P<0.05) (Figure 4).
The triglyceride level of WC and WIF reduced considerably (P>0.05). The WC+R increased with
respect to WC as well as remediated only with respect to control. WIF+R also increased reasonably to WIF
(P<0.05) (Figure 6).

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 Biokemistri Volume 32, Number 1 (2020)

0 .2 5
C o n tro l

0 .2 0 R
WC
0 .1 5 W C+R
m g /g

W IF
0 .1 0
W IF + R

0 .0 5

0 .0 0

S ta rc h C o n te n t

F ig 3 : E ffe c t o f C r u d e o il a n d p a lm fr u it b u n c h a s h o n m a iz e s e e d lin g s

0 .1 5
C o n tro l
R
WC
0 .1 0
W C+R
m g /g

W IF

0 .0 5 W IF + R

0 .0 0

S ta rc h C o n te n t

F ig 4 : E ffe c t o f C ru d e o il a n d p a lm fru it b u n c h a s h o n b e a n s e e d lin g s

62
 S. O. Olubodun et al.

1 .0
C o n tro l

0 .8 R

-1
WC
0 .6 W C+R
g 100 g

W IF
0 .4
W IF + R

0 .2

0 .0

T rig ly c e rid e C o n te n t

F ig 5 : E ffe c t o f C ru d e o il a n d p a lm fr u it b u n c h a s h o n m a iz e s e e d lin g s

0 .8
C o n tro l
R
0 .6
WC
g 1 0 0 g -1

W C+R
0 .4
W IF
W IF + R
0 .2

0 .0

T rig ly c e rid e C o n te n t

F ig 6 : E ffe c t o f C ru d e o il a n d p a lm fru it b u n c h a s h o n b e a n s e e d lin g s

Plants exposed to crude oil normally are exposed to lower oxygen supply, reduced ATP generation,
and uses the fermentation process as a secondary route in plant metabolism for energy production (34). The
non-significant and significant increase in starch contents in the cowpea root of R, WC, WC-R, WIF and
WIF-R observed in the study are not in agreement with earlier reports that a reduction in oxygen supply
may lead to reduction in adenosine triphosphate (ATP) production which invariably leads to accelerated
sugar metabolism and glycolysis (28, 29 & 30). The decrease in lipid content of the cowpea root observed
in the study compares favourably with increase in glucose contents previously reported (31). This may
indicate that triglycerides were the main source of energy in the plant during crude oil exposure and not
starch in the case of cowpea seedling. Thus lipid was converted into glucose under stress condition as lipid
(storage carbohydrate) are dissociated as primary metabolites in stress condition to overcome the energy
demand by the plant for growth and to overcome oxygen deprivation. The decrease in lipid content may be
related to the toxicity of crude oil because starch helps the plant in providing energy in time of emergency
for survival that is if the energy supplied by carbohydrates is not enough or carbohydrate fails to supply
enough energy for survival.

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 Biokemistri Volume 32, Number 1 (2020)

150
C o n tro l
R

P e rc e n ta g e
100 WC
W C+R
W IF
50 W IF + R

G e r m in a t io n

F ig 7 : E ffe c t o f C r u d e o il a n d a s h fr o m p a lm b u n c h o n G e r m in a t io n % o f m a iz e s e e d lin g s

150
C o n tro l
P e rc e n ta g e (% )

R
100 WC
W C+R
W IF
50 W IF + R

G e r m in a tio n

F i g 8 : E f f e c t o f C r u d e o i l a n d a s h f r o m p a l m b u n c h o n g e r m i n a t io n % o f C o w p e a s e e d li n g s

The results suggested that lipid could be used by the growing plants to produce glucose as energy
source to sustain the metabolic activities occurring in the plant (32). The increased starch contents observed
in the fractions when compared to control cowpea may be a switch to triglyceride degradation as lipids are
those compounds which dissociated as primary metabolites in stress condition to overcome the energy
demand by the plant for growth (32). This may indicate that there was a positive relation between lipids
and growth in early stage of development of cowpea seedlings.
The inter-relationships between starch and triglycerides (TAG) biosynthesis are not clear. It has been
postulated that some species which accumulate both starch and TAG in developing embryos, starch
precedes TAG biosynthesis and in some species the level of starch decreases in parallel with TAG
accumulation, suggesting that the TAG may be synthesized from degradation of the starch (35, 36).
Research has shown that environmental stress induces degradation of carbohydrate that seems to be
temporally coordinated with biosynthesis of neutral lipids. The authors suggests that the findings represent
a carbon flow from the starch to the lipids (36). A recent study shows that whereas most starch is made
from assimilated CO2, most TAG are produced from acetate (36 & 37). These studies are consistent with
two parallel biosynthetic mechanisms, one for starch and another for TAG. However, none of these studies
referred to inter-conversions between starch and lipids or between polar and neutral lipids and their potential
contributions to TAG formation.

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 S. O. Olubodun et al.

The results presented in this study suggests that the plants may have responded to crude oil pollution
in two distinct metabolic phases as reported (36). Either the cells continue to divide slowly, photosynthetic
CO2 assimilation activity drops, or the cells accumulate massive levels of starch in the chloroplast,
accounting for over two-thirds of the total assimilated carbon or cell division almost stops, photosynthesis
drops, the starch level reaches a steady state, and TAG is produced, mostly by recycling of starch carbon.
So the general carbon metabolism switched from photosynthetic carbon assimilation to starch degradation
and the carbon reserves gradually change from all starch to progressively increasing TAG levels (36 & 38).
The variations observed between the two plants with or without the presence of APB amendment
implies that these crops has natural variations in their ecological and biological characteristics. Cowpea as
a legume for example, have bacteria in its root nodules which may aid in the degradation of crude oil thus
protecting the seedling (39) while maize as a cereal, may have the ability like other grasses, to be more
efficient for their fibrous root system with extensive root surface area for microbial colonization and dense
rhizosphere (40). This is consistent with previous reports of interspecies differences in sensitivity to crude
oil, and may be related to differences in systemic uptake of oil compounds, nutrient availability, and cell
wall structural differences (41).
The study, therefore, indicates that the starch and triglyceride contents were affected by the various
crude oil fractions, an indication that membrane integrity, energy production and viability may be affected
and that crude oil and its fraction affects starch and triglyceride contents in ways which are species related.
However, APB has the potentials for protecting and maintaining starch and triglyceride contents for the
seedlings in a crude oil polluted environment. The study suggests that APB could be used as fertiliser to
increase the pH and the nutrient contents of acidic soils and thus improve the soil fertility. However, further
studies is needed to elucidate the mechanism of alteration of starch and triglyceride contents in the plant
species.

Acknowledgements
The Authors wish to thank the University of Benin for the opportunity to carry out this research.

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Inhibition of some enzymes implicated in diabetes mellitus

by raw and blanched extracts of African lettuce (Launaea
taraxacifolia)
Bukola C. Adedayo*1, Sunday I. Oyeleye1,2 and Ganiyu Oboh1
1
Functional Foods and Nutraceuticals Unit of 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. Tel: +2348039115201; E-mail address: [email protected]

(Received January 18, 2020; Accepted March 9, 2020)

ABSTRACT: This study evaluates the impacts of blanching on α-amylase, α-glucosidase and lipase activities,
and Fe2+-induced lipid peroxidation (LPO) inhibitory potentials as well as phenolic constituents of African
lettuce (AL) leaves. The results revealed that both raw and blanched of AL extracts inhibited all the enzymes
dose-dependently, but extract from raw AL had the highest α-amylase (IC50 = 0.20 mg/mL), α-glucosidase (IC50
= 0.27 mg/mL) and lipase (IC50 = 0.45 mg/mL) inhibitory potential compared to that of blanched AL (α-
amylase, IC50 = 0.33 mg/mL; α-glucosidase IC50 = 0.52 mg/mL, lipase IC50 = 0.71 mg/mL). Raw AL extract
exhibited highest lipid peroxidation (IC50 = 1.01 mg/mL) inhibitory effect, scavenged DPPH (0.62 mg/mL) and
OH (0.40 mg/mL) radicals than the blanched (LPO, IC50 = 1.43 mg/mL; DPPH, 0.82 mg/mL; OH, 0.75 mg/mL).
Furthermore, catechin, caffeic and ellagic acid, epigallocatechin, rutin, isoquercitrin and quercetin were
identified in addition with gallic and chlorogenic acids and quercitrin in raw AL, while caffeic acid derivative
(13.40 mg/g) was detected in blanched AL only. Conclusively, blanching reduced the antioxidant and
antidiabetic potentials and phenolic contents of African lettuce leaves as evidence in the blanched AL.

Keywords: African lettuce, blanching, enzymes, antioxidant, polyphenols, diabetes mellitus.

Introduction

Diabetes mellitus is a known metabolic syndrome resulting from dysfunction of insulin

production (Ryu et al., 2013; Saravanan and Parimelazhagan, 2014). Deactivation/reduction of α-
amylase, α-glucosidase and lipase activities have been established as control points in the
management of diabetes mellitus (Ademiluyi et al., 2015; Naik et al., 2016). Consumption of dietary
food rich in phytochemicals, as alternative therapeutics is a reliable practice in managing the disease
(Ademiluyi et al., 2015; Adefegha et al., 2015; Adedayo et al., 2015). In many homes today,
consumption of vegetables is on the increase. The reason is because epidemiological studies have
positively linked the intake of vegetables, known to be rich in bioactive compounds such as
polyphenols, with effective management of diabetes mellitus.

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According to Ryu et al. (2013) and Ademiluyi et al. (2015), polyphenols, a secondary metabolite
phytochemicals, possessed insulin-like ability and could also prevent oxidation of biomolecules such
as lipids, protein and genetic materials which, if left unchecked, can induce oxidative stress (Adedayo
et al., 2015; Oboh et al., 2016). However, there are reports on the effects of processing, including
blanching, on the biological effect of polyphenols.
African lettuce (Launaea taraxacifolia, Family: Asteraceae) is a tropical leafy vegetable,
commonly consumed in Nigeria. The leaf is often used in folklore medicine as an alternative remedy
in the treatment of several human ailments, including diabetes (Adebisi, 2004; Adinortey et al., 2012),
but information on the mechanism of action is scarce. In Nigeria, African lettuce is blanched, before
consumption, purposely to increase the acceptability and palatability. However, till date, the effect of
blanching on the functional/nutraceutical values of African lettuce has not being evaluated. This study
aimed at evaluating the effects of blanching on α-amylase, α-glucosidase and lipase activities, and
Fe2+-induced lipid peroxidation inhibitory perennials of African lettuce including their constituents
phenolics

Materials and Methods

Chemical and reagents

Chemicals and reagents used in this study were of analytical grade and the water was glass
distilled. Kenxin (Model KX3400C) model of refrigerated centrifuge and JENWAY UV-visible
spectrophotometer (Model 6305; Jenway, Barlo World Scientific, Dunmow, United Kingdom) were
used to centrifuge and measure absorbance respectively.

Sample processing
Fresh leaves of African lettuce (AL) were harvested from the University Medicinal Farm Garden
between March and April 2018 and authenticated by A. A. Shorungbe (Voucher Number
FUTA/BIO/131). The leaves were divided into two (2) portions; the blanched (BAL) (treated for 5
min at 80°C), and raw (RAL). Both portions (RAL and BAL) were dried at 30°C and blended. The
extracts were prepared and the clear homogenate obtained was used.

Inhibition of α-amylase, α-glucosidase and lipase enzymes assays

Effect of the extracts on hog pancreatic α-amylase (0.5 mg/mL, EC 3.2.1.1) and α-glucosidase
activities was determined using Worthington Biochemical Corp (Worthington, 1993) and Apostolidis
et al. (2007) respectively. On lipase activity, Licia et al. (2006) method was used.

Antioxidant activity
The lipid peroxidation determination was according to the method of Ohkawa et al. (1979). The
method of Halliwell and Gutteridge (1981) was used for hydroxyl radical scavenging ability of the
extracts, while Puntel et al. (2015) method was used for Fe2+ chelating ability of the extracts

Spectrophotometric and HPLC-DAD analysis of phenolic in African lettuce

The total phenol content of the extracts was determined according to the method of Singleton et
al. (1999) and presented as gallic acid equivalent. The total flavonoid content follows Meda et al.
(2005), and calculated as quercetin equivalent. The quantification of phenolic compounds in the
extracts was carried out according to the method described by Adedayo et al (2016). using high
performance liquid chromatography coupled with Diode-Array Detector (HPLC-DAD).

Data analysis
The results of three replicates reading were pooled together and expressed as mean ± standard
deviation. One-way analysis of variance (ANOVA) and least significance difference (LSD) were
carried out. Duncan multiple range tests were used to carry out post hoc analysis. Concentration
needed to scavenge/inhibit 50% of radical/enzyme activity under the described assay conditions (IC50)
was calculated by nonlinear regression analysis. Significance was accepted at p ≤ 0.05.

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Results and Discussion

The effect of the extracts on some enzymes of carbohydrate metabolism are presented in Fig. 1A
and 1B while IC50 values are listed in Table 1. The extracts inhibited α-amylase activity, however, raw
AL extract exhibited higher α-amylase inhibitory potential than the extract from blanched AL. In a
similarly, extract from raw leaf of AL showed the highest α-glucosidase inhibitory potential than the
extract from BAL. The same manner of effect was also observed on pancreatic lipase activity where
extract from raw AL had higher inhibitory potential than the extract from blanched leaf of AL (Table
1)

Table 1. IC50 values of α-amylase and α-glucosidase, Fe2+- induced lipid peroxidation inhibitory
potentials, radicals (DPPH*, OH*) scavenging and Fe2+ chelating abilities of extracts from raw (RAL)
and blanched (BAL) African lettuce (mg/mL)

Parameters RAL BAL

α-Amylase inhibition 0.20±0.01a 0.33±0.04b

α-Glucosidase inhibition 0.27±0.01a 0.52±0.02b
Lipase inhibition 0.45±0.03a 0.71±0.07b
Fe2+ induced lipid peroxidation 1.01 ±0.03a 1.43±0.01b
DPPH* scavenging ability 0.62±0.02a 0.82±0.02b
OH* scavenging ability 0.40±0.05a 0.75±0.03b
Fe2+ chelating ability 0.25±0.01a 0.51±0.04b

Values represent means of triplicate. Values with the same alphabet along the same row are not significantly
different (P > 0.05)

Inhibition of carbo enzymes is one of the therapeutic approaches in managing diabetes as these
delays carbohydrate digestion and ultimately glucose absorption (Kumar et al., 2011; Ryu et al.,
2013; Adefegha et al., 2014; Ademiluyi et al., 2015). The inhibition of α-amylase and α-glucosidase
by the vegetable extract, which is in line with several reports, could be attributed to the presence of
phenolic compounds in the vegetables (Saliu and Oboh, 2013). More so, the higher α-amylase and α-
glucosidase inhibitory potential of the RAL extract correspond to its phenolics, notably, caffeic and
chlorogenic acids, and quercitrin (Oboh et al., 2014; Abirami et al., 2014; Adefegha et al., 2015).
Also, the synergistic effects of these phenolics could have influenced the strongest inhibitory effect of
raw extract.
Reports on inhibition of enzyme, such as pancreatic lipase, which is responsible for more than
70% hydrolysis of dietary fats, is also pivotal in the management of metabolic disorder (Birari and
Bhutani, 2007). Pancreatic lipase is responsible for the digestion and absorption of fatty composition
of food (You et al., 2012). Hence inhibition of pancreatic lipase is a valuable pathway for the
treatment of diet-induced hyperglycaemia in humans. As shown in Fig. 1C and Table 1, AL extracts
inhibited lipase activity, but RAL had the highest effect.

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1A 1B

1C

Figure 1: A-α-Amylase, B-α-Glucosidase and C-Pancreatic lipase inhibitory activity of extracts from raw and
blanched African lettuce. Values represent mean±standard deviation (N=3)

The extracts further inhibited MDA production, and scavenged hydroxyl and DPPH radicals,
chelate Fe2+ (Fig 2A, B and 2 respectively), and the extract from raw leaf had the highest effect (Table
1). In addition. Oxidative stress is responsible for many incidence and progression of diabetes and its
complications (Stephens et al., 2009). The increased level of MDA could be due to iron’s ability to
break down lipid peroxide, which in turn causes radicals formation and favours lipid peroxidation
(Bayir et al., 2006; Ademiluyi et al., 2014). Interestingly, the studied extracts inhibited lipid
peroxidation in the tissue homogenate, scavenged OH radical and, also chelate Fe2+, the effect that
could be linked to the presence phenolic compounds (Oboh et al., 2014; Adefegha et al., 2015).

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 B. C. Adedayo et al.

Consumption of dietary phenolic-rich foods such as the studied sample could be of use to strengthen
endogenous antioxidant (Adefegha et al., 2015; Oboh et al., 2018). The antiradicals and Fe2+ chelating
abilities of the studied extracts could be among the possible mechanisms through which the studied
samples prevent lipid peroxidation.

2A 2B

2C 2D

Figure 2A-Malonyladehyde content, (2B) DPPH radical and (2C) Hydroxyl (OH) radical scavenging abilities
and, (2D) Fe2+ Chelating ability of raw AL (RAL) and blanched AL (BAL). Values represent means of
triplicate.

HPLC-DAD analysis has advantage over colorimetric determination of phenolic compounds as it

reveals accurate information of individual compounds. In this study, gallic acid, catechin, chlorogenic,
caffeic and ellagic acid, epigallocatechin, rutin, isoquercitrin, quercitrin and quercetin were identified
in the studied samples On the account of total phenol and flavonoid contents (Table 2), Raw leaf’s
extract contained higher total phenol than the extract from blanched. Likewise, the total flavonoid
content was higher in the RAL compared to that of BAL. The HPLC-DAD phenolic profiles of
African lettuce (raw and blanched) (Tables 3 and Figure 6a and b) revealed the presence of phenolic
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acids and flavonoids. Gallic and chlorogenic acids and quercitrin were present only in the RAL while
caffeic acid was revealed only in BAL. Furthermore, raw is richer in caffeic acid, epigallocatechin,
quercetin while ellagic acid, rutin, and isoquercitrin were abundantly present in blanched extract when
compared (Table 2).

Raw Blanched

Figure 3. Representative high performance liquid chromatography profile of African lettuce leaves. (A) Raw
leaves (RAW, peaks 1- 10 represents Gallic acid, catechin, chlorogenic acid, caffeic acid,, ellagic acid,
epigallocatechin, rutin, isoquercitrin, quercitrin and quercetin respectively. (B) Blanched leaves (BAL, peaks 1-
8 represents catechin , caffeic acid , caffeic acid derivative , ellagic acid , epigallocatechin, rutin , isoquercitrin
and quercetin respectively..

Several preclinical studies revealed that polyphenols offer protection against many human
ailments most especially those triggered by oxidative stress such as diabetes and cardiovascular
diseases (González-Castejón and Rodriguez-Casado, 2011) via chelation of metals and abstraction of
free radicals whose formations have been linked to normal cellular metabolism (Amic et al., 2003).
However, thermal processing including blanching have been reported to increase/decrease the
phenolic content and antioxidative ability of vegetables (Valverdú-Queralt et al., 2011; Sharma and
Gujral, 2011; Gerard and Roberts, 2014). The decrease/loss of some phenolic constituents (Table 2)
and total phenolic contents (Table 3) in BAL extract could be accrued to the effect of the blanching
process and consequently responsible for the decrease in the enzymes inhibition and antioxidant
potentials. This loss of phenolics in the BAL extract is therefore in line with the report of (Ahmed and
Ali, 2013), that blanching caused loss of phenolics in Cauliflower, and further stated that the loss
could be as a result of covalent binding of amino acid with oxidized phenol and/or polymerization
reaction. Phenolics in vegetables exist in two forms: free and those that form complexes with cell wall
(bound phenolics). Hence, increased cooking temperatures and time causes cell walls disruption and
breakdown of phenolic compounds (Miglio et al., 2007; Ferracane, et al., 2008; Bunea et al., 2008;
Ahmed and Ali, 2013; Sharma and Gujral, 2011)

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Conclusion
Inhibition of key enzymes linked to diabetes and oxidative stress by AL, could be one of the
mechanisms behind its use in folkloric medicine. However, this study has shown that blanching
causes a reduction of its potentials, in relation to loss of some phenolic constituents as indicated in the
results with BAL. Therefore, blanching could reduce functional and nutraceutical values of AL.

Conflict of Interest
No Conflict of interest

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BKR 20200031/32108

Features, Evaluation and Treatment of Coronavirus

Disease 2019 (COVID-19)*
Marco Cascella1; Michael Rajnik2; Arturo Cuomo3; Scott C. Dulebohn; Raffaela Di
Napoli4.
1
Istituto Nazionale Tumori - IRCCS - Fondazione Pascale, Via Mariano Semmola 80100, Napoli. Italy
2
Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
3
Istituto Nazionale Tumori - IRCCS - Fondazione Pascale. Napoli, Italy
4
Institut Jules Bordet, Brussels, Belgium

Published Online March 20, 2020.

Introduction

According to the World Health Organization (WHO), viral diseases continue to emerge and
represent a serious issue to public health. In the last twenty years, several viral epidemics such as the
severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 to 2003, and H1N1 influenza in
2009, have been recorded. Most recently, the Middle East respiratory syndrome coronavirus (MERS-
CoV) was first identified in Saudi Arabia in 2012.
In a timeline that reaches the present day, an epidemic of cases with unexplained low respiratory
infections detected in Wuhan, the largest metropolitan area in China's Hubei province, was first reported
to the WHO Country Office in China, on December 31, 2019. Published literature can trace the
beginning of symptomatic individuals back to the beginning of December 2019. As they were unable
to identify the causative agent, these first cases were classified as "pneumonia of unknown etiology."
The Chinese Center for Disease Control and Prevention (CDC) and local CDCs organized an intensive
outbreak investigation program. The etiology of this illness is now attributed to a novel virus belonging
to the coronavirus (CoV) family.
On February 11, 2020, the WHO Director-General, Dr. Tedros Adhanom Ghebreyesus, announced
that the disease caused by this new CoV was a "COVID-19," which is the acronym of "coronavirus
disease 2019". In the past twenty years, two additional coronavirus epidemics have occurred. SARS-
CoV provoked a large-scale epidemic beginning in China and involving two dozen countries with
approximately 8000 cases and 800 deaths, and the MERS-CoV that began in Saudi Arabia and has
approximately 2,500 cases and 800 deaths and still causes as sporadic cases.

*This article was reproduced, with permission, from NCBI Bookshelf (https://www.ncbi.nlm.nih.gov/books/NBK554776/). It
is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution, and reproduction in
any medium or format, as long as appropriate credit is given to the original author(s).

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 Biokemistri Volume 32, Number 1 (2020)

This new virus seems to be very contagious and has quickly spread globally. In a meeting
on January 30, 2020, per the International Health Regulations (IHR, 2005), the outbreak was declared
by the WHO a Public Health Emergency of International Concern (PHEIC) as it had spread to 18
countries with four countries reporting human-to-human transmission. An additional landmark
occurred on February 26, 2020, as the first case of the disease, not imported from China, was recorded
in the United States.
Initially, the new virus was called 2019-nCoV. Subsequently, the task of experts of the
International Committee on Taxonomy of Viruses (ICTV) termed it the SARS-CoV-2 virus as it is very
similar to the one that caused the SARS outbreak (SARS-CoVs).
The CoVs have become the major pathogens of emerging respiratory disease outbreaks. They are
a large family of single-stranded RNA viruses (+ssRNA) that can be isolated in different animal
species.[1] For reasons yet to be explained, these viruses can cross species barriers and can cause, in
humans, illness ranging from the common cold to more severe diseases such as MERS and
SARS. Interestingly, these latter viruses have probably originated from bats and then moving into other
mammalian hosts — the Himalayan palm civet for SARS-CoV, and the dromedary camel for MERS-
CoV — before jumping to humans. The dynamics of SARS-Cov-2 are currently unknown, but there is
speculation that it also has an animal origin (Fig.1).

Fig. 1: Transmission Cycle of SARS CoV 2. Contributed by Rohan Bir Singh, MD; Made with
Biorender.com

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The potential for these viruses to grow to become a pandemic worldwide seems to be a serious
public health risk. Concerning COVID-19, the WHO raised the threat to the CoV epidemic to the "very
high" level, on February 28, 2020. Probably, the effects of the epidemic caused by the new CoV has yet
to emerge as the situation is quickly evolving. On March 11, as the number of COVID-19 cases outside
China has increased 13 times and the number of countries involved has tripled with more than 118,000
cases in 114 countries and over 4,000 deaths, WHO declared the COVID-19 a pandemic.
World governments are at work to establish countermeasures to stem possible devastating effects.
Health organizations coordinate information flows and issues directives and guidelines to best mitigate
the impact of the threat. At the same time, scientists around the world work tirelessly, and information
about the transmission mechanisms, the clinical spectrum of disease, new diagnostics, and prevention
and therapeutic strategies are rapidly developing. Many uncertainties remain with regard to both the
virus-host interaction and the evolution of the epidemic, with specific reference to the times when the
epidemic will reach its peak.
At the moment, the therapeutic strategies to deal with the infection are only supportive, and
prevention aimed at reducing transmission in the community is our best weapon. Aggressive isolation
measures in China have led to a progressive reduction of cases in the last few days. In Italy, in
geographic regions of the north, initially, and subsequently throughout the peninsula, political and
health authorities are making incredible efforts to contain a shock wave that is severely testing the health
system.
In the midst of the crisis, the authors have chosen to use the "Statpearls" platform because, within
the PubMed scenario, it represents a unique tool that may allow them to make updates in real-time. The
aim, therefore, is to collect information and scientific evidence and to provide an overview of the topic
that will be continuously updated.

Etiology

CoVs are positive-stranded RNA viruses with a crown-like appearance under an electron
microscope (coronam is the Latin term for crown) due to the presence of spike glycoproteins on the
envelope. The subfamily Orthocoronavirinae of the Coronaviridae family (order Nidovirales)
classifies into four genera of CoVs: Alphacoronavirus (alphaCoV), Betacoronavirus (betaCoV),
Deltacoronavirus (deltaCoV), and Gammacoronavirus (gammaCoV). Furthermore, the betaCoV genus
divides into five sub-genera or lineages.[2] Genomic characterization has shown that probably bats and
rodents are the gene sources of alphaCoVs and betaCoVs. On the contrary, avian species seem to
represent the gene sources of deltaCoVs and gammaCoVs.
Members of this large family of viruses can cause respiratory, enteric, hepatic, and neurological
diseases in different animal species, including camels, cattle, cats, and bats. To date, seven
human CoVs (HCoVs) — capable of infecting humans — have been identified. Some of HCoVs were
identified in the mid-1960s, while others were only detected in the new millennium.
In general, estimates suggest that 2% of the population are healthy carriers of a CoV and that these
viruses are responsible for about 5% to 10% of acute respiratory infections.[3]

• Common human CoVs: HCoV-OC43, and HCoV-HKU1 (betaCoVs of the A lineage); HCoV-
229E, and HCoV-NL63 (alphaCoVs). They can cause common colds and self-limiting upper
respiratory infections in immunocompetent individuals. In immunocompromised subjects and
the elderly, lower respiratory tract infections can occur.
• Other human CoVs: SARS-CoV, SARS-CoV-2, and MERS-CoV (betaCoVs of the B and C
lineage, respectively). These cause epidemics with variable clinical severity featuring respiratory
and extra-respiratory manifestations. Concerning SARS-CoV, MERS-CoV, the mortality rates
are up to 10% and 35%, respectively.

Thus, SARS-CoV-2 belongs to the betaCoVs category. It has round or elliptic and often
pleomorphic form, and a diameter of approximately 60–140 nm. Like other CoVs, it is sensitive to
ultraviolet rays and heat. Furthermore, these viruses can be effectively inactivated by lipid solvents

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including ether (75%), ethanol, chlorine-containing disinfectant, peroxyacetic acid and chloroform
except for chlorhexidine.
In genetic terms, Chan et al. have proven that the genome of the new HCoV, isolated from a cluster-
patient with atypical pneumonia after visiting Wuhan, had 89% nucleotide identity with bat SARS-like-
CoVZXC21 and 82% with that of human SARS-CoV[4]. For this reason, the new virus was called
SARS-CoV-2. Its single-stranded RNA genome contains 29891 nucleotides, encoding for 9860 amino
acids. Although its origins are not entirely understood, these genomic analyses suggest that SARS-CoV-
2 probably evolved from a strain found in bats. The potential amplifying mammalian host, intermediate
between bats and humans, is, however, not known. Since the mutation in the original strain could have
directly triggered virulence towards humans, it is not certain that this intermediary exists.

Transmission

Because the first cases of the CoVID-19 disease were linked to direct exposure to the Huanan
Seafood Wholesale Market of Wuhan, the animal-to-human transmission was presumed as the main
mechanism. Nevertheless, subsequent cases were not associated with this exposure mechanism.
Therefore, it was concluded that the virus could also be transmitted from human-to-human,
and symptomatic people are the most frequent source of COVID-19 spread. The possibility of
transmission before symptoms develop seems to be infrequent, although it cannot be excluded.
Moreover, there are suggestions that individuals who remain asymptomatic could transmit the virus.
This data suggests that the use of isolation is the best way to contain this epidemic.
As with other respiratory pathogens, including flu and rhinovirus, the transmission is believed to
occur through respiratory droplets from coughing and sneezing. Aerosol transmission is also possible
in case of protracted exposure to elevated aerosol concentrations in closed spaces. Analysis of data
related to the spread of SARS-CoV-2 in China seems to indicate that close contact between individuals
is necessary. The spread, in fact, is primarily limited to family members, healthcare professionals, and
other close contacts.
Based on data from the first cases in Wuhan and investigations conducted by the China CDC and
local CDCs, the incubation time could be generally within 3 to 7 days and up to 2 weeks as the longest
time from infection to symptoms was 12.5 days (95% CI, 9.2 to 18).[5] This data also showed that this
novel epidemic doubled about every seven days, whereas the basic reproduction number (R0 - R naught)
is 2.2. In other words, on average, each patient transmits the infection to an additional 2.2 individuals.
Of note, estimations of the R0 of the SARS-CoV epidemic in 2002-2003 were approximately 3.[6]
It must be emphasized that this information is the result of the first reports. Thus, further studies
are needed to understand the mechanisms of transmission, the incubation times and the clinical course,
and the duration of infectivity.

Epidemiology

Data provided by the WHO Health Emergency Dashboard (March 14, 06.00 am CET) report
142.320 confirmed cases worldwide since the beginning of the epidemic. 5.388 (3.78%) cases have
been fatal.
In china, 81.021 (57%) cases confirmed clinically and in the laboratory, and 3.173 deaths are
reported. In addition to China, there are 61.299 confirmed cases in 129 other countries. The countries
with most cases are Italy (17.660) and the Islamic Republic of Iran (11.364). The epidemiological
scenario, therefore, has drastically changed, as on March 3 about 92% (79.968) of the confirmed cases
were recorded in China, where almost all the deaths were also recorded (2,873, 96.5%). Of note, the
"confirmed" cases reported between February 13, 2020, and February 19, 2020, include both laboratory-
confirmed and clinically diagnosed patients from the Hubei province.
The most up-to-date source for the epidemiology of this emerging pandemic can be found at the
following sources:

• The WHO Novel Coronavirus (COVID-19) Situation Board

• The Johns Hopkins Center for Systems Science and Engineering site for Coronavirus Global Cases
COVID-19, which uses openly public sources to track the spread of the epidemic.

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Pathophysiology

CoVs are enveloped, positive-stranded RNA viruses with nucleocapsid. For

addressing pathogenetic mechanisms of SARS-CoV-2, its viral structure, and genome must be
considerations. In CoVs, the genomic structure is organized in a +ssRNA of approximately 30 kb in
length — the largest known RNA viruses — and with a 5′-cap structure and 3′-poly-A tail. Starting
from the viral RNA, the synthesis of polyprotein 1a/1ab (pp1a/pp1ab) in the host is realized. The
transcription works through the replication-transcription complex (RCT) organized in double-
membrane vesicles and via the synthesis of subgenomic RNAs (sgRNAs) sequences (Fig. 2). Of note,
transcription termination occurs at transcription regulatory sequences, located between the so-called
open reading frames (ORFs) that work as templates for the production of subgenomic mRNAs. In the
atypical CoV genome, at least six ORFs can be present (Fig. 3). Among these, a frameshift between
ORF1a and ORF1b guides the production of both pp1a and pp1ab polypeptides that are processed by
virally encoded chymotrypsin-like protease (3CLpro) or main protease (Mpro), as well as one or two
papain-like proteases for producing 16 non-structural proteins (nsps). Apart from ORF1a and ORF1b,
other ORFs encode for structural proteins, including spike, membrane, envelope, and nucleocapsid
proteins.[1] and accessory proteic chains. Different CoVs present special structural and accessory
proteins translated by dedicated sgRNAs.

Fig. 2: Covid 19, Corona Replication. Contributed by Rohan Bir Singh, MD

Pathophysiology and virulence mechanisms of CoVs, and therefore also of SARS-CoV-2 have
links to the function of the nsps and structural proteins. For instance, research underlined that nsp is
able to block the host innate immune response.[7] Among functions of structural proteins, the envelope
has a crucial role in virus pathogenicity as it promotes viral assembly and release. However, many of
these features (e.g., those of nsp 2, and 11) have not yet been described.

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Fig. 3: Single-stranded RNA genome of SARS-CoV2. Contributed by Rohan Bir Singh, MD; Made
with Biorender.com

Among the structural elements of CoVs, there are the spike glycoproteins composed of two
subunits (S1 and S2). Homotrimers of S proteins compose the spikes on the viral surface, guiding the
link to host receptors.[8] Of note, in SARS-CoV-2, the S2 subunit — containing a fusion peptide, a
transmembrane domain, and cytoplasmic domain — is highly conserved (Fig. 4). Thus, it could be a
target for antiviral (anti-S2) compounds. On the contrary, the spike receptor-binding domain presents
only a 40% amino acid identity with other SARS-CoVs. Other structural elements on which research
must necessarily focus are the ORF3b that has no homology with that of SARS-CoVs and a secreted
protein (encoded by ORF8), which is structurally different from those of SARS-CoV.
In international gene banks such as GenBank, researchers have published several Sars-CoV-2 gene
sequences. This gene mapping is of fundamental importance allowing researchers to trace the
phylogenetic tree of the virus and, above all, the recognition of strains that differ according to the
mutations. According to recent research, a spike mutation, which probably occurred in late November
2019, triggered jumping to humans. In particular, Angeletti et al. compared the Sars-Cov-2 gene
sequence with that of Sars-CoV. They analyzed the transmembrane helical segments in the ORF1ab
encoded 2 (nsp2) and nsp3 and found that position 723 presents a serine instead of a glycine residue,
while the position 1010 is occupied by proline instead of isoleucine.[9] The matter of viral mutations is
key for explaining potential disease relapses.
Research will be needed to determine the structural characteristics of SARS-COV-2 that underlie
the pathogenetic mechanisms. Compared to SARS, for example, initial clinical data show less extra
respiratory involvement, although due to the lack of extensive data, it is not possible to draw definitive
clinical information.

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Fig. 4: SARS- CoV 2 Structure. Contributed by Rohan Bir Singh, MD; Made with Biorender.com

The pathogenic mechanism that produces pneumonia seems to be particularly complex. Clinical
and preclinical research will have to explain many aspects that underlie the particular clinical
presentations of the disease (see Fig. 5). The data so far available seem to indicate that the viral infection
is capable of producing an excessive immune reaction in the host. In some cases, a reaction takes place
which as a whole is labeled a 'cytokine storm'. The effect is extensive tissue damage. The protagonist
of this storm is interleukin 6 (IL-6). IL-6 is produced by activated leukocytes and acts on a large number
of cells and tissues. It is able to promote the differentiation of B lymphocytes, promotes the growth of
some categories of cells, and inhibits the growth of others. It also stimulates the production of acute
phase proteins and plays an important role in thermoregulation, in bone maintenance and in the
functionality of the central nervous system. Although the main role played by IL-6 is pro-inflammatory,
it can also have anti-inflammatory effects. In turn, IL-6 increases during inflammatory diseases,
infections, autoimmune disorders, cardiovascular diseases and some types of cancer. It is also
implicated into the pathogenesis of the cytokine release syndrome (CRS) that is an acute systemic
inflammatory syndrome characterized by fever and multiple organ dysfunction.

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Fig. 5: Clinical Presentation of Patients with CoVID-19. Contributed by Rohan Bir Singh, MD; Made
with Biorender.com

Histopathology

Tian et al.[10] and others reported histopathological data obtained on the lungs of two patients
who underwent lung lobectomies for adenocarcinoma and retrospectively found to have had the
infection at the time of surgery. Apart from the tumors, the lungs of both 'accidental' cases showed
edema and important proteinaceous exudates as large protein globules. The authors also reported
vascular congestion combined with inflammatory clusters of fibrinoid material and multinucleated giant
cells and hyperplasia of pneumocytes.

History and Physical

The clinical spectrum of COVID-19 varies from asymptomatic or paucisymptomatic forms to

clinical conditions characterized by respiratory failure that necessitates mechanical ventilation and
support in an intensive care unit (ICU), to multiorgan and systemic manifestations in terms of sepsis,
septic shock, and multiple organ dysfunction syndromes (MODS). In one of the first reports on the
disease, Huang et al. illustrated that patients (n. 41) suffered from fever, malaise, dry cough, and
dyspnea. Chest computerized tomography (CT) scans showed pneumonia with abnormal findings in all
cases. About a third of those (13, 32%) required ICU care, and there were 6 (15%) fatal cases.[11]

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The case studies of Li et al. published in the New England Journal of Medicine (NEJM) on January
29, 2020, encapsulates the first 425 cases recorded in Wuhan.[5] Data indicate that the patients' median
age was 59 years, with a range of 15 to 89 years. Thus, they reported no clinical cases in children below
15 years of age. There were no significant gender differences (56% male). Clinical and
epidemiological data from the Chinese CDC and regarding 72,314 case records (confirmed, suspected,
diagnosed, and asymptomatic cases) were shared in the Journal of the American Medical Association
(JAMA) (February 24, 2020), providing an important illustration of the epidemiologic curve of the
Chinese outbreak.[12] There were 62% confirmed cases, including 1% of cases that were
asymptomatic, but were laboratory-positive (viral nucleic acid test). Furthermore, the overall case-
fatality rate (on confirmed cases) was 2.3%. Of note, the fatal cases were primarily elderly patients, in
particular those aged ≥ 80 years (about 15%), and 70 to 79 years (8.0%). Approximately half (49.0%)
of the critical patients and affected by preexisting comorbidities such as cardiovascular disease,
diabetes, chronic respiratory disease, and oncological diseases, died. While 1% of patients were aged 9
years or younger, no fatal cases occurred in this group.
The authors of the Chinese CDC report divided the clinical manifestations of the disease by their
severity:

• Mild disease: non-pneumonia and mild pneumonia; this occurred in 81% of cases.
• Severe disease: dyspnea, respiratory frequency ≥ 30/min, blood oxygen saturation (SpO2) ≤ 93%,
PaO2/FiO2 ratio or P/F [the ratio between the blood pressure of the oxygen (partial pressure of
oxygen, PaO2) and the percentage of oxygen supplied (fraction of inspired oxygen, FiO2)] < 300,
and/or lung infiltrates > 50% within 24 to 48 hours; this occurred in 14% of cases.
• Critical disease: respiratory failure, septic shock, and/or multiple organ dysfunction (MOD) or
failure (MOF); this occurred in 5% of cases.[12]

Data obtainable from reports and directives provided by health policy agencies, allow dividing the
clinical manifestations of the disease according to the severity of the clinical pictures. The COVID-
19 may present with mild, moderate, or severe illness. Among the severe clinical manifestations, there
are severe pneumonia, ARDS, sepsis, and septic shock. The clinical course of the disease seems to
predict a favorable trend in the majority of patients. In a percentage still to be defined of cases, after
about a week there is a sudden worsening of clinical conditions with rapidly worsening respiratory
failure and MOD/MOF. As a reference, the criteria of the severity of respiratory insufficiency and the
diagnostic criteria of sepsis and septic shock can be used.[13]

Uncomplicated (mild) Illness

These patients usually present with symptoms of an upper respiratory tract viral infection,
including mild fever, cough (dry), sore throat, nasal congestion, malaise, headache, muscle pain, or
malaise. Signs and symptoms of a more serious disease, such as dyspnea, are not present. Compared to
previous HCoV infections, non-respiratory symptoms such as diarrhea are challenging to find.

Moderate Pneumonia

Respiratory symptoms such as cough and shortness of breath (or tachypnea in children) are present
without signs of severe pneumonia.

Severe Pneumonia
Fever is associated with severe dyspnea, respiratory distress, tachypnea (> 30 breaths/min), and
hypoxia (SpO2 < 90% on room air). However, the fever symptom must be interpreted carefully as even
in severe forms of the disease, it can be moderate or even absent. Cyanosis can occur in children. In this
definition, the diagnosis is clinical, and radiologic imaging is used for excluding complications.

Acute Respiratory Distress Syndrome (ARDS)

The diagnosis requires clinical and ventilatory criteria. This syndrome is suggestive of a serious
new-onset respiratory failure or for worsening of an already identified respiratory picture. Different

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forms of ARDS are distinguished based on the degree of hypoxia. The reference parameter is the
PaO2/FiO2:

• Mild ARDS: 200 mmHg < PaO2/FiO2 ≤ 300 mmHg. In not-ventilated patients or in those managed
through non-invasive ventilation (NIV) by using positive end-expiratorypressure (PEEP) or
a continuous positive airway pressure (CPAP) ≥ 5 cmH2O.
• Moderate ARDS: 100 mmHg < PaO2/FiO2 ≤ 200 mmHg.
• Severe ARDS: PaO2/FiO2 ≤ 100 mmHg.

When PaO2 is not available, a ratio SpO2/FiO2 ≤ 315 is suggestive of ARDS. Chest imaging
utilized includes chest radiograph, CT scan, or lung ultrasound demonstrating bilateral opacities (lung
infiltrates > 50%), not fully explained by effusions, lobar, or lung collapse. Although in some cases, the
clinical scenario and ventilator data could be suggestive for pulmonary edema, the primary
respiratory origin of the edema is proven after the exclusion of cardiac failure or other causes such as
fluid overload. Echocardiography can be helpful for this purpose.

Sepsis

According to the International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3),
sepsis represents a life-threatening organ dysfunction caused by a dysregulated host response to
suspected or proven infection, with organ dysfunction.[14] The clinical pictures of patients with
COVID-19 and with sepsis are particularly serious, characterized by a wide range of signs and
symptoms of multiorgan involvement. These signs and symptoms include respiratory manifestations
such as severe dyspnea and hypoxemia, renal impairment with reduced urine output, tachycardia,
altered mental status, and functional alterations of organs expressed as laboratory data of
hyperbilirubinemia, acidosis, high lactate, coagulopathy, and thrombocytopenia. The reference for the
evaluation of multiorgan damage and the related prognostic significance is the Sequential Organ Failure
Assessment (SOFA) score, which predicts ICU mortality based on lab results and clinical data.[15] A
pediatric version of the score has also received validation.[16]

Septic Shock

In this scenario, which is associated with increased mortality, circulatory, and cellular/metabolic
abnormalities such as serum lactate level greater than 2 mmol/L (18 mg/dL) are present. Because
patients usually suffer from persisting hypotension despite volume resuscitation, the administration of
vasopressors is required to maintain a mean arterial pressure (MAP) ≥ 65 mmHg.

THE PECULIAR HISTORY OF THIS NEW DISEASE

In some patients, the clinical history of this disease occurs with particular characteristics. It
foresees that the patient manifests above all fever, which is not very responsive to antipyretics, and a
state of malaise. A dry cough is often associated. After 5-7 days, older patients with already impaired
lung function begin to experience shortness of breath and increased respiratory rate. In more fragile
patients, however, dyspnea may already appear at the onset of symptoms. On the other hand, in younger
subjects and in those who do not have basic respiratory impairments or other comorbidities, dyspnea
may appear later. In these patients experiencing worsening inflammatory-induced lung injury, there is
a decrease in oxygen saturation (<93%). This seems to be the crucial phase of the disease, from this
point onwards, there may be a rapid deterioration of respiratory functions. The scenario is truly
incredible because for patients who are paucisymptomatic and slightly hypoxic, the first therapeutic
approach is oxygen therapy. Although this strategy is effective, worsening of respiratory failure may
occur in some patients. With the drive preserved, the next step, according to logic, is the NIV. This
therapy has a rapid success by increasing the P/F. In some patients, however, there is a sudden,
unexpected worsening of clinical conditions. Patients collapse under the operator's eyes and require
rapid intubation and invasive mechanical ventilation. However, after 24-48 hours the patient can have
a rapid improvement with an increase in P/F. Operators are therefore tempted to proceed with weaning.

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But very often, after an initial success there is a new worsening of respiratory conditions, such as to
require a new invasive therapy. Therefore, mechanical ventilation has also been suggested for 1-2
weeks.

THE MESSAGE FOR THOSE WHO TREAT THESE PATIENTS IS THEREFORE TO

FOLLOW THE CLINICAL PERFORMANCE FOCUSING ESPECIALLY ON THE VALUES
OF THE SATURIMETRY, RATHER THAN CONSIDERING THE TYPICAL P/F VALUES
SUGGESTIVE OF ARDS.

Evaluation
Most countries are utilizing some type of clinical and epidemiologic information to determine who
should have testing performed. In the United States, criteria have been developed for persons under
investigation (PUI) for COVID-19. According to the U.S. CDC, most patients with confirmed COVID-
19 have developed fever and/or symptoms of acute respiratory illness (e.g., cough, difficulty breathing).
If a person is a PUI, it is recommended that practitioners immediately put in place infection control and
prevention measures. Initially, they recommend testing for all other sources of respiratory infection.
Additionally, they recommend using epidemiologic factors to assist in decision making. There are
epidemiologic factors that assist in the decision on who to test. This includes anyone who has had close
contact with a patient with laboratory-confirmed COVID-19 within 14 days of symptom onset or a
history of travel from affected geographic areas (presently China, Italy, Iran, Japan, and South Korea)
within 14 days of symptom onset.
The WHO recommends collecting specimens from both the upper respiratory tract (naso- and
oropharyngeal samples) and lower respiratory tract such as expectorated sputum, endotracheal aspirate,
or bronchoalveolar lavage. The collection of BAL samples should only be performed in mechanically
ventilated patients as lower respiratory tract samples seem to remain positive for a more extended
period. The samples require storage at four degrees celsius. In the laboratory, amplification of the
genetic material extracted from the saliva or mucus sample is through a reverse polymerase chain
reaction (RT-PCR), which involves the synthesis of a double-stranded DNA molecule from an RNA
mold. Once the genetic material is sufficient, the search is for those portions of the genetic code of the
CoV that are conserved. The probes used are based on the initial gene sequence released by
the Shanghai Public Health Clinical Center & School of Public Health, Fudan University, Shanghai,
China on Virological.org, and subsequent confirmatory evaluation by additional labs. If the test result
is positive, it is recommended that the test is repeated for verification. In patients with confirmed
COVID-19 diagnosis, the laboratory evaluation should be repeated to evaluate for viral clearance prior
to being released from observation.
The availability of testing will vary based on which country a person lives in with increasing
availability occurring nearly daily.
Concerning laboratory examinations, in the early stage of the disease, a normal or decreased total
white blood cell count and a decreased lymphocyte count can be demonstrated. Lymphopenia appears
to be a negative prognostic factor. Increased values of liver enzymes, LDH, muscle enzymes, and C-
reactive protein can be found. There is a normal procalcitonin value. In critical patients, D-dimer
value is increased, blood lymphocytes decreased persistently, and laboratory alterations of multiorgan
imbalance (high amylase, coagulation disorders, etc.) are found.

Treatment / Management

There is no specific antiviral treatment recommended for COVID-19, and no vaccine is currently
available. The treatment is symptomatic, and oxygen therapy represents the major treatment
intervention for patients with severe infection. Mechanical ventilation may be necessary in cases of
respiratory failure refractory to oxygen therapy, whereas hemodynamic support is essential for
managing septic shock.
On January 28, 2020, the WHO released a document summarizing WHO guidelines and scientific
evidence derived from the treatment of previous epidemics from HCoVs. This document addresses
measures for recognizing and sorting patients with severe acute respiratory disease; strategies
for infection prevention and control; early supportive therapy and monitoring; a guideline for laboratory

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diagnosis; management of respiratory failure and ARDS; management of septic shock; prevention of
complications; treatments; and considerations for pregnant patients. Among these recommendations,
we report the strategies for addressing respiratory failure, including protective mechanical ventilation
and high-flow nasal oxygen (HFNO) or non-invasive ventilation (NIV).

Intubation and protective mechanical ventilation

Special precautions are necessary during intubation. The procedure should be executed by an
expert operator who uses personal protective equipment (PPE) such as FFP3 or N95 mask, protective
goggles, disposable gown long sleeve raincoat, disposable double socks, and gloves. If possible, rapid
sequence intubation (RSI) should be performed. Preoxygenation (100% O2 for 5 minutes) should be
performed via the continuous positive airway pressure (CPAP) method. Heat and moisture exchanger
(HME) must be positioned between the mask and the circuit of the fan or between the mask and the
ventilation balloon.
Mechanical ventilation should be with lower tidal volumes (4 to 6 ml/kg predicted body weight,
PBW) and lower inspiratory pressures, reaching a plateau pressure (Pplat) < 28 to 30 cm H2O. PEEP
must be as high as possible to maintain the driving pressure (Pplat-PEEP) as low as possible (< 14
cmH2O). Moreover, disconnections from the ventilator must be avoided for preventing loss of PEEP
and atelectasis. Finally, the use of paralytics is not recommended unless PaO2/FiO2 < 150 mmHg. The
prone ventilation for > 12 hours per day, and the use of a conservative fluid management strategy for
ARDS patients without tissue hypoperfusion (strong recommendation) are emphasized.

Non-invasive ventilation

Concerning HFNO or non-invasive ventilation (NIV), the experts' panel, points out that these
approaches performed by systems with good interface fitting do not create widespread dispersion of
exhaled air, and their use can be considered at low risk of airborne transmission.[17] Practically, non-
invasive techniques can be used in non-severe forms of respiratory failure. However, if the scenario
does not improve or even worsen within a short period of time (1–2 hours) the mechanical ventilation
must be preferred.

Other therapies

Among other therapeutic strategies, systemic corticosteroids for the treatment of viral pneumonia
or acute respiratory distress syndrome (ARDS) are not recommended. Moreover, unselective or
inappropriate administration of antibiotics should be avoided, although some centers recommend it.
Although no antiviral treatments have been approved, several approaches have been proposed such as
lopinavir/ritonavir (400/100 mg every 12 hours), chloroquine (500 mg every 12 hours), and
hydroxychloroquine (200 mg every 12 hours). Alpha-interferon (e.g., 5 million units by aerosol
inhalation twice per day) is also used.
Preclinical studies suggested that remdesivir (GS5734) — an inhibitor of RNA polymerase with
in vitro activity against multiple RNA viruses, including Ebola — could be effective for both
prophylaxis and therapy of HCoVs infections.[18] This drug was positively tested in a rhesus macaque
model of MERS-CoV infection.[19]
In Italy, a great investigation led by the Istituto Nazionale Tumori, Fondazione Pascale di Napoli
is focused on the use of tolicizumab. It is a humanized IgG1 monoclonal antibody, directed against
the IL-6 receptor and commonly used in the treatment of rheumatoid arthritis.
When the disease results in complex clinical pictures of MOD, organ function support in addition
to respiratory support, is mandatory. Extracorporeal membrane oxygenation (ECMO) for patients with
refractory hypoxemia despite lung-protective ventilation should merit consideration after a case-by-
case analysis. It can be suggested for those with poor results to prone position ventilation.

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Prevention

Preventive measures are the current strategy to limit the spread of cases. Because an epidemic will
increase as long as R0 is greater than 1 (COVID-19 is 2.2), control measures must focus on reducing
the value to less than 1.
Preventive strategies are focused on the isolation of patients and careful infection control,
including appropriate measures to be adopted during the diagnosis and the provision of clinical care to
an infected patient. For instance, droplet, contact, and airborne precautions should be adopted
during specimen collection, and sputum induction should be avoided.

The WHO and other organizations have issued the following general recommendations:

• Avoid close contact with subjects suffering from acute respiratory infections.
• Wash your hands frequently, especially after contact with infected people or their environment.
• Avoid unprotected contact with farm or wild animals.
• People with symptoms of acute airway infection should keep their distance, cover coughs or
sneezes with disposable tissues or clothes and wash their hands.
• Strengthen, in particular, in emergency medicine departments, the application of strict hygiene
measures for the prevention and control of infections.
• Individuals that are immunocompromised should avoid public gatherings.

The most important strategy for the populous to undertake is to frequently wash their hands and
use portable hand sanitizer and avoid contact with their face and mouth after interacting with a possibly
contaminated environment.
Healthcare workers caring for infected individuals should utilize contact and airborne precautions
to include PPE such as N95 or FFP3 masks, eye protection, gowns, and gloves to prevent transmission
of the pathogen.
Meanwhile, scientific research is growing to develop a coronavirus vaccine. In recent days, China
has announced the first animal tests, and researchers from the University of Queensland in Australia
have also announced that, after completing the three-week in vitro study, they are moving on to animal
testing. Furthermore, in the U.S., the National Institute for Allergy and Infectious Diseases (NIAID)
has announced that a phase 1 trial has begun for a novel coronavirus immunization in Washington State.

Differential Diagnosis

The symptoms of the early stages of the disease are nonspecific. Differential diagnosis should
include the possibility of a wide range of infectious and non-infectious (e.g., vasculitis,
dermatomyositis) common respiratory disorders.

• Adenovirus
• Influenza
• Human metapneumovirus (HmPV)
• Parainfluenza
• Respiratory syncytial virus (RSV)
• Rhinovirus (common cold)

For suspected cases, rapid antigen detection, and other investigations should be adopted for
evaluating common respiratory pathogens and non-infectious conditions.

Pertinent Studies and Ongoing Trials

Multiple studies globally are investigating the use of remdesivir, a broad-spectrum antiviral.

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Prognosis

Preliminary data suggests the reported death rate ranges from 1% to 2% depending on the study
and country. The majority of the fatalities have occurred in patients over 50 years of age. Young children
appear to be mildly infected but may serve as a vector for additional transmission.

Complications

Long term complications among survivors of infection with SARS-CoV-2 having clinically
significant COVID-19 disease are not yet available. The mortality rates for cases globally remain
between 1% to 2%.

Deterrence and Patient Education

Patients and families should receive instruction to:

• Avoid close contact with subjects suffering from acute respiratory infections.
• Wash their hands frequently, especially after contact with sick people or their environment.
• Avoid unprotected contact with farm or wild animals.
• People with symptoms of acute airway infection should keep their distance, cover coughs or
sneezes with disposable tissues or clothes and wash their hands.
• Immunocompromised patients should avoid public exposure and public gatherings. If an
immunocompromised individual must be in a closed space with multiple individuals present, such
as a meeting in a small room; masks, gloves, and personal hygiene with antiseptic soap should be
undertaken by those in close contact with the individual. In addition, prior room cleaning with
antiseptic agents should be undertaken and performed before exposure. However, considering the
danger involved to these individuals, exposure should be avoided unless a meeting, group event,
etc. is a true emergency.
• Strict personal hygiene measures are necessary for the prevention and control of this infection.

Enhancing Healthcare Team Outcomes

Since the first outbreak of coronavirus (COVID-19) in Wuhan, China, the disease is spreading
worldwide. Individuals at the extreme of ages and those that are immunocompromised are at the most
significant risk. All health care workers should understand the presentation of the disease, workup, and
supportive care. Further, health professionals should be aware of the precautions necessary to avoid the
contraction and spread of the disease. [Level 5]

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