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The present study investigates the phytochemical and
acute toxicity studies of the methanol extract of Napoleonae imperialis
leaves in albino rats. The lorke’s method was used for the acute toxicity study
of which eighteen (18) male albino rats were used. In the first phase, the rats
were divided into three groups of three rats each and received the extract at a
dose of 10 mg/kg, 100 mg/kg and 1000 mg/kg. In the second phase, the rats were
divided into three groups of three rats each and received the extract at a dose
of 1600 mg/kg, 2900 mg/kg and 5000 mg/kg. The animals were observed for general
signs and symptoms of toxicity including mortality over a period of 48 h. The
acute toxicity study (LD50) showed no adverse effect in their general behavior
and mortality at the dose level given, which suggest that the leaf might be
generally regarded as safe with no remote risk of acute intoxication. The
phytochemical analysis showed the presence of phenol (5807.79 ± 210.87),
tannins (71.49 ± 4.79), glycosides (56.97 ± 1.59), alkaloids (607.22 ± 14.42),
reducing sugar (1853.26 ± 178.63), saponin (6.06 ± 0.03), carbohydrates
(15547.83 ± 729.84), flavonoids (2104.38 ± 39.29), steroids (19.44 ± 1.57) and
terpenoids (101.69 ± 3.88). Lately, the usages of phytochemicals are of
particular interests in several disease managements, as they present natural
means to suppressing of genetic transcription via some epigenetics mechanisms.
The presences of some of these biological molecules in this plant justify to a
large extent, some of the ethno- medicinal applications of Napoleonae
imperialis.
Keywords: Acute toxicity, Phytochemical
analysis, Phenol, Flavonoids, Steroids, Epigenetics
INTRODUCTION
Napoleonae
imperialis is a
small, evergreen tropical West African tree and it belongs to the family of
lecythidaceae, native to Africa [1]. It grows averagely to 6 m height with a
dense, and low branching crown. The showy flowers have two inner rows of petal
and vary in color, usually creamy yellow, along the circumference, with the
center varying from red to apricot to purple. They develop either as tender
trunks or from the ancient wood of the branch. The fruit is a berry, dark
orange or reddish brown containing kink a kidney shaped seed. The specie is
popularly cultivated as an ornamental tree [2].
Using Napoleonae
imperialis as a medicinal plant
in an earlier study [3], it demonstrates
antibacterial and wound healing properties in albino rats. In another
experiment [4], prepared an herbal
ointment of the methanol solution of Napoleonae
imperialis and examined its wound healing effect by the excision wound
model on guinea pigs. The result of the experiment indicates that Napoleonae imperialis extract possess a
better wound healing property as compared to the antibiotic used as control.
Other plants that had shown wound healing effect includes the following: Kaempferia galangal, Radix paeoniae,
Prosopis cineraria, Trigonella foenum, Lawsonia alba (Lynthraceae), Pterocarpus
santalinus, Clerodendum seratum, Ginkgo biloba, Euphorbia hirta, Cecropia
pellata, Cathranthus roseus, Sesamum indicum, Lycopodium serratum, Morinda
clitrifolia, Arternanthera sessilis, etc.
MATERIALS &
METHODS
Plant material collection
A fresh leaf of the plant Napoleonae
imperialis were locally sourced in Abia State, Nigeria and was identified at the Plant Science and Biotechnology
Department, Michael Okpara Uiversity of Agriculture, Umudike, Nigeria. The
fresh leaves were washed and dried under shade at room temperature, using a
blender; the leaves were blended into powder.
Extraction
The powdered leaves of Napoleonae imperialis (100 g) were soaked in methanol for 48
h and the extract filtered using a Whatman
no. 1 filter paper, the filtrate was allowed to evaporate to dryness, under a
water bath with a temperature set at 40°C.
Animals
Male albino rats of mean weight
of 130 g were used for the study. The animals were kept in the animal house
(Michael Okpara University of Agriculture, Umudike, Nigeria), allowed to acclimatized
for two (2) weeks, and were
used, following the approval of institutional animal ethical committee. Commercial pellet diet (Vital growers mash; Grand
Cereals and Oil Mills, Nigeria) and water were given to the animals ad libitum.
Phytochemical analysis of the methanol
extract of Napoleonae imperialis leaves
The preliminary phytochemical
screening of the methanol extract of Napoleonae
imperialis leaves were carried out, to ascertain the presence of
phytochemicals, those detected were quantified. Both qualitative and
quantitative analyses were done using standard methods previously described [5].
Acute Toxicity
Studies
Lethal dose (LD50) determination
was conducted using the Lorke’s method. Nine mice, divided into 3 groups. The
first group received the extract (i.p.) at a dose of 1000 mg/kg b.wt,
group 2 received the extract at a dose of 100 mg/kg b.wt (i.p.), while
the last group received the extract at the dose of 10 mg/kg body weight.
Animals were observed for general signs and symptoms of toxicity including mortality
over a period of 24 h. In the second phase 9 mice were divided into 3 groups.
The extract was administered at a dose of 1600, 2900, and 5000 mg/kg b.wt (i.p.)
respectively. Based on the result of the first phase, the final LD50
was calculated as the square root of the geometrical mean of the highest
non-lethal dose and the lowest lethal dose [6].
Using the formu
la
below, the LD50 was calculated:
D0 =
Highest dose that gave no mortality,
D100 =
Lowest dose that produced mortality
RESULTS
The present study investigated the
phytochemical properties of Napoleonae
imperialis. The Table 1 and Table 2 show the results of the
qualitative and quantitative composition of the powdered leaf extract.
Acute
toxicity and lethality (ld50) test
Oral administration of
up to 5000 mg/kg body weight of methanol extract of Napoleonae imperialis leaves to mice caused no death in the two stages of the test. Thus,
oral LD50 of the extract in mice was
estimated to be greater than 5000 mg/kg body weight, which suggests that the
leaf may be generally regarded as safe with a remote risk of acute
intoxication. The high degree of safety is also consistent [7] and its popular
use as herbs in some part of Nigeria. The Table 3 shows the lethal
dosage of the extract, using albino rats as experimental animals.
DISCUSSION
These stud(ies)y
revealed that, the oral administration of 5000 mg/kg body weight of the Napoleonae
imperialis methanol leaf extract, did not affect the rat and thus no acute
toxicity or instant death in any of the rats treated with high dosage during
the observation period. The median lethal dose (LD50) of the present
study is in agreement with earlier studies [8,9]. These two earlier studies
reported the LD50 of this plant to be greater than 4000 mg/kg and
2000 mg/kg b.wt respectively. The results from the present study suggested that
oral administration of the methanol extract of Napoleonae imperialis could be considered safe, which may be
attributed to low or absence of toxic constituents in the methanol extract.
In the present
study, phytochemical analysis of the methanol leaf extract of Napoleonae
imperialis showed the presence of alkaloids, tannins, terpenoids, reducing
sugar, glycosides and flavonoids in moderate quantities, while saponins, acidic
compounds and steroids were in low amount whilst, carbohydrates and phenols
were present in abundance, all these bioactive components have been reported previously to
have hepatoprotective and antioxidant activities [10,11,12].
Experimental
evidence had shown earlier that flavonoids possess antimicrobial, antibacterial
and HIV-inhibitory activities [13]. The alkaloids are responsible for reducing
the blood pressure and are thus known for the hypotensive effect of Napoleonae
imperialis leaf extract
[14]. Plants composed of saponins are considered to possess antioxidant, anti-cancer,
anti-inflammatory and anti-viral effects [15]. Other studies have attributed
some of these activities to the presence of alkaloids, flavonoids, polyphenols
and reducing sugars [15]. The presences of these biological molecules justify
some of the reasons, for the ethno-medicinal applications of Napoleonae
imperialis [2].
Phytocompounds as epigenetic regulators
Current understanding clearly indicated that
the genome contains information in two forms, i.e., genetic and epigenetic.
Whilst the genetic information gives the blueprint for the transcription and
translation of all the proteins necessary for both prokaryotic and eukaryotic
cells, the epigenetic information provides instructions on the general
direction such as; when, where and how the genetic information should be used.
With high throughput screening and available
molecular targeting techniques, precision targeting is now possible. Thus,
using such, the modulation of DNA
methylation, histone and chromatin modification, an epigenetic directional
processes in cancer via phytocompounds that altered expression of tumor
suppressor genes have been identified and discussed [16, 17].
Foods and herbal medicine high in flavonoids,
phenols and other bioactive compound characterized in the Napoleonae imperialis leaves, have been implicated in altering
histone acetylation, thus modulating gene expression [16]. Gene expression is known to be regulated
by epigenes, which occur following post-translational modification (PTM) of
histone proteins, involving; acetylation, ADP ribosylation. methylation,
ubiquitinylation, phosphorylation and sumoylation. Of these processes,
methylation and acetylation of histone, all, which are potential targets for
bioactive compound, seems to be the most common PTM event associated with
carcinogenesis [17]. These processes, underscore the role bioactive compound
could play in managing complex diseases such as cancer (which possess variant
phenotypes), if and when properly harnessed. A major drawback of phyto extracts
are the likelihood of adulteration with toxic agents such as heavy
metals and other toxicants such as insect toxins or animal products etc.
Some classes of
enzymes referred to as histone acetyltransferases (HATs) are involved in
catalyzing histone acetylation processes whilst, deacetylation is catalyzed by
histone deacetylases (HDACs). While HATs transfer acetyl groups onto the
ε-amino group of lysine (K); HDACs remove these acetyl groups from K. It is
known that acetylation of histones leads to an open chromatin structure
enabling transcription factors to bind to DNA, via some remodeling, whereas
deacetylation would leads to transcriptional repression due to chromatin
condensation [17].
Phytocompounds are thought to modulate these processes (Figure 1), via
activation or deactivation of some cellular enzymes.
The impact of
natural compounds on mammalian epigenome (nutri-epigenetics) is rapidly
emerging (Figure
1). However, with the advent of genomics, there remain challenges (such as;
interpretation of effects and its impacts on genetic variants), in using
phytocompound to target genomic Single Nucleotide Polymorphisms (SNPs) that
have phenotypic outcome. In many disease processes such as cancer and aging
for, which DNA damage is associated, epigenes modifications are implicated, so
also, in diseases involving DNA damages due to mutations such as in sickle cell
disease (SCD), [see 18 and references therein]. However, the precise role of
bioactive compound and the mechanisms by, which they may be involved in the
suppression of genetic transcription via epigenetics mechanisms such as
hypermethylation at CpG islands and histone modifications remain an area of active
research [19]. Here, using the Figure 1, we postulate putative areas
where and how phyto active compounds could modulate and thereby, be implicated
in the complex interplay signaling activities leading to gene
expression/regulation (Figure 1).
CONCLUSION
The
present study investigated the phytochemical properties of Napoleonae imperialis. The study establishes the low or
absence of toxic constituents in the methanol extract.
The use of phytochemicals is of particular
interests in several, disease management. Many of these phytochemicals
establish their disease curative potency via multiple processes and mechanisms.
These characteristics, as well as their generally low toxicity, position these
phyto-compounds as crucial in attempts to find less expensive curative drugs in
both resource poor environment and in developed economics. Moving forward,
there are needs for more functional studies to understand the biochemistry that would reveal the
mechanisms and pathways by, which active compounds of plants origin affects
candidate genes.
CONFLICT OF INTERESTS
We declare that there is no conflict of interests.
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