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Vol. 14(7), pp. 229-239, August, 2020
DOI: 10.5897/AJPP2016.4591
Article Number: 518E4E764447
ISSN: 1996-0816
Copyright ©2020
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJPP
African Journal of Pharmacy and
Pharmacology
Full Length Research Paper
Pharmacognostic and toxicological evaluation of the
leaves of Piper guineense Schum. and Thonn
(Piperaceae)
Tavs A. Abere1 ⃰, Mumuni Sumaila1 and Obi Ifeoma Stephanie2
1Department of Pharmacognosy, Faculty of Pharmacy, University of Benin, Benin City. PMB 1154, Edo State, Nigeria.
2Department of Pharmacognosy, Faculty of Pharmacy, Madonna University, Elele, Rivers State. Nigeria.
Received 16 May, 2016: Accepted 24 August, 2016
The leaves of Piper guineense Schum. and Thonn (Piperaceae) are used in ethnomedicine for the
management/treatment of various diseases. The objective of this study was to establish the
pharmacognostic and toxicological profiles of P. guineense to help practitioners in making choices,
and to assist scientists involved in the research of the Piper genus. The fresh leaves were examined for
their macroscopic and microscopic properties. Numerical (quantitative analysis) and phytochemical
evaluations were carried out using standard methods. Acute toxicity profile of the plant, including LD
50 was investigated using mice while sub-acute toxicity to determine effects of the plant leaf extract on
some major organs was investigated for 30 days in rats. The macroscopic, microscopic and numerical
features observed in the leaves of P. guineense could aid in sample identification. Glycosides, alkaloids
and phenolics were among the secondary plant metabolites present. Oral doses ≤ 8000 mg/kg did not
lead to death of the animals. Sub-acute toxicological evaluations at the doses of 250 and 500 mg/kg
showed mild congestion in all the target organs, except spleen, where at 500 mg/kg, in addition to mild
stromal oedema, there was mild follicular activation and moderate hyperplasia of sinus histiocytes.
These results could aid researchers and practitioners in the investigations and consumption of the
leaves of P. guineense. Its overall safety profile needs to be further evaluated and care should be taken
on prolonged usage.
Key words: Piper guineense, Piperaceae, pharmacognostic standardization, toxicological.
INTRODUCTION
The standardization of herbal medicines is the process
involving a series of laboratory experiments that reveal
and assemble a set of inherently peculiar characteristic.
These characteristics include constant parameters that
are definitive, with qualitative and quantitative values or
specific, unique and unshared features on the basis of
which similar herbal medicines, claimed to be the same,
can be compared for the purpose of authenticity,
genuineness, purity, efficacy, safety, repeatability,
reproducibility and the overall quality assurance (Elujoba,
*Corresponding author. E-mail: willams_alves@hotmail.com. Tel: (81) 99957-6266.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
230 Afr. J. Pharm. Pharmacol.
1999).
Klein (1996) stated that medicinal plants have the
potential to be harmful, postulating that it is dependent on
the dose of substance consumed. However, probably
because medicinal plants are natural, a greater
proportion would have to be consumed to cause harm
when compared with orthodox drugs. Therefore,
determination of acute and sub-acute toxicity profiles of
medicinal plants is essential to guide herbal medicine
practitioners and researchers (Abere et al., 2014).
Piper guineense Schum. and Thonn belongs to the
Piperaceae family (Macmillan, 1984). Common names
include African black pepper, Benin pepper, Guinea
cubeb pepper or Ashanti pepper. It is also known as uririe
(Urhobo), Iyere (Yoruba) and Uziza (Ibo). It is native to
the tropical and subtropical world (Nwinyi et al., 2009). It
is a highly valued ingredient in Africa because of its
varied and numerous uses. The ethanol leaf extract of P.
guineense has significant anti-inflammatory and diuretic
properties (Omodamiro and Jimoh, 2014). Udoh et al.,
(2012) reported that consuming the leaves of P.
guineense have the ability to interfere with the female
reproductive system by affecting the activities of the
uterine muscles. The antifertility effects of the aqueous
extract on wistar rats had earlier been reported by
Mbongue et al. (2005). Ekanem et al. (2010) had
attributed these effects to the activities of alkaloidal
amides on the uterine muscles. The piperine-amide
present in the leaf is responsible for its insecticidal
activity against Zonocerus variegatus (Nwinyi et al.,
2009). Antiparasitic, antimicrobial and antifungal activities
of the leaves and seeds of P. guineense have also been
reported (Ekanem et al., 2010).
In view of the challenges confronting the practice of
herbal medicines, provision of information on the toxicity
of these products will assist and encourage herbal
medicine practitioners and researchers (Adefemi et al.,
1988). This study on the leaves of P. guineense was
geared towards establishing its pharmacognostic and
toxicological profiles to aid in differentiating it from similar
species.
MATERIALS AND METHODS
Preparation of plant extract
P. guineense Schum. and Thonn (Piperaceae) leaves were
collected in Benin City, Nigeria. Authentication was done by the
plant curator at the Department of Pharmacognosy, Faculty of
Pharmacy, University of Benin, Benin City where a voucher
specimen was lodged. The leaves were dried at ambient
temperature for 72 h and powdered.
Animals
Albino mice of Swiss origin (21. 44 ± 1. 02 g) and Wistar male rats
(239. 00 ± 14. 02 g) were sourced from the Department of
Pharmacology and Toxicology, Faculty of Pharmacy, University of
Benin, Benin City. Animals used for the experiments were handled
with care and procedures duly followed international standards for
use of animals in experiments (National Institute of Health, 2002).
The ethical approval of the study was part of the comprehensive
work design approved by the College of Medicine, University of
Benin Animal Ethics Committee (ADM/F. 22A/Vol. viii/349).
Macro- and micro-morphological examinations
The macroscopic and microscopic chemomicroscopic examinations
of the fresh and powdered leaves of P. guineense were carried out
using standard methods (Wallis, 1985; Evans, 2006; AP, 1986).
Phytochemical investigation
Screening for secondary plant metabolites was carried out
according to previously described methods (Brain and Turner,
1975; Ciulei, 1981; Evans, 2006; Harborne, 1992). These include
chemical tests for tannins, flavonoids, alkaloids, cardiac, saponin,
anthracene and cyanogenetic glycosides.
Numerical values determinations
This involved the quantitative investigations to determine moisture
content, ash values as well as solubility profiles (AP, 1986; BP,
1980).
Toxicological evaluation
Acute toxicity test
The animals (Swiss albino mice) were divided into 9 groups of 5
animals each. They were orally administered the extracts of P.
guineense at doses range of 1-8 g/kg. Mice administered 5 ml/kg of
normal saline served as the control group. On day 1, the animals
were observed for 10 min for every 6 h and for at least 10 min for
the next two days. Lethality as well as gross toxicological features
was recorded daily for each group. These features include state of
hyperactivity, diarrhea, convulsion and pile-erection (Dietrich,
1983). Observation of the animals was continued for the next
fourteen days.
Sub-acute toxicity test
In the test to determine the effects of administering the extract of P.
guineense on key organs in Wistar rats, the procedure of Abere et
al. (2015) was adopted using oral doses of 250 and 500 mg/kg per
day of the extract for 30 consecutive days. 5 ml/kg of normal saline
(vehicle) served as the control. At day 30, the rats were sacrificed
with the hearts, livers, spleen and kidney removed and preserved in
10% formaldehyde solution. The organs were sectioned (6 µ thick)
and embedded in paraffin wax and stained with H&E (Adefemi et
al., 1988).
RESULTS
Macroscopic characteristics of the leaves of P.
guineense
It was a simple leaf with alternate petioles. The shape
Abere et al. 231
Table 1. Numerical data of leaves of P. guineense.
Parameter
Mean ± SEM (% w/w)
Moisture content*
12.50 ± 0.21
Total ash*
7.9 ± 0.11
Acid – insoluble ash†
0.89 ± 0.01
Water – soluble ash†
4.10 ± 0.25
Alcohol – soluble extractive*
8.00 ± 0.08
Water – soluble extractive*
5.80 ± 0.16
*n = 30; † n = 15.
Table 2. Phytochemical constituents of P. guineense
leaves.
Classes of secondary metabolites
Inferences
Tannins
+
Flavonoids
+
Alkaloids
+
Anthracene derivatives
-
Saponin glycosides
+
Cardiac glycosides
+
Cyanogenetic glycosides
-
-= absent; + = present.
was elliptical, venation was pinnate while apex was
acuminate. Fresh leaves were greenish in colour, though
darker in the upper surface. Leaf size was 10.3 cm ± 0.5
(length) and 6.0 cm ± 0.2 (breadth). The fresh leaves had
an aromatic odour and a hot and very pungent taste.
Microscopic characteristics of the leaves of P.
guineense
Microscopical examination of the leaves of P. guineense
showed that anticlinal walls were straight and contained
numerous calcium oxalate crystals scattered all over the
surface of the lamina. Each stoma was surrounded by 2
subsidiary cells with their common walls at right angles to
the long axis of the guard cells (diacytic arrangement).
The transverse section of P. guineense leaf revealed an
upper and lower epidermi consisting of cells that are
similar in sizes. P. guineense had a bifacial surface, that
is, the two surfaces were not identical structurally, thus
the arrangement was dorsiventral. The mesophyll
consisted of 2 parts, viz, the spongy mesophyll containing
a crystal sheath (clusters crystals of calcium oxalate) and
the palisade mesophyll. The vascular bundle, consisting
of xylem and phloem tissues was surrounded by layers of
fibres.
Chemomicroscopic characteristics of the leaves of P.
guineense
Chemomicroscopic examination of P. guineense leaves
showed the presence of starch, lignin, calcium oxalate
crystals, tannins and cellulose.
Numerical values of P. guineense leaves
The results of the quantitative analysis of P. guineense
leaves are presented in Table 1.
Phytochemical screening
The phytochemical investigations of P. guineense leaves
showed the presence of tannins, flavonoids, alkaloids,
saponins and cardiac glycosides (Table 2).
Toxicological evaluation
In the acute toxicity study, P. guineense extract caused
no death up to the oral dose level of 8000 mg/kg body
weight in mice, thus the LD 50 was < 8g/kg. The animals
232 Afr. J. Pharm. Pharmacol.
A
B
Figure 1. Photomicrograph of the heart of rats administered with normal saline for 30 days showing
myocardium A, pierced by small cardiac vein B (H&E 10x).
did not show any significant changes in their behaviour,
mood, movement, agility and posture. The frequency of
stooling was normal. There was absence of wet stools;
rather, stools were dry and rounded. The animals did not
show signs of convulsion or exhibit writhing.
Administration of the extract of P. guineense for 30
days in rats did not produce any major toxicological
symptoms nor were deaths recorded. General
histopathological analysis of the heart (Figure 2) in the
250 mg/kg P. guineense treated group showed mild
transmural oedema, vascular congestion and infiltrates of
chronic inflammatory cells. There was no difference at
the dose of 500 mg/kg (Figure 3). In the liver, at 250 and
500 mg/kg (Figures 5 and 6), there were mild portal
vascular congestion and hypertrophy, in addition to mild
periportal infiltrate of chronic inflammatory cells. The
spleen at 250 mg/kg showed mild stromal oedema and
mildly activated sinus histiocytes (Figure 8) while at 500
mg/kg (Figure 9), in addition to mild stromal odema and
follicular activation, there were also moderate hyperplasia
of sinus histiocytes. The kidney showed mild interstitial
vascular congestion and hypertrophy (Figures 11 and
12). Figures 1, 4, 7 and 10 served as controls for the
heart, liver, spleen and kidney’ at the end of the result for
toxicological evaluation.
DISCUSSION
Piper guineense is very similar to other species of the
Genus Piper, especially Piper nigrum, (pepper of
commerce) which is the source of black and white
peppers. The Piperaceae family has well over 700
species (Nwinyi et al., 2009). The presence, nature and
spread of pharmacognostic characters in medicinal plants
aid in their identification. It is pertinent to note that the
establishment of pharmacognostic parameters and
standards of a medicinal plant are important before it can
be included in an Herbal Pharmacopoeia (Prajapati and
Patel, 2010). The morphological features described for
the leaves of P. guineense could therefore, serve as a
basis for the identification and further investigation of the
plant.
The numerical values of the leaves of P. guineense
described could assist in establishing the true identity of
the plant. The moisture content of 12.5% for the leaves of
P. guineense is within the pharmacopoeia limits of water
content for vegetable drugs given as 8 to 14% (AP,1986).
Abere et al. 233
A
B
C
Figure 2. Photomicrograph of the heart of rats administered with 250 mg/kg extract of P.
guineense for 30 days showing mild transmural oedema A, mild vascular congestion and
dilatation B and mild infiltrate of chronic inflammatory cells C (H&E x 40).
A
Figure 3. Photomicrograph of the heart of rats administered with 500 mg/kg extract of P.
guineense for 30 days showing mild transmural oedema A (H&E x 10).
234 Afr. J. Pharm. Pharmacol.
A
C
B
Figure 4. Photomicrograph of the liver of rats administered with normal saline for 30 days
showing central vein A, hepatocytes B and sinusoids C (H&E x 10).
A
B
Figure 5. Photomicrograph of the liver of rats administered with 250 mg/kg extract of P.
guineense for 30 days showing mild portal vascular congestion and hypertrophy A and mild
periportal infiltrate of chronic inflammatory cells B (H&E x 40).
Abere et al. 235
A
B
Figure 6. Photomicrograph of the liver of rats administered with 500 mg/kg extract of P. guineense
for 30 days showing mild portal vascular congestion and dilatation A and mild infiltrates of chronic
inflammatory cells B (H&E x 40).
B
A
Figure 7. Photomicrograph of the spleen of rats administered with normal saline for 30 days
showing white pulp A and red pulp B (H&E x 10).
236 Afr. J. Pharm. Pharmacol.
A
A
Figure 8. Photomicrograph of the spleen of rats administered with 250 mg/kg extract of P.
guineense for 30 days showing mild stromal oedema A and mildly activated sinus histiocytes B
(H&E x 40).
C
B
A
Figure 9. Photomicrograph of the spleen of rats administered with 500 mg/kg extract of P.
guineense for 30 days showing mild stromal oedema A and mild follicular activation B and
moderate hyperplasia of sinus histiocytes C (H&E x 40).
Abere et al. 237
C
B
A
Figure 10. Photomicrograph of the kidney of rats administered with normal saline for 30 days
showing glomeruli A and tubules B separated by interstitial space C (H&E x 10)
Figure 11. Photomicrograph of the kidney of rats administered with 250 mg/kg extract of P.
guineense for 30 days showing mild interstitial vascular congestion and hypertrophy A (H&E x 10).
A
238 Afr. J. Pharm. Pharmacol.
A
Figure 12. Photomicrograph of the kidney of rats administered 500 mg/kg extract of P. guineense for 30 days
showing mild interstitial vascular congestion and hypertrophy A (H&E 40x).
1986). The leaves of P. guineense can be stored in tightly
covered containers at ambient temperatures without
much concern to the deterioration of the active
ingredients. The values of the acid-insoluble ash (0.89 ±
0.01) and water-soluble ash (4.10 ± 0.25) gave an
indication that P. guineense can be used directly as
powdered drugs.
The administered dose of the herbal drug primarily
determines its safety. Acute and sub-acute toxicological
studies of herbal medications, especially the
determination of the median lethal dose (LD 50) are very
desirable, as this could give an index of the dose
regimen. Where the herbal formulation is to be
administered for more than 3 days, it becomes necessary
to subject them to sub-acute toxicity test (Abere et al.,
2014). The main reason is to determine the likely organs
that could be susceptible to toxicity by the herbal drug on
prolonged usage. Histopathological effects of the
administration of 250 and 500 mg/kg per day of the
extracts of P. guineense on rats showed no significant
differences at low and high doses in the liver, kidney and
heart, except with the spleen, where there was mild
stromal oedema and mildly activated sinus histiocytes at
the dose of 250 mg/kg. At the dose of 500 mg/kg, in
addition to mild stromal oedema, there was mild follicular
activation and moderate hyperplasia of sinus histiocytes.
Effects on the other organs were majorly mild.
CONCLUSION
Scientific data that could aid the standardization of P.
guineense Schum. and Thonn (Piperaceae) have been
provided. Any medicinal product that is claimed to be P.
guineense, but whose leaves have characteristic that are
significantly different from the standards presented could
be rejected. In the light of the toxicological results, care
should be taken when using the drug for a long period.
CONFLICT OF INTERESTS
The authors have none to declare.
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