Journal of Phytomedicine and Therapeutics
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Tarfa et al
JOPAT Vol 21(1), 754 - 771, Jan – June. 2022 Edition
ISSN2636 - 5448 https://dx.doi.org/10.4314/jopat.v21i1.3
Evaluation of safety of aqueous extract of Tapinanthus sessillifolius parasitic on Psidium
guava
Florence D.Tarfa, 2Onanuga C.O,3Obodozie-Ofoegbu. O . and 4Adoga G.I
1*
Department of Medicinal Chemistry and Quality Control (MC & QC), National Institute for
1, 2,3
Pharmaceutical Research and Development (NIPRD), Idu Industrial Area, P.M.B 21 Garki, Abuja Nigeria
Department of Biochemistry,College of Medicine,University of Jos,Nigeria.
4
Abstract
Plants products have been a source of food and medicines since antiquity. Tapinanthus
sessillifolius commonly known as African mistletoe is used in lifestyle preventive medicine to
enhance wellbeing and for treatment of various diseases. The heavy metals, macro elements were
determined using Flame Atomic Absorption Spectrophotometer and some physio-chemicals
were also evaluated for consistency of the extract. The acute and subchronic toxicity studies of
aqueous fresh leaf extract of Tapinanthus sessillifolius was evaluated in albino mice and rats.
This is to determine its safety profile by evaluating its effects on feed and water intake, body
weight, relative organ weight and changes in some biochemical parameters after 21 days daily
oral administration to rats. The results, estimated LD50 to be greater than 2000 mg/kg/bw. The
extract had no adverse effect on the efficiency of food and water consumption. Relative organ
weight and the biochemical parameters tested were not significantly different p<0.05 when
compared to untreated animals. This was supported by histopathological studies of the organs
where no adverse lesions were observed on tissues. However, there were lymphatic aggregates
infiltration in one of the lungs rat treated with 800mg/kg. The toxic heavy metals, lead, cadmium
and arsenic were not detected while moisture and ash were 7.02 % and 10.2% respectively
falling within the permissible limit of WHO and RDA. Repeated oral administration of fresh leaf
extract of Tapinanthus sessillifolius is relatively safe.
Keywords: Tapinanthus sessillifolius, Psidium guajava, safety, heavy metals, biochemicals.
©2007 The authors. This work is licensed under the Creative Attribution 4.0 International license.
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Introduction
Infestation of mistletoes on smaller trees
The use of medicinal plants as remedies for
lead to poor growth and productivity and
treatment of diseases, fitness, wellness and
eventual death of host plants, especially
longevity are becoming more popular with
during unfavorable weather conditions.
the emergence of COVID -19 pandemic and
Ethnomedicinal uses of mistletoes had for a
other chronic ailments. Mistletoe with the
very long time been in the hands of very few
common name(s) – bird lime, all heal,
herbal practitioners who claimed general use
devil’s fuge, Iscador is an all heal plant
to sorcery and magical powers to treat
[1,2,3]. Mistletoe is a general term for
mental conditions, sterility, and health
woody shoot parasites in several plant
families,
especially
Loranthaceae
problems
and
belong
to
the
ones majorly used in herbal medicine for the
treatment of headache, rheumatic pain,
Loranthaceae are Helixanthera, Berhautia,
Globimetula,
urino-genital
leaves, stems, berries and flowers are the
family
Loranthaceae (5,6). The seven genera of the
Englerina,
with
system, rheumatism and pain. [4,8]. The
Viscaceae [4,5]. Most genera of African
mistletoes
associated
hypertension [10,11] ulcers [12] and cancers
Agelanthus,
[13,14]. They are also acclaimed to possess
Tapinanthus and Phragmanthera with about
hypoglycemic [15,16] lipid lowering [17]
five or more species are recognised in West
and antibacterial effects [18].
Africa [7]. Worldwide, approximately 1500
species of mistletoe have been identified
The chemical information readily available
[7,8.]. Mistletoes are hemi-parasitic shrubs
includes the structural polysaccharide and
growing on dicotyledonous trees such as
protein in Phragmanthera capitata leaf cell
Albizzia leb beck, Terminalia mantaly,
wall, the viscotoxins, carbohydrates and
Terminalia catappa, Khaya senegalensis,
cytotoxic lectins. Compounds like alkaloids,
and
flavonoids, cyanogenic glycoside (linmarin
Theobroma cacao [7]. Mistletoe attaches
gallate) and walbruside were isolated [19].
itself to the host by modified roots otherwise
Flavonoids namely, quercetin, catechin,
known as “haustorium” [5,6,7,]. Haustorium
qercitrin, rutin and avicularin have been
ensures the continuity of the macro elements
isolated from the leaves of Globimimetula
sodium (Na), potassium (K), calcium (Ca)
braunii [19,20]. The present study is to
from the host to the parasite plant [8,9].
evaluate the acute and subchronic toxicity
Citrus
grandis,
Cola
acuminata
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effects of the aqueous fresh leave extract of
Moisture - The determination of
on
moisture content was based on the
Psidium guajava host and also determine
method of the Association of Official
some quality standard makers of ash,
Analytical Chemist (AOAC) [20]. It
moisture and toxic heavy metals.
is an indirect distillation method
Tapinanthus
growing
A.
sessilifoliuss
(evaporation of moisture). One gram
Materials and methods
(1.0 g) of the sample powder was
Plant material
weighed on aluminum foil on the
The leaves of T. sessilifolius were harvested
automated moisture analysis pan
from the host, Psidium guajava in Jos,
(Model MB 200, OHAUS Florham
Plateau State of Nigeria. The plant material
PK.USA) and set at 105 °C for 3
was identified and authenticated by Prof. Z.
hours
O. Gbile (Consultant Taxonomist, UNDP).
moisture content of the sample was
Voucher specimen (No. FHI 105336) was
obtained from the moisture balance
deposited at Forestry Research Institute,
B.
Ibadan and at NIPRD, Idu, Abuja Nigeria.
after
which
percentage
Total Ash - The total ash was also
determined
by
AOAC
[20].
Porcelain crucible was washed and
Preparation of extract
Extraction
of
placed in the muffle furnace for 10
Plant
minutes to dry. After which it was
Tapinanthus
sessillifolius
removed and placed in a desiccator
The fresh plant material was cold extracted
containing active desiccant to avoid
with distilled by disruption of the cell wall
moisture contact and allowed to cool.
using blender. The extract was filtered
After cooling, it was weighed (M1). 2
through muslin cloth then filtered again
g of each of the sample were
through
and
weighed in the crucibles (M2) and
lyophilized using Lyovac GT2 (Germany).
was placed in the Muffle furnace at a
The yield was found to be (10.53 % w/w).
550 oC and allowed to stand 3 hours
Whatman
filter
No.
42
for complete combustion to ash to be
Physical Evaluation
achieved. The ash samples were
Proximate analysis of extract
removed placed in the desiccator for
cooling and the weighed (M3). The
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percentage ash was calculated using
Atomic
Absorption
Spectrophotometer
the formula:
(AAS) using air acetylene as fuel. The slit
of 0.5 and appropriate lamps and their
wavelengths were
used. The mean value
and the standard deviation of each element
C.
was determined by the AAS computer.
Total proteins were estimated by the
Samples were analyzed in triplicate. Results
method of AOAC [20]
obtained in part per million (PPM) were
converted
Phytochemical screening
preliminary
phytochemical
Animals
flavonoids, alkaloids and terpenes [21,22].
Swiss albino mice (20 – 30 g) and Wistar
Elemental analysis
rats (150 – 200 g) of either sex obtained and
Plant samples were prepared for elemental
maintained at the Animal Facility Centre
analysis as modified by AOAC [20]. The
(AFC)
dried plant extract was ashed in oven
of
the
National
Institute
for
Pharmaceutical Research and Development,
electric muffle furnace maintained at 400 C
o
Abuja were used. All animals were housed
and 420 oC, for about 6 - 7 h to destroy all
under standard conditions of temperature
organic materials in the sample. The
(25 ± 2 oC) and light approximately (12/12 h
crucible containing pure ash was then taken
light/dark cycle) and fed on standard diet
out of the furnace and kept in a desiccator.
and water ad libitum. These animals were
Thereafter the ash was digested with triple
acids:
as
In-vivo toxicity studies
screening for phenols, tannins, saponis,
mixture
gram
were determined by flame flourimeter.
and Agrawal and Paradhavi (2007) were
for
milligram/100
indicated below. Sodium and potassium
The standard methods of Harbone (1998)
used
to
approved for use by the ACE committee
sulfuric
after reviewing the protocol for good
acids:sulphuric:perchloric acid (11:6:3) to
laboratory practice and animal handling,
obtain a clear solution. The solution was
which is in compliance with the National
then made to 25 ml with double distilled
Institutes of Health Guide for the Care and
water and read up with the flame technique
use for Laboratory animals (Publication No.
of Hitachi Model 80-80 polarize Zeeman
85-23, revised 1985(23).
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��� =
Acute toxicity tests
The
oral
acute
toxicity
(LD50)
����ℎ������������������ℎ������
�100
���������ℎ����ℎ������
��� =
was
determined in mice by a modified Lorke’s
������������������������ℎ������
�100
���������ℎ����ℎ������
Animals
[24] model. The test was carried out in five
were
observed
for
signs
of
abnormalities during the whole treatment.
groups, each consisting of five (5) mice.
Animals were fasted overnight after of food
Group I – IV were administered with
the last dose, but allowed free access to
varying doses of extract at 10, 100, 1000 and
water. Blood samples were obtained by
2000 mg/kg i.p., while group V served as
retro-orbital puncture, using capillary tubes
control and was administered normal saline
for blood biochemical studies after which
at 10 ml/kg. Signs and symptoms of toxicity
the animals were anesthetized by chloroform
was observed over a period of 24 h. Death
inhalation. The internal organs were isolated,
and sign of toxicity within this period were
weighed and fixed in formaldehyde for
monitored was recorded.
histopathological investigation. The relative
organ weight (ROW) was calculated thus:
Sub-Chronic toxicity Studies
Test Guidelines with slight modifications
��� =
according to the Organization for Economic
Cooperation and Development (OECD, 407)
[25]. Healthy rats of either sex were
������������ℎ��������(�)
�100
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Statistical analysis: Data were expressed as
randomly divided into four groups (n= 5).
Mean ± SEM Data for toxicity studies were
Animals received vehicle orally (water;
analyzed by ANOVA followed by Dunnett’s
control group) and aqueous extract in doses
post hoc test for multiple comparisons and
of 200, 400 and 800 mg/kg/day for 21
p<0.05 was considered significant.
consecutive days. Body weight was recorded
weekly as D1, D7, D14, D21. Food
Results
consumption and water intake were daily
Phytochemical
monitored. Daily feed (DFI) and Daily water
analysis
intake (DWI) were determined [30] as:
758
and
Physicochemical
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The
extract
of
protein and fats that were present are shown
the
in Table 2. Table 3 represent the mineral
polyphenols, terpenoids saponin, glycosides.
elements and heavy metals that were not
Alkaloids were not detected as shown in
detected.
secondary
Table
1.
showed
the
metabolites
Moisture,
Table 1.
presence
such
ash,
as
carbohydrate,
Phytochemical compounds of aqueous fresh leaf extract of Tapinanthus
sessilifolius on P. guajava host plant
Phytochemicals
Remarks
Polyphenols
Flavonoids
++
++
Terpenoids
Saponins
++
Alkaloids
++
-
++ = Positive; - = Not detected
Table 2.
Some physico-chemical parameters of aqueous fresh leaf extract of
Tapinanthus sessillifolius on P.guajava host plant
Parameter
Ash
Moisture
Lipids
Protein
Carbohydrate
Fibre
Concentration
(%)
10.02
7.02
16.32
5.56
54.8
7.3
WHO
<8
<10
-
-
-
-
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Table 3:
Mineral and heavy metal content of aqueous fresh leaf extract of
Tapinanthus sessillifolius
Elements
Na
(mg/100g 1.22 ±
dry
0.01
weight)
RDA mg
WHO
<2300
K
32.1 ±
0.2
Mg
Ca
10.95 ± 50.55 ±
0.01
0.42
Fe
Zn
Pb
Cd
As
7.89 ±
0.02
13.42 ± 0.009 ±
0.02
0.01
ND
ND
0.4-5.1 30-40
2001300
0.2-27
2-15
-
-
-
-
-
-
50
10
-
-
-
RDA means recommended daily allowance (DRI, Food and Nutrition Board, 2000)
WHO = World Health Organization
ND = Not detected
Effect of leaf extract T. sessillifolius on
Effects on sub-chronic toxicity studies in
acute oral administration in mice
rats
The oral acute administration of the extract
The
at varying doses of 10, 100,1000, and 2000
sessillifolius at doses of 200, 400 and 800
mg/kg showed no any signs of toxicity. The
mg/kg bw. for 21 days did not show
behavioural pattern was similar in treated
significant difference in body weight when
groups and control animals. No death was
treated animals were compared to normal
recorded in all animals.
The acute lethal
control group even though there is increase
dose (LD50) was therefore estimated to be
in weight across all groups (Table 4). Also,
greater than 2000 mg/kg body weight.
there was no significant change in food and
760
daily
oral
administration
of
T.
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water intake with administration of the
Microscopic examination of the tissues of
extract. The relative organ weights of liver,
the internal organs did not show any change
kidney, lungs, spleen and pancreas treated
in the structure of the cells of liver, kidney
rats were similar to control with no
spleen and pancreas of the treated animals
significant difference between the groups
that could be ascribed to the treatment of
(Table 5). Treatment with the extract did not
rats with the aqueous leaf extract of T.
affect the levels of serum glucose or lipid
sessillifolius.
profile as all the parameters testes were
aggregates were observed in one of the lung
within normal range and not significantly
tissue in treated animals at doses of 800
different from untreated group (Table 6).
mg/kg which were not present in control
However,
lymphocytic
animals (Figure 1).
Histological analysis
Table 4.
Effect of aqueous leaf extract of Tapinanthus sessilifolius (TS) on relative
organ weight (g) in normal rats
Organ (g)
TS
(mg/kg)
Liver
Kidney
Lungs
Spleen
Pancreas
Heart
Control
3.30 ± 0.10
0.96 ± 0.11
0.75 ± 0.14
0.37 ± 0.30
0.153 ± 0.20
0.35 ± 0.21
200
3.16 ± 0.20
0.91 ± 0.12
0.69 ± 0.13
0.34 ± 0.11
0.16 ± 0.09
0.32 ± 0.06
400
3.21 ± 0.11
0.89 ± 0.20
0.72 ± 0.21
0.33 ± 0.02
0.151 ± 0.06
0.33 ± 0.12
800
3.32 ± 0.11
0.94 ± 0.10
0.77 ± 0.12
0.32 ± 0.11
0.153 ± 0.2
0.33 ± 0.12
Data are expressed as Mean ± SEM (n = 5). All Values in test group are not significantly
different from control (p<0.05) Two-way ANOVA Dunnett post hoc.
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Table 5:
Effect of Aqueous leaf extract of Tapinanthus sessilifolius (TS) on glucose and
lipid profile in normal rats.
Lipid profile
(mg/dl)
TS
(mg/kg)
Glucose
Cholesterol
HDL-cho
LDL-cho
Triacylglycerol
Control
94.34 ± 2.1
97.00 ± 1.5
39.10 ± 1.3
39.70 ± 0.9
109.50 ± 1.3
200
95.82 ± 5.2
92.80 ± 5.2
38.90 ± 1.7
39.10 ± 1.5
102.50 ± 1.5
400
93.25 ± 2.2
89.80 ± 4.2
40.20 ± 0.8
39.10 ± 1.2
95.50 ± 1.3
800
98.33 ± 3.1
91.34 ± 1.7
39.1 ± 2.0
39.70 ± 1.5
107.03 ± 1.2
Data are expressed as Mean ±SEM (n = 5). All values in test group are not significantly different
from untreated control (p<0.05). Two-way ANOVA Dunnett post hoc.
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Table 6.
Effect of aqueous leaf extract of Tapinanthus sessilifolius (TS) on some
biochemical parameters in normal rats.
TS
(mg/kg)
ALT(UI)
AST(UI)
Ratio
Creatine
(UI)
ALP
T.BIL
Control
20.31± 0.70
39.6 ± 0.1
0.33
0.37 ± 0.30
0.153 ± 0.20
2.40 ± 0.31
200
19.5 ± 0.10
38.5± 0.12
0.45
0.34 ± 0.12
0.160 ± 0.09
2.32 ± 0.41
400
19.80 ±1.6
39.50± 0.2
0.51
0.33 ± 0.2
0.154 ± 0.11
3.12 ± 0.21
22.4 ± 0.5
40.20 ±0.1
0.55
0.32 ± 0.1
0.151 ± 0.1
3.20 ± 0.4
800
Data are expressed as Mean ± SEM (n = 5). All values in test group are not significantly
different from untreated control (p<0.05). Two-way ANOVA Dunnett post hoc.
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Control
TS (800 mg/kg)
A - Normal Lung
Figure 1
B - Focal lyphocytic aggregate
Representative slides of the histological analysis of the lung tissues of control
(A) and treated (B) animals (Mag 20x10)
Discussion
Plants are a rich source of macro elements
essential for human beings and they
Toxicological studies are generally done to
contribute to vital body function growth [29].
determine the level of exposure and prevent
In this study, the inorganic essentials
the potential risk associated with the use of
elements such as sodium (Na), potassium
medicinal plants. The low level of moisture
(K), calcium (Ca) and the trace elements that
content that falls within the acceptable limit
include iron (Fe), magnesium (Mg) and zinc
of 10 % will not promote microbial growth
and
subsequent
biodegradation
(Zn)
and
which
are
required
for
various
metabolic processes in man were detected
instability of the bioactive components of
and the values falls within the WHO limits
the plants [27,28]. Fibre content of 7.3% is
[43]. Adding to the integrity and safety of
good for proper motility and faecal excretion.
the extract is the absence of heavy metals
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like Lead (Pb), Cadmium (Cd) and Arsenic
nutritional composition might have played a
(As). These metallic elements were not
significant
detected in this plant material as reported for
degenerative diseases and processes [34].
other herbals [29,30].
Decrease in weight or excessive increase can
According to reports by Rosidah 2009 [31]
be a pointer to toxicant in the system.
administration of 500 - 5000, and 5000 -
However, the treatment with fresh extract
15000 mg/kg body weight is relatively safe
the weight gain is not different from
and non-toxic respectively. Therefore, the
untreated even though not with food and
lethal dose of greater than 2000 mg/kg of
water efficiency and relative organ weight.
this study is relatively safe. The repeated
The astringency and pungency of the fresh
administration of doses up to 800 mg/kg to
extract would have caused the moderate
normal rats did not produce observable toxic
consumption especially at higher dose of
effects at the doses tested. This is consistent
800mg/kg body weight and the possibility of
with other reports which showed that
the mild lymphatic aggregates infiltration
mistletoes extracts are relatively non-toxic
observed in the lungs histopathology of one
[30,31,32]. However, in handling the plant
in this treated group and this may be during
material from raw material to extract,
oral administration liquid entered the lung.
physicochemical reference like moisture and
The liver and kidney play vital role in
ash are critical so as to avoid deterioration,
biotransformation and filtering blood from
microbial and fungal growth that can be
digestive tract to rest of the body [36]
introduced as toxicants [33]. The extract is
Therefore,
rich in polyphenols that are strong radical
inflammation
scavengers or antioxidants [34] but lack
mitochondrial cells. It is known that
alkaloids which was reported by other
elevated
studies on same host and difference species
conjunction with a rise in bilirubin level are
and this could be due to season and location
considered
of harvest [30;32] This is in agreement to
hepatotoxicity linked to oxidant stress [35].
the chemotaxanomic profiles [41].
Increase in the aminotransferases in serum
The quality of many foods depends on the
indicates cellular injury that leads to
concentration and type of minerals and
leaching of enzymes to serum before its
biomolecules they contain. The extracts
manifestation with clinical histopathology
765
role
any
against
toxicants
and
a
can
injury
transaminase
as
variety
of
cause
to
activities
marker
index
the
in
of
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Tarfa et al
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[36]. Reduced clearance due to increased
The work was supported by NIPRD
proliferation of cells, increased rate of cell
postgraduate
turn over and increased cell damage and
thankful to Mr Sunday Dzarma for technical
increase in enzyme synthesis [37,38] This
assistance , Mrs Faustina Nwachikwu , Mr
increase was not the case in this studies
Bamaiyi Wayas (of blessed memory) and
since the enzyme ratios were less than one
Ali Sale of the Animal Facility Centre of
because ratio of one indicates injuries and
Department
damage to liver [39]. It has been frequently
Toxicology, NIPRD.
reported that in the development of liver
oxygen
species
Pharmacology
are
and
often
1. Abayomi, S., Eyitope, O. and Adedeji,
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O. (2013). The role of medicinal plants
Lipotropic
in
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(ROS),
of
Authors
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