European Journal of Medicinal Plants
8(2): 87-96, 2015, Article no.EJMP.2015.093
ISSN: 2231-0894
SCIENCEDOMAIN international
www.sciencedomain.org
Pharmacognostic Evaluation of Spigelia anthelmia
Linn (Loganiaceae)
O. Taiwo Elufioye1* and A. Omolola Olaifa1
1
Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Nigeria.
Authors’ contributions
This work was carried out through collaboration between the two authors. Author OTE designed the
study, wrote the protocol and wrote the final manuscript. Author AOO did the analyses of the study,
the literature searches and wrote the first draft of the manuscript. Both authors read and approved the
final manuscript.
Article Information
DOI: 10.9734/EJMP/2015/16490
Editor(s):
(1) Marcello Iriti, Faculty of Plant Biology and Pathology, Department of Agricultural and Environmental Sciences,
Milan State University, Italy.
Reviewers:
(1) Anonymous, India.
(2) Anonymous, India.
Complete Peer review History: http://www.sciencedomain.org/review-history.php?iid=1084&id=13&aid=8923
th
Original Research Article
Received 4 February 2015
nd
Accepted 2 March 2015
Published 21st April 2015
ABSTRACT
Aims: To establish the pharmacognostic standard for S. anthelmia with a view to assisting in the
standardization and quality control of medicinal products from the plant.
Study Design: Pharmacognostic assessment of Spigelia anthelmia.
Place and Duration of Study: Department of Pharmacognosy laboratory, University of Ibadan,
Nigeria. Between 2013 and 2014.
Methodology: Chemo microscopic evaluation and determination of physicochemical properties
(moisture content, ash values, extractive values) of the powdered whole plant as well as studies of
the macroscopic and anatomical sections of the leaf were carried out using standard methods.
Results: Evaluation of the macro and microscopic characters showed that the stem is greyish
green with smooth bark, the leaves are opposite, simple and entire. The leaf epidermis is straight
with numerous starch grains and calcium oxalate crystals. The stomata are anomocytic while the
trichome is uniserate. The physichochemical parameters for the whole plant are: Moisture content
11.0±0.05%, total ash 14.67±0.05%, acid insoluble ash 3.33±0.01%, sulphated ash 11.0±0.05%,
alcohol soluble extractive 0.25±0.06% and water soluble extractive 0.69±0.02%.
Conclusion: The results of this research provided information which can be included in official
monograph of the plant for its proper identification.
_____________________________________________________________________________________________________
*Corresponding author: E-mail: [email protected];
Taiwo and Omolola; EJMP, 8(2): 87-96, 2015; Article no.EJMP.2015.093
Keywords: Spigelia anthelmia; pharmacognostic evaluation; chemomicroscopy; physichochemical
parameters.
that the leaves of this plant possess
bronchospasmolytic
and
anti-inflammatory
properties [9,12,13]. Also, extract of the plant has
been reported to cause neuromuscular blockade
producing dose-related myotonia and muscular
paralysis of rapid onset, [14]. It inhibits hatching
of egg and larval development of sheep and goat
gastrointestinal nematodes [15]. It has also been
reported that caution is needed not to apply high
doses, as this may cause convulsions. Because
of the toxicity of the plant, it is essential that
immediately after a dose is taken to treat worm
infections, a strong purge is taken as a chaser
[16].
1. INTRODUCTION
Plants have long been used by man to maintain
health and well-being [1] Ancient Egyptians, for
example, chewed willow bark to relieve fever and
headaches. Thousands of years later, scientist
discovered that this back contains salicylic acid,
a non- steroidal anti-inflammatory agent.
Atropine, used to relax muscles of the eyes, was
originally obtained from the deadly night shade
plant. Fox cloves contain digitoxin which is used
clinically to treat irregular heartbeat. Today, plant
based substances are still contribution to
medicine especially in the area of drug discovery.
In Nigeria and many other African countries,
application of medicinal plants especially in
traditional
medicine
is
currently
well
acknowledged and established [1]. However, the
challenge of standardization of herbal medicinal
products posses a treat to its wide acceptance.
Standardization of herbal products which cut
across proper identification (of its raw materials),
collection and processing will enhance better
patience acceptance of such products.
Pharmacognosy as a field of study is concerned
with description and identification of crude drugs
both in whole state and in powder. This branch of
Pharmacognosy finds importance, particularly for
pharmacopoeia and quality control purposes [2].
Parameters obtained from pharmacognostic
evaluation of medicinal plants have been useful
in their identification and differentiation of the
various species [17]. This research is aimed at
providing pharmacognostic standard for S.
anthelmia which can be included in the
monograph of the plant for proper identification.
2. MATERIALS AND METHODS
2.1 Collection of Plant Material
The plants materials i.e. the leaves and the stem
were collected in June, 2013 in Ibadan, Nigeria.
The plant was identified and authenticated by Dr.
Afilaka of Forest Research Institute of Nigeria
(FRIN). Voucher specimen (FHI NO-109818)
was deposited at the
Department
of
Pharmacognosy herbarium. The whole plant was
air dried, powdered and stored in an appropriate
container until required for use.
S. anthelmia Linn is an annual plant of about 60
cm high with a scarcely branched stem and short
stalked, feather-like lobed leaves sets in whorls
of four. The spikes with small purple or bright red
flowers come out of the whorl. The fruit is a twolobed capsule with warty seeds [3,4]. The plant is
used in ethnomedicine for treatment of coronary
heart diseases, headaches, neuralgia and
migraine, as well as iritis, chronic catarrh,
inflammation of mucus membrane (especially
respiratory tract), facial pain, teeth, eye and
temple. It is useful in the management of
difficulty in breathing. It also exerts powerful
action on nervous system and in sinus infection.
It is valued by some practitioners in cardiac
palpitation and endocarditis [5-7]. In northern
amazoni, a warm infusion of the root was used to
bath children as tranquilizer and tonic to induce
sleep [6,7]. The leaves are popularly used in the
management of asthma and helminthiasis in
several parts of Africa [8,9].
2.2 Macroscopic Evaluation
The macroscopic features and organoleptic
properties of the fresh leaf and stem as well as
powdered whole plant were evaluated using
standard methods [18].
The microscopic evaluations of the transverse
section of the fresh leaf and stem as well as
powdered whole plant were carried out [18,19].
The powdered whole plant was cleared by
adding few drops of chloral hydrate and heating
gently over Bunsen burner. A drop of glycerol
was added to the slides to prevent crystallization
of the sample. The mount was then examined
under microscope. This procedure was repeated
for the different slides used in the microscopic
examination.
Anthelmintic [10,11] and cardio-protective
activities have been reported. It has been shown
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Taiwo and Omolola; EJMP, 8(2): 87-96, 2015; Article no.EJMP.2015.093
and in case of picric acid reddish strands in case
of millions reagent indicates the presence of
protein.
2.3 Chemo Microscopic Evaluation
The powdered whole plant was mounted on the
microscope slides and observed under
compound microscope for the presence of
chemical substances like; cellulose, tannins, fat
and oils, starch, lignin, calcium oxalate and
calcium carbonate [19].
2.11 Test for Oils
The powdered plant was mounted in Sudan IV
reagent. Pinkish coloration is an indication of the
presence of oils.
2.4 Cellulose Test
2.12 Test for Alkaloids
The powdered whole plant was mounted in N/50
iodine solution followed by 66% sulphuric acid. A
blue coloration indicates the presence of
cellulose.
The powdered plant was mounted in
Dragendorff’s
solution.
Purple
coloration
indicates the presence of alkaloids.
2.5 Lignin Test
2.13 Test for Terpenoids
The powdered plant was mounted in
phloroglucinol
followed
by
concentrated
hydrochloric acid; a red coloration indicates
lignifications.
The powdered plant was mounted in
Anisaldehyde in H2SO4/MeOH and heated at
105°C/10 min. If purple coloration is observed, it
indicates positive result.
2.6 Tannins Test
2.14 Determination of Physicochemical
Constants
The powdered plant was mounted in ferric
chloride
solution
and
observed
under
microscope. Blue-black coloration indicates the
presence of tannins.
2.14.1 Determination of moisture content
The moisture content was determined by loss on
drying method [18]. Two grams of powdered
plant was measured accurately and placed into a
clean evaporating dish of a known weight. The
dish was then transferred into a pre-heated oven
which was set at 105°C. After an hour, the
weight of the powdered drug and evaporating
dish was determined and returned into the oven.
The weighing was done repeatedly at 30 mins
intervals until constant weight was achieved. The
moisture content was then calculated with
reference to the original weight of the powdered
sample as follows;
2.7 Starch Test
The powdered plant was mounted in N/50 iodine.
Bluish coloration indicates presence of starch.
2.8 Calcium Oxalate Crystals Test
The powdered plant was cleared in chloral
hydrate solution; presence of calcium oxalate
crystals is seen as bright structures of definite
shapes and sizes. On addition of 80%
hydrochloric acid and viewing under microscope,
disappearance of calcium oxalate crystals
confirms their presence.
% moisture content: weight of moisture x 100
Original weight of the sample
2.14.2 Determination of total ash value
2.9 Calcium Carbonate Test
Five grams of the powdered plant was accurately
weighed and transferred into a pre-weighed
crucible. The crucible and its content were gently
heated over a Bunsen burner flame to burn the
plant material completely to ash. The crucible
was then covered with lids and then placed in a
furnace at 600°C for two hours. After cooling, the
crucible was then placed in the desiccators and
reweighed. The percentage of ash values was
The powdered plant was mounted in glycerol.
The slide was irrigated with acetic acid solution.
Evolution of gas indicates the presence of
calcium carbonate [18].
2.10 Protein Test
1% picric acid and million’s reagent were used.
Yellowish strands on the structure (microscopic)
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Taiwo and Omolola; EJMP, 8(2): 87-96, 2015; Article no.EJMP.2015.093
calculated
from
the
pharmacopoeia of India [20].
procedures
in
solvent. 25 mL of the filtrate was collected,
transferred to a weighed thin porcelain dish and
evaporated to dryness on a water bath. It was
then dried completely in an oven at 90°C/105°C
to constant weight and final weight was then
determined [18].
% Ash value = weight of residual ash
x 100
Original weight of Sample
2.14.3 Determination of acid insoluble ash
% Alcohol-soluble extractive value =
After obtaining the total ash, 25 ml of dilute HCL
was used to wash total ash into a crucible and
covered with a watch glass and boil for 5 min.
The solution was then cooled and rinsed through
an ash less filter paper, the residue was then
washed with hot water until the filtrate became
neutral.
weight of residue in 25 mLx 100
25
2.15.2 Water-soluble extractive value
The procedure above was repeated but water
was used instead of 90% ethanol [18].
Filter paper was then placed in silica crucible and
dried on a hot plate and then incinerated by
gradually increasing the heat in a muffle furnace
at 450°C for 2 h (or oven dried at 105°C). The
crucible and contents was then cooled and final
weight was noted and recorded [18,19].
2.16 Statistical Analysis
All data are expressed as mean+standard error
of mean (SEM) of three replicates.
3. RESULTS
% Acid Insoluble Ash =
3.1 Macroscopic Description
weight of residual ash x100
Weight of original sample
The macroscopic and organoleptic evaluation
showed the presence of the features and
characteristic shown in Table 1.
2.14.4 Determination of sulphated ash
Two grams of the powdered whole plant was
weighed accurately into a tarred crucible and the
weight was determined. The sample was
moistened with sulphuric acid and thereafter
heated gently at a temperature as low as
practicable until the sample was thoroughly
charred. After cooling the residue was moistened
with small amount of sulphuric acid (usually 1
mL), heated gently until white fumes were no
longer evolved and ignited at 550°C to 650°C.
Care was taken to ensure that flames were not
produced at any time during the procedure. The
crucible was cooled in the desiccators, weighed
accurately and percentage residue was
calculated [18].
Table 1. Macroscopic and organoleptic
features of Fresh S. anthelmia whole plant
Evaluating feature
Bark
Fracture
Fractured surface
Odour
Taste
Shrinkage
Colour
Texture
Leaves
% Sulphated Ash = weight of residual ash x100
Weight of original sample
Characteristic
Smooth
Transversely
Granular
Faint
Bitter
Channelled and branched
Greyish-green
Horny
Opposite, simple and
entire
3.2 Microscopic Description
2.15 Determination of Extractive value
This involves the description of different
microscopic character of the plant such as leaf
content, stomata, trichomes etc.
2.15.1Alcohol- insoluble value
Five grams of the powdered whole plant was
accurately weighed into 250 mL conical flask,
100 mL of 90% ethanol was added and
stoppered. The flask was kept aside for 24 h with
frequent shaking for the first 6 h, allowed to stand
for 18 h and then filtered rapidly to avoid loss of
3.3 Chemo Microscopic Features
This involves the determination of certain
constituents in the plant by appropriate chemical
tests.
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3.4 Determination
Values
of
right angles to the third axis. The fresh stem
(Fig. 2) has vascular bundles with starch grains,
fibre and calcium oxalate crystals. The fibres
were observed to compose of spindle-shaped (or
elongated) cells with pointed ends and thick
walls. In addition to the above, the powdered
whole (Fig. 3) plant also showed the presence of
cork cells and phloem. The cork cells appeared
as compact mass of cells which are rectangular
in shape and are arranged in regular radial rows
and are lignified. The structural element of the
phloem which includes sieve tube, companion
cells, phloem parenchyma and secretory cells
were observed. The cells are elongated and
isodiametric and are arranged in linear shape.
Physicochemical
The following results were obtained from the
physicochemical evaluation.
4. DISCUSSION
Interest in herbal drugs has recently increased.
This is because of the belief that herbal
medicines are safe, inexpensive and have no
adverse effects [21]. However, complete
acceptance of herbal alternative medicines is still
facing some obstacles due to lack of proper
documentation
as
well
as
appropriate
standardization and quality control processes. It
is a fact that the therapeutic efficacy of medicinal
plants depends on the quality and quantity of its
chemical constituents and that the misuse of
medicine or natural products in general started
with wrong identification [22]. Thus, proper
identification of the starting plant materials for the
preparation of herbal drug is very imperative to
ensure some level of standards for such
products. Proper identification can be achieved
through pharmacognostic and phytochemical
studies. Thus, it is very essential to lay down
pharmacognostic specifications for medicinal
plants which are of use in various herbal drugs
[22].
Chemo microscopic evaluation of the powdered
whole plant, as shown in Table 2 revealed the
presence of cellulose, lignin, tannins, starch, oils,
and calcium oxalate. The presence of other
metabolites like alkaloids, terpenoids and
proteins were also observed. These metabolites
have shown various pharmacological actions and
may thus be responsible for the activities
associated with the plant.
Physicochemical parameters, Table 3, such as
moisture content, ash values (including total ash,
acid insoluble ash and sulphated ash) as well as
extractive values (alcohol soluble extractive and
water soluble extractive) were determined for the
powdered whole plant.
Spigelia anthelmia is a plant that has found
various uses in ethnomedicine. The findings of its
pharmacognostic evaluation could therefore be
of use in its proper identification, collection and
further investigation.
Moisture content evaluates the level of water
present in the material. High water content is
uneconomical and could promote the growth of
microorganisms leading to degradation and
spoilage. Also, in conjunction with suitable
temperature, moisture may lead to activation of
enzymes [17]. The moisture content observed for
S. anthelmia is 11.0±0.05% which falls within the
pharmacopoeial limits for water content (8-14 %)
for vegetable drugs [23].
The macroscopic features showed that the
leaves are opposite, simple and entire. The bark
is smooth and transversely fractured. The plant is
greyish-green in colour with faint odour and bitter
taste.
Microscopically, the fresh leaves (Fig. 1) have
straight walled epidermis with diagnostic
uniserrate trichomes and anomocytic stomata.
There is also the presence of starch grains and
calcium oxalate crystals. The starch granules are
of varying sizes and contain eccentric helium
with well-defined striation while the calcium
oxalate crystals were observed as shining prism
crystals of various distinctive shapes and sizes.
The crystals were seen clustered and confined to
the rows of parenchymatous cells and they have
three unequal axes with two lateral axes at right
angle to one another, but only one of these is at
Ash values are used to determined quality and
purity of crude drugs [22]. It gives an idea of the
earthy matter or the inorganic composition and
other impurities present along with the drug [24].
Water soluble ash is used to estimate the
amount of inorganic compounds present while
acid insoluble ash consist mainly of silica and
indicate contamination with earthy materials [22].
The total ash, acid insoluble ash and sulphated
ash values for S. anthelmia are 14.67±0.05%,
3.33±0.01% and 11.0±0.05% respectively. This
suggests minimal level of contamination.
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Taiwo and Omolola; EJMP, 8(2): 87-96, 2015; Article no.EJMP.2015.093
(a)
(bi)
(bii)
(c)
(d)
(e)
(f)
Fig. 1. Microscopic features of the fresh leaf of S. anthelmia
(a) uniserrate trichome (bi) bundle sheath (bii) leaf epidermis (c) starch grain (d) fibre (e) Anomocytic stomata
cells (f) calcium oxalate crystals
92
Taiwo and Omolola; EJMP, 8(2): 87-96, 2015; Article no.EJMP.2015.093
(a)
(b)
(c)
Fig. 2. Microscopic features of the fresh stem of S. anthelmia
(a) fibres (b) calcium oxalate crystals (c) vascular bundles with starch grains
Table 2. Chemo microscopic evaluation of the
powdered whole plant
Parameter
Cellulose
Lignin
Tannins
Starch
Oils
Calcium oxalate crystals
Alkaloids
Terpinoids
Proteins
Table 3. Physicochemical values of
S. anthelmia whole plant
Result
+
+
+
+
+
+
+
+
+
Evaluative parameter
Moisture content
Total ash value
Acid insoluble ash
Sulfated ash
Alcohol-soluble extractive
Water-soluble extractive
Percentage
11.0% ±0.05
14.67%±0.05
3.33% ±0.01
11.0%±0.05
0.25%±0.06
0.69%±0.02
Estimation of extractive values reflects the
amount of active metabolites of a plant material
as extracted with given solvents. Extractive
values are primarily useful in the determination of
+: present
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Taiwo and Omolola; EJMP, 8(2): 87-96, 2015; Article no.EJMP.2015.093
exhausted and adulterated drugs [25]. The
alcohol- soluble and water –soluble extractive
values of S. anthelmia were 0.25±0.06% and
0.69±0.02% respectively. Ash values and
extractive values can be reliable aids for the
detection of adulteration [24].
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 3. Microscopic features of powdered whole plant of S. anthelmia
(a) cork cells (b) Phloem parenchyma (c) Fibres (d) Starch grains (e) Calcium oxalate crystals (f) Uniserrate
trichome
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Taiwo and Omolola; EJMP, 8(2): 87-96, 2015; Article no.EJMP.2015.093
9.
5. CONCLUSION
Proper identification of plant material is crucial to
the standardisation and quality of products from
such plants. The various parameters reported for
S. anthelmia provides a number of information
which may be included in official monograph for
its proper identification.
10.
11.
CONSENT
It is not applicable.
ETHICAL APPROVAL
12.
It is not applicable.
COMPETING INTERESTS
Authors have
interests exist.
declared
that
no
13.
competing
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