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131
Document heading doi:10.1016/S2221-1691(14)60221-5 襃 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.
A pharmacobotanical study of two medicinal species of Fabaceae
Mubo A Sonibare1*, Tolulope A Oke1, Mike O Soladoye2
1Department of Pharmacognosy, Faculty of Pharmacy University of Ibadan, Ibadan Nigeria
2Department of Biological Sciences, Faculty of Science Bowen University, Osun State, Nigeria
Asian Pac J Trop Biomed 2014; 4(2): 131-136
Asian Pacific Journal of Tropical Biomedicine
journal homepage: www.elsevier.com/locate/apjtb
*Corresponding author: Mubo A Sonibare, Senior Lecturer, PhD, Department of
Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria.
Tel: 00234-8134901273
E-mail: sonibaredeola@yahoo.com
Foundation Project: Supported by the University of Ibadan Senate Research Grant
(Grant No. SRG/FP/2010/4A).
1. Introduction
Medicinal plants have great applications in folk medicine
within the African region; there is heavy reliance on them in
alleviating various disease conditions with the consequent
risk of gradual but imminent extinction[1-5]. Many plants
are investigated for the development of phytocompounds
useful in drug development[6]. There is no doubt that these
natural compounds from plants had contributed positively to
the health care delivery system in many rural communities
in Africa[7]. However, a review of literature revealed that
there are still seemingly insurmountable problems of mis-
identification in many medicinal plant groups. This has
increased the quest for search for other efficient methods
for the identification of medicinal plants and herbal
medicines[8-10].
Thus, with the increasing trend in the use of medicinal
plants, botanicals or herbal preparations particularly in
PEE R REVIEW ABSTRAC T
KEYWORDS
Leaf epidermis, Trichomes, Stomata, Anatomy, Medicinal plants, Nigeria
Objective: To carry out a pharmacobotanical study of Lonchocarpus cyanescens (Schum & Thonn)
Benth (L. cyanescens) and Leptoderris micrantha Dunn (L. micrantha) which are two key medicinal
plants from the family Fabaceae.
Methods: The epidermal peel was obtained by soaking the leaf in concentrated nitric acid
(HNO3) in a petri dish. Both surfaces were carefully mounted on clean glass slides and dehydrated
by ethyl alcohol, and stained with safaranin O for 2 min. Transverse sections of plant leaf
were obtained by free hand sectioning. Phytochemical screening for various constituents was
carried out on the powdered leaves. Other parameters such as, moisture content, ash value, acid
insoluble ash, water-soluble ash, water and alcohol extractive values were obtained by standard
techniques.
Results: The distinctive features of the species include: the presence of stomata on both surfaces
of L. cyanescens and the absence in L. micrantha. Presence of larger epidermal cells in both upper
and lower surfaces of L. cyanescens [(35.25依1.64)伊(31.25依2.36), (43.0依2.63)伊(39.5依5.11)] respectively
compared to L. micrantha. Glandular multicellular trichomes are present in L. micrantha but
absent in L. cyanescens. Numerous trichomes surround the transverse section of the leaf of L.
micrantha but absent in L. cyanescens. Preliminary phytochemical screening showed that both
species contain secondary metabolites such as alkaloids, anthraquinones, cardiac glycosides,
tannins, saponins, steroids and flavonoids.
Conclusions: The microscopic and phytochemical data provided in this study are useful for the
standardization of the medicinal plants.
Contents lists available at ScienceDirect
Peer reviewer
Abiodun E Ayodele, Professor,
Department of Botany, University of
Ibadan, Ibadan, Nigeria.
Tel:
+
23408023255482
E-mail: bayodele@yahoo.com
Comments
It is a good paper reporting the
anatomical and pharmacognostic
features of two Nigerian medicinal
plants, thus ensuring the correct
identification of both plants when
collected in fragmentary forms
and screened for subsequent drug
development.
Details on Page 135
Article history:
Received 17 Oct 2013
Received in revised form 29 Oct , 2nd revised form 12 Nov, 3rd revised form 5 Dec 2013
Accepted 20 Jan 2014
Available online 28 Feb 2014
Mubo A Sonibare et al./Asian Pac J Trop Biomed 2014; 4(2): 131-136
132
developing countries where orthodox medical care is neither
readily accessible nor affordable, it is only proper to ascertain
quality control standards in the herbal products from these
plants before they are subjected to human utilization[11]. The
two medicinal plants treated in this study are important in
African ethno medicine. The two plants have been mentioned
in our recent ethnobotanical survey as part of the plants used
in treating psychosis in Nigeria[12,13]. Other medicinal uses
of Leptoderris micrantha (L. micrantha) include its use for
dropsy, swellings, oedema, gout, and pulmonary troubles[14].
The root of Lonchocarpus cyanescens (L. cyanescens) is used to
teat arthritis and root and stem decoction is given to females
after childbirth and also used to treat hernia[15].
Anatomical and micromorphological characteristics of
leaves have played an important role in plant taxonomy,
especially of particular groups at generic and specific levels.
Studies in this field have attracted the attention of plant
morphologists and systematists to resolve taxonomic conflicts
in different groups of plant[16,17]. Although many studies have
been conducted in this area in other plants for the purpose of
correct identification of plant[18-22], little is known about the
anatomy and micromorphology of the medicinal plants under
study. Apart from a sketchy description of L. micrantha, little
or no work is available on its pharmacognostic features. The
same is true for L. cyanescens.
Therefore, in view of the wide medicinal applications
of the two plants under investigation, the present study
was undertaken in order to document information on
micromorphological features of the plants which would help in
their identification and authentication as well as provide basic
pharmacognostical data required for a herbal pharmacopoeial
compilation.
Thus, this paper reports on the macro and micro
characters of L. cyanescens and L. micrantha leaves, their
specific physical and chemical standards which may be
useful as quality control parameters in the Nigerian herbal
pharmacopoeia.
2. Materials and methods
2.1. Plant collection and authentication
L. cyanescens was collected at the main site of Faculty
of Veterinary Medicine, University of Ibadan, Nigeria.
while L. micrantha was collected at Iddo local Government
along Eruwa road in Ibadan, Nigeria. The specimens were
identified and authenticated by Mr. O. A. Osiyemi at the Forest
Herbarium, Ibadan (FHI), Nigeria where vouchers were also
deposited under FHI number 109689 for L. cyanescens and FHI
number for L. micrantha, respectively.
2.2. Gross morphology
Morphological studies were carried out by observation of
plant parts on the field with naked eyes and with the use of a
hand lens where necessary.
2.3. Surface tissue preparation
For epidermal studies, Shultze’s method of maceration with
improved technique[23] was followed. Leaves were taken in
petri dishes, covered with 4 mL of concentrated nitric acid
and kept under sun for 30 min. Adaxial and abaxial epidermal
peels obtained with the use of camel hair brush were rinsed
in distilled water severally, bleached with one to two drops of
chloral hydrate for 30 seconds. To remove chlorophyll, stained
in 1 % aqueous Safranin O solution for 3 min and mounted in
dilute glycerol for observation.
2.4. Transverse section
Leaf transverse sections were made with free hand
sectioning using surgical scapel. Briefly, scalpel was used to
cut 3 cm伊4 cm of the plants through the midrib region. The
small portion was inserted into a 3 cm伊4 cm section of unripe
pawpaw to enhance easy cut. The transverse sections obtained
were cleared using sodium hypochlorite 3.85% M/V and stained
with Safranin O reagent and rinsed with 70% alcohol. Glycerol
was added as mountant. The tissue distribution through the
midrib was examined under low power magnification and was
photographed with compound microscope fitted with a digital
camera.
2.5. Phytochemical screening
Fresh plant leaves were air-dried, powered with a blender
and stored in cellophane bags for phytochemical screening.
Phytochemical screening on the powdered leaves for various
constituents such as alkaloids, anthraquinone, cardiac
glycoside, glycoside, tannins, terpenoids, saponins and
steriods flavonoids, phlobatannins etc. was done using the
standard procedure[24].
2.6. Moisture content
The moisture content of the drugs (air dried plants) was
determined. Briefly, powdered drug (2 g) was transferred into
a china dish and distributed evenly to a depth not exceeding
10 mm. The loaded plate was heated at 105 °C in hot air oven
and weighed at different time intervals until a constant weight
was obtained. The difference in weight after drying and initial
weight is the moisture content. Same experiment was repeated
six times for precision and percent moisture for the sample
was calculated.
2.7. Total ash value
Powdered drug (2 g) was weighted accurately into a tarred
silica crucible and incinerated at 450 °C in muffle furnace
until free from carbon. The crucible was cooled to room
temperature and weighed. Percentage of ash was calculated
with reference to air dried substance.
伊100
% Ash value = Weight of residual ash
Original weight of sample
Mubo A Sonibare et al./Asian Pac J Trop Biomed 2014; 4(2): 131-136 133
2.8. Acid insoluble ash
Ash obtained from total ash was boiled with 25 mL of 2 N
HCl for few minutes and filtered through an ashless filter
paper. The filter paper was transferred into a tarred silica
crucible and incinerated at 650 °C in muffle furnace until
free from carbon. The crucible was cooled and weighted.
Percentage of acid insoluble ash was calculated with reference
to air dried substance as:
% Acid insoluble ash= Weight of residual ash
Weight of original sample 伊100
2.9. Water soluble ash
Ash obtained from total ash was boiled with 25 mL of
distilled water for few minutes and filtered through an ashless
filter paper. The filter paper was transferred into a tarred
silica crucible and incinerated at 450 °C in muffle furnace
until free from carbon. The crucible was cooled and weighed.
Percentage of water soluble ash was calculated with reference
to air dried substance.
2.10. Alcohol-soluble extractive value
About 5 g of the powdered drug was accurately weighed into
250 mL stoppered conical flask. A hundred milliliters of 90%
ethanol was added and tightened firmly. The flask was shaken
using a mechanical shaker for 6 h, and then allowed to stand
for 18 h. The extract was filtered quickly (by suction filtration).
The weight of a clean, heated and cooled flat-bottomed
evaporating dish was accurately determined. Tweenty
milliters of the filtrate was evaporated to dryness. The residue
was dried to constant weight at 105 °C in an oven and the final
weight determined. The alcohol soluble extractive value was
calculated thus:
% Alcohol soluble extractive value= Weight of residue in 20 mL
20 伊100
2.11. Water-soluble extractive values
About 5 g of powdered drug was treated with 100 mL water
at in a stoppered flask with frequent shaking during first
6 h using electrical shaker and allowed to stand for 24 h.
Temperature was maintained at 45 °C during entire process.
Extract was filtered and 10 mL of filtrate was evaporated in a
tarred dish at 105 °C and weighed. Water soluble extractive
value was calculated as:
% Water soluble extractive values= Weight of residue in 20 mL
20 伊100
3. Results
3.1. Macroscopic characteristics
The two medicinal plants: L. cyanescens and L. micrantha
differ in their morphological features. In L. cyanescens leaf
shape is lanceolate, margin is entire, surface is glabrous,
midrib is prominent with papery texture. In L. micrantha, leaf
is oblanceolate in shape, margin is entire, apex is acute, surface
is glabrous, petiolate with sessile flower and flat, fruit is broadly
elliptic.
3.2. Epidermis
Leaf epidermal studies of L. micrantha and L. cyanescens were
carried out in search of anatomical characters of taxonomic
value that may contribute to the proper identification of the
plants. Qualitative and quantitative features of the epidermal
morphology of the plants are recorded in Table 1. The epidermal
cells on the adaxial surface of L. micrantha have straight
anticlinal walls and unicellular trichomes (Figures 1 A and B),
while on the adaxial surface of L. cyanescens, epidermal cells
have wavy/undulating anticlinal walls (Figures 1 C and D).
trb trb
tr
ec ec
A
C
B
D
Figure 1. Adaxial epidermis of L. micrantha and L. cyanescens. (400伊)
A&B: Adaxial epidermis of L. micrantha; C&D: Adaxial epidermis of L.
cyanescens. trb: Trichome base; tr: Trichome; ec: Epidermal cell.
The abaxial surface of L. micrantha has straight to curve walls
and peltate trichomes. No stomata were observed on either
of the surfaces in this species (Figures 2 A and B). Epidermal
cells are smooth to slightly wavy on the abaxial surface of
L. cyanescens (Figure 2C). Both surfaces have many stomata,
paracytic and anisocytic on the adaxial and abaxial surfaces,
respectively (Figure 2D). However, fewer stomata and trichomes
were seen on the adaxial surface on L. cyanescens compared
to the numerous seen on its abaxial surface. Multicellular
Table 1
Combined qualitative and quantitative features of the epidermal morphology of L. micrantha and L. cyanescens.
Taxa Leaf
surface Stomatal type Epidermal cell shape Anticlinal wall
pattern Trichome type Trichome
length (μm)
Trichome
width (μm)
Epidermal cell
length (μm)
Epidermal cell
width (μm)
Stomatal
length (μm)
Stomatal
width (μm)
L. micrantha Adaxial Absent Polygonal Straight Unicellular 232.5依29.5 26.0依5.8 27.0依1.7 21.0依1.7Absent Absent
Abaxial Absent Polygonal/Irregular Straight/Curved Glandular, Peltate 338.8依47.3 19.3依1.5 25.0依1.4 22.5依1.6Absent Absent
L. cyanescens Adaxial Paracytic Irregular Undulate Unicellular 253.0依57.2 18.0依3.2 35.1依1.6 31.1依2.4- -
Abaxial Anomocytic Irregular Undulate Multicellular, Uniseriate 102.3依4.1 12.0依0.7 43.0依2.6 39.5依5.1 15.0依1.1 12.8依1.1
Mubo A Sonibare et al./Asian Pac J Trop Biomed 2014; 4(2): 131-136
134
uniseriate trichomes were observed on the abaxial surface.
Mucilage was observed on the abaxial epidermal cells of both
plants.
mu
tr
ec st
A
C
B
D
Figure 2. Abaxial epidermis of L. micrantha and L. cyanescens. (400伊)
A & B: Abaxial epidermis of L. micrantha; C & D: Abaxial epidermis of L.
cyanescens showing smooth to slight wavy epidermal wall and anisocytic
stomata. mu: Mucilage; tr: Trichome; ec: Epidermal cell; st: Stomata.
3.3. Leaf transverse section
Transverse sections of leaf blades of L. cyanescens and L.
micrantha are shown in Figures 3 A and B. The transverse
section of L. cyanescens appeared concave on the abaxial and
convex on the adaxial surface. Cuticle is moderately thick and
the vascular bundle is arc-shaped. The xylem vessel is lignified
as it picks up stain with safranin reagent. The transverse section
of L. micrantha has numerous unicellular trichomes, covered
with moderately thick cuticle. The vascular bundle is arc
shaped with xylem inside and phloem outside. Variations were
found in shapes and numbers of vascular bundles, trichomes
and presence of mucilage. Also, numerous peltate trichomes
were seen in L. micrantha while these were absent in L.
cyanescens.
tr
xy xy
bs
ph
cu col
AB
Figure 3. Transverse section of the leaf through the midrib regions of L.
micrantha and L. cyanescens. (100伊)
A: L. micrantha; B: L. cyanescens. tr: Trichome; xy: Xylem; bs: Bundle sheath;
col: Collenchyma; cu: Cuticle.
3.4. Physicochemical constant and phytochemical data
The physicochemical constants recorded for the two medicinal
plants: L. micrantha and L. cyanescens include: moisture
content, total ash, acid insoluble ash, water insoluble ash, water
extractive value, acid extractive value as presented in Table 2.
Phytochemical data appeared in Table 3.
Table 2
The analytical values in respect of physicochemical constant for L.
micrantha and L. cyanescens.
Physico-chemical
constants
Mean analytical values
L. micrantha L. cyanescens
Moisture content 6.500
依
6.500 14.000
依
2.800
Total ash 8.000
依
0.000 14.500
依
2.100
Acid insoluble ash 4.500
依
0.700 7.500
依
0.700
Water insoluble ash 10.000
依
1.400 7.750
依
1.100
Water extractive value 0.146
依
0.020 0.121
依
0.001
Acid extractive value 0.450
依
0.100 0.550
依
0.100
Table 3
Phytochemical screening results of the powdered leaves of L .
micrantha and L. cyanescens.
Phytoconstituents L. micrantha L. cyanescens
Alkaloids
+ +
Anthraquinones
+ +
Cardiac glycosides
+ +
Tannins
+ +
Saponins
+ +
Steroids
+ +
Flavonoids
+ +
Phlobatannins - -
+
: Present; -: Absent.
4. Discussion
The variations observed in the epidermal morphology of L.
micrantha and L. cyanescens from the family Fabaceae could be
used to distinguish the species. Anatomical features are widely
used in systematics for identification, for placing anomalous
groups in satisfactory position in classification and for
indicating patterns of relationship that may have been observed
by superficial convergence in morphological features[25]. Non-
glandular unicellular trichomes on both adaxial and abaxial
surfaces are considered interesting and the density of hairs was
more abundant on the abaxial surface for both plants. The high
density of thick and coated hairs probably serves to reduce the
rate of transpiration in plants and this buttresses the importance
of trichomes in taxonomy as a diagnostic tool[26].
The adaxial and the abaxial surfaces of both plant species
have comparable sizes of epidermal cells. Few stomata and
trichomes were seen on the adaxial surface on L. cyanescens
compared to the numerous seen on its abaxial surface. Stomata
were not observed on the adaxial and abaxial surfaces of L.
micrantha although there are numerous trichomes on both
surfaces. Thus, stomata character and trichomes can aid
classification and identification of these plants.
The two medicinal plants appear to be rich in secondary
metabolite, widely used in traditional medicine to manage
or cure various ailments. Phytochemical screening showed
that both species contain metabolites such as alkaloids,
anthraquinones, cardiac glycosides, tannins, saponins,
steroids and flavonoids. The presence of the various secondary
metabolites may support the folkloric claims on the medicinal
Mubo A Sonibare et al./Asian Pac J Trop Biomed 2014; 4(2): 131-136 135
potential of these plants as anti-inflammatory, antispasmodic,
analgesic and antidiuretic.
Physical constant parameters and quantitative evaluation
such as moisture content, total ash value, acid insoluble ash,
water insoluble ash, water and acid extractive values which
form part of the parameters evaluated in standardizing herbal
medicines, were also determined in this study. Low moisture
content is reported to indicate less chances of microbial
degradation of plant drugs during storage[27]. This is in line
with our observations in this study, the moisture content value
of L. cyanescens and L. micrantha are respectively, (14.0依2.8) %,
w/w and (6.0依6.5)%, w/w. The general requirement of moisture
content in crude drug is that, it should not be more than 14%[28],
thus the values obtained in this study are within the accepted
range. A standard that is expressed as “not more than” value
is recommended for total ash and acid insoluble ash values
while water insoluble ash is expressed as “not less than”
value. Based on the results obtained for L. cyanescens and L.
micrantha, the total ash should not be more than 14.5依2.1 and
8.0依0.0 respectively with the acid insoluble ash values of not
more than 7.5依0.7 and 4.5依0.7 respectively. The water insoluble
ash should not be less than 7.75依1.1 and 10依1.4 respectively.
For water extractive and acid extractive values which are also
expressed as “not less than”, the results obtained indicate that
L. cyanescens and L. micrantha should not be less than 0.121
依0.001 and 0.146依0.020 respectively for water extractive values
and 0.55依0.10 and 0.45依0.10 respectively, for acid extractive
values.
Anatomical studies on the two medicinal plants
revealed sharp generic variations in sizes and types
of stomata, shapes of epidermal cells on the adaxial
and abaxial surfaces, sizes and types of trichomes, all
of which could be employed in species identification.
Correct identification of medicinally important
species is necessary for their sustainable and effective
utilization as well as for detecting adulteration of
plant drugs[29,30]. Thus, micromorphological character
variations observed in our study had provided the basis
for the identification of these plants highlighting their
taxonomic relationships in line with report on other
plants[31-33]. Leaf epidermal and anatomical features
such as stomata, trichomes and other markers have
proved useful and of great taxonomic significance[34-36].
In our study of the micromorphological features and
phytochemical screening of the leaves of L. micrantha
and L. cyanescens, we are able to draw the following
conclusions: Firstly, stomata are absent on both adaxial
and abaxial surfaces of L. micrantha. Secondly, the
leaves of both plants contain alkaloids, anthraquinones,
cardiac glycosides, tannins, saponins, steroids,
flavonoids which may be responsible for the therapeutic
effects of the two plant species.
The morpho-anatomical features observed in the two
medicinal plants are sufficiently distinctive and may
be used at specific level for delimitation of plants. The
different features peculiar to each of the medicinal
plants provide the basis for its detailed assessment and
form part of the catalogue of characters that may be
employed in proper identification of these plants and
as quality control standards which can be used for the
compilation of herbal pharmacopoeial.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
The authors are grateful to Mr. OA Osiyemi for plant
identification and to Mr. AA Adeniran for technical
assistance in slide preparation. This study is supported
by the University of Ibadan Senate Research Grant
(Grant No. SRG/FP/2010/4A).
Comments
Background
A major problem in traditional medicine is the
misidentification of drug plants particularly when these
are bought directly from the local markets. There is
therefore the need to use other characters apart from the
macrocharacters to ascertain the authenticity of such
crude drug samples.
Research frontiers
The study provides information on the microcharacters
and pharmacognostic features of L. cyanescens (Schum
& Thonn.) Benth. and L. micrantha Dunn.
Related reports
Some related researches are Taxonomic importance of
anatomical and micromorphological features in plants
(Kahraman and Celep, 2010, Saheed and Illoh, 2010),
and Plants are investigated for the development of
phytocompounds useful in drug development (Cornara et
al., 2013).
Innovations and breakthroughs
Additional information on useful characters for
the authentication of samples of the two important
medicinal plants.
Applications
It provided some means of authentication of crude
drug samples for new drug development.
Peer review
It is a good paper reporting the anatomical and
pharmacognostic features of two Nigerian medicinal
plants thus ensuring the correct identification of
both plants when collected in fragmentary forms and
screened for subsequent drug development.
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