J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2015, 4(1), 5-11 ISSN:23254513( PRINT) ISSN 2325 - 453X (ONLINE )

Research Article 5

Comparative Evaluation of some Selected Bioactive Constituents in the

Leaves and Bark of Avicennia marina (Forsk.) Veirh from the Sudanese
Red Sea Coast
Nahid A. Osman1* and Faiza A. Abkar1
1

Faculty of Marine Science and Fisheries, Red Sea University, Port Sudan, Sudan. P. O. box 24.
* Corresponding author (nahidcoast@yahoo.com).
(Received: August 27, 2014; Accepted: January 06, 2015)

Abstract-The
presence
of
selected
phytoconstituents in the leaves and bark of
Avicennia marina (Forssk.) Veirh collected from a
stand along the Sudanese Red Sea coast is assessed
with regular phytochemical methods. The
qualitative screening revealed that both plant parts
contained alkaloids, flavonoids, sterols, and
tannins. Quantitatively, the bark contained more
alkaloids (12.75%), flavonoids (4.5%), and tannins
(34.6%) than the leaves which contained 11.63%,
3.6% and 15.90% respectively. The findings were
correlated with the prevailing environmental
conditions in the study area. The potentiality of this
tree as a possible source of phytochemicals with
pharmaceutical prospective was also discussed.
Index terms: Phytochemicals, Avicennia marina,
leaves, bark, Sudan

I.

INTRODUCTION

Mangroves thrive under stressful and extreme

tropical environmental conditions [1] such as
high solar radiation, temperature, salinity, and
anaerobic conditions that may have unfavorable
effects on the photosynthesis of these plants.
Hence, mangroves have evolved special
adaptation to survive these conditions.
Adaptations of mangrove plants to their harsh
habitat occur at morphological, physiological,
and biochemical levels spanning canopy
architecture, salt regulation, and oxygen
conversion [2]. Although the main function of
these adaptations is to counteract the
unfavourable effects of the aforementioned
abiotic stresses, it has resulted in unique
attributes which distinguish mangroves among
other plants.
One of these attributes are the secondary
metabolites produced by the mangroves which
have been used traditionally by medicinal local
practitioners due to their proved medicinal values
[3,4]. Contemporary research has verified the
traditional knowledge on mangrove and has

revealed that mangrove plants contain a wide

array of novel bioactive metabolites with
pharmaceutical potential [5], however, of
varying efficiency on inhibition of pathogens and
tumors.
Along the Red Sea coasts mangroves survive in
an extreme tropical arid environment with
significant solar radiation, insufficient and
temporal freshwater influx, and highly saline
seawater ( 40). The true mangrove plant
Avicennia marina (Forsk.) Veirh is the keystone
species of the mangrove stands along the Red
Sea coast of Sudan. The plant that was recently
placed in the family Acanthaceae is commonly
known as the grey or white mangrove. It is a
small tree or shrub (4 to 7m in height) grows in
the intertidal areas receiving freshwater
discharges along the tropical and subtropical sea
coasts.
Although A. marina has been reported as a
medicinal plant in both conventional medicine
[6,7] and contemporary pharmacognosy research
on mangrove plants little research was done to
investigate phytoconstituents of the Sudanese
species. Recently, Mouafi et al. [8] reported the
presence of seven phytochemicals from A.
marina leaves collected from the Sudanese Red
Sea coast and the ability of its extract to inhibit
the growth of some antibiotic resistant bacteria
and one fungi species.
Recognizing the paucity of information on
quantitative values of phytochemicals of
Sudanese A. Marina the present paper aims to
compare the availability of selected bioactive
constituents in leaves and bark of this true
mangrove tree and to determine their relative
concentrations in these parts. Besides, the paper
aimed to quantify these bioconstituents for
comparison with relevant values of equivalent
plants from the wet tropical region to help

J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2015, 4(1), 5-11 ISSN:23254513( PRINT) ISSN 2325 - 453X (ONLINE )

understand the influence of environmental

conditions on the synthesis of these compounds.
II. MATERIALS AND METHODS
The experimental work was based on the
recommendations given by Harbone [9]. The
qualitative screening tested the presence of 4
phytochemical groups of compounds namely:
alkaloids, flavonoids, sterols, and tannins. The
quantitative measurements determined the
content of 3 of the 4 tested phytochemicals
namely: alkaloids, flavonoids, and tannins.
Regular phytochemical
procedures were
followed for the qualitative and quantitative
analysis. Experiments were performed in
replicates and\or triplicates to ensure reliability
of the results.
Study area
The leaves and bark of Avicennia marina were
collected from a mangrove stand located 8
kilometers south of Port Sudan harbor between
latitudes E 19 35 40 and E 19 36 40 and
longitudes N 37 14 54 and N 37 14 55.
Plant materials
Mature healthy leaves and the stem bark of
healthy trees were collected from the stand and
washed with tap and distilled water respectively.
The samples were then dried in the shade at
room temperature, milled to a homogenous
powder and kept in tight container.
Extraction
The serial exhausted extraction method [10] was
followed to extract plant materials. Ten grams of
plant tissues were first macerated in 100 ml of
ether for 3 days at room temperature with
frequent shaking. The mixture was centrifuged
(10000g/10m) and filtered with Whatman No. 1
filter paper into a clean glass vial and kept at 4
C until further analysis. Then the residues were
successively
extracted
with
chloroform,
methanol, and distilled water respectively.
Qualitative screening of bioactive constituents
The extracts were tested for the presence of
alkaloids, flavonoids, sterols, and tannins
phytochemical groups of compounds. This was

6
based on development of colouration and
precipitation upon addition of certain chemical
reagents to the plant parts extracts.
Test of alkaloids
The presence of alkaloids in the plant extract was
tested with Wagner`s reagent following the
procedure described by Sabri, et al. (2012) [11].
Ten ml of the extract were evaporated to dryness.
Two ml of 2% HCl acid solution were added to
the dry residues. Few drops of the reagent were
added. Formation of reddish brown precipitate
indicates the presence of alkaloids.
Test of flavonoids
This test was done as described in Pamar et al.
(2012) [12]. Few drops of NaOH were added to 2
ml of extract. The formation of intense yellow
colouration that changed to colourless when few
drops of dilute HCl were added indicates the
presence of flavonoids.
Test of sterols
Two ml of concentrated sulphuric acid were
added to 2 ml extract [13]. Formation of red
precipitate indicated the presence of sterols.
Test of tannin
Tannins were detected according to the methods
described in Ugochuhwu et al. (2013) [14]. To 1
ml of extract solution, 1 ml of 3% ferric chloride
solution was added. Development of brownish
green or a blue black coloration indicated the
presence of tannins.
Quantitative determination of bioactive
constituents
The crude concentration of three of the four
tested phytochemical groups of compounds was
gravimetrically determined in the leaves and bark
of A. marina on dry weigh basis. The
concentration of alkaloids was determined
according to Harbone (1973) as described by
Agoreyo et al. (2012) [15]. The method of
Kocip-Abyazan (1994) described by Eleazu et al.
(2012) [16] was performed to determine the
concentration of flavonoids. Tannins were
quantified with the methods illustrated by Vetter
and Barbosa (1995) [17]. Below are the
descriptions of the procedures performed.

J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2015, 4(1), 5-11 ISSN:23254513( PRINT) ISSN 2325 - 453X (ONLINE )

Moisture content
Two grams of the plant tissue were incubated in
an oven at 105 C until the sample reached a
constant weight. Moisture content (MC) was
determined gravimetrically with the following
formula.
MC (%) = weight of sample after oven
drying/weight of sample/100
Alkaloids
Five grams of the sample was weighed into a 250
ml beaker and 200 ml of 10% acetic acid in
ethanol were added. The mixture was covered
and allowed to stand for four hours. Then filtered
and the extract was concentrated on a water bath
to one-quarter of the original volume.
Concentrated ammonium hydroxide solution was
added drop wise to the extract until the
precipitation was complete. The whole solution
was allowed to settle and the precipitate was
filtered, washed with dilute ammonium
hydroxide solution, dried and weighed. The
content of alkaloids was determine with the
following formula
Alkaloids (%) = weight of precipitate X100.
weight of sample-MC
Flavonoids
Ten grams of the plant sample were extracted
repeatedly with 100 ml of 80% aqueous
methanol at room temperature. The mixture was
filtered through whatman filter paper No 42 (125
mm). The filtrate was later transferred into a
crucible and evaporated over a water bath to
dryness and weighed when constant weight was
achieved. The total content of flavonoids was
determined as follows.
Flavonoids (%) = weight of precipitate X100.
weight of sample-MC
Tannins
Two grams of plant tissue were extracted with
100 ml distilled water for one hour at 90 C. the
mixture was decanted through a filter paper and
the residue was extracted again. The 2 filtrates
were combined, allowed to cool down, and made
up to 500 ml with distilled water. One hundred
ml of this solution were transferred to a beaker
and 10 ml of 40% formaldehyde and 5 ml of
concentrated sulphuric acids were added. The
mixture was refluxed for 30 minutes, allowed to
cool down and filtered. The precipitate was dried
and weighed. Tannin content was obtained with
the following formula.
Tannins (%) = weight of precipitate X100.
weight of sample-MC

III. RESULT AND DISCUSSION

7
Alkaloids, flavonoids, sterols, and tannin were
detected and measured in both leaves and bark of
Sudanese A. marina.
Presence of the bioactive constituents in the
leaves and bark of A. marina
The presence of phytochemical compounds of
Sudanese A. marina (Forssk.) Veirh varied in the
different extracts of leaves and bark (Table 1). In
the leaves extracts, alkaloids were present in both
methanolic and distilled water extracts.
Flavonoids were detected only in the distilled
water extract. Sterols were absent from the ether
extract of the leaves and were detectable in the
chloroform, methanol, and distilled water
extracts. Presence of tannins in the leaves was
observed in both methanolic and distilled water
extracts. Among the bark extracts, alkaloids were
only soluble in the methanolic one. Flavonoids
were detectable in both methanolic and distilled
water extracts. The sterols and tannins of the A.
marina bark were soluble in all the solvents
employed for the extraction. The variation in the
presence of the phytochemical compounds in the
different extracts may largely be attributed to the
solubility of these compounds in the solvents
used for extraction. The solubility of any
phytochemical in a given solvent is influenced
by many factors. Some of these factors are
related to the physicochemical properties of the
phytochemical compound and the other may be
related to the solvent characteristics. Examples
of the factors related to the phytochemical
compound properties are polarity, the functional
group of the phytochemical, and its molecular
weight. During extraction the solvent diffuses
into the solid plant materials and dissolve
compounds with similar polarity [10]. Applying
this to the currently obtained solubility pattern of
phytoconstituents in the extracts of leaves and
bark of A. marina may suggest that the leaves
and bark would contain the same phytochemical
groups of compounds; however their chemical
structures and type may differ. For example, and
within the context of this study, the leaves may
contain 2 types of alkaloids one soluble in
methanol and the other is soluble in distilled
water. Bark seems to contain one group of
alkaloids soluble in methanol. This may also be
applicable for flavonoids of both leaves and bark.
Likewise, the detection of sterol in 3 leaves

J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2015, 4(1), 5-11 ISSN:23254513( PRINT) ISSN 2325 - 453X (ONLINE )

extracts and 4 bark extracts could also indicates

that Sudanese A, marina contain an array of
sterols that remains to be investigated. Similarly,
the solubility of tannins in 2 different extracts of
the leaves and in all the 4 different extracts of the
bark may preliminary indicate the diversity of
tannins compounds produced by this tree and
signify its potentiality as a medicinal plants and

8
highlight the need for further research to
characterize these bioactive compounds.
The bioactive contents in the leaves and bark
of A. marina
In general the bark of the Sudanese A. marina
(Forssk.) Veirh contained greater concentrations
of the tested phytochemical groups of
compounds compared to the leaves (Table 2)

Table 1
Presence of the bioactive constituents in the leaves and bark of A. marina from Sudan.
Phytochemical groups of compounds Presence of phytochemicals in leaves and bark of A. marina
Leaves
Bark
E
C
M
W
E
C
M
W
Alkaloids.
+
+
+
Flavonoids.
+
+
+
Sterols.
+
+
+
+
+
+
+
Tannins.
+
+
+
+
+
+
Percentage yield (%)
2.43
1.6
1.72
2.94
3.45
1.78
1.5
2.13
E: ether, C: chloroform, M: methanol, W: distilled water.

Table 2
Content of the alkaloids, flavonoids, and tannins in the leaves and bark of A. marina from Sudan.
Alkaloids (%) Flavonoids (%) Tannins (%)
A. marina part
Leaves
Bark

11.50.15
12.730.025

3.550.05
4.500.0

Comparatively the bark of A. marina contained

more alkaloids (12.75%), more flavonoids
(4.5%) and higher tannins percent (34.60%) than
the leaves which contained 11.63%, 3.6% and
15.90% respectively.
Presence of alkaloids has been reported from
species of the genus Avicennia including A.
marina [8,18,19]. Species of this genus from
Australia were considered as a poor source of
alkaloids [2]. The present values of the
concentrations of alkaloids of the leaves and bark
of the Sudanese A. marina indicate that this tree
could represent a potential source of alkaloids,
particularly when compared with the values of
alkaloids obtained for some medicinal plants. For
example the value of 1.04% alkaloids in the
medicinal plant Sida acuta (Malvaceae) was the
maximum value reported by Edeoga et al (2005)
[20] among the 10 medicinal plants investigated.
This is also similar to the value of 1.13%
reported by Khan et al (2011) [21] for Adhatoda

15.850.05
34.650.07
vasica.
Possible
explanations
for
the
comparatively high level of alkaloids in the
tissues of Sudanese A. marina may almost
certainly be related to biotic and abiotic stress
conditions. The latter is manifested in the harsh
environmental condition of aridity prevailing in
the plant habitat. Recent studies and reviews
reported the protective role of higher plant
secondary metabolites against abiotic stresses
including drought, high salinity, UV light
exposure,
high
and
low
temperatures
[22;23;24,25;26].
Under
these
stressing
conditions, reactive oxygen species (ROS) are
excessively released [26]. ROS cause disturbance
in plant cell homeostasis and eventually leads to
cell death. Therefore, plants suffer from these
stresses generate high oversupply of bioactive
compounds to prevent the damaging effect of
ROS. Elevated levels of alkaloids, flavonoids,
and phenolics were reported from plants
subjected to drought and salt stress conditions

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[25,27,28.29]. In some instance, an increase of

187% in total alkaloids content in Catharanthus
roseus plant subjected to drought stress was
reported [30]. The extreme tropical arid climate
prevailing in the Sudanese Red Sea coast may
has produced a significant bearing on the
composition and content of A. marina bioactive
constituents including alkaloids. It seems that the
Sudanese species may have accumulated
alkaloids to survive these conditions. This is in
conformity with Selmar (2008) [24] who
discussed the analogous quality differences
observed between equivalent medicinal plants
grown in moderate Atlantic climate and those
grown in semi-arid region with regard to
secondary products contents. The content of
these products is less in the former group of plant
compared to the second one.
Under biotic stress conditions the plants use
alkaloids as a chemical defense against
hervbivory and pathogens [31]. Alkaloids tend to
be accumulated in the plant part to be protected
at sufficient concentration to be an adequate
defense. For example quinolizidine and
pyrrolizidine alkaloids are concentrated in the
peripheral layers of the stems of Lupinus and
Senecio and are 10 to 20% higher than the mean
value of the whole stem [32]. In the present
communication alkaloids were extracted from
the most vulnerable parts of the Sudanese A.
marina, leave and bark that are subjected to
camel browsing and extreme solar radiations.
Some alkaloids exert pharmacological activities
in human and lower animals. In human they
influence the nervous system particularly the
action of the chemical transmitters [32].
Alkaloids have many other pharmacological
benefits including antihypertension, anticancer,
antimalarial, antiarrhythmic, antibiotic and
antiseptic activities.
The values of flavonoids obtained for both leaves
and bark of A. marina in this study are
remarkably higher than those reported for other
mangroves from wet tropical region [33] where
the concentration of flavonoids in the bark of
Aegceras corniculatum was 0.44%, Bruguiera
gymnorrhiza was 0.26%, Cynometra iripa was
0.49% and that of Lumnitzera racemosa was
0.89%. The higher levels of flavonoids in the
Sudanese mangrove species may largely be an
adaptation to the extreme solar radiation in the
Red Sea region that is known to experience some
of the hottest and most arid conditions which
occur in any marine area on Earth [34]. This is

9
also in conformity with Bandaranayake (1994)
[2] who reported that tropical mangroves have
high concentration of flavonoids in the leaf
epidermis and that these compounds perform a
protective role against the damaging effects of
ultraviolet-B radiation. It has been suggested that
a flavonoids-DNA complex provide mutual
protection against oxidative damage of harmful
solar radiation [35] through acting as a screen in
the epidermal cell layer thus adjusting the
antioxidant system at both cell and whole
organism levels. Accordingly, flavonoids are of
great pharmacological benefits to human health
particularly in preventing and curing of ailments
resulting from the free radicals and inflammatory
agents.
Plant sterols know as phytosterols have several
bioactive properties with possible benefits for
human health [36]. They contribute to lowering
serum cholesterol level and were reported to
produce anti-inflammatory, anti-bacterial, antiartherosclerotic, anti-oxidant, anti-ulcerative, and
antitumor influences. Besides, phytosterols serve
as precursor for the production of hormone
pharmaceuticals.
Similarly, the concentration of tannins in
Sudanese species is noticeably higher that its
concentration in Aegceras corniculatum (5.08%),
Bruguiera gymnorrhiza (3.70%), Cynometra
iripa (5.22%), and that of Lumnitzera racemosa
(10.27%). The members of the genus Avicennia
were reported to exhibit an exceptionally strong
positive reaction for tannins tests in qualitative
phytochemical screening [2]. Additionally,
drought stressed plants were reported to yield a
10% higher amount of phenolic compounds [24].
The biological role of tannin in plant is related to
protection against infection, insect and animal
hervbivory. In traditional healing tannincontaining plant extracts were used as astringent,
diuretics, antiinflamatory, antiseptics, and
haemostatic pharmaceuticals [37]. Recently
compounds representative of tannins were found
to have antiviral, antibacterial, and anticancer
activities in biological tests.
IV. CONCLUSION
The search for new lead compounds for the
development of new pharmaceuticals has
become increasingly important especially with
the drug resistance exhibited by disease causing
agents.
Surviving
in
extremely
harsh
environmental conditions as well as being

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subjected to anthropogenic stresses, the Sudanese

A. marina may contain novel pharmacological
therapeutics. Further investigations on the
isolation, characterization and biological assays
of the bioactive constituents of this tree may help
to provide a template for new potent drugs.
Acknowledgements
The authors are grateful to Zeinab Osman, Ph. D
of wood chemistry, Sudan National Center of
Research for reviewing the manuscript.
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