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ORIGINAL ARTICLE
Discovery Phytomedicine 2021, Volume 8, Number 4: 167-174
www.phytomedicine.ejournals.ca Discovery Phytomedicine 2021; 8(4): 167-174. doi: 10.15562/phytomedicine.2021.183 167
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ABSTRACT
Senna alata L. is a widely distributed medicinal plant mainly used
to treat fungal infections. The objectives of the present study
were to investigate the phytochemical constituents, antioxidants,
thrombolytic, anticoagulant and anthelmintic potentials of the
aqueous (AE) and ethanolic (EE) extracts of Senna alata leaves. The
major phytochemical classes were checked through qualitative
screening. Quantitative assays were employed to determine the
total phenolic, avonoid, avonol, tannin, and protein contents.
Antioxidant potential was revealed through the DPPH scavenging
assay. The extracts were applied to dissolve blood clots to evaluate the
thrombolytic potential. Prothrombin time (PT) and activated partial
thromboplastin time (aPTT) tests indicated the potential anticoagulant
activity. The anthelmintic potential was evaluated by using aquarium
worms (Tubifex tubifex). The presence of 9 dierent classes of
phytochemicals indicated the chemical diversity of the extract. In the
quantitative determination, EE was found to contain higher quantities
of phytochemicals than AE. The highest DPPH scavenging activity
(89.44%) was displayed by the EE at its 800 µg/mL concentration.
The IC50 values of EE and AE were 61.02 µg/mL and 142.42 µg/mL,
respectively. During the thrombolytic potential evaluation of EE and
AE, 37% and 27% clot lysis abilities were observed respectively. EE
paralyzed the aquarium worms at 4 min and killed them at 6 min.
The leaves of Senna alata have the potentials for being utilized for
medicinal purposes other than their traditional use as an antifungal
agent. These ndings can pave the way for the exploration of herbal
remedies with better ecacy.
Keywords: anthelmintic; clot lysis; cassia alata; Dadmordon; phytomedicine; prothrombin time
INTRODUCTION
Oxidative stress arises from the altered levels of
reactive oxygen species (ROS) or oxygen- free radi-
cals which can cause many human diseases. ese
free radicals are generated within the body which
may be responsible for cell and tissue damage
during infections and illness.1 Antioxidants can
inhibit the oxidation of other molecules and also
can prevent or repair the detrimental eects of
oxygen free radicals. Natural antioxidants provide a
protective eect and counteract the progression of
many diseases and disorders.2
rombus or blood clot formation in the circula-
tory system is known as thrombosis. It causes blood
ow blockage and results in severe consequences
such as strokes and heart attacks which oen lead
to death. During thrombosis, several drugs are
used to dissolve the blood clots. In addition to
thrombosis, coagulation factors sometimes act in
an uncontrolled manner and their aberrant action
within the blood vessels can give rise to adverse
consequences.3 For the treatment of these throm-
boembolic disorders, anticoagulant agents are used.
rombolytic agents are used for the dissolution of
blood clots. Anticoagulants are generally used as an
acute and intensive care approach for the inhibition
of the intrinsic and extrinsic pathways in the blood
clotting cascade.4 erefore, both thrombolytic
agents and anticoagulants are important in ghting
cardiovascular complications.
Parasitic worms are the contributing factors to
some of the most common and prevalent chronic
infections in developing and underdeveloped
countries. ese peoples are the main victims of
helminthiasis where children are more susceptible
and vulnerable.5 Synthetic anthelmintics have some
major disadvantages including lack of availability,
high price, and risk of misuses that can cause resis-
tance to drugs.
Medicinal plants have been utilized for safe
and eective remedies to diseases for thousands of
years. Plants contain certain chemical substances
known as phytochemicals which are responsible for
their medicinal values. Among the phytochemicals,
secondary metabolites are attributed to dierent
therapeutic purposes and also act as leads for the
synthesis of novel drugs.6 Modern research now
focusing on natural remedies due to several draw-
backs of synthetic drugs. e demand for natural
medicine is continuously increasing and that’s why
researchers have devoted themselves to exploring
Cell Genetics and Plant Biotechnology Laboratory, Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka- 1342,
Bangladesh.
*Correspondence to:
Dr. Abdullah MohammadShohael,
Professor, Department of
Biotechnology and Genetic
Engineering, Jahangirnagar
University, Savar, D haka- 1342,
Bangladesh, Phone:
+8801841391712,
amshohael@juniv.edu
ORCID ID: https://orcid.
org/0000- 0003- 3879- 2464
Cite This Article: Ahmed, S.,
Rahman, F.B., Shohael. A.M. 2021. In
vitro analysis of phytoconstituents
and bioactivities of Senna
alata L. leaf extracts. Discovery
Phytomedicine 8(4): 167- 174. DOI:
10.15562/phytomedicine.2021.183
Volume No.: 8
Issue: 4
First page No.: 167
RH_Author: XXX
Doi: Discovery Phytomedicine.2021.183
Original Article
In vitro analysis of phytoconstituents and
bioactivities of Senna alata L. leaf extracts
Sium Ahmed, Faisal Bin Rahman, Abdullah Mohammad Shohael*
168 Discovery Phytomedicine 2021; 8(4): 167-174. doi: 10.15562/phytomedicine.2021.183 www.phytomedicine.ejournals.ca
In vitro analysis of phytoconstituents and... Abdullah Mohammad Shohael, et al.
the potential activities of medicinal plants.7 ere
are many previous reports of antioxidant, thrombo-
lytic, anticoagulant, and anthelmintic capabilities of
plant extracts. ese studies aid in the discovery of
new agents to ght associated diseases with better
ecacy and fewer side eects.
Senna alata L. (S. alata) is commonly known as
Dadmordon in Bangladesh and is found all over the
country.8 e plant is also known as Cassia alata,
ringworm shrub, Candle brush, Candlestick, etc.9
Leaves of this plant have been traditionally used
to treat ringworm infections. e plant is native to
Central America and now growing in many coun-
tries of Southeast Asia, Africa, and North America.
S. alata is an important medicinal plant and
proved to have numerous pharmacological uses
including digestive, dermatologic, anti- infectious,
anti- diabetic, anti- inammatory properties, etc.10
Leaves of this plant are popular among traditional
medicine practitioners to treat skin disorders and
fungal infections. e leaves are found to be useful
in the treatment of convulsion, syphilis, gonor-
rhoea, heart failure, abdominal pains, stomach
problems, fever, asthma, etc.9,10
S. alata has been extensively investigated for
their antimicrobial activities while their other
potentials remained in the dark. In the backdrop of
this situation, we have designed our experiments to
update the existing knowledge of phytochemicals
present as well as the antioxidant capacity in their
aqueous and ethanolic extracts. Besides, the throm-
bolytic, anticoagulant and anthelmintic activities
were investigated which were rarely explored before
as per the best of our knowledge.
MATERIALS AND METHODS
Plant material and extract preparation
e leaves of Senna alata L. were collected from
the herbal gardens of Cell Genetics and Plant
Biotechnology Laboratory at the Department
of Biotechnology and Genetic Engineering,
Jahangirnagar University, Dhaka- 1342, Bangladesh
(23°53’14” N 90°15’56” E).
e fresh leaves were collected from the plant
and thoroughly washed with distilled water to
remove any dirt or unwanted substances. e leaves
were then shade dried for 7 days and nally dried in
a hot air oven (JSR, Korea) at 45°C days for 24 h. A
mechanical grinder (Philips, Netherlands) was used
to obtain ne powder from the dried leaves. e
powder (10 gm) and 100 mL of each solvents (etha-
nol and distilled water) were taken in conical asks
which were placed in an orbital shaker. e shaker
performed mixing at 120 RPM for 72 h at room
temperature. e obtained extracts were ltered
using Whatman® no. 1 lter paper. e ltrates
were concentrated at 45°C using an evaporator. e
concentrated extracts were used to prepare stock
solutions (100 mg/mL) namely aqueous extract
(AE) and ethanolic extract (EE). Qualitative and
quantitative determinations of phytochemicals
were done with the extracts at a concentration of
1 mg/mL.
Qualitative determination of phytochemicals
Qualitative tests were done for determining the
presence of secondary metabolites such as alka-
loids, coumarins, glycosides, avonoids, phenols,
resins, saponins, tannins, and terpenoids according
to the methods described previously.11,12
Quantitative determination of
phytochemicals
e total phenolic content was estimated using
Folin–Ciocalteu reagent by following a method
previously described.13 Briey, 0.1 mL of extracts
and standard were taken in test tubes. en, 2.5
mL of deionized water was added to each tube.
Immediately 0.1 mL Folin–Ciocalteu reagent (2 N)
was added to the tubes. Aer proper mixing, the
tubes were kept for 6 min at room temperature.
Finally, 0.5 mL of sodium carbonate solution (20%)
was added to the tubes. e tubes were kept for 30
min at room temperature. A UV- visible spectro-
photometer (T60 UV- Visible Spectrophotometer,
PG Instruments Ltd., United Kingdom) was used to
measure the absorbance at 760 nm. Gallic acid was
used as the standard.
Total avonoid content was determined by
following a method previously described.13 First,
0.25 mL of extracts and standard were taken in test
tubes. en, 1.25 mL of distilled water was added to
each tube. Immediately, 0.75 mL of sodium nitrite
(5%) solution was added. e tubes were kept for
6 min at room temperature and aer that 0.15 mL
of aluminum chloride solution (10%) was added to
the tubes. e tubes were kept for another 5 min and
then 0.5 mL of sodium hydroxide (1 M) was added.
e volume of the mixture was adjusted to 2.5 mL by
adding distilled water. e absorbance was measured
at 510 nm. (+)- catechin was used as the standard.
Total avonol content was determined by follow-
ing a method previously described.14 First, 2 mL of
the samples and standard were taken in test tubes.
en, 2 mL of aluminum chloride (2%) was added
to each of the tubes. Immediately, 3 mL of sodium
acetate solution (5%) was added to the tubes. e
test tubes were kept at 20°C for 2.5 hours in a refrig-
erated incubator (Hysc, Korea). e absorbance
was measured at 440 nm. Quercetin was used as the
standard.
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In vitro analysis of phytoconstituents and... Abdullah Mohammad Shohael, et al.
e tannins were determined using the Folin-
Ciocalteu reagent by following a method previ-
ously described.11 First, 0.1 mL of the samples and
standard were taken in test tubes. en, 7.5 mL
distilled water was added to each of the tubes. Aer
that, 0.5 mL of Folin- ciocalteu reagent was added.
Immediately, 1 mL of sodium carbonate solution
(35%) was added to the tubes. e total volume was
adjusted to 10 mL by adding distilled water. e
tubes were kept at room temperature for 30 min.
e absorbance was measured at 725 nm. Tannic
acid was used as standard.
Total proteins were estimated by following the
method reported by Bradford.15 First, 0.1 mL of
samples and standard were taken into test tubes.
en, 0.9 mL of Bradford reagent was added to
each of the tubes. e tubes were kept for 2 min.
e absorbance was measured at 595 nm. Bovine
Serum Albumin (BSA) was used as the standard.
Determination of antioxidant potential
Antioxidant activity was determined in terms of
DPPH (2,2- diphenyl- 1- picrylhydrazyl) free radical
scavenging assay where ascorbic acid was used as
the standard.12 Five Dierent concentrations (800,
400, 200, 100, 50 μg/mL) of extracts and standard
were prepared and taken in test tubes. To the tubes,
3 mL of DPPH solution (0.004%) was added. e
tubes were kept for 30 min in a dark chamber at
room temperature. e absorbance was measured
at 517 nm. e percent scavenging activity was
calculated according to the following formula-
Percent scavenging activity (%) = [(AC- AS)/ AC] ×100
Here, AC - absorbance of the control
AS - absorbance of the sample
In vitro thrombolytic activity
In vitro thrombolytic activity was determined by
the ability of extracts to lyse blood clots.16 Five
mL of whole blood (vein) was drawn from young,
healthy and disease- free human volunteers (n=5).
In previously weighed microcentrifuge tubes, 500
μL of blood was taken in each tube. For blood clot
formation, the tubes were kept in an incubator
(Hysc, Korea) at 37°C for 45 min. Aer incubation,
the remaining serum from each tube was decanted
without aecting the clot. e tubes with the clots
were weighed to determine the weight of the clot.
Aer that, to the tubes containing only the blood
clots, 100 μL of extracts of two dierent concentra-
tions (5, 10 mg/mL) was added. streptokinase was
used as the positive control and distilled water was
used as the negative control. To observe the clot
lysis, the tubes were kept in an incubator (Hysc,
Korea) at 37°C for 90 min. Aer the incubation,
generated uid was decanted from every tube. e
tubes were weighed to determine the dierence in
weight aer clot lysis. e thrombolytic activity was
expressed as the percentage of clot lysis according
to the following formula-
Percent clot lysis (%) = (Weight of released clot /
Weight of total clot) × 100
In vitro anticoagulant activity
Prothrombin time (PT) test and activated partial
thromboplastin time (aPTT) test were employed
to study the anticoagulant activity. using the
romboplastin LI kit by Agappe diagnostics
Switzerland GmbH and aPTT kit by Analyticon
Biotechnologies AG, Germany according to the
methods described previously.16 Five mL of whole
blood (vein) was drawn from young, healthy and
disease- free human volunteers (n=2). In sterile
15mL falcon tubes, 9 parts of blood and 1 part
of 3.2% trisodium citrate solution (0.109 M)
were mixed properly. e platelet- poor plasma
was obtained from the blood by centrifuging the
tubes at 3000 rpm for 15 min. e PT Reagent was
Pre- warmed at 37°C for 10 min. In microcentri-
fuge tubes, 100 μL of plasma was taken and incu-
bated at 37°C and for 3 min. To the tubes, 100μL
extracts of three dierent concentrations (1.0,
0.5 and 0.25mg/mL) were added. en, a 200 μL
pre- warmed PT reagent was added. e clotting
time was recorded by a timer. In the case of aPTT,
Calcium Chloride (0.025M) was pre- warmed at
37°C for 10 min in a water bath. In microcen-
trifuge tubes, 50 µL of plasma was taken. To the
tubes, 50 μL extracts of three dierent concentra-
tions (1.0, 0.5 and 0.25 mg/mL) were added. e
tubes were incubated at 37°C for 2 min. Finally,
50 µL pre- warmed Calcium Chloride (0.025M)
was added to the tubes. e clotting time was
recorded by a timer.
In vitro anthelmintic activity
e anthelmintic activity was determined by the
ability of extracts to paralyze and kill the exper-
imental aquarium worm (Tubifex tubifex).17 e
worms were purchased from a local aquarium shop
situated at Kataban, Dhaka, Bangladesh. Five mL of
extracts at 4 dierent concentrations (50, 25, 12.5,
6.25 mg/mL) were taken in small beakers. In each
beaker, 20 worms were placed. e time for paraly-
sis and death was measured using a stopwatch. e
mean time for paralysis (min) corresponds to the
time when the movement of the worm was stopped
except for vigorous shaking and the mean time of
death (min) corresponds to the time when no sign
of movement was observed even with shaking and
placing to slightly warm water. Levamisole was
170 Discovery Phytomedicine 2021; 8(4): 167-174. doi: 10.15562/phytomedicine.2021.183 www.phytomedicine.ejournals.ca
In vitro analysis of phytoconstituents and... Abdullah Mohammad Shohael, et al.
used as the positive control and distilled water was
used as the negative control.
Statistical analysis
All data were presented as the mean ± standard
error of the mean (SEM) for three independent
biological replications. e analysis was carried out
using GraphPad Prism 6.0.
RESULTS
Qualitative determination of phytochemicals
Qualitative testing can indicate the presence
or absence of the secondary metabolites in the
extracts. e results of the qualitative determina-
tion of phytochemicals are presented in Table 1.
Qualitative testing was done for 9 dierent
phytochemical classes, where presence was detected
for all compounds in both AE and EE.
Quantitative determination of
phytochemicals
Quantitative tests of phytochemicals were done for
ve dierent types of chemical classes. e results
of the quantitative determination of phytochem-
icals are given in Figure 1. e phytochemical
contents were higher in EE than AE for all of the
compounds. Total phenolic content was 87.67 mg/g
in the EE which was slightly higher than the total
phenolic content in AE (62.61 mg/g). e total
avonoid contents were low in both the extracts,
where the content in EE (22.43 mg/g) was found
to be higher than in the AE (18.73 mg/g). Both the
extracts exhibited a similar quantity of avonols
where the content in EE (11.68 mg/g) was higher
than the content in AE (10.52 mg/g). Total tannin
content was lowest in terms of all the compounds
tested. EE (12.37 mg/g) had higher tannin content
than the AE (7.53 mg/g). Total protein contents
were 37.73 mg/g for EE and 33.40 mg/g for AE.
Determination of antioxidant potential
e results of the antioxidant assay and the IC50
values are given in Figure 2. According to the assay,
EE has better antioxidant capacity in comparison to
the AE. e highest scavenging activity (89.44%)
was displayed by the EE at its 800 µg/mL concentra-
tion while at the same concentration AE scavenged
78.47% DPPH free radicals (Figure 2A). e scaveng-
ing activity was measured at 5 dierent concentra-
tions where at the lowest concentration (50 µg/mL)
58.13% and 54.04% scavenging activity was
achieved by EE and AE respectively (Figure 2A).
We have calculated the IC50 value by linear regres-
sion analysis (Figure 2B). e EE extract showed
excellent IC50 value while compared to the AE. e
IC50Value for EE was 61.02 µg/mL while AE showed
a much higher IC50 Value (142.42µg/mL) compar-
atively (Figure 2B). Ascorbic acid as a control
compound scavenged 95.7% DPPH free radicals at
its 800 µg/mL concentration. e IC50 value of the
scavenging activity for ascorbic acid was found as
12.37 µg/mL (Figure 2A and 2B).
In vitro thrombolytic Activity
e thrombolytic activity of AE and EE in terms of
percent clot lysis (%) are presented in Figure 3. EE
at its 10 mg/mL concentration, achieved the highest
(37%) clot lysis activity. e second highest value
(32.59%) was shown by EE at its 5 mg/mL concen-
tration. e AE also has a considerable thrombolytic
activity where at 10 mg/mL concentration 26.64%
Table 1 Qualitative determination of phytochemicals for aqueous
extracts (AE) and ethanolic extracts (EE) of Senna alata
leaves
Phytochemical
classes
Extracts AE EE
Alkaloids + +
Coumarins + +
Glycosides + +
Flavonoids + +
Phenols + +
Resins + +
Saponins + +
Tannins + +
Terpenoids + +
Indication: + = Present; - = Not detected.
Table 2 Prothrombin Time (PT) and activated Partial
Thromboplastin Time (aPTT) of aqueous extracts (AE) and
ethanolic extracts (EE) of Senna alata leaves
Samples Concentrations
Prothrombin
time (s)
Activated partial
thromboplastin
time (s)
Standard time of clotting without
anticoagulants 10- 15 25- 39
Sample without extracts 12±0.70 28±0.70
AE
1 mg/mL 26±2.12 41±2.12
0.5 mg/mL 23±0.70 33±0.70
0.25 mg/mL 21±0.70 31±0.70
EE
1 mg/mL 28±0.70 47±0.70
0.5 mg/mL 26±0.00 42±0.00
0.25 mg/mL 24±1.41 36±1.41
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In vitro analysis of phytoconstituents and... Abdullah Mohammad Shohael, et al.
clot lysis was achieved and at 10 mg/mL concentra-
tion 23.37% clot lysis was achieved. Besides, 81.67%
thrombolytic activity was obtained for the positive
control streptokinase. Distilled water as the negative
control performed 3.90% clot lysis.
In vitro anticoagulant activity
e results of the anticoagulant activity are shown
in Table 2. Both the results of the prothrombin time
(PT) test and the activated partial thromboplas-
tin time (aPTT) test are incorporated. AE and EE
successfully extended the time of coagulation so that
they are eective as anticoagulants., Comparatively,
the EE extended time more eectively than the
AE. For the PT test, the time of clotting for normal
blood samples without applying anticoagulant was
12- 15 s. While the extracts were added, the time of
clotting increases to 28 s for EE and 26 s for AE at
1 mg/mL extract concentration. e PT time was
found 26 s and 23 s for EE and AE respectively at
0.5 mg/mL extract concentration and 24 s and 21 s
for EE and AE respectively at the extract concentra-
tion of 0.25 mg/mL.
For aPTT test, the time of clotting for a normal
blood sample without applying anticoagulant was
25- 39 s. While the extracts were added, the time
extends to 47 s for EE and 41 s for AE at 1 mg/mL
extract concentration. For EE, the aPTT time was
found 42 s at 0.5 mg/mL extract concentration.
e rest of the extract concentrations for both AE
and EE couldn’t be able to extend the time than the
reference range. However, the times were higher
than the sample containing no extracts.
In vitro anthelmintic activity
e results of the anthelmintic activity are displayed
in Figure 4. e EE showed the quickest time for
paralysis (4 min) at its 50 mg/mL concentration
which was very close to the time of paralysis (5 min)
recorded for AE. At 25 mg/mL extract concentra-
tion, the times of paralysis recorded were 27 min
and 49 min for EE and AE respectively. e highest
time of paralysis (103 min) was recorded for AE at
its 6.25 mg/mL concentration. e lowest time of
death (6 min) was observed both for EE and AE at
their 50 mg/mL concentration. e highest time
of death (162 min) was observed for AE at its 6.25
mg/mL concentration. e worms were paralyzed
within 4 min and killed at 10 min when the positive
control levamisole (1 mg/mL) was applied. In the
case of distilled water (negative control) no sign of
paralysis and death was observed.
DISCUSSION
Human beings are using medicinal plants for a long
time to achieve good health and prevent diseases.
Extensive research on medicinal plants, identica-
tion and characterization of active pharmacological
Figure 1 Quantitative determination of phytochemicals in the aqueous
extracts (AE) and ethanolic extracts (EE) of Senna alata leaves. (A)
Total phenolic content (mg GAE/g of extract) (B) Total avonoid
content (mg CE/g of extract) (C) Total avonol content (mg QE/g
of extract) (D) Total tannin content (mg TAE/g of extract) (E) Total
protein content (mg BSAE/g of extract)
Figure 2 Antioxidant activity in terms of DPPH free radical scavenging
assay of aqueous and ethanolic extracts of Senna alata leaves. IC50=
half maximal inhibitory concentration; AA= Ascorbic Acid; AE=
Aqueous Extract; EE= Ethanolic Extract
Figure 3 rombolytic activity of aqueous and ethanolic extracts of Senna
alata leaves. SK= Streptokinase; AE= Aqueous Extract; EE=
Ethanolic Extract; DW=Distilled Water
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In vitro analysis of phytoconstituents and... Abdullah Mohammad Shohael, et al.
agents have opened new frontiers in safe and eec-
tive treatments. Plant produce many chemical
compounds that serve dierent functions and
known as phytochemicals.12 Among them, second-
ary metabolites serve functions other than growth
and reproduction. ese secondary metabolites
have many useful implications in health and medi-
cine. e qualitative testing of phytochemicals
can indicate the presence or absence of second-
ary metabolites or bioactive components in the
plant extracts.16 In the present study, as alkaloids,
coumarins, glycosides, avonoids, phenols, resins,
saponins, tannins, and terpenoids were found to
be present which indicated the chemical diversity
of the leaf extracts as well as supports the medici-
nal eects they provide. e concerned plant parts
and the solvent used for extraction oen act as a
contributing factor for the presence and absence of
phytochemicals.11 Aer the qualitative determina-
tion, some of the important compounds were quan-
tied, namely the phenols, avonoids, avonols,
proteins, and tannins. ese chemical compounds
are attributed to dierent pharmacological eects
and are widely explored in dierent plants. In many
studies, the phenolic compounds were termed as the
contributing factor for the medicinal plant’s antiox-
idant activity.18 Flavonoids are another class of plant
secondary metabolite that have been implicated
in the treatment of many diseases and disorders.
ey are also active against reactive oxygen species
and oxidation of low- density lipoproteins which
in turn contribute to the reduction of thrombotic
tendency19 vegetables, and grains. Divided into
several subclasses, they include the anthocyanidins,
pigments chiey responsible for the red and blue
colors in fruits, fruit juices, wines, and owers; the
catechins, concentrated in tea, the avanones and
avanone glycosides, found in citrus and honey;
and the avones, avonols, and avonol glycosides,
found in tea, fruits, vegetables, and honey. Known
for their hydrogen- donating antioxidant activity as
well as their ability to complex divalent transition
metal cations, avonoids are propitious to human
health. Computer- controlled high- performance
liquid chromatography (HPLC. Flavonols are a class
of dietary avonoids and can act as antioxidants.20
Tannins are water- soluble polyphenols available in
many plants. ey showed various activities such
as antioxidant, antimicrobial, anti- mutagenic and
health- healing eects.21 is is worth mention-
ing that these compounds can provide medicinal
eects alone as well as their synergistic eects are
responsible for most of the health benets. Ita and
Ndukwe (2017) reported the phenolic content as
78.21 mg GAE/g and 46.3 mg GAE/g and avonoid
content as 39.29 mg QE/g and 26.17 mg QE/g in
the ethanolic and aqueous root extracts of S. alata,
respectively.6 In contrast to this study, extracts from
the leaf of S. alata contain higher phenol content
and lower avonoid content than the root extracts.
e DPPH radical scavenging assay is a widely
used and the most popular technique to estimate the
antioxidant ability of compounds.22 In the present
study, the extracts displayed excellent antioxidant
activity. It is worth mentioning that the scavenging
eect provided by EE is considerably higher than
the AE where the IC50 value for EE is very promis-
ing for further investigations. It is reported that S.
alata root extracted with water and ethanol exhib-
ited an IC50 value of 61.15 µg/mL and 45.18µg/mL.6
While in our studies, EE showed a lower IC50 value
than that observed for root extracts. So, it can be
said that ethanolic leaf extracts have better antiox-
idant capacity. erefore, it is evident that the leaf
extracts also showed a similar antioxidant response
and thereby may have good potential for being
utilized against oxidant damage.
Unusual clot formation in the arteries and veins
causes many health problems including cardiovas-
cular disease. rombin forms blood clots from
brinogen. A tissue plasminogen activator acti-
vates plasminogen to form plasmin which lyses the
blood clots.3 Several studies have provided evidence
of signicant thrombolytic activity of medicinal
plants available in Bangladesh.24,25 In our study of
thrombolysis, both extracts were eective in clot
lysis which suggests that S. alata extracts can be
utilized as a thrombolytic agent also. Based on the
result, it can be said that the ethanolic extract is
promising in thrombolysis. Mannan etal. (2011)
evaluated the thrombolytic potential of S. alata
seed oil extracts and found 37.92% clot lysis activity
Figure 4 Anthelmintic activity of aqueous and ethanolic extracts of Senna
alata leaves. LS= Levamisole: AE= Aqueous Extract; EE= Ethanolic
Extract; DW=Distilled Water
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In vitro analysis of phytoconstituents and... Abdullah Mohammad Shohael, et al.
at 10 mg/mL concentration.25 So, it is evident that
the leaves extract has a more powerful eect than
the seed oil extract. Rahman etal. (2013) reported
31.61% clot lysis activity by the ethanolic extracts of
Senna sophera.26
Anticoagulants are used mainly to counteract
arterial and venous thrombotic problems. Plants
have been investigated to nd their anticoag-
ulant potential because they contain bioactive
compounds that may aect coagulation processes.27
In line with the thrombolytic activities, plant
extracts have eects on the intrinsic and extrinsic
pathway factors to inhibit the blood coagulation
cascade.28 e PT and aPTT tests are used to moni-
tor the coagulation and anticoagulant treatments
and to detect blood- clotting disorders.29 PT assay
is concerned with the extrinsic pathway of coag-
ulation and sensitive to the level of coagulation
factor VII. e aPTT test is associated with the
monitoring of intrinsic coagulation pathway and
sensitive to the coagulation factors VIII, IX, XI, and
XII. In search of that potential, our study reported
the excellent anticoagulant potential of S. alata leaf
extracts. e prolongation in time in both PT and
aPTT suggests the utility of the extracts as antico-
agulant and mark them potential candidates for
future endeavors. It is also evident that the S. alata
leaf extracts have considerable eects on both the
intrinsic and extrinsic pathway factors.
e present study illustrates that both the
extracts aect the motility and mortality of the
experimental worms Tubifex tubifex. As negative
control had no eects on the motility and mortal-
ity, it is quite evident that the helminths were
negatively aected by the eect of extracts. ere
was a dose- dependent ecacy in treated worms
where the increased concentration caused more
destructive eects. In a study conducted by Kundu
etal. (2012), the ethanolic extract of S. alata leaves
moderately aected the motility of Hymenolepis
diminuta.7 Another study performed by Roy and
Lyndem (2019) demonstrated ethanolic extracts of
three species of Senna leaves on Paramphistomum
gracile and found Concentration- dependent
eects on movement and death in individual and
combination treatment. ey also reported that the
extract from S. alata caused the earliest paralysis in
comparison to the other two Senna species tested.30
CONCLUSION
In our study, we have demonstrated the qualitative
and quantitative phytochemical analysis and the
eect of the extracts of S. alata leaves as antioxi-
dant, anthelmintic, thrombolytic and anticoagulant
agents. It is evident from the study that ethanol
performed better as a solvent than water as the
phytochemical contents were higher in ethanolic
extracts. Moreover, potential bioactivities were
also better for ethanolic extracts than the aque-
ous extracts. However, in general, S. alata leaves
extracts have a potential antioxidant, anthelmintic,
thrombolytic and anticoagulant activity which
should be further investigated for understanding
the underlying mechanisms. e present study
sheds light on some of the unexplored potentials
which turn out to be interesting from the perspec-
tive of medicinal importance. e leaves can be
useful alongside their conventional utilization as
antifungal agents. Besides, further investigations
can be employed for the identication of particu-
lar bioactive compounds responsible for specic
functions.
AUTHORS’ CONTRIBUTIONS
SA and AMS led the conceptualization and initial
design of the study. FBR collected and prepared the
samples. SA and FBR performed the experimental
investigations. SA performed the data analysis,
visualization and draed the initial manuscript.
AMS supervised and edited the nal manuscript.
All authors read and approved the nal manuscript.
ACKNOWLEDGMENTS
e current research was partially supported by
the research grant provided by GARE (Grant
for Advanced Research in Education LS2016165
No. 37.20.0000.004.033.020.2016.7725) funded
by the Ministry of Education, Bangladesh and
Special Allocation in Science and Technology
of Ministry of Science and Technology (No.
39.00.0000.09.06.79.2017/ES- 99), Bangladesh. e
authors acknowledge the signicant contributions
and knowledgeful insights provided by the labora-
tory members.
CONFLICT OF INTEREST
e authors have no conicting interests.
ETHICAL ISSUES
e experimental protocols for the thrombolytic and
anticoagulant study by using human blood in the
assays were approved by the Biosafety, Biosecurity
and Ethical Clearance Committee, Jahangirnagar
University. e blood was collected from human
volunteers aer obtaining their informed consent.
174 Discovery Phytomedicine 2021; 8(4): 167-174. doi: 10.15562/phytomedicine.2021.183 www.phytomedicine.ejournals.ca
In vitro analysis of phytoconstituents and... Abdullah Mohammad Shohael, et al.
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