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OPENACCESS Pakistan Journal of Nutrition
Phytochemical Screening and Pharmacological Activities of the
Ethanolic Stem Extract of Cleome gynandra
1,2Farjana Yasmin, 2Nor Adlin Yusoff and 1Amir Hossain
1Department of Pharmacy, ASA University Bangladesh, 1207 Dhaka, Bangladesh
2Cluster of Integrative Medicine, Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Bertam, Penang, Malaysia
Objective: This study aimed to identify the phytochemical compounds of the ethanolic stem extract of
to determine the antioxidant, membrane stability and analgesic activities of CGEE
Materials and Methods: Phytochemical analysis was
conducted qualitatively. Content of total phenolic compounds and tannins was determined using Folin Ciocalteau reagent. Antioxidant
activity was evaluated using the 1,1-diphenyl-2-picrylhydrazyl assay, membrane stabilizing activity was tested using hypotonic solution
and heat-induced hemolysis and analgesic activities were evaluated using the acetic acid-induced writhing test in mice. Results: CGEE
is rich in tannins, flavonoids, steroids, glycosides, acidic compounds and proteins. The total phenolic and tannin contents were
237.92 mg gallic acid equivalents (GAE)/100 g and 21.07 mg GAE/100 g of dried plant extract, respectively. CGEE also displayed promising
DPPH free radical scavenging activity, with an IC50 value of 9.62 :g mLG1. In the hypotonic solution-induced hemolysis test, the extract
showed 43.67, 42.85 and 38.66% inhibition at 0.5, 1.0 and 2.0 mg mLG1, respectively, whereas the standard resulted in 30.57% inhibition.
In the heat-induced hemolysis test, 1 mg mLG1 of extract resulted in 84.43% inhibition of hemolysis. Furthermore, in the analgesic activity
test, CGEE doses of 250 and 500 mg kgG1 body weight resulted in good inhibition of the writhing reflex (40.87 and 53.92%, respectively),
while the standard drug (diclofenac sodium) at 25 mg kgG1 body weight resulted in inhibition of 64.35%. Conclusion: Results of this study
suggest that the stem extract of
possesses antioxidant, membrane stabilizing and analgesic activities.
, antioxidants, stem extract, membrane stabilizing, oxidative stress
Received: September 30, 2019 Accepted: January 21, 2020 Published: March 15, 2020
Citation: Farjana Yasmin, Nor Adlin Yusoff and Amir Hossain, 2020. Phytochemical screening and pharmacological activities of the ethanolic stem extract
. Pak. J. Nutr., 19: 153-159.
Corresponding Author: Farjana Yasmin, Department of Pharmacy, ASA University Bangladesh, 1207 Dhaka, Bangladesh
Cluster of Integrative Medicine, Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Bertam, Penang, Malaysia
Copyright: © 2020 Farjana Yasmin
. This is an open access article distributed under the terms of the creative commons attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Competing Interest: The authors have declared that no competing interest exists.
Data Availability: All relevant data are within the paper and its supporting information files.
Pak. J. Nutr., 19 (4): 153-159, 2020
Oxidative stress is a factor that leads to the onset of
numerous chronic diseases, such as hypertension, diabetes,
cancer and neurodegenerative disorders1,2. In healthy
individuals, the production of free radicals is balanced by the
antioxidative defense system. Imbalance occurs when
equilibrium favors free radical generation due to the
depletion of antioxidant levels, thus causing oxidative
stress3. The inflammatory reaction is part of the bodys
protective response to foreign organisms, including viruses,
dust particles and pathogens4. Studies have shown that
inflammatory reactions are positively related to the presence
of free radicals and establishment of the oxidative stress
condition. During the inflammatory reaction, free radicals and
reactive oxygen species (ROS) such as non-radical hydrogen
peroxide, nitrite oxide, superoxide and hydroxyl are
overproduced. The excess free radicals and ROS can cause
injury to the tissue by damaging macromolecules and
inducing lipid peroxidation of membranes5, thereby leading
to the onset and progression of various inflammatory
associated diseases. Tissues, consequently, require
antioxidants to neutralize and decompose these free radicals
and ROS. Antioxidants protect cells from damage caused by
these free radicals by inhibiting or delaying cellular damage,
mainly through free radical scavenging properties6. Membrane
stabilization is the process of maintaining the integrity of
biological membranes, such as those of erythrocytes and
lysosomal membranes, against osmotic and heat-induced
lysis7. Stabilization of lysosomal membranes is important for
limiting the inflammatory response by inhibiting the release
of lysosomal constituents of activated neutrophils, such as
bactericidal enzymes and proteases, which cause further
tissue inflammation and damage upon extracellular release8.
Therefore, plants with antioxidant and membrane stabilizing
properties should offer more significant protection against
inflammatory induced diseases than plants without these
Currently, numerous scientific studies are focused on
discovering natural drugs from medicinal plants due to the
promising therapeutic effects of phytochemicals and their less
severe side effects.
is a genus of flowering
plant in the family Cleomaceae. The genus includes about 170
species of herbaceous annual or perennial plants and shrubs9.
Nutritional analyses have found that
is high in
micronutrients, including $-carotene, folic acid, ascorbic
acid, vitamin E, oxalic acid, iron and calcium. For centuries,
has been incorporated in Ayurvedic medicine to
treat various illnesses, such as gulma (tumor, irregularity or
diverticulosis), krmiroga (worm infection), asthila (prostate
enlargement), kandu (pruritus) and karnaroga (ear
infections)10. R ecen t scie ntif ic stu dies have a lso re port ed
the pharmacological activities of different parts of this
plant and they include antioxidant11,12, antidiabetic13,
anticarcinogenic and anti-inflammatory14 properties. These
activities are attributable to the high concentrations of
bioactive compounds present, namely flavonoids, tannins,
glucosinolates and iridoids15.
Despite extensive literature on the pharmacological
, to date there is no information
available about the possible pharmacological effect of the
stem extract of
. Previous studies of this
plant focused mainly on the leaves. Therefore, the goal of
this study was to assess the antioxidant, analgesic and
membrane stabilizing activities of the ethanolic stem extract
Results of this study will provide an
evidence-based validation on the folkloric use of this plant.
MATERIALS AND METHODS
Chemicals: Sodium carbonate and Folin-Ciocalteu (FC)
reagents were obtained from Merck (Darmstadt, Germany).
1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium dichromate,
gallic acid (GA), ascorbic acid (AA), aluminium chloride,
sulphuric acid, nitric acid, sodium hydroxide,
ethylenediaminetetraacetic acid (EDTA), diclofenac sodium,
sodium nitrous, Tween-80 and ferrous chloride were
purchased from the Sigma Chemical Co., St. Louis, MO, USA.
was collected from
Bandarban, Bangladesh in July 2018. The plants were washed
and dried under direct sunlight for one week. The plant was
identified by the taxonomist at the Bangladesh National
Herbarium, Mirpur-1, Dhaka-1216 (voucher specimen no:
Preparation of the extract: Two hundred grams of dried stem
were ground into a fine powder using a grinder machine. The
powder was then extracted with 1500 mL of ethanol.
Thereafter, the mixture was filtered using cotton followed by
Whatman (No. 1) filter paper. The obtained filtrate was
evaporated to dryness in open air to yield the crude ethanolic
extract, which was denoted as CGEE. It was kept at 4EC until
used for further analysis.
Qualitative phytochemical screening: Th e f ol l ow in g r e ag en ts
were used to quantitatively screen for the presence of
th e ph yto che mic al s: R edu cin g su gar was i den tif ied us ing
Pak. J. Nutr., 19 (4): 153-159, 2020
Fehlings solution and Benedicts reagent; alkaloids with
Mayers and Dragendroffs reagent; saponins with distilled
water; glycosides with sodium hydroxide solution; steroids
with sulphuric acid; tannins with ferric chloride and potassium
dichromate; and gum content with Molish reagent16.
Measurement of total phenolic content: The FC technique
was used to estimate the phenolic content in CGEE17
test, an aliquot of CGEE or a positive control was mixed
with 2 mL of FC reagent (1:10, v/v) and 2 mL of sodium
carbonate (75 g LG1). The tubes were shaken for 15 sec and
incubated for 20 min at room temperature for colour
development. Absorbance was recorded at 750 nm using an
UV spectrophotometer. The total phenolic contents were
expressed as mg of gallic acid equivalents (GAE) per 100 g of
the dried extract.
Measurement of total tannin content: Total tannin content
was also measured using FC reagent18. To estimate the total
tannin content, 0.1 mL of CGEE or a positive control was mixed
with 7.5 mL of distilled water and 0.5 mL of FC reagent. The
mixture was thoroughly mixed and kept for 5 min. Next, 1 mL
of 35% sodium carbonate was added, followed by dilution
with 10 mL of distilled water. The mixture was incubated at
room temperature for 30 min. Absorbance was recorded at
725 nm using the UV spectrophotometer. The total tannin
contents were expressed as mg of GAE per 100 g of the dried
DPPH Scavenging assay: The antioxidant activity of the
extract was estimated using the DPPH free radical scavenging
assay19. DPPH solution at the concentration of 0.04% (w/v) was
prepared in ethanol. A volume of 1 mL of CGEE at different
concentrations was mixed with 3 mL of DPPH solution. The
mixture was shaken thoroughly and placed aside in the dark
for the 30 min reaction period at room temperature. After
incubation, the absorbance of the mixture was recorded at
517 nm using the UV spectrophotometer. The following
equation was used to calculate the DPPH free radical
DPPH free radical scavenging activity (%) = ×100
where, A0 is the absorbance of the control (DPPH solution
without sample) and A1 is the absorbance of the plant
extract/positive control. Ascorbic acid was used as the positive
control. The percentage of scavenging activity was then
plotted against log concentration and a graph for the half-
maximal inhibitory concentration (IC50) was created.
Membrane stabilization assays
Hypotonic solution-induced hemolysis: The membrane
stability activity of the extract was evaluated using hypotonic
solution-induced hemolysis. The test sample consisted of a
stock erythrocyte suspension (0.50 mL), 4.5 mL of hypotonic
solution (50 mM NaCl) in 10 mM sodium phosphate buffer
saline (pH 7.4) and either CGEE (2.0 mg mLG1) or acetyl salicylic
acid (0.1 mg mLG1). The mixtures were incubated for 10 min at
room temperature. After incubation, the mixtures were
centrifuged for 10 min at 3000 g and the supernatant was
collected. The absorbance of the supernatant was measured
at 540 nm using the UV spectrophotometer. The percentage
inhibition of either hemolysis or membrane stabilization was
calculated using the following equation18:
(OD OD )
Inhibition of hemolysis (%) = 100
where, OD1 is the optical density of the hypotonic buffered
saline solution alone (control) and OD2 is the optical density of
the test sample in hypotonic solution.
Heat-induced hemolysis: For this analysis, 5 mL of isotonic
buffer containing 1.0 mg mLG1 of different concentrations of
C GE E w e re a dd e d t o tw o se t s o f ce n tr i fu g e t u be s 21. The vehicle
control group was prepared with the same amount of extract.
Erythrocyte suspension (30 mL) was added to each tube and
inverted gently. One set of tubes was incubated at 54EC for
20 min in a water bath and the other set of tubes was placed
into an ice bath at 0-5EC. The mixtures then were centrifuged
at 1300 g for 3 min. The supernatant was collected and
the absorbance was recorded at 540 nm using the UV
spectrophotometer. The percentage of inhibition or
acceleration of hemolysis was calculated using the following
Inhibition of hemolysis (%) = 100
where, OD1 is the optical density of the unheated test sample,
OD2 is the optical density of the heated test sample and OD3
is the optical density of the heated control sample.
Analgesic activity assay
Acetic acid-induced writhing technique: Twenty Swiss
albino mice (18-22 g) were used in this study. Animals were
divided into four groups of five and fasted for 2 h before
commencement of the test. The control group (Group I)
wa s tr eate d wi th 1 % Twe en- 80 sol ution dis solv ed i n wate r
Pak. J. Nutr., 19 (4): 153-159, 2020
cavenging activity (%)
Log concentration (µg mL )
(10 mL kgG1). The positive control group (Group II) was treated
with diclofenac sodium at a dose of 25 mg kgG1 body weight.
Group IIIs and IV were treated with CGEE at doses of 250 and
500 mg kgG1 body weight, respectively. Thirty minutes after
the treatment, 0.7% acetic acid was injected intraperitoneally.
Five minutes after the injection of acetic acid, the number
of abdominal constrictions (writhing) within the 15 min
observation period was recorded22.
Statistical analysis: Mean±standard error of the mean (SEM)
was used to present the data. All parameters were evaluated
for their significance level by correlation and regression
analysis and t-tests (p<0.05) also were used. Microsoft Excel
2016 was used for both statistical analysis and graphical
Phytochemical screening of CGEE: Phytochemical screening
of CGEE indicated the presence of reducing sugar, combined
reducing sugar, tannins, flavonoids, glycosides, proteins and
steroids (Table 1).
Total phenol and tannin content of CGEE: The total amounts
of phenol and tannin in CGEE were calculated from the linear
regression equation of gallic acid standard calibration curves
(y = 0.113x -0.201, R2 = 0.8626 and y = 0.014x-0.010, R2 = 0.947,
respectively) and expressed in GAE. Total phenol and total
tannin contents in CGEE were 237.92±0.0129 mg GAE/100 g
and 21.07±0.0004 mg GAE/100 g of CGEE.
DPPH free radical scavenging activity of CGEE: Antioxidant
activity of CGEE was evaluated using the DPPH free radical
scavenging method. The IC50 values of CGEE and ascorbic acid
were 9.62 and 5.51 µg mLG1, respectively (Fig. 1).
Membrane stabilization activity of CGEE: Data in Table 2
show that CGEE inhibited lysis induced by the hypotonic
solution, as indicated by the high percentage inhibition of
hemolysis (3.66, 42.85 and 43.67%) recorded for doses of 2, 1
and 0.5 mg mLG1, respectively. In the heat-induced hemolysis
test, inhibition of hemolysis was 84.43% at the concentration
of 1 mg mLG1 CGEE.
Analgesic effect of CGEE
Writhing test: Table 3 shows the effect of different
concentrations of CGEE on the acetic acid-induced writhing
reflex in mice. CGEE (250 and 500 mg kgG1) and the positi ve
Fig. 1: DPPH scavenging activity of CGEE and ascorbic acid
Table 1: Qualitative phytochemical screening of ethanolic stem extract of
Phytochemical groups CGEE
Reducing sugar +
Combined reducing sugar +
Acidic compounds -
+: Presence, -: Absence
Table 2: Effect of different concentrations of CGEE on hypotonicity induced
haemolysis of erythrocyte membrane
Treatments Concentration of hemolysis (%)
Hypotonic medium (control) 50.0 mM 0.00
Standard acetyl (salicylic acid) 0.1 mg mLG130.57
CGEE 2.0 mg mLG138.66
1.0 mg mLG142.85
0.5 mg mLG143.67
control, diclofenac sodium (25 mg kgG1 body weight),
significantly (p<0.01) inhibited abdominal writhing in
mice compared to the control group. The writhing inhibition
of CGEE occurred in a dose-dependent manner, as the
percentage of inhibition increased when the concentration of
In this study, phytochemical compounds of CGEE
were evaluated using qualitative and quantitative assays.
Pharmacological activities of CGEE, namely antioxidant,
analgesic and membrane stability properties, were also
studied. The results indicated that CGEE contains a reducing
sugar, combined reducing sugar, tannins, flavonoids, alkaloids,
glycosides, proteins and steroids but it lacks saponins, acidic
Pak. J. Nutr., 19 (4): 153-159, 2020
Table 3: Effect of different concentrations of CGEE on acetic acid-induced writhing reflex in mice
Animal groups Mean of writhing Writhing±SEM (%) Writhing inhibition (%) t-test (p-value)
Negative control (1% Tween-80) 4.60 100.00±2.12 0.00
Diclofenac-Na (25 mg kgG1) 1.64 35.65±1.39 64.35 5.8320**
CGEE (250 mg kgG1) 2.72 59.13±1.39 40.87 4.4100**
CGEE (500 mg kgG1) 2.12 46.08±0.93 53.92 5.3600**
ethanolic stem extract; **p<0.01, SEM: Standard error of mean
compounds and gums (Table 1). CGEE contained a good
amount of total phenolic and total tannin contents that were
comparable to the standards (Fig. 1).
Plants produce various secondary metabolites as their
natural defense23. Among these secondary metabolites, few
are toxic to animals and most of them possess various
therapeutic properties. For example, glycosides and flavonoids
have strong antidiabetic and antioxidant activities24. Different
studies have proposed that various sorts of polyphenol
compounds, such as phenolic acids, flavonoids and tannins,
have numerous biological effects, including antioxidant
activity25,26. In agreement with these statements, our results
showed that CGEE possessed antioxidant properties and acted
by scavenging DPPH free radicals. Elmastas
.28, reported that phenolic compounds contain
hydroxyl groups that may contribute to antioxidant activity
and play a critical role in scavenging free radicals. Other
studies have reported the positive correlation between the
amount of phenolic contents and antioxidant activity of a
plant29,30. Moreover, tannins are generally defined as naturally
occurring polyphenol compounds of high molecular weight
that can form a complex with proteins. Tannins are an
important source of protein in animals but the amounts of
tannins present vary and are very changeable and their effects
on animals range from valuable to toxic to lethal31.
Health risks can be prevented by consuming foods
containing antioxidants and analgesics32 which can have
positive effects on cell structures such as the cell membrane.
The robustness of a cell depends on the wholeness of its
membranes. One way to test the effect of a compound on
membrane stability is to expose red blood cells to a hypotonic
or heated medium. Membrane stabilizers should protect the
membrane against injury and elicit anti-inflammatory effects33.
Compounds with me mb ra n e- st a bilizing properties are well
known for their ability to interfere with the initial phase of
the inflammatory reaction that prevents the release of
phospholipase, which stimulates the formation of
inflammatory mediators34. Our results indicated that CGEE
contains phenols and phenolic compounds inhibit
prostaglandin cyclooxygenase as well as inflammatory
mediators35. Results of our experiments showed that CGEE at
a concentration of 1 mg mLG1 better prevented hypotonic
solution-induced and heat-induced lysis of the human
erythrocyte membrane compared to the standard acetyl
salicylic acid (0.1 mg mLG1). This finding suggests that CGEE
may possess good membrane stabilizing activity.
The anti-nociceptive acetic acid-induced writhing reflex
model was used to identify the analgesic activity of CGEE
Intraperitoneal injection of acetic acid creates a pain
sensation36 and the writhing reflex in animals by activating the
chemo-sensitive nociceptors37 . Acetic acid-induced twisting
represents the pain sensation by triggering the localized
inflammatory response. CGEE treatment resulted in significant
inhibition of writhing compared to the standard drug
diclofenac sodium (Table 3). The polar compounds present in
the plant extract may explain the observed analgesic activity.
The 500 mg kgG1 dose had the highest inhibition rate, the
inhibitory effect of the positive control was greater and the
inhibitory effects were statistically significant (control vs. CGEE
250 mg kgG1, p<0.01 and control vs. CGEE 500 mg kgG1,
p<0.05). These results demonstrate that CGEE at the given
doses significantly reduced the acetic acid-induced writhing
reflex in mice.
In summary, we have demonstrated that CGEE has
antioxidant, membrane stabilizing and analgesic properties.
The results of this study provide a scientific basis for the
utilization of this plant in folk medicine for the treatment of
oxidative disorders. We can also use it to prevent cell lysis.
This study, however, had some limitations. For example, all
tests were conducted using an
models is needed to validate
the findings. Additionally, in-depth study is required to
elucidate the underlying mechanisms responsible for the
antioxidant, anti-inflammatory and membrane stabilizing
properties of CGEE.
This study reports that CGEE has potential antioxidant
activity, free-radical scavenging activity, an analgesic effect
and a membrane stabilizing effect. However, these preliminary
results do not explain the actual mechanisms for the
various pharmacological actions, thus more in-depth studies
(including isolation and identification of active compounds
Pak. J. Nutr., 19 (4): 153-159, 2020
and in-depth pharmacological mechanistic assays) are
required to elucidate the exact mechanisms of action of the
extract and its active compound(s).
The authors are thankful to the Department of Pharmacy,
ASA University, Bangladesh for providing laboratory facilities,
chemicals and reagents and contributory support. We also
give our heartfelt thanks to the International Centre for
Diarrheal Disease and Research, Bangladesh for providing trial
mice and bacterial strains. Our special thanks go to the
Bangladesh National Herbarium for identification of the plant.
No other institution/foundation provided us with any sort of
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