Asian Journal of Green Chemistry 7 (2023) 258-268

Asian Journal of Green Chemistry

Original Research Article View Article Online │ View Journal

Study of Anti-Thrombocyte Activity of Cassia fistula Seeds Extract

and It’s Total Phenolic and Flavonoid Content, In Vitro Antioxidant
and Anti-Inflammatory Activities
Fazle Rabbi Shakil Ahmeda, Mst. Jesmin Sultanab,* , Afroza Sultanaa, Md. Ferdous Aloma
aDepartment of Pharmacy, Khwaja Yunus Ali University, Enayetpur, Chouhali, Sirajganj-6751, Bangladesh
bDepartment of Materials Science and Engineering, University of Rajshahi, Rajshahi-6205, Bangladesh

ARTICLE INFORMATION ABSTRACT

Submitted: 20 May 2023 The purpose of this study was to measure the total phenol and flavonoid
Revised: 22 June 2023 content and assess the antioxidant and anti-thrombocyte activity in ethanol
Accepted: 24 June 2023 extracts of Cassia fistula seeds. Aluminum chloride was used to calculate the
Available online: 8 July 2023 amount of flavonoids, and Folin-Ciocalteu reagent was used to calculate the
total amount of phenolic compounds using a spectrophotometric method.
Manuscript ID: AJGC-2305-1393
The in vitro antioxidant activity of the analyzed extracts was evaluated
Checked for Plagiarism: Yes
utilizing the DPPH approach. The total phenolics and flavonoid contents in
Language Editor:
the seeds extract Cassia fistula 246 ± 0.08 mg GAE/g dw and 118 ± 0.001 mg
Dr. Fatimah Ramezani
QE/g dw, respectively. The obtained results concluded that it may be
Editor who approved publication:
considered a good amount of phenolic and flavonoid compounds. The in
Dr. Zeinab Arzehgar
vitro anti-inflammatory properties showed the highest percentage
DOI: 10.22034/ajgc.2023.398285.1393 inhibition of protein denaturation was 54% for 45 µg/ml and 70% for the
reference drug diclofenac sodium at a similar dose. The minimum inhibition
of hemolysis 58% was observed at 50 µg/mL of Cassia fistula seeds extract
KEYWORDS and 81% for the same dose of standard aspirin. The results presented that
Cassia fistula the ethanol seeds extract of Cassia fistula has potential anti-thrombocyte
Phytochemicals activity. IC50 values were used to express the antioxidant activity of the
Anti-thrombocyte investigated extracts. The IC50 values was found to be 8.90 µg/ml for ethanol
Antioxidant seeds extract which is comparable to that of ascorbic acid (IC50 = 6.73
Anti-inflammatory µg/ml) a well-known standard antioxidant. Among these results, the lower
the IC50 showed the higher the free radical scavenging activity. Among these
findings, the stronger the free radical scavenging activity was seen the lower
the IC50 indicated. Based on all of the findings, we concluded that the anti-
thrombocyte antioxidant and anti-inflammatory activities may be caused by
phytochemicals identified in C. fistula extract.
© 2023 by SPC (Sami Publishing Company), Asian Journal of Green
Chemistry, Reproduction is permitted for noncommercial purposes.

Corresponding authors, email: [email protected], [email protected] (M.J. Sultana).

Tel.: 088-01776800915.
Study of Anti-Thrombocyte Activity of … 259

Graphical Abstract

Introduction and the plant extracts were found to have

strong antibacterial, antifungal, anti-
As sources of remedies, medicinal plants are inflammatory, and antioxidant effects [6].
frequently employed as complementary It has been reported on the chemical
therapeutic tools for the prevention or analysis of various C. fistula components. It was
treatment of several diseases. Very common discovered to include proanthocyanidins,
plant Cassia fistula L., noted for its therapeutic phenolic compounds, and flavonoids [7].
effects. Cassia fistula Linn, a member of the Several pharmacological activities of C.
Caesalpiniaceae family and popularly named fistula extracts have been reported, including
the "golden shower tree" (Bengali: Bandor anti-inflammatory [8], wound healing
lathi), is well-known for its therapeutic properties [1] antioxidant [9], antibacterial
benefits. It is found in a number of areas, [10], and anticancer activity [11].
including Brazil, West Indies, China, South However, there hasn't been any information
Africa, and Asia [1]. published about a thorough pharmacological
Cassia fistula is one of the herbs that are analysis of Cassia fistula seed extracts. Based
most frequently used in Unani and Ayurvedic on the literature review, the current study used
medicine. It has been advised to utilize Cassia seeds extract that had been subjected to
fistula to treat diabetes, leucoderm, pruritus, phytochemical analysis. The same extracts
and haematemesis. Cassia fistula has also been were used for their anti-thrombocytic activity,
found to be helpful for treating skin conditions, anti-inflammatory effects, and antioxidant
liver issues, and tuberculous glands [2]. characteristics (which had not been explored
Extracts relieve constipation, piles, and in earlier investigations).
detoxifier [3]. Furthermore, the total content of phenolic
Numerous biologically significant and flavonoid of C. fistula seeds extract. The
substances were isolated and identified from seeds of C. fistula will gain valuable qualities as
various plant parts [4, 5]. a result of these findings.
The observations were made utilizing
various solvent extracts and plant components,
F.R. Shakil Ahmed et al. 260

Experimental Professor Dr. AHM Mahbubur Rahman

Materials and Methods (Taxonomist), Department of Botany,
University of Rajshahi, Bangladesh. The
Plant samples (seeds) collection and voucher number of the plant Cassia fistula is FK
preparation for extract 239.
The collected seeds were cleaned, shade
Cassia fistula seeds were procured from
dried for two weeks, and ground by a
Kamarkhand, Sirajganj on 1st June 2022. The
mechanical grinder (Figure 1).
plant was identified and verified by

Figure 1. Processing of Cassia fistula seeds powder

Dried ground seeds were soaked in ethanol Determination of the total content of the
to extract the phytochemicals Whatman No. 1 phenolic compound
filter paper and a Buckner funnel were used to
The total content of the phenolic compound
filter the extracts. Using a rotary evaporator
of the tested seed extracts was identified
and lowered pressure at 40 °C, the filtrate was
spectrophotometrically with Folin-Ciocalteu
concentrated to dryness to determine the
reagent according to the aforementioned
crude extract yield.
procedure [15] with slight modifications. Folin
Phytochemical analysis Ciocalteu reagent (5 mL) and 4 mL 7.5%
Na2CO3 solution were mixed with dilute Cassia
Phytochemical analysis was carried out fistula seeds extract (1 mL) or Gallic acid (a
utilizing accepted techniques from the common phenolic component).
literature [12, 13]. Following a 15-minute standing period, the
Using the test of different chemical groups mixes were given a colorimetric analysis at 765
found in the extract of P. minima, nm to measure their total phenolic content.
phytochemical screening was done [14]. Gallic acid solutions in methanol and water
Our study is now focused on the chemical (50:50, v/v) at concentrations of 15.62, 31.25,
groups that can be found in the seeds extract of 62.5, 125, 250, and 500 mg mL-1 were used to
Cassia fistula, specifically steroids, tannins, create the standard curve. On a
alkaloids, quinine, flavonoids content, spectrophotometer, in contrast to a blank (5
saponins, terpenoids, glycosides, mL of Folin-Ciocalteu reagent and 4 mL of
anthraquinone, proteins, phenols, 7.5% Na2CO3 were added in 1 mL of methanol);
carbohydrates, and gum are all present. the absorbance was measured at 765 nm. The
Study of Anti-Thrombocyte Activity of … 261

total content of the phenolic compound of the In vitro anti-inflammatory effects

samples was calculated using the calibration
curve equation, and the findings were The protein denaturation approach was
represented as mg of Gallic acid equivalents used to treat inflammation using the ethanoic
per g of dried weight extracts (mg GAE/g dw), seeds extract of Cassia fistula [17].
which is a typical reference substance. 2.8 milliliters of phosphate-buffered saline
(PBS), 0.2 milliliters of fresh hen egg albumin,
Determination of the total content of the and 2 milliliters of various test extract
flavonoid strengths with final concentrations of 15, 20,
25, 30, 35, 40, and 45 µg/mL were all included
The total content of the flavonoid in the 5 mL reaction mixture. The same amount
compound of the extracts was determined of double-distilled water was utilized as a
using spectrophotometric analysis, and the control. The mixtures were then heated for five
process was based on the complexes that minutes at 70 °C after 15 minutes of incubation
formed between the flavonoids and aluminum at 37 °C. After cooling, using a T60 visible
[16]. spectrophotometer (PG Instruments Limited),
0.5 mL of a 1:10 mg mL-1 Cassia fistula seeds their absorbance at 660 nm was assessed. To
extract was combined with methanol (1.5 mL), test the absorbance at a final concentration of
0.1 mL of 10% aluminum chloride, sodium (15, 20, 25, 30, 35, 40, and 45 µg/mL),
acetate (0.1 mL of 1 M), and double distilled diclofenac sodium was employed as a standard
water (2.8 mL). It stayed at room temperature medication and treated uniformly [18].
for thirty minutes, and a spectrophotometer By applying the formula below, the
was used to measure the reaction mixture's percentage (%) inhibition for protein
absorbance at 510 nm. Quercetin solutions in denaturation was determined [19].
methanol were prepared at concentrations of
500, 250, 125, 62.5, 31.25, and 15.62 mg mL-1
to create the calibration curve.
Abs control − Abs sample
Rotein denaturation inhibition percentage = × 100
Abs control
Determination of anti-thrombocyte activity of Cassia fistula or aspirin (for reference drug)
at various concentrations (50, 100, 200, 400,
Five milliliters of blood from healthy and 800 μg/mL, respectively) made up the test
human participants who had not taken any solution. The test control solution consisted of
NSAIDs before the test was obtained for the 1 mL of phosphate buffer, 0.5 mL of 10% HRBC,
experiment. Following that, it was combined and 2 mL of distilled water in isotonic saline.
with an equal volume of the anticoagulant Hemoglobin content was calculated
ethylene-diamine-tetraacetic acid (EDTA), and spectrophotometrically at 560 nm after assay
centrifuged at 3000 rpm. mixtures were centrifuged at 3000 rpm for 20
Isosaline was employed to clean the packed minutes and incubated at 37 °C for 30 min [20].
cells, and red blood cells were suspended at By assuming the control produced 100% of the
10% v/v and used for observation. 0.5 mL of hemolysis, the percentage of hemolysis was
10% HRBC, 1 mL of 0.15 M phosphate buffer estimated. The formula below was used to
(pH 7.4), 2 mL of hypotonic saline, and 0.5 mL project the hemolysis percentage:
F.R. Shakil Ahmed et al. 262

Percentage of hemolysis =
Abs sample
× 100
found for the genus of Artemisia, which
Abs control indicated the presence of monoterpenes,
sesquiterpene [22].
According to the spectroscopic approach, the
antioxidant effects of crude extracts and The total content of phenolic and flavonoids
positive controls (ascorbic acid) on DPPH
radicals were calculated [21]. By measuring the reducing capacity of the
An equal amount of the 60 mM DPPH phenolic component, the total phenolics
solution and each aliquot of the crude extracts component concentration in the analyzed
(1.5 mL each) were combined. All the extracts was measured
combinations were vigorously mixed and spectrophotometrically, and the results were
allowed to stand for 20 minutes in the dark at derived using the equation for the standard
room temperature. curve: (y = 0.0885x + 0.0049, R² = 0.9758)
The absorbance of the reaction solutions obtained from the calibration curve (Figure 2)
was determined spectrophotometrically at 517 where, x represents the concentration of
nm. Ethanol was use as negative control. The extract from Cassia fistula seeds and y
percentage of DPPH decolorizations of the represents absorbance at 750 nm.
samples was calculated using the following The total phenolics content in the seeds
equation: extract of Cassia fistula is 246 ± 0.08 mg GAE/g
dw (Table 2). It can be concluded from the data
1 − (ABS sample )
% Decolorization = × 100 that there may be a sufficient amount of
1 − (ABS control )
phenolic chemicals.
The production of chelates and the
The effective concentration at which 50% of
displacement of absorption bands as a result of
the radicals in DPPH were scavenged was
a complex formed by flavonoids with metal
known as the IC50 value. This was determined
ions, such as Al3+, serve as the basis for
by interpolating with a linear regression
estimating the total quantity of flavonoids
analysis. IC50 was determined from %
present. The total amount of flavonoid was
inhibition vs. log conc. graph. A lower IC50 value
calculated with the crystalline aluminum
denoted higher antioxidant activity.
chloride and crystalline sodium acetate
Results and Discussion reagent. Quercetin was used as standard. The
total flavonoid content was determined as mg
Phytochemical analysis QE/g of dried plant material using the equation
for the standard curve: y = 0.0951x - 0.0091, R2
An examination of the phytochemical
= 0.9787 where, Y represents the absorbance
composition of the seeds of C. fistula indicated
at 510 nm and x is the concentration of sample
the presence of nine significant
extract of Cassia fistula (Figure 3). The total
phytoconstituents, including alkaloids, tannins,
flavonoid contents in the seeds extract of
flavonoids, saponins, terpenoids, quinine,
Cassia fistula 118 ± 0.001 mg QE/g dw (Table
anthraquinone, phenols, carbohydrates, and
2). It can be concluded from the data that there
proteins.
may be a sufficient amount of flavonoid
In addition, the absence of steroids, gum,
compounds.
and glycosides was noted in the outcome. This
is depicted in Table 1.A similar result was also
Study of Anti-Thrombocyte Activity of … 263

Table 1. Phytochemical analysis of Cassia fistula seeds extract

The phytochemicals' Ethanol extract Name of the Ethanol extract
names Phytochemicals
Glycosides - Gums -
Flavonoids + Steroids -
Saponin + Carbohydrates +
Tannins + Quinone +
Protiens + Alkaloids +
Terpenoids + Anthraquinone +

(+) = Presence, (−) = Absence

0.6
Absorbance vs Concentration
y = 0.0885x + 0.0049
0.5 R² = 0.9758
0.4
Absorbance

0.3

0.2

0.1

0
15.62 31.25 62.5 125 250 500
Concentration (mg/ml)
Absorbance Gallic acid Linear (Absorbance Gallic acid)

Figure 2. Graphical representation of absorbance of Gallic acid

Table 2. The total content of phenolic and flavonoids of the seed extract from Cassia fistula
Test Sample Absorbance Total phenolics Absorbance at Total flavonoids
at 750 nm content 510 nm content
(Average ± (mg GAE/g dw) (Average ± SD) (mg QE/g dw)
SD)
Ethanol extract of 0.223±0.08 246 ± 0.08 0.122±0.0011 118 ± 0.001
Cassia fistula seed

In vitro anti-inflammatory activity The RBCs hemolysis was significantly

inhibited at all doses of Cassia fistula seeds
Egg albumin was used to conduct in vitro extract (50, 100, 200, 400, and 800 μg/ mL)
anti-inflammatory activities. Using the seeds of
(Table 3). At different doses, the lowest
Cassia fistula, the highest percentage of protein
inhibition of hemolysis 58% was observed 50
denaturation inhibition was observed as 54%
µg/mL of Cassia fistula seeds extract and 81%
for 45 μg/mL and 70% for the same dose of the
for the same dose of standard aspirin. The
standard medication diclofenac sodium. Figure
results presented that the ethanol seeds
4 presents a summary of the outcomes.
extract of Cassia fistula has potential Anti-
thrombocyte activity.
Determination of anti-thrombocyte activity
F.R. Shakil Ahmed et al. 264

Figure 3. Graphical representation of absorbance of Quercetin

Figure 4. Inhibition of protein denaturation activity of Cassia fistula seeds extract. The data are shown as
mean ± SEM (p < 0.05)

Table 3. Percentage of hemolysis inhibition at various concentrations

Percentage of hemolysis
inhibition
Concentration(μg/mL) % Inhibition of %Inhibition of
Cassia fistula seeds extract Aspirin
50 58 81
100 68 87
200 77 90
400 84 94
800 88 96
The data are shown as the Mean ±SEM of three replicates (n=3). One-way ANOVA was used to evaluate the data.
At P< 0.05, values were deemed significant.
Study of Anti-Thrombocyte Activity of … 265

Antioxidant activity of Cassia fistula seeds percentage oxidative inhibition values of seed
extract extract evaluated at various doses are shown in
Figure 5 along with the results. IC50 was
The antioxidant activity of the seeds of
determined from % inhibition vs. log conc.
Cassia fistula extracted in 95% ethanol was
Graph (Figure 6).
examined using the DPPH free radical
These results make it evident that the
scavenging test technique. The most
ethanol seed extract of Cassia fistula has an IC50
mentioned method for evaluating the
value of 8.90 µg/mL, which is equivalent to
antioxidant activity of numerous plant-based
ascorbic acid's IC50 value of 6.73 µg/mL, as a
medications is the DPPH (1, 1-diphenyl-2-
well-known standard antioxidant. A similar
picrylhydrazyl) assay.
result was also found for the methanolic
This approach is based on the decrease of
extracts of Ephedra sarcocarpa which showed
the colored free radical DPPH in ethanolic
the high antioxidant activities and the IC50
solution by various sample concentrations.
value in the DPPH assay 4.6 mg/mL [23].
IC50, or 50% oxidative inhibitory concentration,
On the other hand, Artemisia kulbadica
was used to measure antioxidant activity. Ten
extracts was exhibited the moderate values
different concentrations of extract (1, 1.95,
DPPH radical scavenging activity IC50= (422.4
3.90, 7.81, 15.62, 31.25, 62.2, 125, 250, and 500
± 2.4) μg/mL [24].
μg/mL) were prepared in ethanol solvent for
Among these results, thus the free radical
this experiment. Ethanol without extract
scavenging activity was higher the lower the
served as the control, and ascorbic acid served
IC50 revealed. However, this was noticed that
as the standard. Using a UV-Visible
the extract has a lower antioxidant activity of
spectrophotometer, absorbance was
Cassia fistula seeds extract than standard
determined at wavelength 517 nm. Each
ascorbic acid.
experiment was carried out three times. The

Figure 5. Absorbance vs. concentration graph for (a) ethanol extract of Cassia fistula and (b) standard
(Ascorbic acid)
F.R. Shakil Ahmed et al. 266

Figure 6. % inhibition vs. concentration graph for (a) ethanol extract of Cassia fistula seed and (b) standard
(Ascorbic acid)

Conclusion Department of Materials Science and

Engineering, University of Rajshahi, Rajshahi,
According to the research that was given, C. Bangladesh. Furthermore, they would like to
fistula seed extracts have a high concentration thank Md. Rubel Hossain (Department of
of phenolic and flavonoid components, which Library Science, Khwaja Yunus Ali University,
directly contributes to their significant Sirajganj, Bangladesh) for checking similarity
antioxidant activity. These findings using their software.
demonstrate the efficacy of traditional
medicinal practices and point to the possibility Disclosure Statement
for C. fistula seeds to be used as an antioxidant
and anti-inflammatory agent. It is obvious that No potential conflict of interest was reported
the seeds of C. fistula may contain some novel by the authors.
compounds that have the potential to be
Funding
sources of novel anti-inflammatory, anti-
thrombocyte, and antioxidant drugs based on This research did not receive any specific grant
the research into the effects of anti- from funding agencies in the public,
inflammatory, anti-thrombocyte, and commercial, or not-for-profit sectors.
antioxidant behaviour of ethanol seed extract.
Authors' Contributions
Acknowledgements
All authors contributed to data analysis,
The authors would like to thank Khwaja Yunus drafting, and revising of the article and agreed
Ali University, Sirajganj, Bangladesh. Also, they to be responsible for all the aspects of this
present their sincere gratitude to the work.
Study of Anti-Thrombocyte Activity of … 267

Orcid [12]. Trease G.E., Evans W.C. 13th edn. Bailliere

Tindall, London, 1989, 176 [Google Scholar],
Mst. Jesmin Sultana [Publisher]
https://orcid.org/0009-0009-9316-9609 [13]. Govind P., Madhuri S. Indian. Drugs., 2006,
43:869 [Google Scholar], [Publisher]
References
[14]. Ahmed F.R.S., Sultana A., Sultana M.J. Saha
[1]. Kumar V.P., Chauhan N.S., Padh H., A. Bull. Natl. Res. Cent., 2022, 46:94 [Crossref],
Rajani M. J. Ethnopharmacol., 2006, 107:182 [Google Scholar], [Publisher]
[Crossref], [Google Scholar], [Publisher] [15]. Singleton V.L., Orthofer R., Lamuela-
[2]. Dutta A., De B. Indian. J. Pharmacol. Sci., Raventos R.M. In Methods in Enzymology,
1998, 60:388 [Publisher] 1999, 299:152 [Crossref], [Google Scholar],
[3]. Agarwal S.S., Paridhavi M. Ahuja [Publisher]
Publishing House, 2005, 281 [Publisher] [16]. Chang C., Yang M., Wen H., Chern J. J. Food
[4]. Thirumal M., Surya S., Kishore G. Crit. Drug Analaysis, 2002, 10:178 [Crossref],
Rev. Pharmaceut. Sci., 2012, 1:43 [Publisher] [Google Scholar], [Publisher]
[5]. Sultana M.J., Ahmed F.R.S., Alam M.T. Asian [17]. Sahoo A.K., Dash U.C., Kanhar S.,
J. Green Chem., 2019, 3:518 [Crossref], [Google Mahapatra A.K. Toxicol, 2017, 4:274 [Crossref],
Scholar], [Publisher] [Google Scholar], [Publisher]
[6]. Gupta R.K. CBS publishers and distributors, [18]. Sultana M., Nibir A.I.S. Ahmed F.R.S. Beni-
1st edition., 2010, 116 Suef Univ. J. Basic. Appl. Sci., 2023, 12:26
[7]. Luximon R.A., Bahroun T., Soobrattee M.A., [Crossref], [Google Scholar], [Publisher]
Aruoma O.I. J. Agric. Food. Chem., 2002, [19]. Dineshkumar G., Radha V., Sri Kumaran N.
50:5042 [Crossref], [Google Scholar], Int. J. Green. Pharm., 2018, 12:S528 [Crossref],
[Publisher] [Google Scholar], [Publisher]
[8]. Sharma D.K., Sharma S.K., Lonare M.K., [20]. Hossain R., Rahman M.A., Rafi M.K.J.,
Kaur Rajdeep, Dumka V.K. Pharmacological Siddique T.A., Noman A.A., Makki A., Alelwani
Evaluation of Antipyretic, Analgesic and Anti- W., Hajjar D., Tangpong J. Notulae Botanicae
inflammatory Activities of Ethanolic Extract of Horti Agrobotanici Cluj-Napoca, 2020, 48:1155
Cassia fistula. Indian Journal of Animal [Crossref], [Google Scholar], [Publisher]
Research, 2022, 56:483 [Crossref], [Google [21]. Myint, Phyu P., May T.S., Htet H.H. Journal
Scholar], [Publisher] of Pharmacognosy and Photochemistry, 2017,
[9]. Irshad M., Singh M., Zafaryab M., Rizvi 6:528 [Crossref], [Publisher]
M.M.A. Int. J. Med., 2012 [Crossref], [Google [22]. Rustaiyan A., Ezzatzadeh E. Asian. J. Chem.,
Scholar], [Publisher] 2011, 23:1774 [Google Scholar], [Publisher]
[10]. Irshad M., Aijaz A., Zafaryab M., Ahmad [23]. Rustaiyan A., Javidnia K., Farjam M.H.,
F., Manzoor N., Singh M., Rizvi M.M.A. Nat Prod Aboee-Mehrizi F., Ezzatzadeh E. Journal of
Commun., 2013, 8:261 [Crossref], [Google Medicinal Plants Research., 2011, 5:4251
Scholar], [Publisher] [Crossref], [Google Scholar], [Publisher]
[11]. Irshad M., Mehdi S.J., Al-Fatlawi A.A., [24]. Ezzatzade E., Farjam M.H., Rustaiyan A.
Zafaryab M,. Ali A., Ahmad I., Singh M., Rizvi Asian. Pacific. J. Tropical. Disease., 2012, 2:S431
M.M.A. J. Biol. Active. Prod. Nat., 2014, 4:158 [Crossref], [Google Scholar], [Publisher]
[Crossref], [Google Scholar], [Publisher]
F.R. Shakil Ahmed et al. 268

How to cite this manuscript: Fazle Rabbi

Shakil Ahmed, Mst. Jesmin Sultan, Afroza
Sultana, Md. Ferdous Alom. Study of Anti-
thrombocyte Activity of Cassia fistula Seeds
Extract and It’s Total Phenolic and Flavonoid
Content, In Vitro Antioxidant and Anti-
Inflammatory Activities. Asian Journal of
Green Chemistry, 7(4) 2023, 258-268.
DOI: 10.22034/ajgc.2023.398285.1393