Pharmacological Research - Modern Chinese Medicine 9 (2023) 100338

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Pharmacological Research - Modern Chinese Medicine

journal homepage: www.elsevier.com/locate/prmcm

GC-FID spectroscopic analysis and antioxidant activities of methanolic

fraction of Cassia tora leaves
C. Chinelo Nkwocha *, 1, O. Joshua Felix 2, N. Rosemary Idoko 3
Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: Introduction: From time immemorial, different parts of Cassia tora (Jue ming zi) have found application in Chi­
GC-FID nese medicine. There is a reputation for the therapeutic properties of the plant’s leaves, seeds, and roots. The
Bioactive compounds objective of this study is to ascertain the antioxidant properties and bioactive compounds found in Cassia tora
Antioxidants
leaf.
Phytochemicals
FRAP
Materials and method: Phytochemical and antioxidant studies were carried out using standard analytical tech­
niques. Bioactive compounds were analysed using GC FID.
Results: Results of the quantitative phytochemical analysis of the methanolic fraction of Cassia tora leaves
indicated the presence of saponins (71,760±943.2µg/ml), terpenoids (47,466.70±46.2µg/ml), tannins
(21,253.30±46.2µg/ml), flavonoids (14,682.70±40.3µg/ml), phenols (30,986.70±46.2µg/ml), steroids
(13,557.30±24.4µg/ml), alkaloids (9770.67±9.2µg/ml), glycosides (5434.67±139.8µg/ml). Using ferric
reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and total antioxidant capacity (TAC)
to analyze the antioxidant properties of the methanolic fraction of Cassia tora leaves, it was discovered that the
higher the scavenging power of the fraction, the lower the concentration of the standard (ascorbic acid). In
comparison to the standard (ascorbic acid), the fraction had the maximum antioxidant capacity at 6.86mg/ml.
Total antioxidant power in the fraction ranged from 1.78mg/ml to 16mg/ml. Twenty (20) bioactive chemicals
were found in the methanol fraction of Cassia tora leaves, according to the results of the gas chromatography
flame ionization detector (GC-FID) investigation.
Conclusion: The findings of this current study demonstrate that the leaves of Cassia tora are rich in numerous
bioactive compounds and are natural sources of antioxidants.

1. Introduction and a variety of alkaloids are examples of commonly identified

Humanity has benefited greatly from plants and herbs as a source of
food, folk medicines, and beginning materials for the creation of new
synthetic pharmaceuticals. They can create a vast range of chemical
substances that are used for biological processes and defence against
predators like insects, fungi, yeasts, bacteria, viruses, and other diseases
[1]. The mechanisms by which chemical elements in plants exert their
effects on the human body are the same as those that are widely known
to apply to the chemical elements in conventional medications [2].
Therefore, in terms of how they function, herbal medications are similar
to prescription drugs. Phenols, tannins, flavonoids, lignans, terpenes,
plant-based secondary metabolites [3]. Natural products have become
extremely important for the development of polypharmacological drugs
for Intrusion Detection Systems (IDS), cancers, and neurological disor­
ders because they are better models with ideal pharmacokinetic­
s/pharmacodynamics properties [4] and frequently contain biologically
relevant molecular scaffolds and pharmacophore patterns that have
evolved as preferred ligand-protein binding motifs [5].
A well-known medicinal herb called Cassia tora Linn. is widely
distributed in India, China and other tropical nations. It is a semi-wild
annual herb that is commonly grown throughout Southeast Asia,
India, Northern Australia, and the Americas [6]. It is an annual herb

* Corresponding author.
E-mail address: chinelo.nkwocha@unn.edu.ng (C.C. Nkwocha).
1
234 8063482535.
2
234 8080439460.
3
234 9052538198.

https://doi.org/10.1016/j.prmcm.2023.100338
Received 23 October 2023; Received in revised form 15 November 2023; Accepted 22 November 2023
Available online 6 December 2023
2667-1425/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
C.C. Nkwocha et al.
with a fetid fragrance that can reach a length of 30 to 90 cm. Because the
plant is hermaphrodite, both the male and female flower parts are
produced. The paripinnate, up to 10-centimetre-long leaves of Cassia
tora, which are roasted in oil and consumed in some regions of India,
have an obovate shape. Corymbose raceme flowers are present. They
contain five petals and five sepals, making them pentamerous. Accord­
ing to Kim et al. [7], they are yellow, and their blossoming season is from
August to September after the Indian monsoon. The seeds are 3–4 mm s
thick and around 1 cm long. They have an oblong shape and are dark
brown. They taste harsh and are renowned for having significant ther­
apeutic potential. Early chemical ingredient investigations on two spe­
cies of plants in the genus Cassia—Cassia tora and Cassia
obtusifolia—were conducted, and it was discovered that their chemical
profiles were noticeably different [7]. In traditional Ayurvedic and
traditional Chinese medicine, its usage has been described as an anti­
oxidant, antimicrobial, antihepatotoxic, antidiuretic, antidiarrhoeal and
antimutagenic plant. It has also been suggested to improve visual acuity
[8]. Its seeds are reputed in Chinese medicine as vision-improving,
antiasthenic, asperient, diuretic and an effective agent in lowering
cholesterol and reducing blood pressure [9].
Nowadays, the "one drug, multitarget" approach to drug develop­
ment has replaced the "one drug, one target" approach [10]. Due to the
awful adverse effects of synthetic pharmaceuticals, the bulk of the
world’s population relies on medicinal plants as their primary form of
therapy [11]. Additionally, individuals used natural cures and re­
searchers studied the pharmacology of plants because of their potential
to treat diseases and the need for their study in sacred books [12]. Given
this, the study focuses on the phytochemical and polyphenolics present
in Cassia tora leaf extract as well as its antioxidant properties. This will
add to the literature on the plant’s medicinal properties and provide
background information for researchers who wish to carry out more
advanced research on the pharmacological aspects of the plant espe­
cially in Chinese medicine.
2. Materials and methods
2.1. Materials
2.1.1. Collection and authentication of plant material
In March 2023, fresh Cassia tora leaves were harvested in Orba,
Udenu Local Government Area, Enugu State, Nigeria. Mr Alfred Ozioko,
a botanist at the Bio-resources Development and Conservation Pro­
gramme (BDCP) Research Centre in Nsukka, Enugu State, Nigeria,
identified and verified the leaves. The specimen was given the voucher
number Intercedd/27,651 before being placed in the BDCP Herbarium.
Weight of fractions(hexane)(g)
Percentage Yield of hexane fraction =
Weight of Dried Pulverized Sample (g)
2.1.2. Chemicals and reagents
All chemicals used for the study were of analytical grade and were
products of Sigma Aldrich, USA, British Drug House (BDH) England,
Burgoyne, India, Harkin and England, Qualikems India, Fluka Germany,
May and Baker England. The assays were carried out using commercial
kits which were products of Randox, USA and Teco (TC), USA. They
include hydrochloric acid chloroform, Alkaline pirate, 50% m/v potas­
sium ferricyanide, 0.5% Formaldehyde, folin reagent (1/10 dilution),
vanillin reagent, 60% H2SO4, 2% NaCO3, Chromogen, 10% ALCL3, Po­
tassium acetate, formaldehyde, Conc H2SO4, sodium carbonate,
aluminium chloride, potassium acetate, 5% phosphomolybdic acid,
chromogen, sodium phosphate, potassium ferricyanide, trichloroacetic
Pharmacological Research - Modern Chinese Medicine 9 (2023) 100338
acid, ferric chloride, 3.5% Na2CO3, phosphomolybdenum reagent
(28Mm sodium phosphate and 4Mm ammonium molybdate in 0.6M
H2SO4), ammonium molybdate, potassium hydroxide, sodium sulphate,
pyridine, ethanol, methanol, 0.18 Mm (0.005%) methanolic solution of
DPPH, ethyl acetate, n-hexane. All materials were used without further
purification.
2.1.3. Instrument and equipment
The equipment used was obtained from the Department of
Biochemistry, University of Nigeria, Nsukka, and other scientific shops
in Nsukka, Enugu State, Nigeria. They included: conical flasks(Pyrex,
England), water baths (Gallenkamp, England), beakers(Pyrex, England),
weighing balance(Metler HAS, U.S.A), Whatman No 1 Filter papers, test
tubes (Pyrex, England), measuring cylinder (Pyrex, England), Muslin
cloth, flat bottom flask, glass funnel (Pyrex, England), spectrophotom­
eter (spectronic 20D, Germany), micro-pipettes (Perfect, USA), refrig­
erator (Thermocool, England), centrifuge (Vickas Ltd, England) and
rotary evaporator, 10L gallon, gas chromatography equipped with a
flame ionization detector (Buck M910), fractionation column
2.2. Methods
2.2.1. Preparation of plant material
Fresh Cassia tora leaves were weighed (1065.15g) and allowed to air
dry at room temperature. They were then weighed and kept in a jar with
a screw top after being ground into a fine powder. By soaking the
powdered leaves in 3.5L of 100% methanol for 72 h, the leaves were
extracted. The material was subsequently filtered using a muslin cloth
and Whatman grade 1 filter papers. When the filtrate was ready for
further examination, it was stored in a sterile screw-capped vial and
concentrated by vacuum evaporation. Following the formula below, the
weight of the dried, ground-up leaves before maceration and the weight
of the crude extract after concentration were used to calculate the%
yield of the methanol extract:
Weight of CrudeExtract(g)
Percentage Yieldof Crude Extract = x 100
Weight of DriedPulverizedSample (g)
2.2.2. Fractionation of Cassia tora leaf crude extract
The methanol crude extracts were subjected to fractionation using
column chromatography by slurry method. The extract was mixed with
silica gel of pore size 200- 400 mesh, packed into the column and
fractionated with n-hexane, ethyl acetate and methanol in order of
increasing polarity.
x 100
Percentage Yield of ethyl acetate fraction
Weight of fractions(ethyl acetate)(g)
= x 100
Weight of Dried Pulverized Sample (g)
Percentage Yield of methanol fraction
Weight of fractions(methanol)(g)
= x 100
Weight of Dried Pulverized Sample (g)
2
C.C. Nkwocha et al.
2.2.3. Quantitative phytochemical analysis of the methanolic fraction of
Cassia tora leaves
According to Harborne [13], quantitative phytochemical analysis
was performed on the methanolic fraction of Cassia tora leaves to
determine the phytochemical components.The methanol fraction was
measured and 0.8g of the methanol fraction was dissolved into 50ml of
absolute methanol. The mixture was filtered to get a clear solution
which was used for further phytochemical analysis. Conc. Of stock =
0.8g/50ml (vol. of solvents) = 0.016g/ml = 16mg/ml and 0.5ml of the
filtrate was used for each phytochemical analysis.
2.2.3.1. Test for glycosides. The prepared extract (0.5ml) was trans­
ferred into a test tube and 0.2ml of alkaline pirate was added followed
by 2.3ml of distilled water. The mixture was boiled for 5 min, allowed to
cool and absorbance was read at 490nm against the blank. The con­
centration was calculated using C (mg/ml) = C1X where
C1=concentration of stock (16mg/ml), X= absorbance.
2.2.3.2. Test for saponins. The prepared extract (0.5ml) was transferred
into a test tube containing 1ml of vanillin reagent. Then 1.5ml of 72%
H2SO4 was added and incubated for 2 h at room temperature- controlled
water bath. After cooling in cold water, the absorbance was read at
544nm against the blank. The concentration was calculated using C(mg/
ml) = C1X where C1=concentration of stock (16mg/ml), X= absorbance.
2.2.3.3. Test for alkaloids. The prepared extract (0.5ml) was added to
1.5ml of 60% H2SO4 after 5 min,1ml of 0.5% formaldehyde introduced
into the test mixture and incubated for 2 h at room temperature
Absorbance was read at 565nm against distilled water. The concentra­
tion was calculated using C(mg/ml) = C1X where C1=concentration of
stock (16mg/ml), X= absorbance.
2.2.3.4. Test for tannins. A measured volume (0.5ml) of the extract was
added to 0.2ml of Folin reagent (1/10 dilution). Following this, 2.2ml of
distilled water and 0.1ml of 3.5% Na2CO3 was added. After 5 min the
absorbance at 640nm against the blank. The concentration was calcu­
lated using C(mg/ml) = C1X where C1=concentration of stock (16mg/
ml), X= absorbance.
2.2.3.5. Test for flavonoids. A measured volume (0.1ml) of 10% ALCL3
was added to 0.5ml of the methanol fraction. Then 0.1ml of 1M potas­
sium acetate and 2.3ml of distilled water was added and absorbance was
read at 415nm after 30 min of incubation at room temperature. The
concentration was calculated using C(mg/ml) = C1X where
C1=concentration of stock (16mg/ml), X= absorbance.
2.2.3.6. Test for terpenoids. The extract (0.5ml) was pipetted into a test
tube. 0.5ml of 5% phosphomolybdic acid and 1.5ml of concentrated
H2SO4 was added. After incubation for 30 min at room temperature,
absorbance was read at 490nm against the blank. The concentration was
calculated using C(mg/ml) = C1X where C1=concentration of stock
(16mg/ml), X= absorbance.
2.2.3.7. Test for phenols. The extract (0.5ml) was transferred into a test
tube containing 0.2ml of 1/10 dilution Folin reagent. 0.2ml of 2%
Na2CO3 was added and allowed to stand for 30 min at room tempera­
ture. After the addition of 2.1ml of distilled water, the absorbance was
read at 640nm against the blank. The concentration was calculated
using C(mg/ml) = C1X where C1=concentration of stock (16mg/ml), X=
absorbance.
2.2.3.8. Test for steroids. A measured volume of the extract, 0.5ml was
pipetted into a test tube containing 1ml of chromogen and 1.5ml of
distilled water was added. The mixture was incubated at room tem­
perature for 30 min and absorbance was read at 550nm against the
3
Pharmacological Research - Modern Chinese Medicine 9 (2023) 100338
blank. The concentration was calculated using C(mg/ml) = C1X where
C1=concentration of stock (16mg/ml), X= absorbance.
2.2.4. In vitro antioxidants assays of Cassia tora leaves
The in vitro antioxidant activities of the methanolic fraction of Cassia
tora leaves were carried out according to the method of Prieto et al. [14];
Singh et al. [15]; and Alachaher et al. [16]..
2.2.4.9. Ferric reducing antioxidants power (FRAP) of Cassia tora leaves
methanol crude extract. Different concentrations of the fraction 0.05,
0.1, 0.15, 0.2, and 0.25ml were pipetted into the test tubes and made up
to 0.5ml with sodium phosphate buffer (0.1M, pH 6.6) and potassium
ferricyanide (1.0mL, 1.0%) was added. The mixture was incubated at
50◦ C for 20 min. Then 1mL of 0.5% trichloroacetic acid was added and
centrifuged at 4000rpm. The upper layer of the solution (1.0mL) was
diluted with 1.0mL of distilled water and ferric chloride (0.1ml) was
introduced. The absorbance was measured at 700nm against the blank.
%Scavengingactivity = [(A700nm test − − A700nm blank) / A700nm test]X10
2.2.4.10. Diphenyl-1-Pickrylhydrazyl radical(dpph) scavenging activity of
methanol crude extract of Cassia tora leaves. Different concentrations
(0.05, 0.1, 0.15, 0.2, and 0.25ml) of the extract were taken in different
test tubes and made up to 0.5ml with methanol. Then 1ml of 0.18mM
(0.005%) methanolic solution of DPPH was added to these tubes and
shaken vigorously. The tubes were allowed to stand in the dark at room
temperature for 30min. The control was prepared as above without any
extract, and Methanol was used for the baseline correction. Changes in
the absorbance of the samples were measured at 517nm. Radical scav­
enging activity was expressed as the inhibition percentage and was
calculated using the following formula:
%scavengingActivity = [(A517 Ctrl − A517 treatment) / A517 Ctrl]X10
2.2.4.11. Total antioxidants capacity (TAC) of methanolic fraction of
Cassia tea leaf. This was carried out according to the method described
by Prieto et al. [14]. Different concentrations (0.05 - 0.25ml) of the
Cassia tora leaves methanolic fraction were taken into test tubes. 3ml of
phosphomolybdenium reagent (28mM sodium phosphate and 4mM
ammonium molybdate in 0.6M H2SO4, then was incubated for 90min. at
95◦ C, after cooling to room temperature, the absorbance was taken at
695nm against a black. Radical scavenging activity was expressed as the
inhibition percentage and was calculated using the following formula:
%scavengingActivity = [(A695 Ctrl − − A695 treatment) / A695 Ctrl]X100.
2.2.5. Analysis of bioactive compounds by gas chromatography – flame
ionization detector (GC-FID)
According to Buss et al. [17] and Kelly et al. [18], the analysis was
done on the methanolic fraction of Cassia tora leaves using a Gas
Chromatography - Flame Ionization Detector (GC-FID) system (Buck
Scientific M910)
Gas chromatography – flame ionization detector (GC-FID) analysis
was carried out using methanol fraction of Cassia tora leaves to identify
and quantify all bioactive compounds present in the samples.
2.2.5.12. Extraction of bioactive compounds. A quantity, of 0.2g of the
fraction was weighed and transferred in a test tube and 15ml ethanol
and 10ml of 50% m/v potassium hydroxide were added. The test tube
was allowed to react in a water bath at 60◦ c for 3hrs 00mins. After the
reaction time, the reaction product contained in the test tube was
transferred to a separatory funnel. The tube was washed successfully
with 20ml of ethanol, 10ml of cold water, 10ml of hot water and 3ml of
hexane, which was all transferred to the funnel. These extracts were
combined and washed three times with 10ml of 10%v/v ethanol
aqueous solution. The ethanol solvent was evaporated. The sample was
C.C. Nkwocha et al.
solubilized in 1000µl of pyridine of which 200µl was transferred to a vial
for analysis.
2.2.5.13. Quantification of bioactive compounds by GC-FID. Analysis of
bioactive compounds was performed on a BUCK M910 Gas chroma­
tography equipped with a flame ionization detector. A RESTEK 15 meter
MXT-1 column (15m× 0.15um) was used. The injector temperature was
280◦ C with splitless injection of 2µl of sample and a linear velocity of
30cms‾1, Helium 5. 0pa.s was the carrier gas with a flow rate of
40mlmin‾1. The oven operated initially at 200◦ C, it was heated to 330◦ C
at a rate of 3◦ C min‾1 and was kept at this temperature for 5min. The
detector operated at a temperature of 320◦ C. Phytochemicals were
determined by the ratio between the area and mass of internal standard
and the area of the identified phytochemicals. The concentration of the
different phytochemicals expressed in µg/g.
2.3. Statistical analysis
The results of the statistical analysis of the data were presented as
Mean±Standard Deviation. Microsoft Excel was used for the statistical
analysis. The statistical analysis tool in Microsoft Excel was used to
create line and bar charts.
3. Results
3.1. Percentage yield of methanol extract of Cassia tora leaves
The extraction of 566.30g of Cassia tora leaves with methanol gave a
yield of 30.13g; a percentage yield of 5.32% of the starting plant
material.
3.2. Percentage yield of the fractions of Cassia tora leaf methanol extract
The fractionation of the methanol extract of Cassia tora leaves gave a
yield of 8.3g of N-hexane fraction, 12.3g of ethyl acetate fraction and
5.48g of methanol fraction; this gave a percentage yield of 1.47%,
2.17%, 0.97% respectively. The most potent fraction which was the
methanol fraction was used for further analysis.
3.3. Quantitative phytochemical composition of methanolic fraction of
Cassia tora leaves
Phytochemicals found in the methanolic fraction of Cassia tora leaves
are shown in Table 1 below in varied proportions. The result shows
relatively high levels of saponins (71,760±943.2µg/ml), terpenoids
(47,466.70±46.2µg/ml), phenols (30,986.70± 46.2µg/ml), tannins
(21,253.30±46.2µg/ml), flavonoids (14,682.70±40.3µg/ml), steroids
(13,557.30± 24.4µg/ml), alkaloids (9770.67± 9.2µg/ml). Glycosides
(5434.67±139.8µg/ml) were the least phytochemical present in the
methanolic fraction of Cassia tora leaves. The results obtained from
Table 1 show that saponins were the most abundant phytochemical
Table 1
Quantitative Phytochemical Composition of Methanolic Fraction
of Cassia tora leaves.
Phytochemicals Concentration(µg/ml)
Glycosides 5434.67±139.8
Saponins 71,760±943.2
Alkaloids 9770.67±9.2
Tannins 21,253.30±46.2
Flavonoids 14,682.70±40.3
Terpenoids 47,466.70±46.2
Phenols 30,986.70±46.2
Steroids 13,557.30±24.4
Results are expressed as Mean ± SD of three determinations,
(n=3).
4
Pharmacological Research - Modern Chinese Medicine 9 (2023) 100338
present in Cassia tora leaves methanolic fraction.
3.4. In vitro antioxidant activities of methanolic fraction of Cassia tora
leaves
The antioxidant properties of the methanolic fraction of Cassia tora
leaves were determined using Ferric Reducing Antioxidant Power
(FRAP), 2, 2-diphenyl-1-pickrylhydrazyl (DPPH) and Total Antioxidant
Capacity (TAC).
For 2.2-diphenyl-1-pickrylhydrazyl (DPPH) it shows that the small­
est concentration has higher scavenging power. The extract shows
scavenging activity in a concentration-dependent manner with inhibi­
tory concentration that will cause 50 folds scavenging equal to
49.36mg/ml (that at 50% the scavenging activity is at 49.36mg/ml) as
shown in Fig. 1 below;
For ferric reducing antioxidant power (FRAP), the fraction shows
antioxidant properties ranging from 1.78mg/ml to 16mg/ml, as shown
in Fig. 2 below. The fraction exhibited the highest antioxidant power at
6.86mg/ml compared to the standard (ascorbic acid). However, there is
no significant difference between the antioxidant power of the fraction
and ascorbic acid at the concentrations of 4mg/ml and 10.67mg/ml.
According to Fig. 3 below, the proportion for total antioxidant power
(TAC) indicated total antioxidant power ranging from 1.78mg/ml to
16mg/ml. The fraction’s antioxidant power was higher than the refer­
ence (ascorbic acid) at 6.86mg/ml. At doses of 1.78mg/ml and
10.67mg/ml, however, there is no significant difference in the antioxi­
dant capacity of the fraction and ascorbic acid.
3.5. GC-FID identification and quantification of bioactive compounds
Fig. 4 depicts the gc-fid chromatogram that displays the peaks of the
several bioactive substances that were discovered in the methanol
fraction of cassia tora leaves. a total of 20 peaks were found, and using
their peaks and retention time, bioactive substances were recognized
and quantified in the fraction. with a concentration of 22.9290g/ml
(15.96%) and a retention time (rt) of 15.783min, the compound spartein
was discovered to be the most prevalent in the methanolic fraction of
cassia tora leaf, followed by steroids with a concentration of 11.7164g/
ml (8.16%) and rt of 26min. with concentrations of 2.0781g/ml
(1.45%), 2.0623g/ml (1.44%), and 2.0042g/ml (1.40%) correspond­
ingly and retention times of 13.300min, 38.326min, and 40.930min,
respectively, naringenin, catechin, and tannin were the least abundant
components. table 2 lists the substances identified in the methanolic
fraction of Cassia tora leaf along with their retention time (RT), peak
area, peak height, concentration (i.e. external), and percentage (%)
compositions.
4. Discussion
Medicinal plants are thought to be abundant sources of bioactive
substances with the potential for medication development [19]. The
significance of this study lies in the biologically active chemicals present
in the methanolic fraction of Cassia tora leaf and its potential to be used
as a medication sources in Chinese herbal medicine. Numerous phar­
macological and biological effects are displayed by these substances
[20]. Table 1 lists the various amounts of phytochemicals identified in
the methanolic fraction of Cassia tora leaves, with saponins having the
highest concentration (71,760±943.2g/ml) and glycosides having the
lowest (5434.67±139.8g/ml). This is in contrast to the findings of
Rahimullah and Imran [21], who determined that flavonoids were the
most abundant phytochemical in the methanolic extract of Cassia tora
leaves while tannins were the least abundant and terpenoids were not
found. Saponins, the most abundant substance in the methanol portion
of Cassia tora leaves, have antioxidant benefits on the skin and shield it
from UV ray damage. It suppresses the development of malignant cells,
boosts immunological function, decreases cholesterol, acts as an
C.C. Nkwocha et al. Pharmacological Research - Modern Chinese Medicine 9 (2023) 100338

Fig 1. DPPH Scavenging Activities of Methanolic Fraction of Cassia tora leaves (% Scavenging against Concentration). IC50 = 49.36mg/ml.

Fig 2. Ferric Reducing Antioxidant Power of Methanolic Fraction of Cassia tora leaves.

Fig 3. Total Antioxidant Capacity of Methanolic Fraction of Cassia tora leaves.

5
C.C. Nkwocha et al. Pharmacological Research - Modern Chinese Medicine 9 (2023) 100338

result in 50-fold scavenging equal to 49.36mg/ml, or 49.36mg/ml of

Fig 4. GC-FID Graph (Chromatogram) Showing Different Constituents of
Bioactive Compounds Identified in Methanolic fraction of Cassia tora leaves. Y
axis (height), X axis (retention time).
antibacterial, and aids in maintaining membrane integrity. According to
Sandeep and Sumit [22], saponins are structurally complex phyto­
chemicals made up of aglycone moieties. Due to the microheterogeneity
of saponins in nature, isolating them from natural sources is typically a
difficult task [23]. For their numerous nutritional purposes and health
advantages, antioxidants originating from food and medicinal plants are
being studied more and more [24]. A variety of diseases are fought off by
antioxidants found in food and medicinal plants [19]. In this study,
2.2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant
power (FRAP), and total antioxidant capacity (TAC) were used to assess
the antioxidant qualities of Cassia tora leaves. The smallest concentra­
tion in 2.2-diphenyl-1-pickrylhydrazyl (DPPH) has the highest scav­
enging power. The extract demonstrates scavenging activity that is
concentration-dependent, with an inhibitory concentration that will
scavenging activity at 50%. Similar to the outcomes of Sirappuselvi and
Chitra’s [25] antioxidant test on Cassia tora leaves, they reported that
the DPPH radicals were reduced in a concentration-dependent manner.
The findings demonstrated that Cassia tora leaves had strong in vitro
antioxidant activity, which may be ascribed to the phenolics found in the
methanolic fraction of Cassia tora leaves. The DPPH scavenging assay is
based on the production of the non-radical form of standard stable free
radicals, which leads to the decrease of stable free radicals in methanol
solution in the presence of hydrogen-donating antioxidants [26]. The
flavonoid and phenolic contents of plant extracts, in particular, are
responsible for the antioxidant properties of such extracts [27]. Ac­
cording to Fig. 2, the fraction in ferric reducing antioxidant power
(FRAP) exhibits antioxidant qualities ranging from 1.78mg/ml to
16mg/ml. In comparison to the standard (ascorbic acid), the fraction
had the maximum antioxidant capacity at 6.86mg/ml. At concentrations
of 4mg/ml and 10.67mg/ml, however, there is no significant difference
in the antioxidant capacity of the fraction and ascorbic acid. In these
data, C. tora shows a trend in activity, which Ahmed et al. [28] also
observed. The fraction in the total antioxidant capacity (TAC), which
ranged in total antioxidant power from 1.78mg/ml to 16mg/ml as
shown in Fig. 3, had the highest antioxidant power when compared to
the reference standard (ascorbic acid), at 6.86mg/ml. However, the
percentage has a considerable antioxidant capacity.
Fig. 4 displays the GC-FID chromatogram with the peaks represent­
ing the numerous bioactive substances that were discovered in the
methanol fraction of Cassia tora leaves. With the use of their peaks and
retention durations, 20 bioactive chemicals were identified and quan­
tified in the fraction as a whole. Steroids and spartein were determined
to be the two substances that were most prevalent in the methanol
fraction of Cassia tora leaves. This is contrary to the findings of Anyebe
et al. [29] on the bioactive compounds identified in Cassia tora leaf. He
reported flavonoids as the most abundant phytochemical. The result of
this study merely indicates that C. tora leaves are abundant in steroid
and alkaloid compounds. Plant alkaloids have been employed as active
ingredients in numerous herbal therapies due to their wide spectrum of
pharmacological effects [30]. The most prevalent substance, spartein, an
alkaloid, has been used as an analgesic, antiarrhythmic, anticholinergic,
stimulant, anticancer, antimalarial, and antihypertensive drug[31].
Spartein has been shown in prior research to have anti-arrhythmic ef­
fects, and lower blood pressure, and heart rate [32]. Epicatechin, pre­
sent in the methanolic fraction of Cassia tora leaves, has been shown to

Table 2
Bioactive Compounds Identified in the Methanolic fraction of Cassia tora leaves using GC-FID.
Phytoconstituents PK RT PkA PkH Concentration % Composition

Proanthocyanin 1 0.116 3681.8254 411.025 5.1784 ppm 3.61%

Naringin 2 2.223 6793.2211 528.999 8.4542µg/ml 5.89%
Cardiac glycoside 3 3.950 8180.0436 637.037 5.0760µg/ml 3.53%
Anthocyanin 4 6.893 4490.5282 350.840 5.7768µg/ml 4.02%
Ribalinidine 5 10.593 4339.0384 337.681 3.7213µg/ml 2.59%
Naringenin 6 13.300 4917.9070 385.129 2.0781µg/ml 1.45%
Sparteine 7 15.783 12,794.3857 919.800 22.9290µg/ml 15.96%
Rutin 8 19.516 7077.0506 566.914 8.7832µg/ml 6.12%
Cyanogenic glycoside 9 19.616 5530.7470 549.868 7.4338 ppm 5.18%
Flavanones 10 22.293 4749.7578 372.508 8.1471ppm 5.67%
Steroids 11 26.000 6830.6364 529.572 11.7164ppm 8.16%
Epicatechin 12 29.493 4459.3978 349.793 6.6901µg/g 4.66%
Phytate 13 33.753 3207.2056 252.910 4.3108µg/ml 3.00%
Flavone 14 34.206 5931.6368 458.604 7.3616µg/ml 5.13%
Oxalate 15 37.260 6523.5508 508.835 10.3036µg/ml 7.17%
Catechin 16 38.326 9393.7324 732.257 2.0623µg/ml 1.44%
Resveratrol 17 39.586 4412.0147 345.669 3.3526 ppm 2.33%
Tannin 18 40.930 3451.2339 270.975 2.0042µg/ml 1.40%
Sapogenin 19 42.086 6000.1311 470.143 9.8605µg/ml 6.87%
Ephedrine 20 42.943 6524.2634 510.968 8.3931µg/ml 5.84%
Total 119,288.3077 143.6331µg/ml

Key: PK = peak; RT = Retention Time (min); PKA = Peak Area (cm2); PKH = Peak Height (ev).

6
C.C. Nkwocha et al.
have anti-inflammatory, antioxidant, anti-diabetic, and anti-cancer
properties [33]. Additionally present in the fraction, flavones have
been shown to interact with proteins and bind to human serum albumin,
facilitating transport mediated by plasma [34]. These bioactive com­
pounds present in Cassia tora leaves may be responsible for their usage in
Chinese medicine for biological activities.
5. Conclusion
This study has demonstrated that the Cassia tora leaf is a rich source
of biologically active substances, particularly phenolics (30,986.70
±46.2µg/ml) and flavonoids (14,682.70±40.3µg/ml), which are sig­
nificant sources of antioxidants for therapeutic use. The TAC, FRAP, and
DPPH activities in plant leaves demonstrate considerable antioxidant
properties as well. Based on their phytochemical makeup and the exis­
tence of bioactive substances with the potential to be used for thera­
peutic purposes, this suggests that the leaves of Cassia tora have specific
pharmacological properties and a variety of biological activities.
5.1. Limitations
This study has some limitations. We did not study the effect of the
methanolic fraction of Cassia tora leaves on some biochemical parame­
ters (antioxidant, anti-diabetic, anti-cancer) to support or confirm its
application for those purposes. Further studies are needed to ascertain
the effect of its fractions on biochemical parameters and its mechanism
of action.
Declaration of Competing Interest
Authors declare no conflict of interests.
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