Journal of Pharmacognosy and Phytochemistry 2025; 14(1): 316-321

E-ISSN: 2278-4136
P-ISSN: 2349-8234
www.phytojournal.com Phytochemical profiling, antioxidant activity and
JPP 2025; 14(1): 316-321
Received: 09-12-2024 Antitumor activity of Eryngium foetidum leaves
Accepted: 13-01-2025
extract against Ehrlich ascites carcinoma (EAC)
Mahmud Ismail
Department of Biochemistry and cell line
Molecular Biology, University of
Rajshahi, Rajshahi, Bangladesh

Tasnima Kamal
Mahmud Ismail, Tasnima Kamal, Azmin Akter and Asma Ul Husna
Department of Biochemistry and Biswas
Molecular Biology, University of
Rajshahi, Rajshahi, Bangladesh DOI: https://doi.org/10.22271/phyto.2025.v14.i1d.15254
Azmin Akter
Department of Biochemistry and Abstract
Molecular Biology, University of Eryngium foetidum is a well-known medicinal plant and its leaf is commonly used as food. This study
Rajshahi, Rajshahi, Bangladesh was conducted to investigate the Phytochemical profiling, antioxidant activity and anticancer activity of
Eryngium foetidum leaves against the Ehrlich Ascites Carcinoma (EAC) cell line in Swiss albino mice.
Asma Ul Husna Biswas The anticancer efficiency of Eryngium foetidum leaves methanolic extract (EFLME) was assessed in
Department of Biochemistry and Swiss albino mice bearing EAC cells, with the extract administered at doses of 25, and 50 mg/kg body
Molecular Biology, University of
weight/day. This study also investigates the total phenolic, total flavonoid, total flavonol and antioxidant
Rajshahi, Rajshahi, Bangladesh
activity by total antioxidant capacity compared to ascorbic acid. We also evaluate the free radical
scavenging assays of EFLME using DPPH and ABTS, with Ascorbic Acid as a standard. Eryngium
foetidum leaves exhibit promising antitumor activity, inhibiting EAC cell growth by 34.27±7.78% and
58.45±4.36% at doses of 25, and 50 mg/kg body weight/day, respectively, compared to control EAC-
bearing mice. The result of the DPPH and ABTS assay indicated notable antioxidant activity. Our work
showed that a variety of bioactive phytonutrients with potent antioxidant activity and growth-inhibiting
effects against EAC cell lines are naturally present in Eryngium foetidum leaves.

Keywords: Phytochemical, antioxidant, anticancer, EAC, Eryngium foetidum

1. Introduction
Oxidative damage is caused by free radicals [1], which are responsible for many diseases such
as atherosclerosis, cancer, inflammatory joint disease, asthma, etc. [2]. The effects of free
radicals are neutralized by antioxidants.
Scientific study shows that Phytochemicals such as polyphenols, flavonoids and carotenoids
are reported as antioxidants, which can prevent or reduce oxidative damage [3, 12]. In addition,
they have antiviral, anti-inflammatory, and anticancer effects [4, 5, 6].
Plants are the natural source of antioxidants, which play a significant role in preventing aging
and several diseases linked to stress, such as lipid peroxidation and active oxygen species [7].
Eryngium foetidum, commonly referred to as Bilati Donia in Bangladesh, is a biennial,
pungently-smelling herb renowned for its medicinal properties in tropical regions [8].
Belonging to the family Apiaceae. The Eryngium foetidum plant serves as traditional
ethnomedicine to treat diabetes, rheumatism, and several respiratory, inflammation and
stomach disorders [13]. In addition to being used in food, it is a crucial component of the
cosmetics and perfumery industries [9]. In international trade marketplaces, essential oils have a
significant economic value [10]. Specifically, Eryngium foetidum leaves are used for Fever, flu,
diabetes, hypertension, constipation diuretic, and anti-convulsant [11, 12]. Moreover, the
previous study reported that the Eryngium foetidum plant has Anthelmintic activity, Anti-
convulsant activity, Antibacterial activity, Antimalarial activity, and Anti-diabetes activity [8],
yet no anticancer activity of E. foetidum against EAC cell lines has been reported. Due to
higher ethnopharmacological activity, depth studies on E. foetidum phytochemical analysis,
Corresponding Author: antioxidant activity and anticancer activity are in demand. In this study, we aimed to
Mahmud Ismail investigate the phytochemical composition, antioxidant activity and anticancer efficiency of
Department of Biochemistry and Eryngium foetidum leaves methanolic extracts.
Molecular Biology, University of
Rajshahi, Rajshahi, Bangladesh

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2. Materials and Methods 2.3.3 Determination of total flavonols content: 3.0 ml (50
2.1 Chemicals and reagents g/L) of sodium acetate solutions and 2.0 ml of 2 percent AlCl 3
Folin-ciocalteu reagent (FCR), Quercetin, Sodium acetate, were added to 0.5 ml of sample/standard. 2.5 hours were
HCl, methanol, chloroform, concentrated sulphuric acid, spent at 20°C measuring the absorbance at 440 nm. A final
Sodium carbonate, Gallic acid, Catechin, Sodium hydroxide, concentration of 1 mg/ml was used to evaluate the extractives
Ascorbic acid, DPPH (2, 2- diphenyl-1-picryl-hydrazyl), and standard. Quercetin equivalent (QU) was used to express
ABTS. (2’-azino-bis (3- ethylbenzthiazoline-6-sulphonic acid, the total flavonoid concentration, with one milligram of QU
were purchased from Sigma, MO, USA. Aluminum chloride per gram of dry extract [17].
and Sodium nitrite were purchased from Carl Roth, Germany.
All other chemicals used in this study were of analytical 2.4 In vitro Antioxidant assay
grade. 2.4.1 Determination of Total Antioxidant Capacity (TAC)
To determine the TAC of samples and standards. A reaction
2.2 Sample collection and preparation mixture of 0.6M sulfuric acid, 28 mM sodium phosphate, and
For this study, Eryngium foetidum leaves were collected from 1% ammonium molybdate was combined with 3 ml of
a local area near the Mymensingh. samples/standard at varying concentrations. The mixture was
The Eryngium foetidum leaves were first cleaned with clean then incubated at 95°C for 10 minutes. After cooling to room
water to get rid of any dirt that stuck to them. Then, the fresh temperature, the absorbance was measured at 695 nm using a
leaves were allowed to dry under the shed. After completing spectrophotometer against a blank. The reference was
full drying, all of the leaves were ground into a coarse powder ascorbic acid. One milliliter of the reaction mixture and the
using a grinding machine (FFC-15, China), and they were corresponding volume of the same solvent used for the
kept for later use in an airtight container. samples/standard made up a typical blank solution. This
The technique described by Aktar, S. et al. 2019 [14] was used solution was then incubated at 95°C for ninety minutes,
to prepare the Eryngium foetidum methanolic leaf extracts. In during which time the absorbance at 695 nm was measured
[18]
short, 333 milliliters of methanol were used to soak 100 .
grams of powdered leaves in a sealed bottle, and the mixture
was shaken for a full day. After the dissolved fraction was 2.4.2 DPPH free radical scavenging assay
separated, the residue was mixed with an additional 100ml of An ascorbic acid reference control was employed. To put it
methanol for 24 hours. At 39°C, the mixture was filtered and briefly, 3 ml of DPPH solution (0.1 mM) in methanol was
allowed to evaporate under low pressure using a rotary combined with 1 ml of different concentrations of extract and
evaporator. ascorbic acid. After giving the reaction mixture a good vortex,
it was allowed to sit at room temperature for half an hour in
2.3 Quantitative analysis of phytochemicals the dark. Finally, a UV spectrophotometer was used to assess
2.3.1 Determination of total phenolics each extract's absorbance at 517 nm. The following formula
300µl of the extracts/standard at various concentrations were was used to determine each sample's free radical scavenging
dissolved in methanol, and 2.25 ml of the Folin-Ciocalteu activity:
reagent (previously diluted with water 1:10, v/v) was added. [{A0 - A}/ A0] × 100 is the DPPH Radical Scavenging Rate
The mixture was then allowed to stand for five minutes at (%).
room temperature before 2.25 ml of 6% sodium carbonate where "A" represented the tested sample’s, ultimate
was added. The tubes were vortexed to initiate the reaction absorbance following a 30-minute incubation period, and
and then let to stand at 25°C for ninety minutes to develop "A0" (Control) represented the absorbance of the DPPH blank
color. Next, absorbance was determined using a UV solution. The concentration which caused a half-maximal
spectrophotometer set at 725 nm. Standard and extract reduced DPPH radical level (IC50) was determined. The
samples were assessed at a final concentration of one percentage (%) of inhibition was plotted against
milligram per milliliter. The gallic acid equivalent, or GAE, concentration, and IC50 was calculated from the nonlinear
was used to express the total phenolic contents [15]. regression [16].

2.3.2 Determination of total flavonoid content 2.4.3 ABTS free radical scavenging assay
In a test tube, 500µl of the extract and 2250µl of distilled The ABTS scavenging efficacy of the extract was assessed
water were combined. 0.150µl of a 5percent NaNO 2 solution using Re et al. (1995) with certain modifications. To create
was then added, and the mixture was allowed to sit at room the radical cation, combine 2.45 mM potassium persulfate
temperature for six minutes. Subsequently, 1.0 ml of 1M (1/1, v/v) with 7 mM ABTS stock solution. Allow the mixture
NaOH was added after 300µl of a 10% AlCl3.6H2O solution to sit for 4-16 hours, or until the reaction was finished and the
had been added and left to stand for an additional 5 minutes. absorbance remained stable. Next, 3.0 ml of ABTS+ solution
Next, a vortex was used to thoroughly mix the material. At was combined with 1 ml of sample at different concentrations.
510 nm, the absorbance was promptly measured with a A positive control was employed, which was ascorbic acid
[19]
spectrophotometer. The results were determined and reported . The following formula was used to get the scavenging
as milligrams of catechin equivalents (mg CAE/gm) for each rate: ABTS Radical Scavenging Rate (percent) = [{A0-A}/
gram of dried sample [16]. A0] × 100
where A was the tested sample's absorbance after six minutes
For the plant samples, the total flavonoid content was of incubation, and A0 (control) was the absorbance of the
calculated using the formula: (C x V) ÷ m, where C is the ABTS blank solution.
catechin concentration from the calibration curve in mg/ml, V
is the volume of plant extract in ml, and m is the weight of 2.5 In vivo antitumor activity
dried plant extract in grams. 2.5.1 Test animals: Swiss albino mice 5-7 weeks old,
weighing 23-25 grams were collected from the laboratory of
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Molecular Oncology at the Biochemistry and Molecular Eryngium foetidum had considerable antioxidant activity,
Biology department, University of Rajshahi, Rajshahi-6205, which is concentration-dependent.
Bangladesh.
3.2.1 DPPH scavenging activity
2.5.2 Cell line The inhibition efficacy of DPPH free radical scavenging
Transplantable tumor cells (Ehrlich Ascites Carcinoma) were activity was found to be dose-dependent for Ascorbic acid
used in this study for searching the antineoplastic activity of and EFLME. The IC50 values were determined to be
the test sample. The EAC cells were first provided kindly by 40.049±0.69 µg/ml for EFLME, and 13±1.25 µg/ml for
the Indian Institute of Chemical Biology (IICB), located in ascorbic acid. Compared to ascorbic acid, the IC50 value was
Kolkata, India. greater. standard. At 400 µg/ml, ascorbic acid demonstrated
around 95% inhibition, while EFLME displayed
2.5.3 Determination of cell growth inhibition approximately 89% inhibition at the same dosage. The
In vivo, tumor growth inhibition was assessed using the percentage inhibitions of EFLME are presented in Figure 2
method described by Perveen, R. et. Al. 2012 [20]. To and the IC50 value is shown in Table 2.
determine the cell growth inhibition of the test compound,
2×106 EAC cells/ml were inoculated into three groups 3.2.2 ABTS scavenging activity
(control, 25mg/kg body weight and 50mg/kg body weight) of Ascorbic acid and EFLME were found to have dose-
mice (5 in each group) on day ‘’0’’. Treatment was started dependent suppression of the ABTS free radical scavenging
after 24 hours of EAC cell inoculation and continued for five activity. The IC50 values were determined to be 67.97±5.59
days, where group 1 used as a control received no doses. µg/ml for EFLME, and 28.83±1.26 µg/ml for ascorbic acid.
Groups 2 and 3 received the test compound into the Compared to the Ascorbic acid the IC50 value was higher. At
intraperitoneal cavity using a 1 ml syringe at the doses of a concentration of 80 µg/ml, ascorbic acid demonstrated
25mg/kg body weight and 50mg/kg body weight of mice. approximately 82% inhibition, while EFLME demonstrated
Mice in each group were sacrificed on day six. 5 ml of normal approximately 51% inhibition at the same dose. The
saline (0.98% NaCl solution) was injected into the percentage inhibitions of both are presented in Figure 2 and
intraperitoneal cavity using a 5 ml syringe and tumor cells the IC50 value is shown in Table 2.
were harvested using a 3 ml syringe to draw peritoneal fluid.
The peritoneal fluid was diluted ten times with normal saline 3.4 Cell Growth Inhibition
solution and trypan blue dye (0.4%) and then counted by a Treatment with Eryngium foetidum leaves methanolic extract
hemocytometer. The total number of viable cells in every resulted in dose-dependent inhibition of Ehrlich ascites
group of mice was counted and compared with the control carcinoma (EAC) cell growth in mice. On day 6 after tumor
(EAC bearing only) group. The mice grouping was as inoculation, the number of EAC cells in the control group was
follows: Eight groups of EAC tumor-bearing mice were used- (4.972±0.905) ×107.
Group 1 (Control EAC): Untreated, Group 2: Treated with In the EFLME treatment group, the number of EAC cells was
EFLME 25 mg/kg dose/day, Group 3: Treated with EFLME significantly reduced to (3.268±0.39) ×10 7 (p< 0.01), and
50 mg/kg dose respectively (2.066±0.22) ×107 (p< 0.001) at doses of 25, and 50 mg/kg
The cell growth inhibition was calculated using the following body weight, respectively shown in Figure 3 and Table 3.
formula: Treatment with EFLME resulted in dose-dependent inhibition
of EAC cell growth. EFLME exerted growth inhibition of
% Cell growth inhibition = (Cm - Tm / Cm) x 10 34.27±7.78%, and 58.45±4.36%, respectively, at doses of 25
and 50 mg/kg body weight shown in Figure 4 and Table 3.
Where, We also study the morphology of EAC cells for both the
Cm = Mean of the number of tumor cells of the six mice of treatment group and control group by optical microscope. The
the control group. treatment group showed higher apoptosis and lesser
Tm = Mean of the number of tumor cells of the six mice of proliferation compared to the control group which is shown in
the treated group. Figure 5.

3. Results 4. Discussion
3.1 Quantitative Analysis of Phytochemicals Despite having a high mortality rate, cancer is one of the
Total phenolics, total flavonoids and total flavonol contents of deadliest diseases in the world, and current treatment options
EFLME were determined 78.80± 12.08 mg/g of dry extract as do not adequately address this problem [21]. As a result,
GAE, 202.26± 2.17mg/g of dry extract as CAE and 307.09± researchers and drug manufacturers are always looking for
3.40 mg/g of the dry extracts as QUE, respectively shown in safer natural substances to treat cancer. The anticancer
Table 1. The standard curve of gallic acid, catechin and properties of our present experimental plant, E. foetidum,
quercetin is shown in Figure 1. were demonstrated by a variety of bioassays, including
antioxidant activity, phytochemical analysis, and cell growth
3.2 In vitro Antioxidant activity inhibition assay. Our morphological studies of EAC cells also
3.3.3. Determination of total antioxidant capacity suggest that the leaf extracts of E. foetidum can inhibit EAC
The total antioxidant activity of EFLME was assessed by the cell growth.
phosphomolybdenum method using Ascorbic acid (A.A) as It was established that phytochemicals are responsible for
standard. The total antioxidant activity of Eryngium foetidum antioxidant activity [22], Antioxidants can reduce free radicals
and standard ascorbic acid are shown in Figure 2. Ascorbic (oxidative stress) which cause tumors [23]. The presence of
acid showed an absorbance of 0.88± 0.04 at a concentration of antioxidant activity in an extract or particular chemical
400 µl/ml, whereas EFLME showed 0.44± 0.03 at the same demonstrates anticancer action through a distinct mechanism
[24]
concentration. Our results demonstrated that extracts of . In the previous study, researchers found several
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potentialities of E. foetidum leaf such as Anthelmintic According to a prior study, phytochemicals cause the P 53 gene
activity, Anti-convulsant activity, Antibacterial activity, to become overexpressed, which can damage DNA in cancer
Antimalarial activity, and Anti-diabetes activity [8], however, cells [27]. In our study, strong correlations have been observed
no anticancer activity of E. foetidum against EAC cell lines between the outcomes of phytochemical analysis, antioxidant
has been reported. According to our findings, E. foetidum leaf activity, cell growth inhibition, and cellular morphology
methanolic extracts exhibit strong antioxidant activity. observation. These correlations provide strong evidence for
The plant extracts effectively inhibited the development of the modification of genetic construction and apoptotic death
EAC cells because they exhibited strong antioxidant activity. of EAC cells treated with E. foetidum leaf extracts. We
Globally, EAC cells are employed as experimental tumor therefore firmly believe that the different phytocompounds
models for cancer research [25]. The EFLME exhibited present in the E. foetidum leaves methanolic extract have the
satisfactory cell growth inhibition. The EFLME not only potential to be anticancer agents. Additional research is
inhibited the EAC cell's growth but also represented required to identify the bioactive substances that stop EAC
morphological features of program cell death (apoptosis). cell proliferation will be extremely beneficial for current
While control mice displayed normal, round-shaped cells and medicine.
a regular nucleus in optical microscopy, optical microscopy of
EAC cells from mice treated with EFLME revealed Table 1: Phytochemical contents of EFLME
significant morphological changes, including cell membrane Total phenolic 78.80± 12.08mg of GAE/g a
blebbing, cell shrinkage, chromosomal condensation, and Total flavonoids 202.26± 2.17mg of CAE/g
nuclear fragmentation in the nucleus (shown in Figure 5). The Total flavonol 307.09± 3.40 mg of QUE/g
morphological changes observed in EAC cells are indicative GAE: Gallic acid equivalent; CAE: catechin equivalent; QUE:
of apoptosis, which is the body's usual mechanism for quercetin equivalent
a Data were expressed as mean ± standard deviation (SD). (n=3)
eliminating unnecessary and inappropriate cells through a
sequence of events that spare healthy cells. The absence of the
apoptotic mechanism is responsible for the growth of tumors, Table 2: Free radicals scavenging activity of EFLME and Ascorbic
which in turn causes aberrant cell division and the acid in different assays
development of cancer [26]. This experiment is considered to IC50 values a (μg/ml)
be an effective indicator for any type of cancer therapy and Experiment ABTS DPPH
prohibition because we observed nuclear shrinkage, EFLME 67.97±5.59 40.05±0.70
chromatin condensation, and the development of apoptotic Ascorbic Acid 28.83±1.26 13±1.25
bodies in EAC cells of the treatment group (shown in Figure a IC50 value: Effective concentration required for 50% of a

5). maximum inhibition

Table 3: Effect of the EFLME on EAC cell growth inhibition (in vivo)
Nature of Number of EAC cells in mice on % of cell growth
Name of Exp. Dose in mg/kg/day (i.p.)
the Drug day 6 after tumor cell inoculation inhibition
Control (EAC cell-bearing mice) - - (4.972±0.905) ×107 ------
25 (3.268±0.39) ×107 ** 34.27±7.78
EFLME Plant Extract
50 (2.066±0.22) ×107 *** 58.45±4.36
The number of mice in each case (n=5); the results were shown as mean ± SD. Where significant values are *p<0.05, **p<0.01 and ***p<0.001

Fig 1: Standard curve of gallic acid(A), catechin(B) and quercetin(C) for the determination of total phenol, total flavonoid and total flavonol

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Fig 2: Total antioxidant capacity (A), Determination of DPPH(B) and ABTS(C) free radical scavenging activity of Ascorbic Acid and EFLME.

Fig 5: The control (A) and EFLME-treated (B) cells are shown in the
optical microscopic view, respectively. When examined under an
optical microscope, the Control group's nucleuses were round and
normal-looking, and arrows indicated the apoptotic properties of
cells treated by EFLME

5. Abbreviations
℃: Celsius degree; EAC: Ehrlich Ascites Carcinoma;
EFLME E. foetidum leaves methanolic extract; DPPH: 1,1-
diphenyl-2-picrylhydrazyl; ABTS: 2,2'-azino-bis-3-
ethylbenzthiazoline-6-sulphonic acid; SD: Standard
Deviation; GAE: gallic acid equivalent; CAE: catechin
Fig 3: Number of viable EAC cells in mice on day 6 after tumor cell equivalent; QUE: quercetin equivalent
inoculation
6. Acknowledgements
Not Applicable.

7. Competing interests
The authors declare that they have no competing interests.

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