SJA_41_5_300-314

Introduction

Malaysia’s diverse flora has long been central to indigenous medicinal practices, reflecting the region’s cultural and ecological richness (Tan et al., 2020). Among these botanical resources, Eryngium foetidum L., commonly known as ‘Ketumbar Jawa’ or spiny coriander, has drawn considerable attention for its dual role in culinary and therapeutic applications. Belonging to the Apiaceae family, the plant is widely used in Southeast Asia and Latin America, where its serrated leaves and pungent aroma are valued in cuisine, while its medicinal properties have been traditionally employed to relieve digestive disorders, fevers, and inflammatory conditions (Hemachandra et al., 2021). Despite the benefits of medicinal plants, heavy metal contamination in soil poses a quality risk that bioremediation can help to address (Al-Jaberi, 2024).

Botanically, E. foetidum is a perennial herb that typically grows up to 30–40 cm in height, characterized by a basal rosette of elongated, lanceolate leaves with spiny-toothed margins. The leaves, measuring 10–20 cm long, are leathery in texture and emit a strong coriander-like odor when crushed. The plant produces small white to greenish flowers arranged in dense, conical umbels surrounded by spiny bracts, which are distinctive morphological features of the genus (Rodrigues et al., 2022; Hemachandra et al., 2021). It thrives in moist, shaded habitats but can also be cultivated under tropical agricultural conditions, making it accessible both as a culinary herb and a source of medicinal compounds (Rodrigues et al., 2022).

The pharmacological potential of E. foetidum is closely linked to its diverse phytochemical composition, which includes essential oils, flavonoids, tannins, alkaloids, and phenolic acids (Rodrigues et al., 2022; Leitão et al., 2023). In particular, polyphenols and flavonoids have been associated with antioxidant, antimicrobial, and anticancer activities (Rodrigues et al., 2022). Antioxidants mitigate oxidative stress by neutralizing free radicals, thereby reducing the risk of chronic diseases, while antiproliferative and antimicrobial activities position E. foetidum as a candidate for cancer prevention and infectious disease management (Sruthi and Danya, 2021).

Despite these promising findings, most previous studies have been limited to single extraction methods or narrow sets of bioassays. For instance, Hernández-López et al. (2023) examined the ethnobotany, phytochemistry, and biological activities of Eryngium species in Mexico but did not evaluate how post-harvest processing influences its bioactivity. Furthermore, little is known about the combined influence of solvent type and drying temperature on the phytochemical profile and biological activity of E. foetidum. Since extraction solvents such as ethanol and methanol can yield different classes and concentrations of compounds (Sasidharan et al., 2010; Ndiaye et al., 2024), while drying conditions strongly affect the stability of bioactive metabolites (Gąsecka et al., 2020). The study of baobab seed oil revealed that different extraction processes modified its chemical composition and reduced sensitive antioxidants, demonstrating the impact of processing on bioactive compounds (Ndiaye et al., 2024). Similarly, in E. foetidum, drying and extraction methods are critical, necessitating a systematic approach to identify the best conditions for preserving phytochemicals.

To address this gap, the present study investigates the effects of drying temperature (40 °C and 60 °C) and extraction solvent (ethanol and methanol) on the phytochemical composition and biological activities of Malaysian-grown E. foetidum leaves. By assessing antioxidant, antiproliferative, and antimicrobial properties in a unified experimental framework, this study provides new insights into the therapeutic potential of E. foetidum and supports its future development as a source of herbal remedies.

Material and Methods

Extraction of E. foetidum leaves

The fresh mature leaves of E. foetidum were collected from Kampung Amir, Alor Lintang, 22200 Besut, Terengganu (5°46’36.6”N 102°32’08.1” E). The fresh and healthy leaves of E. foetidum were washed with running tap water to clean all the dirt on the surface of the leaves and rinsed with tap water. The leaves were chopped into smaller pieces and divided into two group of samples that dried in different drying temperature (40°C and 60°C) for 72 hours in incubator. The dried samples of each temperature were ground into a coarse powder by using a blender. Then, the samples of each temperature were soaked with ethanol (EtOH) in a conical flask and stirred. The mixture was left for three days. The samples were stirred with a spoon daily. After three days, the extracts were filtered using filter paper. The filtered samples were concentrated by using rotary evaporator to obtain the ethanol crude extracts with different drying temperature and then stored at 4˚C. The process was repeated by using methanol (MeOH) as extraction solvent to obtain methanol crude extracts with different drying temperature. At the end of the extraction process, four E. foetidum leaf extracts with different combination of drying temperature and extraction solvent were obtained as follow: Ethanol extraction at 40°C (EtOH:40°C), Ethanol extraction at 60°C (EtOH:60°C), Methanol extraction at 40°C (MeOH:40°C), and Methanol extraction at 60°C (MeOH:60°C).

Total phenolic content (TPC)

The TPC of the E. foetidum leaf extract were determined using Folin-Ciocalteu’s colourimetric technique (Zin et al., 2018). A stock solution of gallic acid of 1 mg/mL was prepared by dissolving gallic acid in methanol. The standard solution was prepared at six different concentrations: 0, 20, 40, 60, 80 and 100 µg/mL. A total volume of 60 µL of sample or standard was added into an Eppendorf tube, followed by 40 µL of methanol. Approximately 200 μL of Follin Ciocalteu reagent was added and vortexed thoroughly. Then, 800 µL of 7.5 % sodium carbonate (Na2CO3) was added and incubated for 2 hours at room temperature. About 200 µL of the prepared mixture was transferred into a 96-well plate. The absorbance was measured using a microplate reader at 765 nm. Three measurements were taken. The gallic acid standard curve was used to calculate the total phenolic content using the following formula, where C is the total phenolic content (mg of GAE / g of plant extract), c is the concentration of gallic acid (mg/mL) established from the standard curve, V is final volume of plant extract, and m is the weight of the plant extract. The results were measured in milligrams of gallic acid equivalent per gram of extract (mg GAE/g).

C=cV/m

Total flavonoid content (TFC)

The TFC of E. foetidum leaves extract was determined by using a previously reported (Zin et al., 2018). A stock solution of quercetin at a concentration of 1 mg/mL was prepared by dissolving it in methanol. Subsequently, the stock solution was used to prepare standard solution at six different concentrations: 0, 20, 40, 60, 80 and 100 µg/mL. Then, a mixture was prepared by combining 140 µL of sample extracts or standard, 260 µL methanol, 150 µL 1 M potassium acetate, and 150 µL 10% aluminium chloride. At room temperature, the solution was incubated for 30 minutes. About 200 µL of prepared mixture were transferred into 96 well plate. The absorbance was measured using microplate reader at 415 nm. Three measurements were taken. The quercetin standard curve was used to calculate the total flavonoid content using the following formula, where C is the total flavonoid content (mg of QE / g of plant extract), c is the concentration of quercetin (mg/mL) from the standard curve, V is the final volume of plant extract, and m is the weight of the plant extract. The results were given in milligrams of quercetin equivalents per gram (mg QE/g) of samples extract.

C=cV/m

2,2-Diphenyl-1-Picry-Hydrazyl-Hydrate (DPPH) Assay

The previously reported method was adopted (Athilah et al., 2020). A stock solution of the extract was prepared at a concentration of 5 mg/mL in DMSO. A blank solution comprising DMSO and DPPH was made as a control. Ascorbic acid was served as the standard. In 96-well plates, 150 µL of 0.1 mM DPPH working solution were added to the 50 µL sample or standard of different concentrations: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The mixture was thoroughly mixed and left in the dark for 30 minutes. A microplate reader was used to measure the change in absorbency at 517 nm. Three measurements were taken. The percentage of DPPH scavenging activity was calculated as follows where, Ac = absorbance of the control and As = absorbance of the sample.

Inhibition (%) = [(As – Ac) / As] × 100

Ferric reducing antioxidant power (FRAP) assay

FRAP assay was determined according to past study method (Athilah et al., 2020). A stock solution of the extract was prepared at a concentration of 5 mg/mL in DMSO. A blank solution comprising DMSO and FRAP working solution was made as a control. Ascorbic acid was served as the standard. First solution which is 0.3 M acetate buffer pH 3.6 was prepared by combining 0.16 g sodium acetate and 100 mL 0.28 M glacial acetic acid. The solution pH was adjusted with 1 M NaOH or 1 M HCl. Second solution which is 0.01 M of TPTZ solution was prepared by mixing 0.31 g of TPTZ in 100 mL of 40 mM HCl. For 0.02 M ferric (III) chloride (solution 3) was prepared by diluting 0.32 FeCl3 with 100 mL distilled water. To make FRAP reagent, solution 1, solution 2 and solution 3 in 10:1:1 ratio was combined and heated in a water bath at 37°C for 30 minutes. 150 µL FRAP reagent were combined with 50 µL sample extract or standard with different concentrations of 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The absorbance was measured at 593 nm immediately using microplate reader. The percentage of inhibition was calculated with the following equation where As = absorbance of the sample and Ac = absorbance of the control.

Inhibition (%) = [(As – Ac) / As] × 100

2,2’-azino-bis (3- ethylbenzthiazolin-6-sulfonic acid) (ABTS) assay

ABTS radical scavenging activity was determined according to the past study method (Asraoui et al., 2021). A stock solution of the extract was prepared at a concentration of 5 mg/mL in DMSO. A blank solution comprising DMSO and ABTS working solution was made as a control. Ascorbic acid was served as the standard. The ABTS stock solution was prepared by reacting 7 mM ABTS aqueous solution with 2.45 mM aqueous solution of potassium persulfate (K2S2O8) and allowed the mixture to react in the dark at room temperature for 12–16 h. Then the ABTS+ stock solution was adjusted with methanol to obtain an initial absorbance value of 0.7 ± 0.02 at 738 nm. Approximately 50 µL of the extract sample was mixed with 150 µL of ABTS+ diluted solution, and the mixture was incubated for 6 min. A microplate reader was used to measure the change in absorbance at 738 nm. The percentage of inhibition was calculated according to the following equation, where: Ac is the absorbance of the control, and As is the absorbance of the sample.

Inhibition (%) = [(Ac – As)/Ac] × 100

Discuss diffusion method

Disc diffusion method was determined according to previously reported method (Dutta et al., 2017). The bacterial strains used (Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922) were obtained from the Microbiology Laboratory, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin (UniSZA). S. aureus and E. coli suspension cultures were prepared in MH broth and adjusted to 0.5 McFarland turbidity standard. The surface of the MH agar plate was streaked with the bacterial inoculum. Sterile filter paper discs, with diameter 6 mm were placed on the MH agar surface using sterile forceps and then impregnated with 20 µL of the E. foetidum leaf extracts with five different concentrations (20, 40, 60, 80, 100 mg/mL). Disc impregnated with DMSO was used as negative control. For positive control, antibiotic Gentamicin and Ciprofloxacin were used. The plates were incubated at the 37°C for 24 hours. The diameter of the clear zones of inhibition around each disc was measured.

Cell culture

The human breast adenocarcinoma cell line (MCF-7) was used for this study and provided by the UniSZA. The cells were grown in a T25 flask supplied with Roswell Park Memorial Institute (RPMI) growth media, containing 10% fetal bovine serum (FBS). The cells were incubated at 37°C in a 5% CO2 incubator. The cells were grown until confluence and subculture at 2 to 3 days intervals.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

The effects of the E. foetidum leaf extracts on cell proliferation were measured by MTT assay based on the ability of viable cells to convert tetrazolium salt into purple formazan. Briefly, 100 µL of 2 × 105 cells/mL of cells were seeded into 96-well plates overnight. The cells were then treated with 100 µL of different concentrations E. foetidum leaf extracts ranging from 0 to 2000 µg/mL. The cells also were treated with different concentration of positive control Etoposide ranging from 0 to 100 µg/mL. The treated cells were incubated in an incubator at 37°C for 72 hours in a 5% CO2 incubator. After incubation time, 20 µL of 5 mg/mL MTT reagent were added and incubated for 4 hours in the dark to allow the mitochondrial dehydrogenases of viable cells cleaved the tetrazolium ring and yield purple formazan crystal which is insoluble in an aqueous solution. After 4 hours incubation time, the half of the media was removed and 100 µL of DMSO were added to the wells before incubating for another 10 minutes. The absorbance was measured at 570 nm using a microplate reader. The half-maximal inhibitory concentration (IC50) value was computed from a graph of percentage cell viability versus the concentration of the extracted sample with the help of software GraphPad Prism 9.

Morphological observation

The cells were seeded in a 12-well plate at a concentration of 2 × 105 cells/mL and incubated overnight in a humidified 5% CO2 incubator at 37°C. The cells were than treated at moderate dose (IC50) of E. foetidum leaf extracts or control Etoposide before being incubated at 24, 48, and 72 hours. The untreated cells were used as negative control for comparison. The morphological changes of the treated and untreated cells were be observed using a phase contrast microscope.

Statistical analysis

The statistical analysis of data was performed by using software GraphPad Prism version 9. All experiments were performed in triplicate and each data represents mean ± standard deviation (SD). Comparisons of the group were done using one-way or two-way Analysis of Variance (ANOVA), followed by the Tukey post-test for multiple comparisons (* p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; ns for no significant). One-way ANOVA analysis was performed for data of TPC, TFC, DPPH, FRAP, ABTS and MTT assay, meanwhile two-way ANOVA was performed for data of antimicrobial assessment.

Results and Discussion

Total phenolic content (TPC) and total flavonoid content (TFC) of E. foetidum leaf extracts

The evaluation of biological activities of E. foetidum leaf extracts often involves assessing the total phenolic and flavonoid content. Based on Figure 1, the highest TPC was recorded in MeOH:40°C (51.23 mg

GAE/g), which was significantly higher (p < 0.05) than EtOH:40°C (46.12 mg GAE/g) and both 60°C extracts. Similarly, TFC was highest in MeOH:40°C (32.45 mg QE/g), followed by EtOH:40°C (29.88 mg QE/g), with both values significantly higher (p < 0.05) than the 60°C treatments. At lower drying temperature (40°C), EtOH extract shows higher TPC & TFC values than MeOH extract. The choice of solvent can significantly affect the TPC and TFC in plant extracts. Different solvents have varying polarities and extraction efficiencies, which can influence the extraction of phenolic and flavonoid compounds. The effectiveness of ethanolic extract may be attributed to its ability to extract the specific flavonoids and phenolic compounds present in E. foetidum (Dirar et al., 2019). Within the same extraction solvent, EtOH:40°C extract has more TPC & TFC values than EtOH:60°C extract. The results are also similar with methanol solvent, where MeOH:40°C extract has more TPC & TFC values than MeOH:60°C extract. This is because elevated drying temperatures may lead to the degradation of heat-sensitive compounds, contributing to a reduction in both TPC and TFC (ElGamal et al., 2023).

In this study, the E. foetidum leaf dried at 40°C drying temperature and extracted using ethanol appears to be the most effective combination for obtaining extracts with high phenolic and flavonoid concentrations. Several studies have identified the presence of various phenolic compounds in E. foetidum leaf extracts. One study found that the extract exhibited a high concentration of phenolic compounds, including total tannins and total flavonoids (Thi et al., 2020). Another study identified six phenolic compounds in E. foetidum leaf extracts, with chlorogenic acid being the major compound (Leitão et al., 2023). Additionally, the study mentioned that the mixture of water and ethanol yielded the highest contents of flavonols in the extract, represented by quercetin glucuronide (Leitão et al., 2023).

Antioxidant activity of e. foetidum leaf extracts

Antioxidant activity refers to the ability of a substance to neutralize or inhibit the oxidation of other molecules, thereby preventing or reducing the damage caused by free radicals and reactive oxygen species (ROS) (Leitão et al., 2023). E. foetidum, an aromatic and medicinal herb, has been found to exhibit strong antioxidant activity in its essential oils and leaf extracts (Thi et al., 2020). In this study, DPPH, ABTS and FRAP assays were performed to measure the antioxidant activity of E. foetidum leaf extracts.

According to Figure 2, the IC50 results for all antioxidant assays followed a pattern consistent with the phytochemical data. Extracts obtained with methanol demonstrated significantly stronger radical scavenging and reducing activity compared to ethanol extracts. Among drying conditions, 40 °C extracts exhibited higher antioxidant activity than those dried at 60 °C across all assays. Standard ascorbic acid had the lowest IC50 value than all E. foetidum leaf extracts. The lower the IC50 value, the higher the antioxidant activity of the tested sample. The MeOH:40°C extract exhibited the strongest antioxidant activity with IC₅₀ values of 542 µg/mL (DPPH), 72.75 µg/mL (FRAP), and 322.8 µg/mL (ABTS), all significantly lower (p < 0.05) than other extracts, indicating higher antioxidant potential. The results indicate that the MeOH:40°C exhibited the lowest IC50 value among all the extracts followed by EtOH: 40°C. On the other hand, the MeOH: 60°C showed the highest IC50 value, indicating relatively lower antioxidant activity compared to the other extracts. This suggests that the antioxidant activity of E. foetidum is influenced by the choice of solvent and extraction temperature, with the 40°C condition showing higher efficacy compared to 60°C. Ethanol and methanol are commonly used solvents due to their ability to extract a broad spectrum of phytochemicals with antioxidant properties. Methanol extracts generally exhibit superior antioxidant activity compared to ethanol extracts (Dirar et al., 2019). But according to DPPH and FRAP results, MeOH and EtOH extract at 40°C temperature shows no significant different of antioxidant scavenging activity. This indicates that both solvent at 40°C are equally effective in extracting antioxidant compounds E. foetidum plant.

The influence of drying temperature on the antioxidant activity of plant extracts has also been investigated in previous studies. Temperature can affect the stability of bioactive compounds, potentially leading to the degradation of antioxidant molecules. In this study, the lower IC50 value of MeOH and EtOH at 40°C may be due to the preservation of heat-sensitive antioxidants at lower temperatures (Hernández-Bolio et al., 2019). Elevated drying temperatures may lead to the degradation of heat-sensitive compounds, contributing to a reduction in both TPC and TFC

(ElGamal et al., 2023). Likewise, alcoholic extracts of Artemisia absinthium demonstrated strong bioactivity due to the retention of phenolic antioxidants, emphasizing the critical role of solvent and temperature in preserving antioxidant potential in medicinal plants (Alani and Kawan, 2024). Compared with the standard ascorbic acid, it is evident that the leaf extracts of E. foetidum possess antioxidant activity, as indicated by the IC50 values. However, the standard ascorbic acid exhibited a significantly lower IC50 value, emphasizing its stronger antioxidant potential compared to the E. foetidum extracts.

Antimicrobial Activity of E. foetidum Leaf Extracts

The disc diffusion method was used in this study to evaluate the antimicrobial activity of E. foetidum leaf extracts against Staphylococcus aureus and Escherichia coli. The disc diffusion method is a widely employed technique for assessing the antimicrobial susceptibility of microorganisms to various substances, including plant extracts. Based on the antimicrobial results in Figure 3, all E. foetidum extracts displayed significant antimicrobial activity against S. aureus and no notable antimicrobial activity was observed against E. coli. According to Figure 3(A), the EtOH:40°C extract exhibited a zone of inhibition of 15.2 mm, which was significantly higher (p < 0.05) than the EtOH:60°C extract (13.6 mm) at a concentration of 40 mg/mL. Similarly, the MeOH:40°C extract showed a zone of inhibition of 14.8 mm, significantly higher (p < 0.05) than the MeOH:60°C extract (12.9 mm) at a concentration of 20 mg/mL. Lower drying temperatures (40°C) likely preserve more bioactive compounds responsible for antimicrobial activity. Heat-sensitive antimicrobial compounds may degrade at higher temperatures, resulting in reduced effectiveness (Hernández-Bolio et al., 2019). The treatment with fenugreek seed extract led to increased phenolic accumulation and biological effectiveness in date palm seedlings, thereby providing additional support for the critical role of extraction and processing conditions in preserving phytochemical bioactivity (Hamza and Almansour, 2024).

Within the same temperature, EtOH and MeOH extract did not shows any significant difference (p > 0.05) in inhibiting the growth of S. aureus. As the concentration of extracts increased, all E. foetidum extracts also showed no significant difference (p > 0.05) between each other, indicating that different solvents and drying temperatures do not affect the antimicrobial activity of extracts against S. aureus. The antimicrobial activity of E. foetidum against S. aureus has been reported in several studies. The ethyl acetate extract of E. foetidum showed strong antimicrobial activity, and the extract from E. foetidum leaves exhibited an average of 23 mm zone inhibition against S. aureus (Lingaraju et al., 2016). Additionally, the methanolic extract of E. foetidum showed high effective antimicrobial activity against S. aureus (Dalukdeniya and Rathnayaka, 2017; Parham et al., 2020). The absence of inhibitory effects of E. foetidum leaf extracts on E. coli suggests a specific antimicrobial activity against Gram-positive bacteria, such as S. aureus, rather than a broad-spectrum effect. This differential response could be attributed to variations in the cell wall structure and composition between Gram-positive and Gram-negative bacteria. Based on past study, a study on the antibacterial activity of aqueous and methanol extracts of E. foetidum showed mild to moderate antibacterial activity against S. aureus, but no activity against E. coli was observed (Dutta et al., 2017). However, another study reported that the extract from E. foetidum leaves exhibited an average of 20 mm zone inhibition against E. coli and an average of 23 mm zone inhibition against S. aureus (Menchavez et al., 2018).

Antiproliferative activity of E. foetidum leaf extracts against human breast adenocarcinoma cells line (MCF-7)

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to evaluate the effect of E. foetidum leaf extracts against human breast adenocarcinoma cells line (MCF-7). MTT assay is a standard calorimetric assay that used to measure the activity of enzyme That reduced MTT to formazan after cells being treated with a plant extract. Figure 4 displays the IC₅₀ values, indicating the concentration of E. foetidum leaf extracts or etoposide required to reduce the cell population by 50%, while Table 1 provides a summary of these IC₅₀ values for the different treatments. The order of the IC50 value for each sample is as follow: Etoposide (4.6 μg/mL) < EtOH:40°C (394.6 μg/mL) < MeOH:40°C (421.3 μg/mL) < EtOH:60°C (487.2 μg/mL) < MeOH:60°C (502.7 μg/mL). Comparison between E. foetidum leaf extracts shows that the EtOH: 40°C extract exhibits the lowest IC50 value at 394.6 ± 109.80 μg/mL, indicating a relatively higher antiproliferative activity compared to other extraction conditions. However, there is no significant difference (p > 0.05) between EtOH:40°C and MeOH:40°C, indicating that both solvents at 40°C are equally effective in inhibiting MCF-7 cell proliferation. In contrast, the EtOH:60°C and MeOH:60°C extracts demonstrate higher IC50 values (p < 0.05), suggesting a reduced cytotoxic effect on MCF-7 cells under these conditions. The observed variations in IC50 values among different extraction conditions may be attributed to the distinct composition of bioactive compounds extracted under varying solvent types and temperatures. For both EtOH and MeOH extracts, the 40°C condition has a lower IC50 than the 60°C condition, indicating that a lower drying temperature

is more effective in inhibiting MCF-7 cell proliferation. Lower drying temperatures (40°C) appear to be more favourable for preserving bioactive compounds that exhibit cytotoxicity against MCF-7 cells. Conversely, the higher IC50 values for the EtOH: 60°C and MeOH: 60°C extracts may indicate a suboptimal extraction of bioactive compounds or alterations in their composition under prolonged exposure to elevated temperatures. Elevated drying temperatures may lead to the degradation of heat-sensitive compounds, contributing to a reduction in biological activities, including antiproliferative activity (ElGamal et al., 2023). The Etoposide control, a standard chemotherapeutic agent, exhibits a remarkably low IC50 value of 4.6 ± 0.38 μg/mL, reaffirming its potent antiproliferative effect as a positive control. The comparison with Etoposide underscores the potential of E. foetidum leaf extracts to influence MCF-7 cell viability, although at higher concentrations. Several research have been conducted to test the antiproliferative activity of E. foetidum and its constituents. For example, in 2021, Sruthi and Danya studied the cytotoxic activity of the methanolic extract of E. foetidum leaves and discovered that it demonstrated considerable cytotoxicity against MCF-7 (Sruthi and Danya, 2021). Another study established the anticancer potential of E. foetidum essential oil against human prostate adenocarcinoma cell line (PC-3) (Chandrika et al., 2016). The E. foetidum leaf extract exhibited antitumor activity against Ehrlich Ascites Carcinoma in mice, demonstrating therapeutic relevance both in vitro and in vivo (Ismail et al., 2025).

Table 1: The IC50 of E. foetidum leaf extracts and etoposide for MTT assay. Data are represented as mean ± SD, n = 3.

Sample	IC50 (μg/mL)
EtOH:40°C	394.6 ± 109.80
EtOH:60°C	1079.0 ± 85.91
MeOH:40°C	554.6 ± 112.90
MeOH:60°C	1462.0 ± 27.44
Etoposide control	4.6 ± 0.38

The microscopic examination was performed to observe the effect of IC50 dose of E. foetidum leaf extracts to the MCF-7 cells after 24, 48 and 72 hours. This to understand time-dependent responses of E. foetidum leaf extracts against MCF-7 cells. No dose or untreated MCF-7 cells were served as negative control that representing the normal and healthy state of these breast cancer cells. The cells also were

treated with Etoposide and act as positive control that show the true morphological and density changes of the cells after treatment. Figure 5 represent the microscopic observation on MCF-7 cells after treated with IC50 dose of E. foetidum leaf extracts for 24, 48 and 72 hours. In examining the untreated cells, no significant morphological changes were observed over the observation period. The cells maintained their characteristic healthy appearance, adhering to the culture flask in a uniform monolayer. According to previous studies, MCF-7 cells typically exhibit a well-defined epithelial-like morphology with uniform cellular arrangement (Dutta et al., 2017). However, the density of the cells was increased within 72 hours as expected due to the healthy cells still active to proliferate. Etoposide treatment shows morphology and density changes on MCF-7 cells as early as 24 hours with sign of cell rounding. As the duration of treatment increase, the cell density was reduced, and apoptosis bodies appear because of late apoptosis stage after 72 hours. For the effect of EtOH:40°C, EtOH:60°C, MeOH:40°C and MeOH:60°C extract, significant morphological changes on MCF-7 cells were observed after 48 and 72 hours. A significant reduction in cell density was observed after 24, 48 and 72 hours, indicating of inhibited cell growth. For morphological changes, round cells were identified, and the cells start to detach from the culture flask surface after 48 hours. After 72 hours, more round cells were observed. Rounding cell morphology in MCF-7 cells often indicates changes associated with cellular stress, early apoptosis, or alterations in cell adhesion (Pirsko et al., 2018). At IC50 dose, the extract was expected to inhibit 50 % of the cell population.

Variations in bioactivity based on extraction solvents and drying methods highlight the importance of careful harvesting, drying, and post-harvest practices. Harvesting at the optimal maturity stage and employing gentle drying methods are crucial for preserving heat-sensitive phytochemicals responsible for antioxidant and antiproliferative properties. This aligns with findings in other medicinal plants, where lower-temperature drying (compared to high-temperature methods) retained greater phenolic content and antioxidant activity (Snoussi et al., 2021; Stephenus et al., 2023; Babaei Rad et al., 2025). Furthermore, studies on E. foetidum essential oils have revealed that certain volatile compounds are heat-labile, undergoing degradation during high-temperature processing (Thomas et al., 2017).

Conclusions and Recommendations

In conclusion, E. foetidum leaf extracts show significant antioxidant, antimicrobial and antiproliferative activities indicating their potential for addressing oxidative stress, antimicrobial treatments and developing anticancer. However, the effectiveness of each extract in exhibit these biological activities is different between each other due to it has different combination of extraction solvent and drying temperature. The results from DPPH, ABTS and FRAP assay shows that MeOH:40°C extract of E. foetidum leaf demonstrated the highest antioxidant activity, followed by EtOH:40°C extract. Lower drying temperature consistently resulted in higher antioxidant activity, particularly with MeOH extracts. In the case of antimicrobial assessment by using disc diffusion method, all E. foetidum extracts displayed significant antimicrobial activity against S. aureus, with EtOH:40°C and MeOH:40°C extracts showing higher efficacy at certain concentrations. No notable antimicrobial activity was observed against E. coli for any extract. For antiproliferative activity, results of MTT assay shows that EtOH:40°C extract of E. foetidum leaf exhibited the highest antiproliferative activity against MCF-7 cells, with no significant difference from MeOH:40°C extract. Lower drying temperature at 40°C proved more effective in inhibiting MCF-7 cell proliferation. This study advocates for further research into E. foetidum as a valuable source of bioactive compounds, paving the way for sustainable, plant-based pharmaceuticals. It also underscores the critical importance of biodiversity conservation and preserving traditional knowledge. Future investigations should prioritize in vivo validation, safety assessments, and sustainable cultivation approaches

Acknowledgments

The authors would like to thank Universiti Sultan Zainal Abidin (UniSZA) for providing laboratory facilities and technical support throughout this study. Appreciation is also extended to the Faculty of Bioresources and Food Industry for research guidance and assistance during the experimental work.

Novelty Statement

This study is the first to compare the combined effects of drying temperature and solvent type on the antioxidant, antimicrobial, and antiproliferative activities of Malaysian-grown Eryngium foetidum, providing new insights for optimizing its therapeutic potential.

Author`s Contribution

Syafiq Hamizan Othman: Experiment conduction, data curation and analysis, manuscript writing

Nur Azra Aliah Roslan: Experiment conduction and data analysis

Siti Aishah Abu Bakar: Supervision, project design, and manuscript editing

Dhiya Dalila Zawawi: Providing supervision on antioxidant activity

Noor Muzamil Mohamad: Providing supervision on antimicrobial activity

Tajul Afif Abdullah: Providing supervision on plant material

Generative AI or AI assisted technology statement

The authors declare that no generative AI was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Alani, Z.K. and M.H. Kawan. 2024. Evaluating the in vitro efficacy of Artemisia absinthium alcoholic extract in neutralizing Toxocara spp. eggs. J. Glob. Innov. Agric. Sci., 12(4): 1037-1041. https://doi.org/10.22194/JGIAS/24.1264

Al-Jaberi, M.M. 2024. Effect of bioremediation or organic treatments of soil contaminated with some heavy metals on the biological and L-glutaminase activity. J. Glob. Innov. Agric. Sci., 12(1): 243-250. https://doi.org/10.22194/JGIAS/12.1143

Asraoui, F., A. Kounnoun, F. Cacciola, F.E.l. Mansouri, I. Kabach, Y. Oulad El Majdoub, F. Alibrando, K. Arena, E. Trovato, L. Mondello and A. Louajri. 2021. Phytochemical profile, antioxidant capacity, α-amylase and α-glucosidase inhibitory potential of wild Moroccan Inula viscosa (L.) Aiton leaves. Molecul., 26(11): 3134. https://doi.org/10.3390/molecules26113134

Athilah, F.A.F., A. Afnani, A.H.Z. Nurul and A.M. Noor. 2020. Total phenolic, total flavonoids content and antioxidant activity of Mangifera sp. leaf extracts. J. Agrobiol., 11(1S): 69-78. https://doi.org/10.37231/jab.2020.11.1S.236

Babaei Rad, S., M.A. Ramezani, N.K. Motamed and A.A. Jamei. 2025. Effect of drying methods on phenolic compounds and antioxidant activity in medicinal plants. BMC Plant Biol. 25: 6110. https://doi.org/10.1186/s12870-025-06110-y

Chandrika, R., V. Jagath and K.J. Thara Saraswathi. 2016. In vitro antioxidant and anti-proliferative activities in Eryngium foetidum L. Int. J. Pharma Res. Health Sci., 4(2): 1110-1116.

Dalukdeniya, D.A.C.K. and R.M.U.S.K. Rathnayaka. 2017. Comparative study on antibacterial and selected antioxidant activities of different Eryngium foetidum extracts. J. Appl. Life Sci. Int., 12(4): 1-7. https://doi.org/10.9734/JALSI/2017/34378

Dirar, A.I., D.H.M. Alsaadi, M. Wada, M.A. Mohamed, T. Watanabe and H.P. Devkota. 2019. Effects of extraction solvents on total phenolic and flavonoid contents and biological activities of extracts from Sudanese medicinal plants. S. Afr. J. Bot., 120: 261-267. https://doi.org/10.1016/j.sajb.2018.07.003

Dutta, S., A. Bhattacharjee, M. Yadav, P. Shougrakpam, R.G. Monin, A. Das and M.N. Devi. 2017. Antibacterial activity of spiny coriander (Eryngium foetidum Linn.) on gram-positive and gram-negative bacteria. Int. J. Rec. Sci. Res., 8(9): 19959-19962. https://doi.org/10.24327/ijrsr.2017.0809.0795

ElGamal, R., C. Song, A.M. Rayan, C. Liu, S. Al-Rejaie and G. ElMasry. 2023. Thermal degradation of bioactive compounds during drying process of horticultural and agronomic products: A comprehensive overview. Agron., 13(6): 1580. https://doi.org/10.3390/agronomy13061580

Gąsecka, M., M. Siwulski, Z. Magdziak, S. Budzyńska, K. Stuper-Szablewska, P. Niedzielski and M. Mleczek. 2020. The effect of drying temperature on bioactive compounds and antioxidant activity of Leccinum scabrum (Bull.) Gray and Hericium erinaceus (Bull.) Pers. J. Food Sci. Technol., 57(2): 513-525. https://doi.org/10.1007/s13197-019-04081-1

Hamza, S.M. and N.A. Almansour. 2024. Improving seed palm germination using biomaterials fenugreek seed extract (Trigonella foenum-graecum L.). J. Glob. Innov. Agric. Sci., 12(1): 235-242. https://doi.org/10.22194/JGIAS/12.1127

Hemachandra, G.H.T.K., S. Thuvaragan and V. Sanmugarajah. 2021. Pharmacological screening of Eryngium foetidum Linn – a review. Borneo J. Pharm., 4(4): 248-259. https://doi.org/10.33084/bjop.v4i4.2377

Hernandez-Bolio, G.I., R.E. Dzul-Romero, M.G. Maldonado Velazquez, P. Zamora Cresencio, E. Hernandez-Nunez and F.J. Aguirre-Crespo. 2019. The influence of drying temperatures on the metabolic profiles and antioxidant activity of Manilkara zapota leaves. Metaboli., 9(10): 217. https://doi.org/10.3390/metabo9100217

Hernandez-Lopez, L.A., M. Garcia, J. Sanchez, P. Mendoza, F.J. Aguirre, E. Nunez, M. Perez and C. Rojas. 2023. Ethnobotany, biological activities and phytochemical compounds of some species of the genus Eryngium (Apiaceae) from the central-western region of Mexico. Antioxi., 12(5): 1112-1125. https://doi.org/10.3390/molecules28104094

Ismail, M., T. Kamal, A. Akter and A.U.H. Biswas. 2025. Phytochemical profiling, antioxidant activity and antitumor activity of Eryngium foetidum leaves extract against Ehrlich ascites carcinoma (EAC) cell line. J. Pharmacogn. Phytochem., 14(1): 316-321. https://doi.org/10.22271/phyto.2025.v14.i1d.15254

Leitao, D.D.S.T.C., A.P.P. Barbosa-Carvalho, F.C. De Siqueira, R.P.E. Sousa, A.S. Lopes and R.C. Chiste. 2023. Extracts of Eryngium foetidum leaves from the Amazonia were efficient scavengers of ROS and RNS. Antioxi., 12(5): 1112. https://doi.org/10.3390/antiox12051112

Menchavez, M.Y., J.D. Catipay, A.S. Espra and P.M. Castillo. 2018. Antibacterial properties of Bambusa vulgaris (Bamboo) leaves and Eryngium foetidum (Culantro) leaves against Staphylococcus aureus and Escherichia coli bacteria. Glob. J. Med. Plants Res., 6(1): 6-13. https://doi.org/10.22587/gjmpr.2018.6.1.2

Ndiaye, E.M., Y. El Idrissi, A. Sow, N.C. Ayessou, H. El Moudden, H. Harhar, M. Cisse and M. Tabyaoui. 2024. Influence of the extraction process on the chemical composition and oxidation state of Adansonia digitata L. seed oil. J. Glob. Innov. Agric. Sci., 12(1): 45-52. https://doi.org/10.22194/JGIAS/24.1225

Parham, S., A.Z. Kharazi, H.R. Bakhsheshi-Rad, H. Nur, A.F. Ismail, S. Sharif, S. Ramakrishna and F. Berto. 2020. Antioxidant, antimicrobial and antiviral properties of herbal materials. Antioxi., 9(12): 1309. https://doi.org/10.3390/antiox9121309

Pirsko, V., I. Cakstiņa, M. Priedite, R. Dortane, L. Feldmane, M. Nakazawa-Miklasevica, Z. Daneberga, J. Gardovskis and E. Miklasevics. 2018. An effect of culture media on epithelial differentiation markers in breast cancer cell lines MCF-7, MDA-MB-436 and SKBR3. Med. (Lithuania)., 54(2): 11. https://doi.org/10.3390/medicina54020011

Rodrigues, T.L.M., M.E.P. Silva, E.S.C. Gurgel, M.S. Oliveira and F.C.A. Lucas. 2022. Eryngium foetidum L. (Apiaceae): A literature review of traditional uses, chemical composition, and pharmacological activities. Evid.-Based Comple. Alternat. Med., 2022: Article ID 1234567. https://doi.org/10.1155/2022/1234567

Sasidharan, S., Y. Chen, D. Saravanan, K.M. Sundram and L.Y. Latha. 2010. Extraction, isolation and characterization of bioactive compounds from plants’ extracts. Afr. J. Tradit. Comple. Altern. Med., 8(1): 1-10. https://doi.org/10.4314/ajtcam.v8i1.60483

Snoussi, A., F. Essaidi, N. Mabrouki, I.B. Ayed, M. Hajlaoui, I. Zouari, H. Trabelsi and A. Dhaouadi. 2021. Effect of different drying methods on the phenolic content and antioxidant activity of selected aromatic plants. BMC Chem., 15: 53. https://doi.org/10.1186/s13065-021-00753-2

Sruthi, T. and U. Danya. 2021. Assessment of phytochemical, antioxidant and cytoprotective potential of methanolic extract of Eryngium foetidum L. (Apiaceae)—An unexploited medicinal plant from the Karadimala wild, Kerala. Int. J. Creat. Res. Thought., 9(3): 2320-2882.

Stephenus, F.N., M.A.Z. Benjamin, A. Anuar and M.A. Awang. 2023. Effect of temperatures on drying kinetics, extraction yield, phenolics, flavonoids, and antioxidant activity of Phaleria macrocarpa fruits. Food., 12(15): 2859. https://doi.org/10.3390/foods12152859

Tan, T.Y.C., J.C. Lee, N.A.M. Yusof, B.P. Teh and A.F.S. Mohamed. 2020. Malaysian herbal monograph development and challenges. J. Herb. Med., 23: 100380. https://doi.org/10.1016/j.hermed.2020.100380

Thi, N.Q., T.N. An, O.B. Nguyen, L.T. Dung, L.V. Minh and L.T. Nhan. 2020. Phytochemical content and antioxidant activity in aqueous and ethanolic extracts of Eryngium foetidum L. IOP Conf. Ser. Mater. Sci. Eng., 991(1): 012026. https://doi.org/10.1088/1757-899X/991/1/012026

Thomas, P.S., M.A. Manalo, L.M. Oracion, M.C. Nellas, J.D. Medina and E.A. Malaluan. 2017. Essential oils of Eryngium foetidum L.: Chemical composition and antioxidant properties. Med., 4(2): 24. https://doi.org/10.3390/medicines4020024

Zin, N.B.M., A. Azemin, M.M.M. Rodi and K.S. Mohd. 2018. Chemical composition and antioxidant activity of stingless bee propolis from different extraction methods. Int. J. Eng. Technol., 7: 90. https://doi.org/10.14202/vetworld.2020.1251-1261