Evaluating the Antioxidant,Antimicrobial,and Anticancer Effects of Eminium spiculatum Plant Extracts

Abstract

Introduction

Plants have long been used for medicinal purposes, but synthetic drugs emerged with scientific advancements. However, these synthetic drugs often have harmful side effects. Natural products, particularly plants, offer safer alternatives with bioactive compounds that can be used for synthesizing new drugs and developing natural remedies.

Methods

Utilizing an ultrasonic-assisted extraction technique, two distinct preparations of Eminium spiculatum (E. spiculatum) extract were produced. These extracts were obtained using ethanol solutions at concentrations of 100% and 50%, respectively. The biological activities of E. spiculatum extracts were evaluated using chemical assay methods. Specifically, the study assessed the antimicrobial, anticancer, and antioxidant properties of these extracts through standardized chemical techniques. The antimicrobial impact was examined against E.coli, Streptococcus species, as well as Staphylococcus aureus (S. aureus) using the well diffusion method. Furthermore, anticancer tests demonstrated activity against HT29 (colon cancer cells) and MCF7 (breast cancer cells).

Results

The findings revealed complete cell death with both 100% as well as 50% ethanol extracts compared to the control. The extracts exhibited notable antioxidant scavenging capacity against the stable radical DPPH, with values of 298.7 ± 3.3 and 190.8 ± 2.2 µmol Trolox/g for 50% and 100% ethanolic extracts, respectively. These outcomes can be attributed to the extracts’ abundant phenolic content (107.3 ± 3.1 and 99.7 ± 2.0 mg GAE/g for 50% and 100% ethanolic extracts, respectively) and flavonoid content (67.9 ± 2.9 and 45.6 ± 2.8 mg catechin/g for 50% and 100% ethanolic extracts, respectively).

Conclusion

The assessment of E. spiculatum plant extracts revealed significant antimicrobial, anticancer, and antioxidant activities. The extracts demonstrated promising potential for drug development, particularly in the areas of cancer treatment and the production of novel antibacterial drugs. Further exploration of extraction methods to isolate the bioactive compounds could enable E. spiculatum to contribute to the synthesis of safe and effective drugs in the future.

Introduction

Since ancient times, people have used plants for their medicinal benefits, serving various purposes such as food, flavoring, spices, ornamentals, and cosmetics. Medicinal plants contain therapeutic substances that can be used directly or as precursors for drug synthesis. While modern medicine heavily relies on pharmaceutical and synthetic medications, traditional remedies derived from plants and microorganisms continue to play a significant role. Despite their widespread usage, synthetic drugs can have adverse effects.^1-3 However, throughout history, plants and herbs have served as valuable sources of food and remedies for healing wounds, treating diseases, and promoting mental well-being in countries like China, Egypt, India, Greece, and the Middle East.^4-6

Traditionally, medicinal plants were widely used in non-industrialized regions due to their affordability compared to modern medicine. Historical usage of plants was primarily based on instinct rather than scientific knowledge of their medicinal properties. People would utilize whole plants without understanding the specific phytochemicals they contained.^1,7,8 However, as research progressed, it became evident that the active components within plants needed to be extracted and studied individually in laboratories to determine their effects, efficacy, and safety. ⁹ This transition from instinctive to evidence-based knowledge prompted many countries to incorporate these plants into their healthcare systems . Ancient civilizations utilized medicinal plants as primary treatments for various ailments, including epilepsy, infertility, depression, cancer, and psychosomatic disorders. ¹⁰

Palestine is renowned for its rich traditional herbal medicine, which is often considered safer and more effective than synthetic drugs. The country's strategic location along the Mediterranean coast, coupled with its diverse flora, provides a wealth of herbs and plants with pharmaceutical and medical applications. Extracting bioactive components from plants is crucial for drug synthesis, and several extraction procedures, including solvent extraction, distillation, pressing, and sublimation, are used for this purpose. Antioxidants, antimicrobial agents, and anticancer compounds found naturally in plants are of great importance for drug development. Antioxidants protect against harmful free radicals, while antimicrobials combat microorganisms and reduce infection risks.

Cancer is a leading cause of mortality worldwide. This has necessitated the search for natural bioactive compounds that can effectively target cancer cells. Herbal treatments show promise as potential anticancer drugs, providing alternative therapeutic options with fewer side effects than conventional chemotherapy.^11-14

E. spiculatum, an indigenous plant belonging to the Araceae family, thrives in the Middle East, specifically in countries like Palestine, Egypt, Jordan, Lebanon, and Syria (Figure 1).^15,16 While consuming this perennial plant raw can be toxic and cause tongue numbness, it has garnered attention for its medicinal potential.^17,18 The outer part of its lamina features a light background with brown veins, while the inner part showcases a blackish- brown or violet color. Flowering from March to June, it reaches a length of 0.3 meters and can be found in fields, waste grounds, rocks, and sandy semi-deserts. The plant was valued by Egyptian Bedouins, who boiled its corms for consumption, akin to boiled potatoes, and consumed its seeds like peas. E. spiculatum is primarily recognized for its high anticancer activity, as anticancer agents are often associated with the presence of antioxidants. ¹⁹ The current study attempts to examine the antioxidant, antimicrobial, anticancer activities of E. spiculatum plant extracts sourced from Palestine.

Figure 1.

The plant Eminium Spiculatum.

Materials and Methods

Plant Extraction

The aerial components of E. spiculatum were sourced from a local herbal shop in Bethany, Palestine, during the period between March and June 2023, and assigned voucher number Pharm-PCT-278. These aerial parts were carefully dried in a shaded area until a consistent weight was achieved, after which they were pulverized. A total of 10 grams of the ground material was steeped in 100 milliliters of either 100% or 50% ethanol with the aid of ultrasonic treatment for a duration of 3 h. Subsequently, the mixture underwent filtration, and a rotating evaporator concentrated the solvent at a temperature of 47 °C. The resulting residue was then dissolved in methanol to achieve a concentration of 0.1 g/mL. ²⁰

Determination of Total Phenolic Content

The overall phenolic content of the extract was quantified using the Folin-Ciocalteu colorimetric method . The Folin- Ciocalteu reagent was diluted tenfold using distilled water before mixing 0.2 mL of the plant extract with 1.8 mL of the diluted solution. The mixture was allowed to stand for about 5 min before being treated with 1.2 mL of 7.5% NaHCO₃. After a 60-min incubation at room temperature, the samples’ absorbance at 765 nm was measured. The gallic acid standard followed the same procedure. Aqueous solutions with known quantities of gallic acid (ranging from 20 to 500 mg/L) were used to produce the calibration curve. The results were reported as gallic acid equivalents (GAE) per gram of the sample. ²⁰

Determination of Total Flavonoid Content

The aluminum chloride technique combined with spectrophotometric analysis was used to determine the extract's total flavonoid concentration. In a test tube, 1 mL of the extract, 5 mL of distilled water, 0.3 mL of a 5% sodium nitrate solution, and 0.3 mL of a 10% aluminum chloride solution were all mixed. The mixture was left at room temperature for 5 min. After that, 2 mL of 1 M sodium hydroxide was added, and water was used to bring the total volume to 10 mL. Following a thorough vortex, the solution's absorbance was measured spectrophotometrically at 510 nm. To create a standard calibration curve, different concentrations of catechin (20-100 mg/L) were used. The results were reported in mg of catechin equivalents (CEQ) per gram of the sample. ²⁰

Antioxidant Activity: Free Radical Scavenging Activity Using DPPH

To determine if the test sample could neutralize or scavenge the free radical component of the stable DPPH radical, the DPPH antioxidant assay was utilized. First, a 0.062 mM DPPH solution was prepared using 95% methanol as the solvent . This solution was then mixed with 0.1 mL of the extract in a total volume of 3.9 mL. The mixture was vortexed for 10 s and then incubated at room temperature for 30 min. The absorbance of the DPPH solution was measured at 515 nm, with 95% methanol serving as the blank sample. Different Trolox concentrations (20-200 ppm) were used to create a calibration curve. The results were expressed in micromoles of Trolox per gram (µmol Trolox/g). ²⁰

HPLC Analysis

The HPLC analysis was conducted on the extracts obtained from dried E. spiculatum plant samples. The analysis was performed using an ODS column from Waters (XBridge, 4.6 ID×150 mm, 5 μm). The mobile phase consisted of a mixture of 0.5% acetic acid (solvent A) and acetonitrile (solvent B) in a ratio of 80:20 (v/v). The gradient elution method was employed, with the percentage of solvent A decreasing from 100% to 70% over 40 min, then to 40% over 20 min, and eventually reaching 10% over 2 min. After staying at 10% solvent A for 6 min, the mobile phase composition returned to its initial state after 2 min. The HPLC system reached equilibrium with the initial acidic mobile phase (solvent A) for 7 min before injecting the next sample. All samples were filtered using a 0.45μm PTFE filter. The photodiode array (PDA) detector was set to a wavelength range of 210–500 nm. The flow rate was maintained at 1 mL/min, and the injection volume was 20 µL. The column temperature was set to 25 °C.

Antimicrobial Test

To assess the antimicrobial properties of E. spiculatum, the study utilized three bacterial species as test subjects: E. coli, S. aureus, and a Streptococcus species. These microorganisms served as indicators to determine the extract's effectiveness against various bacterial pathogens. A total of 19 grams of agar and 500 mL of distilled water were combined to prepare the Mueller-Hinton agar medium. The mixture was then autoclaved for 15 min at 121 °C. After sterilization, the Mueller-Hinton agar was allowed to cool before being poured into six Petri dishes. After the agar sufficiently solidified, two plates each were inoculated with S. aureus, Streptococcus, and E. coli bacteria. The bacterial inoculum was prepared following the McFarland turbidity standard, a widely recognized method for standardizing bacterial suspension density. A McFarland card, featuring black lines that become distorted at the standard's turbidity, was used periodically to check the inoculum's turbidity. The inoculum was then spread on the agar media using inoculating loops. To test E. spiculatum extracts for antimicrobial properties, the well diffusion method was employed. Wells were created in the agar by pressing and removing the back of a 100 µL pipette tip. These wells were then filled with 100% ethanol extracts of E. spiculatum. The bacteria tested were S. aureus, Streptococcus, and E. coli. The prepared plates were incubated at 37 °C, and results were examined after 24 h.

Anticancer Test

To assess the extracts’ potential cytotoxic effects, two cancer cell lines, HT29 as well as MCF7 acquired from the American Type Culture Collection (ATCC) in the USA, were utilized. The collected cell lines were suspended in RPMI medium as well as incubated for 24 h to achieve adequate settling and adaptation to the culture environment. Five tissue culture plates were prepared for each cancer cell type. These plates were divided into three sections at a ratio of 2:2:1. The first two portions were set aside for the administration of 100% and 50% ethanolic plant extract, respectively, while the third segment acted as a negative control using DMSO.

DMSO-diluted extracts of 100 μL and 50 μL were applied to tissue culture plates for each cell line in the appropriate quantities. Each tissue culture plate of the cell lines received 3 μL of the 100% and 50% ethanolic extracts diluted in DMSO. Following the addition of the extracts, the plates were incubated at a temperature of 37 °C for a duration of 72 h. This 72-h incubation period allowed for the assessment of the cytotoxic impact of the extracts on the cancer cell lines. Various cytotoxicity parameters, such as cell viability, proliferation, and morphology, were subsequently evaluated using techniques such as cell viability assays, flow cytometry analysis, or microscopic observation. The results obtained from these assessments provided valuable insights into the potential cytotoxic effects of the extracts on the cancer cell lines, indicating their potential as anti-cancer agents. Additional analysis, including concentration-dependent effects and determination of IC50 values, may be conducted to further understand the efficacy of the extracts.

Statistical Analysis

This study's results are provided as means ± standard deviation (SD). Statistical analysis was carried out by the Statistical Package for the Social Sciences (SPSS, version 16). Statistical significance was determined with a p-value <0.5.

Results

Antimicrobial Activity

Figure 2 shows the inhibitory effects of the E. spiculatum extract against three different types of bacteria, demonstrating the formation of three distinct zones of inhibition. Among these bacteria, two are classified as Gram-positive. Streptococcus displayed a zone of inhibition measuring 13 mm, while S. aureus exhibited a zone of inhibition measuring 12 mm. Additionally, the extract demonstrated inhibitory activity against a Gram-negative bacterium, E. coli, with a zone of inhibition measuring 14 mm. These findings indicate the potential antimicrobial properties of the E. spiculatum extract, suggesting its effectiveness in targeting and inhibiting the growth of these bacterial strains.

Figure 2.

Iinhibition zones caused by plant extract on the three bacterial cells (a) The inhibition zone of the extract on E.coli (14 mm). (b) The inhibitory zone of the extract on Streptococcus (13 mm). (c) The zone of inhibition of the extract on S. aureus (14 mm).

Antioxidant Activity

The phytochemical and antioxidant activitie observations of the E. spiculatum extract demonstrate the existence of therapeutically active ingredients in the ethanol plant extract, at both 50% and 100% concentration. ¹⁹ Table 1 summarizes the total phenolic and flavonoid content, as well as the antioxidant properties of ethanolic E. spiculatum extracts.

Table 1.

Total Phenolic and Flavonoid Content and Antioxidant Activity of E. spiculatum Extracts*.

Total phenolic content (mg gallic acid equivalent /g)		Total flavonoids content (mg catechin equivalent /g)		Antioxidant activity (µmol Trolox equivalent \g)
50% ethanol extract	100% ethanol extract	50% ethanol extract	100% ethanol extract	50% ethanol extract	100% ethanol extract
107.3 ± 3.1	99.7 ± 2.0	67.9 ± 2.9	45.6 ± 2.8	298.7 ± 3.3	190.8 ± 2.2

*Values are given as Mean ± SD

Total Phenolic Content

The extract with 50% ethanol concentration has a total phenolic content of 107.3 ± 3.1 mg gallic acid/g, while the 100% ethanol extract has a total phenolic content of 99.7 ± 2.0 mg gallic acid/g (Table 1). These quantitative results show that the 50% ethanol extract has a higher extraction efficiency compared to the 100% ethanol extract. The presence of a water-ethanol mixture in the extract improves both the extraction efficiency (due to increased polarity) and the solubility of phenolic compounds (as water enhances solubilization when mixed with ethanol or any alcohol). Consequently, the extraction efficiency increases when water is included in the solvent mixture. ¹

Total Flavonoid Content

The extract with 50% ethanol concentration exhibits a total flavonoid content of 67.9 ± 2.9 mg catechin/g, whereas the 100% ethanol extract has a total flavonoid content of 45.6 ± 2.8 mg catechin/g (Table 1). Similar to the total phenolic content findings, these results indicate that the extract with 50% ethanol is more effective than the 100% ethanol extract in terms of flavonoid extraction.

Aantioxidant Activity

The 50% ethanol extract's antioxidant capacity is measured at 298.7 ± 3.3 µ Trolox/g, while the 100% ethanol extract exhibits an antioxidant activity of 190.8 ± 2.2µ Trolox/g. Consistent with TPC and TFC results, the antioxidant activity is found to be higher in the 50% ethanol extract when compared to the 100% ethanol extract.

HPLC-PDA Profiles of the Extracts

Figure 3 shows the chromatogram of the ethanolic crude extract of E. spiculatum at 280 nm. This wavelength was chosen because the primary peaks exhibited the most absorption at this wavelength. The eluted chemicals were found in the range of 23.06-36.43 min for the 50% ethanol extract and 25.0–35.29 min for the 100% ethanol extract, suggesting the presence of relatively nonpolar compounds. Upon closer inspection, the results reveal that the 50% ethanol extract contains major compounds at 25.8 and 28.4 min. In contrast, the 100% ethanol extract displays one major compound at 29.4 min (Figure 3). These findings provide information about the specific retention times and major compounds present in each extract, thereby contributing to the characterization of the chemical composition of the E. spiculatum extract.

Figure 3.

HPLC-PDA chromatogram of 50% and 100% ethanolic crude extract of E. spiculatum at 280 nm.

Anticancer Activity

During the assessment of the anti-cancer effect of the extracts, the effects were examined on two cancer cell lines: MCF7 and HT29. As a reference point for comparison, a negative control was employed in the study, utilizing DMSO. Importantly, the results revealed that the DMSO control had no impact on the cancer cells. This implies that any observed effects on the cancer cell lines can be attributed to the presence of the E. spiculatum extract itself, rather than the influence of the DMSO solvent.^18,19,21

MCF7- Breast Cancer Cell Line

Following a 72-h incubation period, the Petri dishes were retrieved and subjected to examination using an inverted fluorescent microscope. Figure 4a displays the control culture, where MCF7 cells were cultivated without the inclusion of DMSO or the extract. Figures 4b and 4c exhibit MCF7 cells treated with 100 μL and 50 μL of DMSO, respectively. In both cases, the cells maintained their adherence and were unaffected by the presence of DMSO. These observations demonstrate that DMSO alone does not exert any discernible effect on the MCF7 cell line, reinforcing the notion that any observable effects on the cells can be attributed to sources other than DMSO alone (Figure 5a–d). ¹⁸

Figure 4.

(a) MCF7 control in the RPMI culture media without the addition of DMSO or the plant extract. (b) adherent cells as the MCF7 cells with 100 µl DMSO. (c) Mostly adherent cells of the MCF7 with 50 µl DMSO.

Figure 5.

MCF7 cell line. (a) MCF7 treated with 100 µl of 100% ethanolic extract. (b) MCF7 treated with 50 µl of 100% ethanolic extract. (c) MCF7 treated with 100 µl of 50% ethanolic extract. (d) MCF7 treated with 100 µof 50% ethanolic extract.

HT29 Colon Cancer Cell Line

Figure 6a presents the control culture where HT29 cells were grown without the addition of DMSO or the extract. In Figures 6b and 6c, HT29 cells are depicted with the addition of 50 μL and 150 μL of DMSO, respectively. In both cases, the cells maintained their adherence and were unaffected by the presence of DMSO. These observations confirm that DMSO alone does not exert any discernible effect on the HT29 cell line. ¹⁹

Figure 6.

HT29 cell line. (a) HT29 cell line in control environment (no DMSO or plant extract). (b) HT29 colon cancer cells treated with 50 µL DMSO. (c) HT29 colon cancer cell line treated with 150 µl of DMSO. (d) HT29 treated with 50 µL of 100% ethanol extract (e) HT29 treated with 100 µL of 100% ethanol extract (f) HT29 treated with 50 µL of 50% ethanol extract (g) HT29 treated with 100 µL of 50% ethanol extract.

Discussion

E. spiculatum, a member of the Araceae family, has garnered attention for its potential medicinal properties. This study explores its bioactive compounds and their pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer effects. E. spiculatum extracts contain a diverse array of bioactive compounds, including alkaloids, flavonoids, saponins, terpenoids, phenolic compounds, and tannins. Afifi et al ¹⁹ identified specific compounds such as luteolin, luteolin-7-O-glucoside, isoorientin, vitexin, chrysoeriol-7-O-glucoside, and β-sitosterol in Jordanian E. spiculatum.^2,22 Notably, luteolin exhibited moderate antibacterial activity against E. coli and resistant strains of S. aureus, as well as significant antiproliferative activity against MCF-7 and T47D cell lines. ²

The ethanolic extracts of E. spiculatum demonstrated significant antioxidant potential, attributed to their high phenolic and flavonoid content. These compounds act as free radical scavengers, preventing cellular damage and lipid peroxidation. This antioxidant activity suggests potential applications in treating oxidative stress-related disorders.

Our study demonstrated E. spiculatum's antimicrobial efficacy against both Gram-positive (Streptococcus, S. aureus) and Gram-negative (E. coli) bacteria, with a larger inhibition zone observed for Gram-negative bacteria. This broad-spectrum activity is attributed to alkaloids, flavonoids, and phenolic compounds that disrupt bacterial cell walls, inhibit protein synthesis, and induce oxidative stress. The extract's effectiveness against both Gram-positive and Gram-negative bacteria, including potentially resistant strains, highlights its promise in addressing antibiotic resistance challenges.^1-5

E. spiculatum extracts showed potent anticancer activity against MCF7 breast cancer and HT29 colon cancer cell lines. Both 50% and 100% ethanolic extracts achieved complete inhibition of cell growth at various concentrations (50 μL and 100 μL). The anticancer mechanisms likely involve the induction of apoptosis through increased expression of pro-apoptotic proteins and decreased anti-apoptotic proteins, leading to cell cycle arrest. Terpenoids and alkaloids in the plant may also interfere with cell signaling pathways regulating growth and division.^4,23–25

While this research provides valuable insights into E. spiculatum's therapeutic potential, several limitations should be noted. The study focused on specific cancer cell lines, which may not fully represent the complexity of in vivo scenarios. The antimicrobial and antioxidant evaluations were conducted in vitro, necessitating further investigation in more complex biological environments. Future research should aim to identify specific bioactive components responsible for the observed effects, explore potential adverse effects or toxicity, and conduct in vivo studies to better understand the plant's clinical applicability.

Even with these drawbacks, E. spiculatum shows promise as a source of bioactive compounds with diverse pharmacological activities. Its potential in addressing antibiotic resistance, cancer treatment, and oxidative stress-related disorders warrants further investigation to fully elucidate its therapeutic applications.

Conclusion

Antimicrobial, anticancer, and antioxidant activities of E. spiculatum extracts were investigated in this study. The antimicrobial effect was examined using the well diffusion method. Anticancer activity against HT29 (colon cancer cells) as well as MCF7 (breast cancer cells) was assessed, while antioxidant activity of the extracts was evaluated using the DPPH assay. Total phenolic content as well as total flavonoids content were determined using standard spectrophotometric methods. Extracts from E. spiculatum have a high concentration of phenolic compounds and flavonoids, which provide them with antioxidant and anticancer properties. The presence of these phytochemicals suggests the potential use of E. spiculatum in traditional medicine. Positive findings from the antimicrobial activity tests were obtained against both Gram-positive and Gram-negative bacteria, including those resistant to widely used antibiotics. The antioxidant activity tests demonstrated that the plant extract contains high levels of phenolics, flavonoids, and antioxidants, which can be beneficial in treating various oxidative stress-related diseases. Furthermore, E. spiculatum exhibited inhibitory effects on two cancer cell lines: MCF7 breast cancer and HT29 colon cancer, effectively eliminating both types of cancer cells. Future research is expected to yield promising results and potentially lead to the development of drugs such as antibiotics and antioxidants for the treatment of oxidative stress-related disorders like diabetes, as well as potential therapies for breast and colon cancers. However, further investigation and proper isolation of these essential bioactive compounds are necessary for these advancements to occur.