Isolation, Identification, and in Vitro Antitumor Activity of Polyphenols From Euryale ferox Salisb. Shell

ABSTRACT

The shell of Euryale ferox Salisb., rich in polyphenols, has antitumor potential. We isolated and identified polyphenols from the shell and evaluated their in vitro antitumor activity. Compounds were extracted with 70% ethanol under ultrasound and purified by fractional extraction and silica gel chromatography, yielding three substances. Liquid chromatography–mass spectrometry (LC‐MS) and nuclear magnetic resonance confirmed the three compounds as ellagic acid, corilagin, and geraniin. High‐performance LC quantification showed corilagin and geraniin concentrations of 66.23 and 58.41 mg/g. In vitro experiments on cells showed the ethanol extract had strong inhibitory effects on cell proliferation (IC₅₀: 68.56 µg/mL for HeLa, 78.09 µg/mL for A2780), and cell proliferation, migration, and invasion were regulated by the three compounds, exhibiting time‐ and dose‐dependent characteristics. Corilagin showed the strongest inhibitory effect on HeLa cell proliferation (74.61% at 48 h) and A2780 cell invasion (69.86%). Geraniin inhibited HeLa cell invasion (63.33%) and A2780 cell invasion (61.64%). This study determined the chemical structures of polyphenolic compounds from Euryale ferox Salisb. shell and their impact on tumor cell proliferation, migration, and invasion, offering evidence for developing antitumor drugs and insights for modernizing traditional Chinese medicinal resources.

Polyphenols are isolated from Euryale ferox Salisb. shell and identified as ellagic acid, corilagin, and geraniin. Quantitative analysis shows corilagin (66.23 mg/g) and geraniin (58.41 mg/g) as the major components. These three compounds inhibit the proliferation, migration, and invasion of HeLa and A2780 cells in vitro in a time‐ and dose‐ dependent manner, with corilagin exhibiting the strongest effects. The results support the antitumor potential and valorization of this traditional medicinal resource.

1. Introduction

Euryale ferox Salisb., a perennial aquatic herb of the Nymphaeaceae family, is widely distributed throughout Asia, particularly in China, Japan, and India [1]. Studies show that Euryale ferox Salisb. contains bioactive compounds such as sterols, flavonoids, and polyphenols, which exhibit strong antioxidant properties [2, 3], as well as antimicrobial [3, 4], antidiabetic [5], anticancer [6], and antidepressant effects [7]. For instance, Nam et al. found that Euryale ferox Salisb. extract induces apoptosis in A549 lung cancer cells by inhibiting the activation of protein kinase B (Akt) and tumor protein p53 (p53) [6]. Zhang et al. revealed that Euryale ferox Salisb. polysaccharides increase glucose consumption by upregulating glucose transporter type 4 (GLUT‐4) expression through the phosphatidylinositol 3‐kinase (PI3K)/Akt signaling pathway in insulin‐resistant cells (IR cells), thereby achieving hypoglycemic effects [8]. Additionally, Ahmed et al. reported that 2β‐hydroxybetulinic acid 3β‐oleiate (HBAO) from Euryale ferox Salisb. shell (EFSS) significantly regulates blood glucose and lipid levels in diabetic rats [9]. Furthermore, research indicates that Euryale ferox Salisb. extract exerts antidepressant effects by modulating the AMP‐activated protein kinase (AMPK) pathway to activate cellular autophagy [7].

EFSS, a byproduct of Euryale ferox Salisb. Processing has long been discarded as waste material, posing risks of resource waste and environmental pollution. However, recent research has highlighted the potential of these shells as a valuable resource, particularly due to their bioactive polyphenolic compounds. For instance, Jian et al. demonstrated that polyphenols from EFSS exhibit hepatoprotective effects in mice with non‐alcoholic fatty liver disease induced by a high‐fat diet [10]. Additionally, these polyphenols disrupt the cell wall integrity of Salmonella Typhimurium, showcasing their antibacterial properties [11]. Triterpenoid extracts from the shells also show therapeutic potential against diabetes by addressing insulin resistance [12]. Furthermore, Wu et al. found that complex tannins from EFSS inhibit melanin production, providing a theoretical basis for applications in cosmetics and medicine [13]. Despite these findings, most studies have focused on crude extracts rather than isolating and characterizing specific bioactive polyphenols. Recent advancements, however, have begun to address this gap. For example, Wu et al. isolated corilagin from EFSS polyphenol extracts and demonstrated its immunomodulatory effects by regulating macrophage tolerance to lipopolysaccharides [14]. Zhang et al. extracted three phenolic compounds—gallic acid, epicatechin, and rutin—from the shells and confirmed their efficacy as natural antioxidants in preserving pork sausages [15]. Dai et al. further showed that complex tannins from EFSS inhibit tyrosinase activity. Using nanoliposome technology, these compounds could be developed into novel anti‐browning agents, with potential applications in the fresh‐cut food industry [16].

Despite partial confirmation of the nutritional and pharmacological value of EFSS, its development and utilization remain limited, particularly in the isolation, purification, and mechanistic studies of its polyphenolic components [15].

This study aims to isolate and purify polyphenolic compounds from EFSS and investigate their in vitro antitumor activity. By systematically studying the extraction, separation, and structural identification of polyphenols from EFSS, and their inhibitory effects on tumor cell proliferation, migration, and invasion, this research provides a scientific foundation for the further development and utilization of EFSS. Additionally, it offers new insights into the antitumor research of natural polyphenolic compounds.

2. Experimental

2.1. Materials

Euryale ferox Salisb. was purchased from Wushang Supermarket in 2019 and identified by Professor Xu Ran (Wuhan Polytechnic University). The herbarium specimen (No. 1903070) of this plant is preserved in the Herbarium Center on the fifth floor of the Biochemical Building, Wuhan Polytechnic University. Sephadex LH‐20 dextran gel was obtained from Bio‐Rad. The Cell Counting Kit‐8 (CCK‐8) reagent was purchased from Tongren (Japan). SGC‐7901 (Human gastric cancer cell, RRID: CVCL_0520) and HepG2 (Human hepatocellular carcinoma cell, RRID: CVCL_0027) cell lines were obtained from Biossci (Hubei) biotechnologies Company (China), HeLa (Human cervical cancer cell, RRID: CVCL_0030) and A2780 (Human ovarian cancer cell, RRID: CVCL_0134) cell lines were purchased from the Shanghai Institute of Cell Biology(China). 24‐well 8 µm Transwell cell chambers were obtained from Costar (USA). Matrigel was purchased from Becton, Dickinson, and Company. 5‐Fluorouracil (5‐FU) was obtained from Sigma (USA).

2.2. Methods

2.2.1. Fractionate Extraction and Purification of Polyphenolic Ethanol Extract From EFSS

Dried EFSS was ground and sieved to 200 mesh. Polyphenols were extracted using 70% ethanol at a 1:10 (V:V) ratio via ultrasonic extraction at 60°C for 2 h. After rotary evaporation and freeze‐drying, the ethanol extract was obtained. The freeze‐dried powder was then dispersed in water and subjected to three successive liquid‐liquid partitions with petroleum ether, ethyl acetate, and n‐butanol (1:1, V:V). Each organic layer and the final aqueous layer were concentrated and lyophilized to yield respective fractions, which were then tested for cytotoxic activity using the CCK‐8 assay.

The ethyl acetate fraction was subjected to silica gel column chromatography (60 mesh) using a petroleum ether: ethyl acetate (2:1, V:V) elution system, yielding 10 fractions. Fractions 3, 4, and 6 exhibited the highest cytotoxic activity and were selected for further purification. Fraction 3 was chromatographed on a silica gel column using a gradient elution of petroleum ether: ethyl acetate (100:30–100:5, V:V), yielding five sub‐fractions. Sub‐fraction 3.3 was purified via Sephadex LH‐20 gel permeation chromatography using 30% methanol to afford compound A. Similar procedures applied to fractions 4 and 6 yielded compounds B and C, respectively.

Finally, compounds A, B, and C were dissolved in methanol and further purified by semi‐preparative high‐performance liquid chromatography (HPLC) on a C18 column (150 × 9.4 mm, 5 µm) using 0.1% acetic acid in acetonitrile as the mobile phase at a flow rate of 3 mL/min with an injection volume of 500 µL.

2.2.2. In Vitro Activity Assessment of EFSS Polyphenolic Extract

To identify antitumor‐active components in EFSS ethanol extracts, the CCK‐8 assay was used to evaluate the effects of extracts on HeLa cell proliferation. HeLa cells (4 × 10³ cells/well) were seeded in 96‐well plates and cultured for 12 h to adhere. Various compound concentrations (20, 40, 60, 80, and 100 µmol/mL) were added to the wells and incubated at 37°C for 24 or 48 h. Then, 10 µL of CCK‐8 reagent was added to each well, followed by additional incubation for 3 h. Absorbance was measured at 450 nm.

2.2.3. Structure Elucidation of EFSS Polyphenolic Monomers

For LC‐mass spectrometry (LC‐MS) analysis, 0.10 mg of each compound was dissolved in 1 mL of HPLC‐grade methanol. The LC‐MS analysis was performed using a Waters BEH C18 column (100 × 1.1 mm, 1.7 µm), with a mobile phase consisting of acetonitrile (A) and 0.2% acetic acid aqueous solution (B). The linear gradient elution program was as follows: 0–25 min, 10%–25% A; 25–35 min, 25%–40% A. The flow rate was set at 1 mL/min, and the detection wavelength was 280 nm. The injection volume was 1 µL, and the m/z scan range was 0–1000 to determine the molecular weight and observe fragment ion peaks.

For nuclear magnetic resonance (NMR) analysis, dissolve 5.00 mg of the compound in DMSO‐D6 or Methanol‐D4 and transfer to an NMR tube. The ¹H‐NMR and ¹³C‐NMR spectral ranges are set at −2 to 13 ppm and −20 to 230 ppm, respectively.

2.2.4. Quantification of Polyphenolic Monomers in EFSS Extract

A sample was prepared by dissolving 1.00 mg of the polyphenol extract from EFSS in 1 mL of HPLC‐grade methanol and filtering it through a 0.22‐µm organic membrane. Standard solutions of ellagic acid, corilagin, and geraniin were prepared using the same method and diluted to concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL. HPLC analysis was performed under the following conditions: an Agilent ZORBAX SB‐C18 column (4.6 × 250 mm, 50 µm) was used, with the mobile phase and gradient program matching those in the previous LC‐MS analysis. The injection volume was 20 µL.

2.2.5. Evaluation of Compound Effects on Tumor Cell Proliferation Using the CCK‐8 Assay

The effects of the compounds and EFSS ethanol extract on cell proliferation were evaluated using the CCK‐8 assay. The extract's impact on the proliferation of HeLa, A2780, SGC‐7901, and HepG2 cells was assessed over 48 h. Additionally, the inhibitory effects of the isolated compounds on A2780 and HeLa cells were analyzed during the same period.

2.2.6. Scratch Assay to Determine Compound Effects on Cell Migration

For the scratch assay, six‐well plates were seeded with HeLa and A2780 cells (3 × 10⁶ cells/well) and incubated for 12 h to allow cell adhesion. A sterile 200‐µL pipette tip was used to create a vertical scratch, and the detached cells were removed by washing twice with phosphate‐buffered saline (PBS). Compounds and 5‐FU were added to each well at a concentration of 40 µmol/mL, followed by incubation at 37°C. Initial scratch areas were photographed and measured using an inverted microscope(S₀). After 24 h of incubation, the scratch areas in the treated group and the control group were measured again (S₁). The cell migration inhibition rate was calculated using the formula (1):

$$\mathrm{Cell}\mathrm{migration}\mathrm{inhibition}\mathrm{rate}\left(\%\right)=\left(S_0-S_1\right)/S_0\times 100\%$$ (1)

2.2.7. Transwell Assay to Assess Compound Effects on Cell Invasion

For the Transwell assay, Transwell chambers were coated with 50 µL of a Matrigel: Dulbecco's Modified Eagle Medium (DMEM) (1:9) mixture, incubated at 37°C for 6 h, and then immersed in DMEM for 60 s before use. Compounds and 5‐FU were diluted in serum‐free medium to a concentration of 40 µmol/mL. HeLa and A2780 cells (3 × 10⁶ cells/well) were seeded into the Transwell chambers and cultured in drug‐containing serum‐free medium. The lower chamber contained 500 µL of medium with 20% fetal bovine serum. After incubation at 37°C for 24 h, the medium from the upper chamber was discarded, and the chamber was rinsed twice with PBS and gently wiped with a cotton swab. The chamber was then immersed in methanol for 30 min and stained with 0.1% crystal violet for 15 min. Finally, the cells were photographed and counted under an inverted microscope. The blank control group was denoted S₀, and the experimental group was denoted S₁. The cell invasion rate was calculated using the formula (2):

$$\mathrm{Cell}\mathrm{invasion}\mathrm{rate}\left(\%\right)=S_1/S_0\times 100\%$$ (2)

2.3. Data Statistics and Analysis

All experiments were performed with three independent biological replicates (n = 3), and data are presented as mean ± standard deviation (X ± SD). Cell migration scratch areas and cell invasion cell counts were processed using ImageJ software. Statistical analysis was conducted using GraphPad Prism 8.0: two‐group comparisons by independent‐samples t‐test (*p < 0.05, **p < 0.01, and ***p < 0.001), and multi‐group comparisons by one‐way analysis of variance followed by least significant difference post‐hoc test.

3. Results

3.1. Isolation and in Vitro Antitumor Activity of EFSS Polyphenols

Four fractions obtained from EFSS were evaluated for their impact on HeLa cell proliferation via CCK‐8 assay, as shown in Table 1. While all fractions inhibited HeLa cell growth, the ethyl acetate extract exhibited significantly greater inhibitory effects than the petroleum ether extract, n‐butanol extract, and extract residue across tested concentrations (p < 0.05), with notably higher inhibition rates even at lower dosages. Due to its superior anti—proliferative activity, subsequent purification efforts were concentrated on the ethyl acetate extract.

TABLE 1.

Effects of Euryale ferox Salisb. shell (EFSS) fractionated extracts on HeLa cell proliferation after 24‐h exposure.

Sample	Concentration (µg/mL)	Inhibition rate of cell proliferation (%)
Control	—	0.00 ± 2.75
Petroleum ether extract	20	8.14 ± 2.90 ***
	60	17.29 ± 3.89 ***
	100	30.17 ± 1.71 ***
Ethyl acetate extract	20	23.13 ± 1.76 ***
	60	49.45 ± 1.47 ***
	100	79.13 ± 3.21 ***
n‐Butanol extract	20	20.11 ± 1.54 ***
	60	36.14 ± 1.39 ***
	100	50.17 ± 2.12 ***
Extract residue	20	9.31 ± 1.22 ***
	60	20.13 ± 2.01 ***
	100	27.11 ± 1.76 ***

Note: Results are presented as means ± SD (n = 6); *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control.

After initial separation of the ethyl acetate extract via silica gel column chromatography, the active components were primarily concentrated in fractions Fr.3, Fr.4, and Fr.6 (Table 2), with inhibitory rates of 50.89% ± 3.01%, 43.79% ± 1.26%, and 42.77% ± 1.67%, respectively, all significantly higher than other components (p < 0.05). Further purification of these fractions identified Fr.3.3, Fr.4.2, and Fr.6.4 as highly bioactive. Subsequent purification using gel and semi‐preparative HPLC yielded three compounds: a yellow amorphous powder A (22.013 mg), a white amorphous powder B (27.209 mg), and a white amorphous powder C (17.294 mg).

TABLE 2.

Effects of ethyl acetate‐separated fractions on HeLa cell proliferation after 24‐h exposure.

Sample	Concentration (µg/mL)	Inhibition rate of cell proliferation (%)
Control	—	0.22 ± 2.51
Fr.1	60	2.12 ± 1.41
Fr.2	60	12.13 ± 2.62 ***
Fr.3	60	42.77 ± 1.67 ***
Fr.4	60	50.89 ± 3.01 ***
Fr.5	60	20.12 ± 1.31 ***
Fr.6	60	43.79 ± 1.26 ***
Fr.7	60	15.31 ± 1.78 ***
Fr.8	60	24.90 ± 1.79 ***
Fr.9	60	6.94 ± 2.42 ***
Fr.10	60	9.13 ± 1.03 ***
Fr.3.1	60	1.39 ± 1.04
Fr.3.2	60	14.91 ± 2.17 ***
Fr.3.3	60	46.14 ± 1.22 ***
Fr.3.4	60	20.19 ± 1.46 ***
Fr.3.5	60	17.22 ± 1.48 ***
Fr.4.1	60	22.05 ± 2.13 ***
Fr.4.2	60	61.4 ± 2.09 ***
Fr.4.3	60	10.22 ± 3.49 ***
Fr.4.4	60	8.10 ± 1.73 ***
Fr.6.1	60	9.48 ± 2.19 ***
Fr.6.2	60	7.47 ± 1.21 ***
Fr.6.3	60	16.98 ± 2.89 ***
Fr.6.4	60	47.01 ± 1.54 ***
Fr.6.5	60	20.12 ± 3.04 ***

Note: Results are presented as means ± SD (n = 6); *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control.

3.2. Structural Characterization of Polyphenolic Monomers

Compound A, a yellow amorphous powder, electrospray ionization‐MS (ESI‐MS) m/z: 301 [M—H]⁻; ¹H‐NMR (600 MHz, DMSO): δ 7.46 (2H,s,H5,5'). ¹³C‐NMR (151 MHz, DMSO): 107.69 (C1,1'), 110.26 (C5,5'), 112.32 (C6,6'), 136.40 (C2,2'), 139.55 (C3,3'), 148.12 (C4,4'), and 159.13 (C7,7') (Supporting Information S1). The NMR data were consistent with literature reports [17], confirming that Compound A is ellagic acid, with a molecular weight matching the data. Its structural formula is shown in Figure 1A.

FIGURE 1.

Structural formulas of compounds (A) (Ellagic acid), (B) (Corilagin), and (C) (Geraniin).

Compound B, a white amorphous powder, ESI‐MS m/z: 633[M—H]⁻; ¹H‐NMR (600 MHz, MeOD): δ 3.98 (1H,br,s,H‐2), 4.15 (1H,dd,J = 8.5,11.0 Hz,H‐6), 4.46 (1H,br,s,H‐4), 4.50 (1H,t,J = 8.4 Hz,H‐5), 4.80 (1H,br,s,H‐3), 4.96 (1H,t), 6.37 (1H,s,H‐1), 6.66 (1H,s), 6.69 (1H,s) 7.05 (2H,s). ¹³C‐NMR (151 MHz, MeOD): 62.50 (C‐4), 65.05 (C‐6), 69.52 (C‐2), 71.70 (C‐3), 76.23 (C‐7), 95.04 (C‐1), 108.33 (C‐B5), 110.16 (C‐C5), 110.96 (2C,C‐A2,6), 116.74 (C‐B1), 117.23 (C‐C1), 120.64 (C‐A1), 125.48 (C‐C2), 125.51 (C‐B2), 137.71 (C‐C3), 138.20 (C‐B3), 140.43 (C‐A4), 145.25 (C‐C4), 145.34 (C‐B4), 145.65 (C‐C6), 146.07 (C‐B6), 146.42 (2C‐A3,5), 166.72 (C‐A7), 168.55 (C‐C6), 170.15 (C‐B7) (Supporting Information S1). NMR data consistent with literature reports [18, 19]. Combined with molecular weight information, Compound B was identified as corilagin, with its structural formula shown in Figure 1B.

Compound C, a yellow amorphous powder, ESI‐MS m/z: 951 [M—H]⁻, ¹H‐NMR (600 MHz, MeOD): δ 4.33 (1H, dd), 4.43 (1H,dd, J = 8.0 Hz,10.5 Hz, HB‐6), 4.61 (1H,d), 4.85 (1H,m,HA‐5), 4.95 (1H,m,HB‐6), 5.01 (1H,t,J = 10.5 Hz,H4‐6), 5.20 (1H,s), 5.48 (1H,br,s,HB‐4), 5.51 (1H,br,s), 5.61 (1H,br,s), 6.24 (1H,d), 6.52 (1H,br,s,HB‐1), 6.59 (1H,s,HA‐1), 6.65 (1H,s), 6.88 (1H,s,HA‐3), 7.12 (1H,s,HB‐3), 7.22 (2H,s), 7.37 (1H, s,HD‐3). ¹³C‐NMR (151 MHz, MeOD): 47.46 (C35), 48.17 (C5), 51.26 (C4), 69.38 (C3), 72.25 (C2), 91.27 (C1), 106.83 (C19), 107.35 (C9), 107.54 (C30), 108.99 (C15), 109.47 (C14), 109.67 (C13), 112.57 (C23), 114.86 (C27), 118.70 (C29), 118.82 (C8), 123.60 (C22), 123.98 (C34), 136.2 (C32), 136.78 (C11), 137.31 (C17), 139.43 (C31), 139.48 (C33), 143.94 (C24), 144.17 (C10), 144.24 (C12), 144.72 (C25), 145.11 (C23), 145.15 (C16), 145.21 (C36), 145.51 (C37), 154.21 (C18), 154.65 (C20), 154.81 (C26), 164.21 (C40), 164.90 (C21), 165.02 (C7), 165.5 (C28), 166.06 (C24), 168.65 (C41), 193.72 (C38) (Supporting Information S1). Showed NMR data that aligned with literature data [20, 21]. Based on this and its molecular weight, Compound C was determined to be geraniin, and its structural formula is presented in Figure 1C.

3.3. Quantification of Polyphenolic Monomers in EFSS Extracts

To determine the content of ellagic acid, corilagin, and geraniin in the ethanol extract of EFSS, HPLC analysis was performed (Figure 2). Corilagin was found to have the highest content at approximately 66.23 mg/g, followed by geraniin at about 58.41 mg/g, while ellagic acid had the lowest content at roughly 31.42 mg/g.

FIGURE 2.

High‐performance liquid chromatography (HPLC) profiles of Euryale ferox Salisb. shell (EFSS) polyphenolic extract and mixed standards.

3.4. Effects of EFSS Extracts and Polyphenolic Monomers on Tumor Cell Proliferation

To investigate the antiproliferative effects of EFSS ethanol extract on tumor cells, four cancer cell lines (SGC‐7901, A2780, HepG2, and HeLa) were treated with varying concentrations (50–300 µg/mL) of the extract for 48 h, followed by a CCK‐8 assay for cell viability. The results, depicted in Figure 3, showed dose‐dependent growth inhibition across all cell lines, with stronger effects on HeLa and A2780 cells (IC₅₀ values of 68.56 and 78.09 µg/mL, respectively).

FIGURE 3.

The ethanol extract of Euryale ferox Salisb. shell (EFSS) exerts inhibitory effects on the proliferation of four types of tumor cells over a 48‐h period. Results are presented as means ± SD (n = 6).

To further explore the antitumor bioactivity of the compounds isolated from EFSS, the three compounds were individually applied to two human tumor cell lines, HeLa and A2780, to evaluate their inhibitory effects on cell proliferation after 24 and 48 h. The results showed (Figure 4a,b) that all three compounds significantly inhibited the proliferation of HeLa cells in a time‐ and dose‐dependent manner. The IC₅₀ values at 24 and 48 h indicated that the order of inhibitory effects on HeLa cell proliferation was corilagin > geraniin > ellagic acid. At a concentration of 60 µmol/mL, corilagin exhibited inhibitory rates of (66.55 ± 2.01)% and (74.61 ± 2.31)% on HeLa cell proliferation at 24 and 48 h, respectively, showing no significant difference compared to 5‐Fu. Geraniin demonstrated slightly weaker effects, with inhibitory rates of (46.76 ± 1.92)% and (52.34 ± 2.14)% at 24 and 48 h, respectively. Ellagic acid had the weakest inhibitory effect, with rates of (39.26 ± 2.54)% and (50.92 ± 1.95)% at 24 and 48 h, respectively.

FIGURE 4.

Compounds inhibited the proliferation of HeLa and A2780 cells at 24 and 48 h. (a) The 24 h proliferation inhibition rate of compounds on HeLa cells. (b) The 48‐h proliferation inhibition rate of compounds on HeLa cells. (c) The 24‐h proliferation inhibition rate of compounds on A2780 cells. (d) The 48‐h proliferation inhibition rate of compounds on A2780 cells. Results are presented as means ± SD (n = 6).

However, the effects of the three compounds on A2780 cell proliferation showed slight differences compared to HeLa cells. Corilagin and geraniin inhibited A2780 cell proliferation in a time‐ and dose‐dependent manner, while ellagic acid exhibited some inhibitory effects that did not increase significantly with higher concentrations (Figure 4c,d). The order of inhibitory effects on A2780 cell proliferation was corilagin > geraniin > ellagic acid. At a concentration of 100 µmol/mL, corilagin showed no significant difference in inhibitory effects compared to the positive control 5‐Fu.

3.5. Effects of Polyphenolic Monomers on Tumor Cell Migration

To evaluate the effects of the three natural compounds on tumor cell migration, scratch assays were performed on HeLa and A2780 cell lines. The results (Figure 5) showed varying effects of the compounds on cell migration. Corilagin reduced migration rates to (−8.35 ± 1.35)% and (−21.15 ± 1.65)% in the two cell lines, respectively. Geraniin exhibited migration rates of (−8.21 ± 1.33)% in HeLa cells and (−1.48 ± 1.19)% in A2780 cells. Notably, ellagic acid significantly inhibited migration in HeLa cells (11.16 ± 1.24)% but had no marked effect on A2780 cells. These findings indicate that 5‐FU, corilagin, and geraniin significantly suppressed migration in both cell lines, while ellagic acid may specifically promote migration in HeLa cells. Overall, the effects of these compounds on cell migration showed cell line‐dependent differences.

FIGURE 5.

Ellagic acid, corilagin, and geraniin inhibited the migration of HeLa and A2780 cells. (a) Inhibition rate of the compound on HeLa cell migration. (b) Inhibition rate of the compound on A2780 cell migration. (*p < 0.05; **p < 0.01; ***p < 0.001). Results are presented as means ± SD (n = 3).

3.6. Effects of Polyphenolic Monomers on Tumor Cell Invasion

To investigate the effects of the three compounds on tumor cell invasion, Transwell invasion assays were performed on HeLa and A2780 cell lines. To minimize the impact of cell proliferation inhibition on migration, a drug concentration of 40 µmol/mol was selected for the experiments. The results (Figure 6) showed that all three compounds significantly inhibited the invasion of both HeLa and A2780 cells, with the order of effectiveness being 5‐FU > corilagin > geraniin > ellagic acid. Corilagin and geraniin exhibited significant inhibitory effects on the invasion of both cell lines, with invasion inhibition rates of (66.67 ± 1.69)% and (63.33 ± 1.02)% in HeLa cells, and (69.86 ± 1.33)% and (61.64 ± 1.48)% in A2780 cells, respectively. In contrast, ellagic acid showed a significantly higher inhibitory effect on HeLa cell invasion (38.89 ± 1.01)% compared to A2780 cells (13.69 ± 1.21)%. These findings indicate that 5‐FU significantly inhibited invasion in both cell lines, while ellagic acid specifically promoted invasion in A2780 cells but had minimal effect on HeLa cells. Corilagin and geraniin selectively enhanced invasion in HeLa cells but did not significantly affect A2780 cells, suggesting that the regulatory effects of these compounds on cell invasion are cell type‐dependent.

FIGURE 6.

Ellagic acid, corilagin, and geraniin inhibited the invasion of HeLa and A2780 cells. (a) Invasion rate of compound on HeLa cells. (b) Invasion rate of compound on A2780 cells. (*p < 0.05; **p < 0.01; ***p < 0.001). Results are presented as means ± SD (n = 3).

4. Discussion

As an underutilized biomass resource in the Euryale ferox Salisb. processing industry, the high‐value utilization of EFSS aligns with sustainable development and holds strategic importance for expanding natural antitumor drug sources. This study first systematically reveals the antitumor activity of EFSS polyphenol extracts and their monomeric components—corilagin, geraniin, and ellagic acid—providing critical scientific support for transforming EFSS from agricultural waste to functional ingredients.

Bioactive tracking was used to isolate antitumor components from the ethanol extract of EFSS. The ethyl acetate extract (Fr.3, Fr.4, and Fr.6) showed significantly higher proliferation inhibition in HeLa cells than other fractions (Table 2), likely due to its high content of hydrolyzable tannins. LC‐MS and NMR identified three compounds with typical polyphenol structures. The high content of corilagin (66.23 mg/g) and geraniin (58.41 mg/g) explained the core activity of the ethyl acetate layer.

Ellagic acid, a natural polyphenol, primarily functions as an antioxidant and anti‐inflammatory agent. This study showed some antiproliferative activity against HeLa and A2780 cells, though significantly less than corilagin and geraniin. Specifically, it inhibited HeLa cell proliferation by 39.26% at 24 h and 50.92% at 48 h. While its antitumor effects are weaker than the other two compounds, it still demonstrates cell proliferation inhibition. This activity is likely linked to its strong antioxidant properties, as it can suppress tumor growth by reducing oxidative stress within tumor cells [22]. However, its weaker concentration‐dependent effect on A2780 cells indicates varied mechanisms across different tumor cell types. Some studies suggest that ellagic acid may inhibit tumor cell proliferation and migration by influencing signaling pathways such as PI3K/Akt [23].

Corilagin exhibited the strongest antitumor activity in this study, significantly inhibiting the proliferation of HeLa and A2780 cells. At higher concentrations, it achieved inhibition rates exceeding 70% after 24 and 48 h, comparable to the chemotherapeutic drug 5‐FU. Corilagin also showed marked inhibition of tumor cell migration and invasion, particularly in HeLa cells, where it achieved a 66.67% inhibition rate. These results suggest that corilagin may suppress tumor cell proliferation, migration, and invasion through multiple mechanisms. Previous studies have shown that corilagin can inhibit cell proliferation by causing cell cycle arrest and inducing apoptosis [24]. Chen et al. found that corilagin in the aqueous extract of Polygonum multiflorum upregulates the expression of P53, Bcl‐2, and caspase 3/9 while downregulating Bax in SiHa cells, thereby inducing apoptosis [25]. Additionally, corilagin's antitumor effects may involve reducing reactive oxygen species accumulation in tumor cells through antioxidant actions, thereby slowing tumor growth and spread [26]. Huang et al. demonstrated that corilagin inhibits mouse OS tumor growth by inducing autophagy‐dependent apoptosis [27].

Geraniin, another natural polyphenol with antitumor properties, showed significant antiproliferative, antimigratory, and anti‐invasive effects in both HeLa and A2780 cells, although its potency was less than that of corilagin. In this study, geraniin achieved inhibition rates of 46.76% and 52.34% in HeLa cells after 24 and 48 h, respectively, and exhibited milder but still notable effects in A2780 cells, surpassing ellagic acid in efficacy. Geraniin also demonstrated clear inhibitory effects on cell migration and invasion, indicating its potential to suppress tumor cell infiltration and metastasis. Its antitumor mechanisms may involve enhancing apoptosis and modulating signaling pathways such as MAPK and PI3K/Akt within tumor cells [28].

Notably, the three compounds showed higher sensitivity in HeLa cells than in A2780 cells, which may explain the greater inhibitory effect of the EFSS ethanol extract on HeLa cell proliferation. This difference in sensitivity likely stems from variations in tumor cell surface receptor distribution. HeLa cells, which highly express epidermal growth factor receptors, have been shown to specifically bind with polyphenolic compounds [29]. Moreover, the high selectivity of corilagin for HPV‐positive HeLa cells (Figure 3) may relate to p53 degradation induced by HPV E6/E7 proteins [30]. Research indicates that corilagin can restore p53 function by inhibiting MDM2 ubiquitin ligase activity [31], and the continuous degradation of p53 in HeLa cells makes them more responsive to this pathway modulation. In contrast, the overactivation of the PI3K/AKT pathway in A2780 cells may diminish the inhibitory effects of ellagic acid [32]. It is also important to note that the weak efficacy of ellagic acid against A2780 cells, with an invasion inhibition rate of 13.69%, may be attributed to its low lipophilicity, which results in poor membrane permeability. This is consistent with reports that nano‐encapsulation strategies can significantly enhance bioavailability [33].

5. Conclusion

Natural products, as potential sources for drug development, have garnered extensive attention in the field of cancer therapy. These compounds influence the occurrence and progression of tumors by regulating key biological processes such as cell proliferation, apoptosis, and migration. This study found that corilagin, ellagic acid, and geraniin can significantly inhibit the proliferation and migration abilities of tumor cells, indicating their potential antitumor activity. However, the specific mechanisms of action of these compounds, such as the signaling pathways they regulate, have not been fully elucidated. Future research should further explore their molecular targets. In addition, in vivo experiments are crucial for validating their antitumor effects and evaluating their potential clinical application value. This study provides experimental evidence for the application of natural products in cancer treatment.

Author Contributions

Xinru Jian: writing – original draft, formal analysis, methodology, investigation, validation, and data curation. Tiantian Shan: writing – original draft, methodology, and investigation. Yuhan Yang: methodology and investigation. Xinyi Lu: software and visualization. Haotian Ge: data curation. Longjie Li: Supervision. Hongxun Wang: resources and funding acquisition. Limei Wang: writing – review and editing, conceptualization, resources, supervision, project administration, and funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv70813‐sup‐0001‐SuppMat.pdf

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.