Natural Polysaccharides in Breast Cancer: Fucoidan’s Role in Enhancing Cisplatin Cytotoxicity and Reducing Chemotherapy Resistance (2025)

2.1 Materials

The MDA-MB 231 human breast cancer cell line was supplied by the National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) polycaprolactone (PCL) (80,000 Mn), 2,4,6-Tri (2-pyridyl)-s-triazine (TPTZ), ammonium persulfate ((NH₄)₂ S₂O₈), In vitro Toxicology Assay Kit (XTT based), penicillin-streptomycin and butylated hydroxyanisole (BHA) were purchased from Sigma Aldrich™ (USA). Roswell Park Memorial Institute (RPMI) and fetal bovine serum (FBS) were purchased from GIBCO™ (USA). Acrylamide was purchased from Pharmacia™ (USA). The Total RNA Isolation kit (Hybrid RTM) was purchased from GeneAll™ (South Korea). SYBR® Premix Ex Taq (Tli RNase H Plus) was purchased from Takara Bio™ (Japan). High Capacity cDNA Reverse Transcription Kit was purchased from Applied Biosystems™ (USA). Pierce bicinchoninic acid (BCA) Protein Assay Kit was purchased from Thermo scientific™ (USA). Agarose gel, ethanol 96%, acetic acid (CH₃COOH), trypsin-ethylene diamine tetra acetic Acid (EDTA), and tetramethylethylenediamine (TEMED), Triton X-100, and Tris were purchased from Merck™ (Germany). All other chemicals and solvents were of the highest grade commercially available.

2.2 Sample preparation

S. ilicifolium was collected from the Persian Gulf Beach in Hormozgan province, Iran. The Taxonomy Laboratory at the Faculty of Pharmacy, Mazandaran University of Medical Science, Iran, identified and confirmed the sample to ensure accurate taxonomic classification. The brown algae were meticulously cleaned to remove epiphytes, waste, and necrotic tissue. After rinsing with fresh water, the algae were sun-dried and placed in an oven at 40°C for 2 days to ensure thorough dehydration. Then, a freeze dryer (Christ™, Germany) was employed at −20°C for 3days. The dried algae were then finely cut and ground into a fine powder using a mixer grinder (Yazicilar™, Turkey). The resulting powder was stored in a dry, clean, and sealed container to maintain its integrity.

2.3 Extraction and purification of sodium alginates

Alginate extraction from S. ilicifolium was conducted following a high-temperature alkaline extraction method, according to Bouissil et al. Initially, 85 grams of depigmented and dried algae were subjected to two rounds of treatment with 500ml of 0.1M HCl at 60°C, maintaining a pH of 2.0 with continuous stirring (Drawell™, China) at 250rpm for 2hours. The mixture was centrifuged (Kaida™, China) at 5000rpm for 15minutes at 4°C. The resulting residue, or pellet, was rinsed with distilled water and subsequently treated with a 3% w/v solution of sodium carbonate (Na₂CO₃) at pH11.0 for 2hours at 60°C, again under constant stirring. The supernatants from this process were collected and precipitated using three volumes of ice-cold 96% ethanol. The precipitate was further centrifuged and resuspended in distilled water before being acidified with 6M HCl to a pH below 3.0, precipitating alginic acid. This acid was then redissolved in distilled water and neutralized to pH7.5. The final purification of alginates involved three successive precipitations using ice-cold 96% ethanol. The resulting precipitate was resuspended in distilled water and lyophilized to produce powdered sodium alginates from S. ilicifolium algae [17].

2.4 Extraction and purification of fucoidan

Since fucoidans are polar, hydrophilic, and water-soluble macromolecules, water is an excellent solvent for their extraction. Fucoidan was extracted from 180g of S. ilicifolium powder using 40ml of water heated to 70°C. The extract was then purified through ethanol precipitation. To further remove alginate, 1.5ml of 3M CaCl₂ was added. Lipids and pigments were effectively eliminated using a solvent mixture of acetone and diethyl ether. Finally, the sediment was dried at 40°C for 24hours, yielding a concentrated fucoidan extract [18, 19].

2.5 Preparation of polysaccharides from the algae

About 10g of dried and finely S. ilicifolium algae was subjected to extraction using distilled water heated to 90°C in a water bath for 3hours. Following this initial extraction, the mixture was filtered, and the remaining solid residue underwent a second extraction using distilled water under the same conditions for another 3hours. The combined extracts were then centrifuged to eliminate any impurities. The clear supernatant obtained from this process was concentrated using rotary evaporation to refine the extract further [20].

2.6 Total carbohydrate determination

The phenol-sulfuric acid method was used to quantify the total carbohydrate content in polysaccharides. In this procedure, 2.5ml of 96% sulfuric acid was added to 10μL of the sample at a concentration of 1mg/ml and to a series of standard glucose solutions ranging from 5 to 200μg/ml. Then, 500μL of a 4% phenol solution was quickly added into each tube. The resulting mixture was then incubated in a water bath at 90°C for 5minutes. A standard curve was created using various concentrations of D-glucose to ensure accurate quantification [21].

2.7 Structural analysis of fucoidan and alginate

The UV–Vis absorbance spectrum of alginate extracted from S. ilicifolium was measured using a Double Beam T80 series spectrophotometer (PG Instruments™, China) across the 200–800nm wavelength range. To analyze the composition and block structure of the extracted alginate, ¹H NMR spectroscopy was employed, with the alginate sample prepared at a concentration of 6mg/ml in deuterium oxide (D₂O). For infrared (IR) analysis, the dried alginate sample was finely ground and mixed with anhydrous potassium bromide (KBr) to form pellets. The IR spectra were then recorded at room temperature, covering a frequency range of 650–4000cm⁻¹ using a Fourier-transform infrared (FTIR) spectrometer [22].

2.8 Determination of antioxidant capacity

2.9 Cell culture

MDA-MB 231 cells were cultured in RPMI-1640 medium enriched with 10% heat-inactivated fetal bovine serum (FBS), 100IU/ml penicillin, and 100μg/ml streptomycin. The cultures were maintained in a controlled environment at 37°C with 5% CO₂ to ensure optimal growth conditions. The cells were subcultured twice weekly, starting at approximately 5×10⁵ cells/ml density. Cell viability was assessed using the trypan blue exclusion method, which selectively stains non-viable cells, allowing for accurate viability measurements [25].

2.10 Cytotoxicity evaluation of fucoidan, sodium alginates, and cisplatin on MDA-MB-231 cancer cells

To assess their response, MDA-MB-231 cell lines were exposed to a range of fucoidan (0–800μg/ml), sodium alginates (0–350μg/ml), and cisplatin (0–20μg/ml) concentrations. IC50 was determined using the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay, which measures cell viability based on metabolic activity. In the experiment, 2×10⁴ MDA-MB-231 cells were seeded into each well of a 96-well plate, followed by adding 0.1ml of medium containing different concentrations of fucoidan, sodium alginates, and cisplatin. After incubating the cells for 48hours at 37°C in a 5% CO₂ atmosphere, the XTT reagent was introduced to each well. The absorbance was then measured at 450 and 620nm using a multifunctional ELISA reader. To ensure the reliability of the results, all experiments were repeated three times to ensure accuracy [27].

2.11 Drug interaction analysis in MDA-MB-231 cells

The analysis of drug interactions was conducted using CompuSyn software, which utilizes the modified Chou-Talalay Combination Index (CI) method. This software helps calculate the required doses of drugs, individually and in combination, to achieve specific levels of cytotoxicity. The software calculates a Combination Index (CI) for each cytotoxicity level to evaluate the drug interaction. In the study, MDA-MB-231 cells were treated with 3.6μg (IC₂₀) of cisplatin and various concentrations of fucoidan and sodium alginates-rich extract to assess the interactions. The CI values were used to determine the type of interaction: synergistic (CI<1), additive (CI=1), or antagonistic (CI>1).

The outcomes of these interactions were visualized using several plots:

1. Dose-effect curve: This plot shows the dose-response relationship for both the drug and the extract, individually and in combination.

2. Fa-CI plot: It differentiates between antagonism (CI>1) and synergism (CI<1) based on the Combination Index.

3. Median-effect plot: This plot illustrates the relationship between the drug dose (Log D) and the effect (Log Fa/Fu). The IC₅₀ value is identified where the curve intersects the X-axis.

4. Dose-normalized isobologram: Points on this plot show the interaction between the drug and the extract, indicating synergism, antagonism, or an additive effect based on their position relative to the diagonal line [28].

2.12 Measurement of IL-1 and IL-6 from a THP-1 monocyte cell line stimulated with bacterial lipopolysaccharide (LPS) and treated with non-toxic concentrations of an alginate and fucoidan-rich extract

Human acute monocytic leukemia cells (THP-1) supplied by Pasteur Institute of Iran were cultured in RPMI 1640 medium with 10% fetal bovine serum and 100μM penicillin-streptomycin under conditions of 95% air and 5% CO₂. In the experiment, 4×10⁴ Differentiated THP-1 cells were seeded into each well of a 96-well plate and then exposed to 1μg/ml of bacterial LPS for 24hours along with concentrations of 5 and 50μg/ml of fucoidan-rich extract and with concentrations of 25 and 250μg/ml of alginate-rich extract given in duplicate for each cultured sample. After 48hours, the cells were collected, and the supernatants were stored at −20°C for cytokine analysis. The cell pellets were washed with phosphate-buffered saline (PBS), centrifuged at 1000rpm for 8minutes, and then stored at −20°C. According to the manufacturer’s instructions, IL-1 and IL-6 levels were evaluated using commercially available ELISA kits [29].

2.13 Cytotoxicity evaluation of alginate and fucoidan-rich extract in a 3D culture model

The fabrication and design of the nano-scaffold were performed using an electrospinning device (Fanavaran Nano Meghyas™, Iran). The setup involved an injection flow rate of 1ml/h, a needle distance of 15cm, and a total volume of 10ml. The structural characteristics of the Electrospun Polycaprolactone (PCL) nano-scaffolds were analyzed using scanning electron microscopy (SEM), which provided detailed visualization of the fiber alignment and diameter. To improve the surface properties of the PCL nanofibers, they were treated with O₂ plasma to increase hydrophilicity. This modification was performed for 5minutes at a pressure of 0.4 millibars and a power of 30W in a microwave plasma chamber. The plasma treatment was critical for enhancing cell adhesion and proliferation by making the nanofiber surface more wettable. For sterilization, the PCL nanofibers were treated with 70% ethanol, eliminating microbial contaminants. Post-sterilization, the nanofibers were rinsed three times with phosphate-buffered saline (PBS) to remove any ethanol residues. The nanofibers were then freeze-dried, preserving the scaffold’s structural integrity while maintaining its mechanical properties. Combining SEM analysis, plasma surface modification, ethanol sterilization, and freeze-drying produced high-quality PCL nano-scaffolds ideal for applications in 3D cell culture [30].

2.14 Cytotoxicity evaluation of fucoidan-rich extract and cisplatin on MDA-MB-231 cells in a PCL polymeric nano-scaffold

After determining the IC₅₀ values for both the fucoidan-rich extract and cisplatin in prior experiments, the cells were treated with varying concentrations—ranging from 0 to 1000μg/ml of the fucoidan-rich extract and 2.1μM of cisplatin—under conditions that closely resemble the physiological environment. 3×10⁴ cells were seeded into each well of a 96-well plate and allowed to adhere to a polymeric scaffold for 24hours. Subsequently, different concentrations of the extract and paclitaxel were added in triplicate to each well. An equivalent volume of PBS was added in triplicate for the negative control. The cells were incubated for 48hours at 37°C with 5% CO₂. Following incubation, cell viability was assessed using the XTT assay, with absorbance readings taken at 450 and 620nm using an ELISA reader. The entire experiment was conducted three times to ensure reproducibility and accuracy [31].

2.15 Data analysis and statistics

Results are expressed as the mean±standard deviation (SD). Statistical analyses were conducted utilizing GraphPad Prism version 6 and SPSS version 16. The Kolmogorov-Smirnov test was applied to assess the normality of data distribution. For analyzing the data, paired t-tests were used for normally distributed variables, while the Wilcoxon signed-rank test was employed for non-parametric data. A P-value <0.05 was considered statistically significant in all analyses.

3.1 Total sodium alginates and fucoidan determination

The extraction yields of fucoidan and alginate from the dried algae extract were determined to be 7203 and 441mg per 100 grams of dried extract, respectively. This indicates that 7.2% of the algae extract’s dried weight consisted of fucoidan, while 0.44% was alginate.

3.2 Total carbohydrate determination

The polysaccharide fraction from S. ilicifolium was obtained through a series of processes, including hot water extraction, ethanol precipitation, and dialysis. Using a calibration curve (y=0.0621x+0.0084, r²=0.999) in absorbance of 490nm created with standard glucose solutions, the total polysaccharide content in the fraction was estimated to be 89.39mg/g dried algae weight.

3.3 Antioxidant properties of fucoidan and alginate

3.4 FTIR spectrum of sodium alginate

The FTIR spectrum of sodium alginate (Figure 1) identified various chemical functional groups. A prominent broad absorption band at 3465cm⁻¹ indicates the hydroxyl group’s (‒OH) stretching vibrations. Additionally, peaks within the 2845 to 2930cm⁻¹ range correspond to the stretching vibrations of the methylidyne group (‒CH). Significant peaks observed within 1599±4 and 1410±4cm⁻¹ are associated with asymmetric and symmetric stretching of carboxylate anions (COO‒), respectively. The spectrum also displays a shoulder band at 1083.444cm⁻¹, reflecting C‒C and C‒O stretching vibrations, which suggest cross-linking within the alginate structure. Moreover, the peak at 1022.493cm⁻¹ is attributed to C‒C stretching vibrations, further characterizing the molecular framework.

3.5 FTIR spectrum of fucoidan

The FTIR spectrum of fucoidan revealed several key chemical functional groups (Figure 2). A broad and intense absorption band within the 3000 to 3600cm⁻¹ range signifies the stretching vibrations of hydroxyl groups (‒OH), indicative of hydrogen bonding, commonly associated with polysaccharides. Additionally, absorption peaks between 2845 and 2930cm⁻¹ correspond to the stretching vibrations of the methylidyne (‒CH) group. Further analysis shows spectral peaks between 1610 and 1651cm⁻¹, attributed to carbonyl (C〓O) and carbon-carbon double bond (C〓C) stretching vibrations. These are linked to the presence of uronic acids, suggesting alginic acid as part of the alginate polymers in the sample. Peaks between 1420 and 1445cm⁻¹ are possibly related to C‒H deformation or ‒OH rocking vibrations in monosaccharides. The FTIR spectrum also indicates the presence of C-O-C and C-O-S bonds, with observed peaks ranging from 387 to 1030cm⁻¹. Specifically, a peak near 1078.4cm⁻¹ signifies C‒O, C‒C, and C‒O‒H stretching vibrations in hydrocarbon structures. Lastly, the sharp absorption bands between 729 and 823cm⁻¹ are attributed to C‒O‒S bending vibrations, pointing to sulfate substitution.

3.6 UV-visible analysis

The UV-visible spectrogram of the alginate extract (Figure 3) displayed absorption peaks within the 200–400nm range, which are indicative of carboxylate groups and protein-related components. Peaks observed at 280, 370, and 405nm suggest the presence of phenolic compounds, flavonoids, and their derivatives. The data further highlighted absorption bands between 280 and 410nm, characteristic of aromatic and polycyclic aromatic compounds, including proteins and amino acids, supporting complex organic structures in the extract.

The spectrogram of the fucoidan extract (Figure 4) in the 300 to 400nm range appears abnormal, possibly due to impurities, sample preparation issues, or instrumental problems. Polysaccharides in their pure form are generally optically transparent and do not exhibit absorption in the UV-Vis region. The absorption peak around 230nm may indicate the presence of polyphenols in the sample.

3.7 Cytotoxicity evaluation of cisplatin on MDA-MB-231 cancer cell line

The XTT assay was employed to assess the cytotoxic effects of cisplatin on MDA-MB-231 breast cancer cells. According to the results depicted in Figure 5, different concentrations of cisplatin, ranging from 0 to 20μg/ml, were administered to the cells over 48hours. Cisplatin demonstrated an evident dose-dependent cytotoxicity, with the IC₂₀ and IC₅₀ values calculated at 3.6±0.4 and 15.33±0.9μg/ml, respectively.

3.8 Cytotoxicity evaluation of fucoidan-rich extract and its drug interaction analysis with cisplatin on MDA-MB-231 cancer cell line

The XTT assay was initially conducted to evaluate the impact of a fucoidan-rich extract on the viability of MDA-MB-231 breast cancer cells. As illustrated in Figure 6 (green line), various extract concentrations, ranging from 0 to 800μg/ml, were tested over 48hours. The results revealed that the fucoidan-rich extract induced a dose-dependent cytotoxic effect on the cells, with an IC₂₀ value (the concentration at which 80% of cells remain viable) of 57.1±2.9μg/ml and an IC₅₀ value of 174.9±8.7μg/ml.

Subsequently, the effects of combining different concentrations of the fucoidan-rich extract (ranging from 5 to 400μg/ml) with 3.6±0.4μg/ml (IC₂₀) of cisplatin were analyzed. As shown in Figure 6 (blue line), after 48hours of treatment, the combination significantly enhanced cytotoxicity, reducing the IC₅₀ from 174.9±8.7 to 79.3±4.6μg/ml and lowering the IC₂₀ from 57.1±2.9μg/ml to below 5.0±0.8μg/ml. This indicates a synergistic effect between the fucoidan-rich extract and cisplatin.

The synergistic interaction between the fucoidan-rich extract and cisplatin was validated through CompuSyn software analysis. This software calculated the Combination Index (CI) values for all tested concentrations, with values less than one consistently indicating a synergistic effect (as shown in Table 2). Among the tested concentrations, 200μg/ml demonstrated the highest synergy, with a CI value of 0.28518.

A detailed analysis using CompuSyn software generated several insightful plots (Figure 7).

1. Dose-effect curve (Figure 7A): The combination treatment demonstrated a more substantial dose-dependent effect than the extract alone. The green points, representing the combined treatment of cisplatin and extract, were positioned lower on the graph than the blue line, corresponding to the extract alone. This positioning indicates enhanced cytotoxicity when the treatments are combined.

2. Combination index plot (Figure 7B): All Fa-CI points fell below the line at 1, signifying synergistic effects at all concentrations tested. This confirms that combining treatments is more effective than either treatment alone.

3. Median-effect plot (Figure 7C): The log-transformed data revealed positive slope lines, indicating that higher doses correlate with increased cellular response. The point where the lines intersect the X-axis represents the combined IC₅₀ value, calculated at 79.3±4.6μg/ml.

4. Dose-normalized isobologram (Figure 7D): This plot demonstrated that all combined doses, represented by colored points within the triangle, displayed synergistic effects, with CI values consistently below one, reinforcing the enhanced effectiveness of the combination therapy. The colored points represent the concentration of the combined drug. The closer a point is to the left and bottom, the lower its CI number and the more potent its synergistic effect. Since all five concentrations combined with cisplatin fall within the triangle, each concentration demonstrates a synergistic effect with a CI of less than one.

3.9 Cytotoxicity evaluation of alginate-rich extract and its drug interaction analysis with cisplatin on MDA-MB-231 cancer cell line

As mentioned for fucoidan-rich extract, the XTT assay was used to assess the effects of an alginate-rich extract on the viability of MDA-MB-231 breast cancer cells. Figure 8 (blue line) shows that varying extract concentrations, ranging from 0 to 350μg/ml, were tested over 48hours. The findings indicated that the alginate-rich extract exhibited minimal toxicity toward the MDA-MB-231 cell line, with significant cytotoxicity observed only at concentrations exceeding 280μg/ml, surpassing the IC₂₀ threshold.

The combined effects of the various concentrations of alginate-rich extract (ranging from 0 to 350μg/ml) with 3.6μg/ml of cisplatin (IC₂₀) were evaluated. As shown in Figure 8 (green line), after 48hours of treatment, the combination did not result in any notable increase in cytotoxicity, suggesting that the alginate-rich extract did not enhance the cytotoxic effects of cisplatin in this cell line.

The interaction between the alginate-rich extract and cisplatin was further analyzed using CompuSyn software. The Combination Index (CI) values, calculated across all tested concentrations, revealed that the alginate-rich extract displayed both weak synergistic and antagonistic effects when combined with the IC₂₀ concentration of cisplatin (Table 2). Notably, the 50μg/ml concentration exhibited the strongest synergy, with a CI value of 0.83198 (Table 3).

A detailed analysis using CompuSyn software generated several detailed plots (Figure 9).

1. Dose-effect curve (Figure 9A): The combination of alginate and cisplatin did not increase toxic effects as the alginate concentration was raised. This is evident from the horizontal trend of the green triangles in the dose-effect plot, representing the combination of the alginate extract and cisplatin, which indicates that alginate has an insignificant impact on enhancing cisplatin’s toxicity. Moreover, the red line, which denotes the cytotoxic effect of various concentrations of cisplatin alone, demonstrates the potent toxicity of the drug.

2. Combination index plot (Figure 9B): In the Fa-CI diagram, two data points fell below the line, indicating a synergistic interaction at these two concentrations. However, an antagonistic interaction occurs at three other concentrations of Fa-CI points positioned above the line at 1.

3. Median-effect plot (Figure 9C): The log-transformed data revealed that increasing the dose does not lead to a significant rise in cytotoxic response due to the shallow slope of the combination curve for the drug and the extract. This indicates thatthe tested alginate concentrations did not reach the IC₅₀ threshold. Furthermore, the combination of alginate with cisplatin also failed to achieve an IC₅₀, reflecting thelimited enhancement in cytotoxicity when these two agents are used together.

4. Dose-normalized isobologram (Figure 9D): Doses represented within the triangle display a synergistic effect when combined with cisplatin, while the doses plotted outside the triangle exhibit antagonistic effects. The colored points represent the concentration of the combined drug. The closer a point is to the left and bottom, the lower its CI number and the more potent its synergistic effect. Two concentrations, which lie inside the triangle and close to the diagonal line, show only a weak synergistic effect. In contrast, three concentrations situated outside the triangle demonstrate antagonistic interactions.

3.10 IL-1β and IL-6 cytokine secretion from THP-1 monocyte cell line stimulated with S. ilicifolium fucoidan-rich extract

The impact of non-toxic doses (5 and 50μg/ml) of S. ilicifolium fucoidan-rich extract on cytokine secretion was evaluated using the THP-1 monocyte cell line. Lipopolysaccharide (LPS, 1μg/ml) was used as a positive control to stimulate cytokine secretion. The results revealed that, compared to the untreated control group, both the 5 and 50μg/ml doses of the extract significantly reduced IL-1β cytokine secretion by 14% (p-values=0.0411) and 37% (p-values=0.0022), respectively (Figure 10).

Similarly, for IL-6 cytokine, the 5 and 50μg/ml doses of the extract led to significant reductions of 16% (p-values=0.0411) and 42% (p-values=0.0022), respectively, as shown in Figure 11.

3.11 IL-1β and IL-6 cytokine secretion from THP-1 monocyte cell line stimulated with S. ilicifolium alginate-rich extract

The effect of non-toxic doses (50 and 250μg/ml) of S. ilicifolium alginate-rich extract on cytokine secretion was assessed using the THP-1 monocyte cell line, with lipopolysaccharide (LPS, 1μg/ml) serving as a positive control to induce cytokine production. The findings indicated that, compared to the untreated control group, both the 50 and 250μg/ml concentrations of the extract significantly decreased IL-1β cytokine secretion by 8 and 15%, with p-values of 0.0411 and 0.0022, respectively (Figure 12).

Similarly, the extract significantly reduced IL-6 cytokine levels by 5 and 13% at doses of 50 and 250μg/ml, with corresponding p-values of 0.0411 and 0.0022, as depicted in Figure 13.

3.12 Cytotoxicity evaluation of fucoidan-rich extract on cancer cells and its drug interaction analysis with paclitaxel on MDA-MB-231 cell line in a PCL polymeric nano-scaffold

Cell viability was measured using the XTT assay across a range of extract concentrations to assess the cytotoxic potential of a fucoidan-rich extract on MDA-MB-231 breast cancer cells. As depicted in Figure 14 (green line), after 48hours of treatment with concentrations between 0 and 800μg/ml, the extract exhibited evident dose-dependent cytotoxicity against MDA-MB-231 cells. The IC₂₀, or the concentration required to inhibit 20% of cell growth, was calculated at 262.1±19.7μg/ml, while the IC₅₀ exceeded 800±78.5μg/ml.

Furthermore, as shown in Figure 14 (blue line), the cytotoxic effect was notably enhanced when the extract was combined with 3.6μg/ml cisplatin for 48hours. The IC₂₀ dropped sharply from 262.1±19.3 to 7.2±1.4μg/ml, and the IC₅₀ decreased significantly from over 800±78.5 to 364.5±41.7μg/ml.