Bio-functional and anti-cancer (MCF-7) benzyl isothiocyanate nanoemulsions preparation using green pressurized water and process optimization

Abstract

Benzyl isothiocyanate (BITC), due to high biological activity, especially anti-cancer effect on breast cells has gained more attention in the medicine and pharmaceutical areas. But its low water solubility, environmental instability, and heat sensitivity decrease its applications in aqueous formulation. This study formulated BITC nanoemulsion (BITC NE) using pressurized water (PW) and based on the optimum amounts of BITC (36.5 µL) and Tween 80 (124 µL), to prepare BITC NE with high antioxidant (22.7%), antidiabetic (53.5%) and anti-inflammatory (99.6%) activities. Results indicated that the prepared BITC NE using obtained optimum amounts of its two main component had particle size, polydispersity index and zeta potential values of 180 nm, 0.236 and − 21.32 mV, respectively. Anti-cancer effect of the prepared and optimized BITC NE on breast cancer cell (MCF-7) was also evaluated. Results indicated that as compared to day 3, metabolic activity of MCF-7 cells in the control sample, were increased to 76 and 113% at days 5 and 7 of incubation, respectively. While, BITC NE with its amounts of 10, 50 and 100 µL, decreased that of the cancer cells up to 6.5, 18 and 74.7%, at day 7 of incubation respectively. Results of present study indicated that BITC, due to its dual function, as a natural bioactive compound and anti-cancer agent, is a promising component for further exploration in therapeutic development and functional food research.

Introduction

Benzyl isothiocyanate (BITC) is a naturally phytochemical compound belongs to the isothiocyanate family. It is primarily derived from the enzymatic hydrolysis of glucotropaeolin, a glucosinolate found in plants of the mustard family such as papaya seeds, garlic mustard and radish¹. BITC is characterized by the functional group –N = C=S attached to a benzyl moiety². BITC has attracted significant scientific interest due to its diverse biological activities and potential applications and in pharmaceutical and biomedical researches, it has been investigated for its anti-cancer, antimicrobial, antioxidant, antidiabetic and anti-inflammatory activities³. BITC shows antioxidant activity because it can both directly scavenge free radicals and indirectly boost cellular antioxidant defenses by activating protective enzymes and signaling pathways⁴. BITC has also antidiabetic activity because it improves glucose metabolism, enhances insulin sensitivity, reduces oxidative stress, and modulates signaling pathways that regulate inflammation and antioxidant defenses⁵. Furthermore, BITC exhibits anti-inflammatory activity because it interferes with key molecular pathways that drive inflammation, reduces pro-inflammatory cytokines, and enhances antioxidant defenses that counteract oxidative stress–induced inflammation. Preclinical studies have shown that BITC can help inhibit the growth of cancer cells by activating various signaling pathways, including apoptosis, cell cycle arrest, metastasis, and angiogenesis. It also inhibits tumor growth by inhibiting enzymes involved in metastasis and angiogenesis. In addition to, BITC also has antibacterial, antifungal and antibiofilm activities^6,7. Poor water solubility, chemical instability, and volatility, which reduce BITC bioavailability and make it difficult to maintain consistent concentrations in water-based systems, are the main limitations of BITC for using in aqueous systems⁸.

Nanotechnology-based delivery systems, particularly NEs, have emerged as promising strategies to overcome the limitations of BITC in aqueous formulations. NEs, consisting of oil in water (O/W) dispersions with droplet size less than 1000 nm, are capable to encapsulate BITC within the oil phase, and enhance its solubility and protect that from hydrolytic and oxidative degradations. Furthermore, by preparation of BITC NE, stability of BITC prolongs and its bioavailability improves by facilitating absorption across biological membranes. Moreover, NE systems are capable to control release and target delivery of BITC, which those highly suitable for pharmaceutical, nutraceutical, and food applications^8–10.

Several techniques have been developed to preparation of O/W NEs such as high-pressure homogenization, ultrasounication, solvent displacement, phase inversion and spontaneous methods^11–13. Pressurized water (PW) technique is recognized as an advanced, cost-effective and eco-friendly nanoemultion preparation technique which, without the use of organic solvents and excessive amounts of emulsifier, is capable to prepare NEs with uniform size distribution, with high physico-chemical stability. In fact, under PW conditions (and under controlled temperature and pressure conditions (temperature and pressure higher than 100 °C and 1 bar) water is boiling in its liquid state but, its polarity changes and tends into the polarity of ethanol, methanol and ethanol, which that causes reduction in particle size and increasing in zeta potential value of the formed NE¹⁴. Optimization of the amounts of main components formed O/W nanoemulsion, including emulsifier and non-polar bioactive compound leads to prepared nanoemulsion with desirable physico-chemical properties and biological activities.

According to the best of our knowledge, there is no comprehensive study in preparation of BITC NE using PW method and assessment of its biological and physico-chemical properties which is aligns with the principles of green chemistry utilizing environmentally friendly technique and compounds. Therefore, main objectives of the present study were to (i) green preparation of BITC NEs using PW, (ii) optimization of the process based on response surface methodology to obtain optimum amounts of BITC and emulsifier (Tween 80) for preparation of BITC with highest antioxidant, antidiabetic and anti-inflammatory activities, (iii) assess particle size, polydispersity index, zeta potential, antibacterial and antifungal activities of the prepared BITC using attained optimum conditions, and (iv) evaluate anti-cancer of the produced BITC NE toward breast cancer cells (MCF 7).

Materials and methods

Materials

Benzyl isothiocyanate, 3,5-Dinitrosalicylic acid (DNS), Potassium sodium tartrate tetrahydrate, Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and 2, 2-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Aldrich Co. Steinheim, Germany). Xanthan gum and Arabic gum were bought from Merck Company (Darmstadt, Germany) bovine serum albumin were obtained from Merck (Merck KGaA, Darmstadt, Germany). α-amylase and fetal bovine serum (FBS) were provided from Novozymes Company (Copenhagen, Denmark). RPMI 1640 (Gibco) was purchased from Thermo Fisher Scientific (Waltham, MA USA). Distilled water (DW), methanol, isopropanol and hydrochloric acid and sodium chloride (NaCl) were provided from Dr. Mojallali Company (Tehran, Iran). MCF-7 breast cancer cell line was provided from American Type Culture Collection (ATCC, USA).

Preparation of BITC NEs

Based on the literature studies and our laboratory preliminary tests, BITC NEs using PW method were prepared^14,15. In fact, mixture of Arabic (0.4 g) and Xanthan (0.1 g) gums were added into 400 mL DW and the mixture solution placed in a laboratory heater with stirring, adjusted at 300 rpm and room temperature (30 °C), for 1 h. After preparation of the mixture solution by fully dissolving of both gums in DW, 25 mL of this solution was added into the 13 glass tubes with capacity of 40 mL and different amounts of Tween 80 (T 80), according to central composite design (CCD), were added into each tubes and placed in the laboratory bath ultrasonication device (Para Co., Tabriz, Iran) with constant power (24 W) and frequency (28 kHz), for 10 min. the solution in the tube was aqueous phase and according to the CCD, different amounts of BITC were added into the tubes and after closing the tubes, those were subjected to a laboratory tube shaker for 2 min and finally placed into the laboratory autoclave, set at 120 °C and 1.5 bar, for 30 min. The final products, as BITC NEs, were kept at dark place and room temperature for further analyses.

Physico-chemical analyses

Particle size, size distribution, PDI, zeta potential and its distribution of the produced BITC NEs, were measured using a dynamic light scattering (DLS) particle size analyzer (Malvern Instruments, Zetasizer Nano ZS, Worcestershire, UK) with measurement angle of 90 °, dilution factor of 1:100 V/V (diluted with ultrapure water) and clear disposable zeta cell. For zeta potential measurement Smoluchowski model F(κa) value of 1.50 was used.

Biological activities assessment

Antioxidant activity of the samples were calculated using described method, based on free radical scavenging using DPPH, by Ahmadi and Jafarizadeh-Malmiri¹³. To evaluate the DPPH radical scavenging capacity, 100 µL of the formulated NE was combined with 5 mL of a 50% methanol solution containing 1 mM DPPH. A control was prepared by mixing the DPPH solution with methanol at a 1:1 ratio, without sample. All mixtures were vigorously shaken and incubated in the dark at room temperature (30 °C) for 30 min. The absorbance of both the test and control samples was then measured at 517 nm using a UV–Vis spectrophotometer (Jenway 6705, Staffordshire, UK). However, absorbance of the control was measured rapidly after addition of DPPH (without incubation). Antioxidant activity, expressed as radical scavenging efficiency, was calculated according to Eq. (1).

1

Where I%, Ac and As are the inhibition percent, absorbance of the control and absorbance of the samples, respectively.

In order to measure antidiabetic activity of the prepared BITC NEs, α-amylase inhibition assay was used¹⁶. In this test, 3,5-Dinitrosalicylic acid (DNS) reagent was prepared by dissolving 0.5 g DNS into 10 mL NaOH (5 N) and placed on a laboratory heater with stirring adjusted at 200 rpm and room temperature. After that 15 g of Potassium sodium tartrate tetrahydrate was added slowly into the mixture solution to prepare orange color DNS reagent. On the other hand, starch solution (1% W/V) and α-amylase (1 U) were prepared in the phosphate buffer (0.02% W/V). 2mL of each sample was mixed with 2 ml of enzyme and put in a laboratory incubator, adjusted at 37 °C for 20 min. After that 2 mL of the provided starch solution was added into the mixture solution and placed into the incubator with same conditions. Finally, 4 mL DNS was added into the mixture solution and boiled for 5 min and after decreasing its temperature to the environment, that was mixed with 5 mL of DW and its absorbance was recorded at wavelength of 540 nm. Control sample was also prepared according to the preparation method without prepared BITC NE. The antidiabetic activity, as ability of the NE in α-amylase inhibition, was measured using Eq. (2).

2

Where, Ac and As, are the absorbance of the control and absorbance of the samples, respectively.

Anti-inflammatory activity of the produced BITC NEs was assessed based on percentage inhibition of protein denaturation¹⁷. In this method, 1 mL of bovine serum albumin (1.5% V/V) was into 5 mL sample and the mixture solution was then incubated at 37 °C for 20 min. After that, the solution incubated at 51 °C for another 20 min, and after cooling that to the room temperature its absorbance was recorded at wavelength of 660 nm. Control sample was also prepared without BITC NE. Anti-inflammatory activity, as percentage inhibition of albumin denaturation, was measured using Eq. (3).

3

Where, Ac and As, are the absorbance of the control and absorbance of the samples, respectively.

Anti-cancer assessment

Cytotoxicity of BITC was determined using MTT assay. MCF-7 cells were cultured with a density of 5 × 10⁵ cells/mL in each well of 12-well plates for 3, 5 and 7 days using RPMI 1640 media containing 10% V/V FBS and 1% V/V Pen-strep antibiotic in the presence of the prepared BITC NE. In fact, 10, 50 and 100 µL of the prepared NE were mixed with 990, 950 and 900 µL of the prepared media, respectively and total volume of 1 mL mixture of the media and BITC was put in each well. The plates were then incubated in a humidified incubator with 5% CO₂ at 37 °C. A mixture solution containing 5 mg MTT in 1 mL of the RPMI 1640 media was prepared, diluted (1:10) and filtered through a 0.2 µ m cellulose acetate filter (yellow colour). At different incubation days, cultured sample in the well was withdrawn and 1 mL of the prepared diluted MTT sample was loaded in the well and incubated at 37 °C for 2 h. After that, the MTT solution was removed, and the cultures were solubilized in 1 mL acidic isopropanol (0.04 M HCl in absolute isopropanol) and centrifuged at 1300 rpm for 2 min. Finally, absorbance of the supernatant, with pink colour, was measured at a wavelength of 570 nm, and expresses as optical density (OD). The cell proliferation was also visualized using a laboratory microscope (IX71, Olympus, Tokyo, Japan). Negative control was also provided using culture media, without BITC NE^1,18.

Design of experiments and data statistical analysis

Based CCD and two independent variables, 13 randomized experimental runs were provided (Table 1) and using RSM effects of the independent parameters namely, amount of BITC (10 − 50 µL) and amount of T 80 (100–500 µL) were evaluated on antioxidant (%), antidiabetic (%), zeta and anti-inflammatory (%) activities of the prepared BITC NEs, as responses. Total volume of each run is 25 mL which that was contained DW, Arabic and Xanthan gums, BITC and T80. The responses were correlated to the independent variables using a second-order polynomial equation (Eq. 4) containing constant (A₀), linear (A_i), quadratic (A_ii) and interaction (A_ij) terms¹⁹.

4

Table 1.

Design of experiments using CCD and experimental values of the responses.

Runs	Amount of BITC (µL)	Amount of T 80 (µL)	Antioxidant activity (%)	Antidiabetic activity (%)	Anti-inflammatory activity (%)
1	50	300	*	18.34	99.85
2	30	300	10.4	52.00	99.15
3	30	500	7.5	46.50	*
4	30	300	10.4	52.05	99.18
5	30	300	11.4	50.17	98.61
6	16	160	7.1	57.23	98.72
7	30	300	10.6	*	99.34
8	10	300	6.5	67.46	92.38
9	30	300	9.2	56.88	99.52
10	45	160	*	45.80	99.81
11	16	440	4.4	*	*
12	30	100	16.2	51.94	99.07
13	45	440	22.7	*	99.82

* Out of range.

Coefficient of determination (R²) with P-value of the lack of fit were chosen to assess accuracy of the generated models²⁰. Analysis of variance based on P-value less than 0.05 (P < 0.05) was used to determine significance of the model terms²¹. Numerical and graphical optimizations, based on surface, contour and overlaid plots were stablished to give exact amounts of BITC and T 80 to produce BITC NE with maximum values of the responses²². Experimental design, process modeling, data analysis and optimization of the process in BITC NE preparation were accomplished using Minitab statistical software (v.16, Minitab Inc., PA, USA, www.minitab.com/en-us/products/minitab/). Some values of the responses were out of range due to the human and instrument errors, which those data were excluded from the modeling and optimization procedures and statistical analysis.

Results and discussion

Model generation

Three second order polynomial models were generated according to the CCD and obtained experimental values for the responses (Table 1), to predict antioxidant, antidiabetic and anti-inflammatory activities of the prepared BITC NEs as function of the amounts of BITC and T 80. Table 2 shows the Coefficients of the model’s terms and Table 3 indicates p-values of the model’s terms, R² and p-value of the lack fit of the generated models. Results indicated that linear term of the amount of T 80 had insignificant effect on antioxidant and anti-inflammatory activities of the produced BITC NEs. Furthermore, quadratic term of the smount of T 80 had insignificant effect on all biological activity of the made NEs. In addition to, highest values of R² for the generated models (higher than 95.85%) revealed high appropriateness of the fitted models for correlation of the responses to the independent variables^23,24. Results also indicated that P-values of lack-of-fit for all the fitted models were higher than 0.05 (P > 0.05) which those indicated adequate fitness of the generated models²¹.

Table 2.

Coefficients of the model’s terms.

Term	Antioxidant activity (%)	Antidiabetic activity (%)	Anti-inflammatory activity (%)
A0	14.5099	2.1056	97.7115
A1	-0.6636	2.4259	0.2556
A2	-0.0160	0.2572	-0.0141
A11	0.0283	-0.0247	-0.0068
A22	0.0000	-0.0001	-0.0001
A12	-0.0009	-0.0076	0.0011

A₀: Constant; A₁ and A₂: Linear terms of amounts of the BITC and T 80.

A₁₁ and A₂₂: Quadratic terms.

A₁₂: Interaction term.

Table 3.

P-values of the model’s terms and the values of the coefficient of determination and lack-of-fit for three fitted models.

Term	Antioxidant activity (%)	Antidiabetic activity (%)	Anti-inflammatory activity (%)
A0	0.002	0.886	0.000
A1	0.002	0.008	0.046
A2	0.215	0.007	0.202
A11	0.000	0.028	0.003
A22	0.059	0.317	0.056
A12	0.033	0.002	0.007
R2	99.01	97.68	95.85
Lack-of-fit (p-value)	> 0.05	> 0.05	> 0.05

A₀: Constant; A₁ and A₂: Linear terms of amounts of the BITC and T 80.

A₁₁ and A₂₂: Quadratic terms.

A₁₂: Interaction term.

Effectiveness of independent variables on antioxidant activity of the prepared BITC NEs

Unlike other general antioxidants such as vitamins C and E, that directly donate electrons to neutralize free radicals, BITC antioxidant activity is related to modulating signaling pathways and transcription factors^25,26. As clearly observed in Table 1, antioxidant activity of the prepared BITC NEs changed from 4.4 to 22.7%. Effectiveness of the amounts of BITC and T 80 on this response show in Figs. 1 and 2 (A). According to the Fig. 1A, at constant amounts of T 80, by increasing amount of BITC, antioxidant activity of the prepared NEs increased. Results also indicated that at higher amounts of BITC, by increasing amount of T 80, this activity of the formed NE decreased. It seems that excess emulsifier can shield BITC molecules at the oil–water interface, reduce their accessibility to reactive species. Furthermore, at higher amount of T 80, the emulsifier forms a thicker interfacial layer around BITC, which that reduces BITC exposure to the presented radicals or oxidants in the aqueous phase, and causes its lower antioxidant activity²⁷.

Fig. 1.

Surface plots for antioxidant (A), antidiabetic (B) and anti-inflammatory (C) activities of the prepared BITC NEs as function of the amounts of BITC and T 80.

Figure 2A indicates that prepared BITC NEs using highest amount of BITC and lowest amount of T 80 had highest antioxidant activity. This result indicated the appropriately of the PW technique for preparation of the O/W NEs with high antioxidant activity, because that is based on reduction of water polarity which, it can minimize the amount of used emulsifier^28,29.

Fig. 2.

Contour plots for antioxidant (A), antidiabetic (B) and anti-inflammatory (C) activities of the prepared BITC NEs as function of the amounts of BITC and T 80.

Influences of the amounts of BITC and T 80 on antidiabetic activity of the produced NEs

BITC has antidiabetic activity because it improves glucose metabolism by enhancing glucose uptake in tissues and improving tolerance to high sugar loads, and enhances insulin sensitivity by increasing the responsiveness of cells to insulin and reducing their insulin resistance^5,30. Effects of the amounts of BITC and T 80 on this response show in Figs. 1 and 2 (B). As clearly observed in Fig. 1B, at low and high constant amounts of BITC, by increasing amount of T 80, antidiabetic activity activity of the produced NEs increased and decreased, respectively. On the other hand, at low and constant amounts of T 80, by increasing amount of BITC, antidiabetic activity of the formed NEs increased and decreased, respectively. Presence of curvature in Fig. 1B revealed that interaction term of the amounts of BITC and T 80 had significant (p < 0.05) on the antidiabetic activity of the fabricated NEs and reconfirmed the presented result in Table 3. According to Fig. 2B, prepared BITC NEs using minimum amount of BITC and maximum amount of T 80, had highest antidiabetic activity. The results could be related to the enhanced bioavailability of BITC and synergistic effect of T 80 in the mentioned amounts for both of them. It seems that using high amounts of T 80, NEs with smallest droplet size of BITC formed which, due to highest surface to volume ratio of the formed small droplets of BITC, BITC dispersion and absorption in biological systems increase and its ability to interact with glucose metabolism pathways improves. Furthermore, T 80 itself can improve cell membrane permeability and facilitate BITC uptake^31,32.

Effects of the amounts of BITC and T 80 on anti-inflammatory activity of the made NEs

BITC contains a reactive − N = C = S group that can interact with cellular proteins, especially those involved in redox regulation. This allows it to modulate signaling cascades that control inflammation^33,34. As clearly observed in Table 1, anti-inflammatory activity of the prepared BITC NEs changed from 92.38 to 99.85%. Effectiveness of the amounts of BITC and T 80 on this response show in Figs. 1 and 2(C). According to the Fig. 1C, at constant and low, and high amounts of T 80, by increasing amount of BITC, anti-inflammatory activity of the prepared NEs decreased and increased, respectively. Results also indicated that at constant and low, and high amounts of BITC, by increasing amount of T 80, this activity of the formed NEs decreased and increased, respectively. It seems that at low amounts of BITC, BITC molecules get trapped inside micelles or thick surfactant layers and caused less BITC reach into NF-κB/MAPK pathways. Furthermore, result indicated that, using higher amount of T 80 in the preparation of NE containing high amount of BITC, formed BITC droplet size and polydispersity decrease in the NE which those cause greater interfacial area and improve transfer of BITC to the cell surface³⁵. Presence of curvature in this figure indicated that interaction term of the amounts of BITC and T 80 had significant (p < 0.05) effect on anti-inflammatory of the prepared BITC NE and reconfirmed attained result of Table 3. According to the Fig. 2C, BITC NEs prepared using moderate to high amounts of BITC and T 80. It seems that higher amounts of T 80 provide optimal droplet stabilization, improved bioavailability, and controlled release of BITC by helping BITC to reach target tissues effectively without rapid degradation³⁶.

Optimization and validation of the process for BITC NE preparation

Optimal formulation of the NE, including the accurate ratio of components such as the concentration and amount of oil phase, type and amount of emulsifier, and use of polysaccharide stabilizers, plays a significant role in the final properties of the formed NE³⁷. Fine-tuning these parameters can significantly affect properties such as particle size, size distribution, PDI, stability, antioxidant, antidiabetic, anti-inflammatory, anti-cancer and antimicrobial efficacy³⁸. Therefore, careful formulation design and optimization of component concentrations and amounts are of great importance to achieve a stable NE with desirable biological performance. Graphical optimization based on overlade contour plot for preparation of BITC NE with maximum antioxidant, antidiabetic and anti-inflammatory activities provides (Figure 3A). White area is optimal zone for the amounts of BITC and T 80 for preparation of BITC NE with desirable biological activities. Furthermore, numerical optimization also indicated that using 36.5 µL BITC and 124 µL T 80, as optimum amounts of the independent variables, BITC NE with high antioxidant (22.7%), antidiabetic (53.5%) and anti-inflammatory (99.6%) activities prepared. Results demonstrated that there were insignificant differences between the experimental values of the antioxidant (22±1%), antidiabetic (55±2) and anti-inflammatory (99.5±0.2%) and predicted values of those, and validated the experimental design, modeling and optimization of the BITC NE preparation procedure using CCD and RSM. Results also revealed that the prepared BITC NE using obtained optimum conditions had particle size, PDI and zeta potential of 180 nm (Fig. 3B), 0.236 and -21.32 mV (Fig.3C), respectively. These results were closed to findings of Tina et al.³⁵. They prepared BITC NEs using 4 different emulsifiers with particle size, PDI and zeta potential ranging 120 to 240 nm, 0.15 to 0.30 and -25 to -45 (mV), respectively.

Fig. 3.

Graphical optimization (A), particle size (B) and zeta potential (B) distributions of the prepared BITC NE using obtained optimum amounts of BITC and T 80.

Anti-cancer activity of the prepared BITC NE using obtained optimum conditions

Preclinical studies have shown that BITC can help inhibit the growth of cancer cells by activating various signaling pathways, including apoptosis, cell cycle arrest, metastasis, and angiogenesis. It also inhibits tumor growth by inhibiting enzymes involved in metastasis and angiogenesis^39,40. The cytotoxic effects of the prepared BITC NE, according to the MTT assay shows in Table 4.

Table 4.

OD of culture media containing MCF7 breast cancer cell and different amounts of the prepared BITC NE, at different incubation days.

Sample	Amount of BITC NE (µL)	BITC concentration (µL/mL)	Day 3 of incubation	Day 5 of incubation	Day 7 of incubation
1	10	0.0146	0.122 ± 0.002aB	0.117 ± 0.001bB	0.114 ± 0.002bB
2	50	0.073	0.094 ± 0.003aC	0.084 ± 0.004bC	0.077 ± 0.003bC
3	100	0.146	0.083 ± 0.002aD	0.024 ± 0.003bD	0.021 ± 0.001bD
Control	0	0	0.133 ± 0.004cA	0.234 ± 0.005bA	0.283 ± 0.002aA

Different small and capital letters in the same row and column indicate significant differences (P < 0.05), respectively. Data are the mean ± SD (n = 3).

Results indicated that BITC NE had cytotoxic against MCF-7 cells in a time- and dose-dependent manner. According to the obtained results and as compared to day 3, metabolic activity of MCF-7 cells in the control sample, were increased to 76 and 113% at days 5 and 7 of incubation, respectively. As already mentioned, BITC has low water solubility due to its non-polar nature and it is impossible to use free BITC, as control sample⁸. While, 4 and 6.5% reduction were observed in that of cancer cells, after days 5 and 7 of incubation, when BITC NE with its amount of 10µL was utilized. Furthermore, 50 µL of the BITC NE could reduce metabolic activity of the MCF-7 cells up to 10.6 and 18% at days 5 and 7, respectively. Results also demonstrated that 100 µL BITC NE could significantly (p < 0.05) decreased activity of the MCF-7 cancer cells up to 71 and 74.7% at days 5 and 7, respectively, as compared to that of cells at day 3 of incubation. It seems that release of BITC is one the main reasons of toxicity to the MCF-7 cells. Microscopic images of the cultured MCF-7 cells in the presence of 10, 50 and 100 µL of BITC NE, and without that, as control, at day 7 of storage show in Fig. 4(A – D). As clearly observed in these figures, by increasing amount of the prepared BITC NE in the culture media containing cancer cells, cell viability decreased (Fig. 4A – C). While, MCF-7 cell viability increased in the control sample after day 7 of storage. Presence of spindle-shaped cell in the Fig. 4D could be related to the growth of MCF-7 cells in the control sample. While, in the cultured cell containing BITC NE, the MCF-7 cells had spherical shape and BITC significantly inhibited those metabolic activity and growth². Obtained results were in line with findings of Kadir et al.¹ and Rahaman et al.². They also find that by increasing the concentration of BITC from 0.1 to 100 µM, MCF-7 viability decreased to 0, after 2 days of incubation. Kim et al., also reported that BITC inhibited MCF-7 breast cancer stem cell populations at low micromolar range of 2.5–5 µM⁴¹. In fact, BITC inhibited breast cancer stem cells primarily by inducing oxidative stress, triggering apoptosis, and disrupting stemness-related signaling pathways such as Ron/sfRon, MAPK, NF-κB, and Nrf2^2,41. Sehrawat and Singh found that BITC at low micromolar concentrations, typically 2.5–10 µM, induced apoptosis in MCF-7 cells⁴². Sehrawat et al. also reported that BITC, at plasma-achievable concentrations (2.5–10 µM) rapidly disrupted mitochondrial fusion in MCF-7 breast cancer cells, leading to mitochondrial fragmentation, loss of membrane potential, and apoptosis⁴³. Ngo and Williams isothiocyanates of cruciferous vegetables including BITC, sulforaphane, and phenethyl isothiocyanate consistently inhibited breast cancer cell growth in vitro at low micromolar concentrations of 2–20 µM⁴⁴. Furthermore, Xiao et al. found that BITC at low micromolar concentrations (2.5–10 µM) inhibited growth of MCF-7 breast cancer cells by inducing G2/M cell cycle arrest and apoptosis⁴⁵. Several studies indicated that BITC exerts anticancer activity against MCF-7 breast cancer cells primarily by inducing reactive oxygen species (ROS)-mediated apoptosis, causing cell cycle arrest, and disrupting survival signaling pathways such as MAPK, NF-κB, and AKT^46–50.

Fig. 4.

Cell viability images of the cultured MCF-7 in the presence of 10 µL (A), 50 µL (B) and 100 µL (C) of the prepared BITC NE, and control sample (D).

Conclusion

Present study indicated that PW could effectively prepared and formulated BITC NE using T 80, as emulsifier, and xanthan and Arabic gums, as stabilizers, with small particle size with high uniformity, and highest stability in the colloidal form. Results indicated that RSM was efficient in optimization of the amounts of main components of BITC NE, including T 80 and BITC, to prepare NE with high nutritional and biological activities such as antioxidant, antidiabetic and anti-inflammatory activities. Furthermore, results demonstrated that the prepared BITC using obtained amounts of its main components, had high anti-cancer effect on breast cancer cell (MCF-7) in a time- and dose-dependent manner. Results indicated that by increasing amount of the prepared BITC NE in the cultured media containing MCF-7 cells, cytotoxic effect of BITC also increased, and by increasing the incubation time, BITC releasing also increased, which that could strongly decreased cancer cell viability. Further optimization on other effective parameter in the formation and formulation of BITC NE can develop new supplement and drug formulations.

Author contributions

A.R.: Methodology, Formal analyses, Data curation. S.E.: Supervision, Project administration, Software, Resources, Validation. H. J.-M. Supervision, Conceptualization, Investigation, Writing – review and editing.

Data availability

All data supporting the findings of this study are available within the paper.

Declarations

Competing interests

The authors declare no competing interests.