Spectral Analysis of Bioactive Compounds in Cold Methanolic Extracts of Commercial Broccoli Florets: Antioxidant Analysis and In vitro Cytotoxicity in A549 Lung Cancer Cells

Introduction

Lung cancer, a malignant disease that originates in the cells of the lung, is primarily the result of prolonged exposure to tobacco smoke, although factors such as exposure to radon, asbestos, and certain chemicals also contribute to its development¹. In Saudi Arabia, it is the 5^th most common cancer in men and the 15^th most common in women². Since preventing disease is a higher priority than curing it, dietary choices are an essential aspect of maintaining good health.

Broccoli, scientifically known as Brassica oleracea L. var. italica, belonging to the Brassicaceae family, is of remarkable pharmaceutical importance, as it has antimicrobial properties^3,4 antioxidants, anti-cancer properties⁵, immunomodulatory effects, and antidiabetic properties. It also provides hepatoprotection, cardioprotection, and anti-amnestic benefits. Syed, R. U. et al. (2023) found that cruciferous vegetables such as broccoli are rich in fiber and low in calories, and they contain essential vitamins and minerals, thereby supporting various physiological functions. Research by Jacques et al. suggests that an increase in the consumption of lycopene and beta-carotene may reduce the risk of heart disease⁶ Additionally, a study by Murphy et al. highlights the importance of an increased consumption of fruits and vegetables, which is consistent with dietary recommendations from health and nutrition experts⁷ .The aim of this study is to investigate the bioactive constituents of broccoli, which is readily available in Jazan markets in Saudi Arabia. In addition, the antioxidant properties, and cytotoxic effects of a cold methanolic extract of broccoli florets (CMEB) on A549 lung cancer cells will be investigated, which will provide information about its potential in combating lung cancer.

Methods

Materials

The broccoli was purchased from a commercial market in Jazan, Saudi Arabia. All the chemicals were purchased from Sigma Aldrich, which is supplied by Al Sada trading, Riyadh. All the chemicals used in this study are of analytical grade.

Cold maceration extraction

The broccoli samples were washed thoroughly with fresh water to remove any impurities and were then air dried. After drying, the broccoli was cut into small pieces and air-dried again in the shade for 10 days. The dried broccoli pieces were ground into a coarse powder, which was then collected and stored in an airtight container for further use. The bioactive components of the broccoli floret were extracted by method developed by R.U. Syed et al, 2022.⁸

Gas Chromatography Mass Spectrometry analysis

The presence of major bioactive components in the CMEB was investigated by GC-MS, (Thermo Scientific GC-MS-AS 3000 autosampler – ISQ detector). The GC-MS analysis was performed as reported by R.U.Syed et al, 2022.⁸

Liquid chromatography–mass spectrometry analysis

LC-MS was used to characterize the CMEB. Liquid chromatography was performed on a Shimadzu Exion LC system (Kyoto, Japan). The LC-MS analysis was performed as reported by Menachery et al 2025.⁹

X-ray diffraction analysis

X-ray diffraction (XRD) was utilized to investigate any possible crystalline structures of CMEB. The XRD analysis was performed according to the protocol developed by Rahamat et al 2022⁸.

Molecular docking

The CMEB revealed potential bioactive compounds that were examined through molecular docking studies. These compounds included ethyl iso-allocholate, crinamine, heptadecane, norethandrolone, botulin, cis-vaccenic acid, phytol, n-hexadecanoic acid, lupeol, oleic acid, and astaxanthin. The compounds were subjected to docking processes targeting the catalytic domain of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Notably, SIRT1 is a subject of vast research in oncology; it is known for its multifaceted role in tumor-related signaling pathways. The SIRT1 catalytic domain’s crystal structure, when bound to NAD and an EX527 analog (PDB: 4I5I), served as the reference for the docking studies. This protein structure was optimized by eliminating water molecules, introducing absent hydrogens, determining partial charges, and excising the ligand. A rigid 40 Å × 40 Å × 40 Å grid box, centered on the co-crystallized ligand (EX527 analog), was crafted. Docking studies were performed by using AutoDockTools-4. The authenticity of the docking outcomes was ascertained by redocking the co-crystallized ligand (EX527 analog), yielding a binding free energy and inhibition constant (Ki) of -4.96 kcal/mol and 229.57 uM, respectively.^10-11

Antioxidant studies: DPPH assay

The antioxidant activity of the CMEB was determined using the DPPH free-radical scavenging assay. The final concentrations of 1000, 500, 250, 125, and 62.5 g/mL, respectively, in methanol were used to make the samples. A stock solution of 20 µg/mL concentration of Trolox was prepared in methanol from which five concentrations were prepared, including 1.25, 5, 7.5,10 and 12.5 µg/mL. 100 µL of the CMEB was placed in a 96-well microtiter plate (n=6), and 100µL of freshly prepared DPPH reagent (0.1% in methanol) was added and aspirated three times. The reaction was incubated for 30 minutes at room temperature in the dark. At the end of the incubation period, the resulting reduction in DPPH colour intensity was measured at 540 nm ⁹. The DPPH scavenging effect was calculated using the following equation:

ABTS assay

192 mg of ABTS (2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) were dissolved in distilled water and transferred to a 50 mL volumetric flask, add the volume was completed with distilled water. 1mL of the previous solution was added to 17 μL of 140 mM potassium persulphate, and the mixture was left for 24 hours in the dark. After that, 1mL of the reaction mixture was increased to 50 mL with the additional of methanol to obtain the final ABTS dilution used in the assay. 190 μL of the freshly prepared ABTS reagent was mixed with 10 μL of the CMEB in 96 wells plate (n=6), and the reaction was incubated for 30 min at room temperature in the dark. At the end of the incubation period, the reduction in ABTS color intensity was measured at 734 nm. The data are represented as means ± SD according to the following equation:

In vitro cytotoxicity studies

In this study, A-549 lung cancer cells were obtained from Nawah Scientific Inc. in Mokatam, Cairo, Egypt, and were cultured in DMEM media supplemented with streptomycin, penicillin, and foetal bovine serum under controlled conditions of 37°C at 5% CO₂. The cell viability was determined by the SRB assay in which 5×10^-3 cells were plated in 96-well plates, incubated in complete media for 24 hours, and then treated with CMEB containing media for 72 hours. After exposure to the drug, the cells were fixed with 10% TCA, washed with distilled water, and stained with 0.4% w/v SRB solution. The stained cells were washed with 1% acetic acid; air dried, and then treated with 10 mM TRIS to dissolve the protein-bound SRB stain. Absorbance was measured at 540 nm using a BMG LABTECH-FLUOstar Omega micro plate reader in Ortenberg, Germany. This comprehensive approach allowed for the assessment of cell viability and drug response in the A-549 lung cancer cell line.

Results

Spectral analysis

The GC-MS analysis of the CMEB identified several bioactive compounds, as illustrated in Figure 1 and listed in Table 1. The LC-MS analysis, detailed in Table 2, confirmed the presence of compounds such as ethyl iso-allocholate, heptadecane, norethandrolone, botulin, cis-vaccenic acid, phytol, n-hexadecanoic acid, lupeol, oleic acid, and astaxanthin. These compounds are represented structurally in Figure 2, with the LC-MS chromatogram shown in Figure 3. Ethyl iso-allocholate was found to be a steroidal compound with significant therapeutic potential, showing diverse effects including apoptosis induction and inhibition of metastasis. Norethandrolone was detected with a retention time of 14.26 minutes, a probability index of 85.70%, and accounted for 2.39% of the chromatogram. Crinamine, identified at a retention time of 49.19 minutes and a probability index of 28.67%, comprised 6.08% of the GC-MS chromatogram but was not detected in LC-MS. Heptadecane appeared at 54.88 minutes with a probability index of 20.82%, making up 2.05% of the chromatogram. Betulin was noted at 72.19 minutes with a probability index of 16.19% and 1.24% of the chromatogram. Cis-vaccenic acid was identified at 77.68 minutes with a 16.63% probability index and 1.20% of the chromatogram. Phytol appeared at 75.49 minutes with a probability index of 68.74%, representing 1.16% of the GC-MS chromatogram. N-Hexadecanoic acid was identified at 71.54 minutes with a 37.80% probability index and 1.20% of the chromatogram. Lupeol was found at 117.92 minutes with a 34.80% probability index and 1.20% of the chromatogram. Additionally, 1-Heptatriacotanol, oleic acid, and astaxanthin were detected with varying retention times and probability indices, though 1-Heptatriacotanol was not observed in LC-MS.

Table 1: GC-MS detection of possible bioactive compounds in CMEB

Figure 1: GC-MS chromatogram of CMEB. Click here to View table

Table 2: LC-MS detection of possible bioactive compounds in CMEB

Figure 2: The structure of bioactive compounds of the cold methanolic extract of broccoli through GC-MS and LCMS analyses. Click here to View Figure

Figure 3: LC-MS chromatogram of CMEB and the structure of bioactive compounds The structure of bioactive compounds of the cold methanolic extract of broccoli through GC-MS and LCMS analyses. Click here to View Figure

X-ray diffraction analysis

XRD analysis was employed to characterize the discrete structures of the dried broccoli extract. The analysis conducted after 2 hours revealed distinct particles based on the diffraction peaks observed. Specifically, a cluster of peaks was detected at 65.2°, corresponding to the 2θ value, indicating strong diffraction peaks. The corresponding d-value for this 2θ value was 1.430, with an intensity of 62 counts per second per degree (cps/deg). This suggests the presence of specific biomolecules in the dried broccoli extract. The XRD peaks are consistent with the presence of mineral crystals ¹²

Figure 4: XRD analysis of the cold methanolic extract of broccoli floret (The diffractogram was obtained at 2θ in the range 2°–80°). Click here to View Figure

Molecular docking

Docking the SIRT1 catalytic domain with the bioactive GC-MS detected compounds was achieved using predefined parameters. Multiple docking conformations were generated (Fig. 5). The best conformation was selected and ranked based on the inhibition constant, binding energy, and RSMD. All inhibition constants are in the micro-scale, except for heptadecane and phytol, as seen in Table 3.

Table 3: Docking analysis of possible bioactive GC-MS detected compounds of the cold methanolic extract of broccoli against SIRT1 catalytic domain

Ethyl iso-allocholate exhibits a binding energy of -5.88 kcal/mol and an IC₅₀ of 48.88 µM, reflecting a moderate binding affinity and inhibition potency. It binds to the target protein reasonably well but is outperformed by several other compounds in terms of potency. Crinamine shows a slightly more favorable binding energy of -5.99 kcal/mol and a lower IC₅₀ of 40.46 µM, suggesting it has a stronger interaction with the protein and is more effective at inhibiting the target compared to ethyl iso-allocholate. In contrast, Heptadecane has a significantly less negative binding energy of -3.53 kcal/mol and an IC₅₀ of 2.58 mM, indicating a weak interaction with the target protein and very low potency, as it requires a much higher concentration to achieve inhibition. Norethandrolone and Betulin show intermediate binding energies of -5.54 kcal/mol and -5.48 kcal/mol, respectively, with IC₅₀ values of 87.63 µM and 96.04 µM. These results suggest that both compounds have a moderate binding affinity and are effective inhibitors, though not as potent as crinamine or ethyl iso-allocholate. cis-Vaccenic acid presents a binding energy of -5.93 kcal/mol and an IC₅₀ of 44.82 µM, indicating a strong binding affinity and effective inhibition similar to crinamine. However, Phytol has a binding energy of -3.13 kcal/mol and a high IC₅₀ of 5.06 mM, showing weak binding and very low potency, as it requires a significantly higher concentration to inhibit the target protein.

Lupeol stands out with the most negative binding energy of -6.14 kcal/mol and the lowest IC₅₀ of 31.81 µM, making it the most effective compound in the study. This strong binding affinity and high potency suggest that lupeol is highly effective in inhibiting the target protein even at low concentrations. Conversely, Oleic Acid and n-Hexadecanoic Acid display moderate binding energies of -4.96 kcal/mol and -5.26 kcal/mol, with IC₅₀ values of 232.36 µM and 139.57 µM, respectively. These results indicate that while they bind to the target protein reasonably well, they are less potent compared to compounds like lupeol, requiring higher concentrations for effective inhibition. Overall, lupeol emerges as the most promising compound for further investigation due to its strong binding and potent inhibitory effects, whereas heptadecane and phytol show weaker interactions and lower efficacy.

Antioxidant studies

Antioxidants play a crucial role in protecting the body from the harmful effects of oxidative stress caused by free radicals. In this study, the antioxidant capabilities of CMEB were investigated, as shown in Table 4. The table provides a comparative analysis of the antioxidant activities of the Crude Methanolic Extract of Broccoli (CMEB) and the standard antioxidant Trolox, across two assays: DPPH and ABTS. The IC₅₀ values, expressed in micrograms per milliliter (µg/mL), denote the concentration required to inhibit 50% of the radical activity.

In the DPPH assay, CMEB shows an IC₅₀ value of 699.8 ± 27.99 µg/mL. This high IC₅₀ value indicates that a relatively large amount of CMEB is needed to achieve 50% inhibition of the DPPH radical, suggesting that CMEB has a moderate antioxidant effect. On the other hand, Trolox, the standard, exhibits a significantly lower IC₅₀ value of 7.217 ± 0.309 µg/mL. The ABTS result shows that CMEB has an IC₅₀ value of 185 ± 18.37 µg/mL. This value suggests that CMEB has a considerable antioxidant effect, but it still requires a higher concentration compared to the standard to achieve 50% inhibition of ABTS radicals.

Table 4: The anti-oxidant study of cold methanolic extract of broccoli

*CMEB = Cold methanol extract of Broccoli 

In-vitro cytotoxicity

In vitro cytotoxicity analysis on A549 lung cancer cells showed results at a concentration of 64.5 µg/ml (Fig. 6). Free radical scavenging activity of the crude extract could be one of the reasons the cytotoxic activity against the cancer cells. Hence the extract must be further purified to isolate bioactive compound for obtaining its maximum activity.

Figure 5: The cytotoxicity effect of cold methanolic extract of broccoli on A549 lung cancer cells. Click here to View Figure

Discussion

Spectral analysis

CMEB extract in GC-MS and LC-MS analysis revealed a diverse array of presence bioactive of compounds each with potential therapeutic implications. Ethyl iso-allocholate, a steroidal compound having diverse therapeutic attributes, has garnered significant attention. Thakur and Ahirwar  conducted a study demonstrating that ethyl iso-allocholate, obtained from Trigonella foenum graecum L., induces caspase-dependent apoptosis in cancer cells ¹³. Furthermore, it was found to decrease tumor growth and to inhibit metastasis and angiogenesis in-vivo, while also exhibiting safety for normal tissues. Significant interactions were observed between ethyl iso-allocholate and all the protein targets, with a notable emphasis on caspase-1, indicating its therapeutic potential in terms of anti-inflammatory and anti-tumor effects ¹⁴ .In a previous molecular docking study, ethyl iso-allocholate, derived from medicinal rice known as Karungkavuni, was found to inhibit dihydropteroate synthase in Escherichia coli. This study also highlighted the increased stability of the dihydropteroate synthase-ethyl iso-allocholate complex, as well as its excellent antibacterial properties¹⁵ . Based on reference data from NIST’s integrated library, norethandrolone, a steroid, was detected in the CMEB. It displayed a retention time of 14.26 minutes, had a probability index of 85.70%, and represented 2.39% of the chromatogram. Notably, norethandrolone was also detected in the LC-MS analysis using the negative ionization mode. Crinamine, an alkaloid, was detected alongside ethyl iso-allocholate in the CMEB, displaying a retention time of 49.19 minutes, with a probability index of 28.67% and accounting for 6.08% of the GC-MS chromatogram. However, it remained undetected in the LC-MS protocol.

A prior investigation proposed that crinamine, an alkaloid, exhibited antibacterial properties¹⁶.In another study, it was found that crinamine-induced DNA double-strand breaks without triggering any apoptosis. Additionally, by inhibiting the production of pro-epithelial-mesenchymal transition regulators, it effectively reduces the migration of cervical cancer cells¹⁷.Furthermore, crinamine derived from Crinum asiaticum demonstrated substantial cytotoxicity against DU145 and PANC1 cells.¹⁸

Heptadecane, 9-hexyl- was identified in CMEB at 54.88 min with 20.82% probability index, occupying 2.05% in the chromatogram. Additionally, hepadecane, 9-hexyl- was also determined by the LC-MS analysis using the negative ionization mode. Betulin is a naturally occurring triterpene that is found extensively throughout the plant kingdom. In CMEB, Betulin was identified at a retention time of 72.19 minutes, having a 16.19% probability index, and constituting 1.24% of the chromatogram. Additionally, Betulin was confirmed through the LC-MS analysis using the negative ionization approach. Betulin displays enormous pharmacological potential, such as having anti-inflammatory activity, anti-HIV, anti-cancer, and antibacterial properties ^19-22.Cis-vaccenic acid is an omega-7 fatty acid, otherwise called as (Z)-11-octadecenoic acid. The compound was detected in CMEB at 77.68 min with 16.63 % probability index and occupying 1.20 % in the chromatogram, as well as in the LC-MS analysis negative ionization mode. An earlier report suggested that cis-vaccenic acid inhibits the growth of microbes and lowers cholesterol levels²³.According to earlier research, 11-octadecenoic acid can be a marker for broccoli consumption, and it can reduce non-alcoholic fatty liver disease (NAFLD) and enhance liver health. The same study claimed that eating broccoli regularly would normalize one’s lipid profile.²⁴

Phytol, an acyclic monounsaturated diterpene alcohol, was identified in CMEB at 75.49 min with a probability index of 68.74% and occupying 1.16% in the GC-MS chromatogram, Moreover, phytol was discerned in the LC-MS analysis under the negative ionization mode. Phytol can be found in tocopherols such as vitamins K and E. The aromatic component can be found in cosmetic and non-cosmetic goods and is used in many fragrance compounds.Phytol has shown antinociceptive, anti-inflammatory and antiallergic effects; immunostimulant, the immune modulatory effect of innate and acquired immunity; and antimicrobial and antioxidant activities.^17,25-27

N-Hexadecanoic acid, also known as palmitic acid, was detected in CMEB with a retention time of 71.54 minutes. It had a probability index of 37.80% and accounted for 1.20% of the GC-MS chromatogram. Furthermore, this compound was identified in the LC-MS analysis using the positive ionization mode. An earlier study suggested that palmitic acid can modulate innate immunity by coordinating the immunological activity in inflammatory tissues and controlling the activation of pattern recognition receptors in local innate immune cells. Additionally, protein palmitoylation regulates numerous cellular physiological processes²⁸.A previous study showed that palmitic acid exhibited tumorigenic properties by increasing the growth of colorectal cancer, which is dependent on beta 2-adrenergic receptors (2-AR), and it revealed beta 2-adrenergic receptor expression in the xenograft tissues of a mouse model that carried CRC  ²⁹ .Palmitic acid, on the other hand, has been reported to have anti-androgenic, nematicide, flavoring, antioxidant, and hypocholesterolemic qualities³⁰. An earlier report demonstrated that palmitic acid is one of its main constituents of broccoli, et al. and Olga et al. reported the detection of palmitic acid in broccoli flour prepared from the dried florets, leaves, and stalks of broccoli ^31,32 .A study published in 2021 found that palmitic acid substantially inhibited the growth of prostate cancer cells both in vitro and in-vivo³³.Although numerous studies have indicated that broccoli has many medicinal properties, it has also been shown to have some adverse effects, such as inducing insulin resistance that leads to type 2 diabetes and promoting cancer metastasis, a more aggressive “memory” in cancer cells^34,35.

Lupeol is a phytosterol, which is a naturally occurring pentacyclic triterpenoid identified in CMEB at 117.92 minutes with a probability index of 34.80% and occupying 1.20% of the GC-MS chromatogram. It has also been detected in the LC-MS analysis positive ionization mode. Lupeol has demonstrated anti-inflammatory, anti-acne, and anti-cancer properties.^26,36 In the CMEB, 1-Heptatriacotanol, oleic acid, and astaxanthin were identified, each displaying favorable retention times and probability indices. Yet, their occupancy percentages in the chromatogram were relatively low, as detailed in Table 1. Notably, the LC-MS analysis failed to detect 1-Heptatriacotanol in both positive and negative ionization modes. Conversely, both oleic acid and astaxanthin were identified in the LC-MS analysis using the positive ionization approach.

Remarkably, these molecules have good medicinal properties. 1-Heptatriacotanol has been reported as having an anti-hypercholesterolemic effect ³⁷.Oleic acid is a monounsaturated fatty acid that is showing good therapeutic value by reducing blood pressure, lowering cholesterol, preventing type -2 diabetes, improving brain function, and having immune modulatory effects and anti-cancer properties ^38-40. Astaxanthin was reported as an antioxidant and as having anti-cancer effects that promote healthy skin, improve cardiac health and male fertility, and reduce inflammation and pain. ^41-45

X-ray diffraction analysis

The XRD results provide detailed insights into the crystalline structure of the dried broccoli extract. The distinctive diffraction peaks observed at 65.2° (2θ) with a d-value of 1.430 are indicative of the presence of mineral crystals within the extract. This cluster of peaks reflects the arrangement and spacing of atoms in the crystalline structures, allowing for the identification of specific minerals present in the extract. The intensity of 62 cps/deg associated with these peaks suggests a relatively high concentration of the mineral components, which could play a role in the extract’s overall biological activity and stability. The clear and well-defined peaks observed are characteristic of crystalline materials, confirming that the dried broccoli extract contains mineral crystals with a defined and ordered structure.

Molecular docking

The docking results indicate that hydrogen bonds formed with Thr-368 and Gln-361 are essential in inhibiting the SIRT1 catalytic domain, as seen in the top two performing compounds, ethyl iso-allocate and crinamine. These results indicate that Thr-368 and Gln-361 are essential amino acids in this activity. Ethyl iso-allocate formed three hydrogen bonds with Thr-368, Glu-410, and Gln-361; similarly, crinamine formed two hydrogen bonds with Thr368 and Gln-361. The estimated free energy of binding and inhibition constant (Ki) for ethyl iso-allocholate were -5.88 kcal/mol and 48.88 uM, respectively, and for crinamine were -5.99 kcal/mol and 40.46 uM, respectively.

Both norethandrolone and betulin formed only one hydrogen bond with Ser-365 and had a similar inhibition constant (Ki) that averaged around 91 uM. Cis-Vaccenic acid formed two hydrogen bonds with Gln-421 and 361, which are identical to ethyl iso-allocholate, resulting in a similar inhibition constant at 44.82 uM. In addition, n-Hexadecanoic acid and oleic acid each formed one hydrogen bond with Gln-361. The estimated free energy of binding and inhibition constant (Ki) for n-Hexadecanoic were -.26 kcal/mol and 139.57 uM, respectively, and for oleic acid were -4.96 kcal/mol and 232.36 uM, respectively. Lupeol docking results were superior to the other docked compounds, even though they lacked the hydrogen bonds. Instead, lupeol formed multiple hydrophobic, polar, negative, and positive interactions with the surrounding residues. The estimated free energy of binding of lupeol was -6.14kcal/mol, and the inhibition constant (Ki) was 31.81 uM. The poses were visualized using the ligand interaction diagram from Maestro.

Figure 5: 2D interaction showing hydrogen bonding in magenta arrow, hydrophobic in green, polar in cyan, negative in red and positive in purple against SIRT1 catalytic domain of (A) ethyl iso-allocholate, Click here to View Figure

Antioxidant studies

The antioxidant activity of CMEB was found to be comparatively lower than that of the comparator antioxidant Trolox in both DPPH and ABTS assays. Previous studies have shown that broccoli possesses commendable antioxidant properties, as demonstrated by the DPPH and ABTS assays ^46-48. The antioxidant property of CMEB in this study could possibly be related to the bioactive constituent phytol. A previous study indicated that broccoli leaves and stems are rich in total phenols, and they have remarkable antioxidant and anti-cancer properties. Another study highlighted that the ethanolic extract from selected Malaysian plants exhibited strong antioxidant properties but no cytotoxic properties.⁴⁹

In-vitro cytotoxicity

The cytotoxicity effect of a cold methanolic extract of broccoli on A549 lung cancer cells, with an IC₅₀ value of 64.5 µg/ml, suggests that the extract contains bioactive compounds capable of significantly inhibiting the growth of these cancer cells, highlighting its potential as a source of anti-cancer agents. Using a cold methanolic extraction process helps retain heat-sensitive compounds, which may contribute to the extract’s effectiveness.

Interestingly, broccoli florets showed lower selectivity in targeting HepG2 and SW480 cells than broccoli shoots ⁴⁸.Historical research has shown that broccoli has several therapeutic benefits, including the role it plays as an anti-cancer agent, immunomodulator, antidiabetic agent, antibacterial agent, hepatoprotectant, cardio protectant, and anti-amnesic, and also because of its antioxidant properties.^4,31,50-52 Consistent with these findings, the current research confirms the cytotoxic activity of CMEB against A549 lung cancer cells. To verify the potential of broccoli extracts in cancer treatment, additional studies would be needed to understand which specific phytochemicals are involved and their mechanisms of action against cancer cells at the molecular level, their safety profile, and their effectiveness in-vivo.

Conclusions

Preventive measures, such as dietary habits, can often be implemented through one’s lifestyle. Research shows that eating broccoli, a vegetable with many medicinal properties, may be beneficial due to its content, which consists of compounds such as iso-allocholate, phytol, betulin, astaxanthin, and oleic acid, which are vital in today’s fast-paced world. Broccoli has been shown to have anti-cancer and anti-proliferation effects due to its cytotoxic properties. In addition, its potential for immunomodulation and antioxidant activity cannot be ignored. Therefore, we deduce that the inclusion of broccoli in one’s daily diet can potentially reduce the risk of cancer development and can serve as a complementary treatment, especially for lung cancer, which can have a significant impact on the progression of the disease.

Acknowledgement

The author would like to thank, (Insert university name and Dept. name) for their guidance and support to complete this article.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Author contributions

RUS: Conceptualization, methodology and resources. SSM, RA and MDA: Conceptualization, Methodology, investigation, validation. SSM, MDA, EMA, BNA, ASA, KMA, RA and RUS: Drafting article.

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