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  • Municipal Landscape-Derived Echinacea purpurea: A Green Nano-Biotechnological Approach for Enhanced Anticancer Compound Production

  • narges karimi,1 yasamin haghshenas,2 mojtaba haghshenas,3 saeed ahmadi,4,*
    1. municipality
    2. university
    3. municipality
    4. municipality


  • Introduction: Abstract Background: Plant-derived secondary metabolites, particularly flavonoids, are promising sources for anticancer drug discovery. Echinacea purpurea is renowned for its immunomodulatory and potential anticancer properties. This study investigates a novel approach to enhance the production of these valuable compounds and their subsequent anticancer efficacy by utilizing biosynthesized Zinc Oxide Nanoparticles (ZnO NPs) in in vitro callus cultures. Methods: ZnO NPs were green-synthesized using Allium jesdianum extract and characterized by XRD, SEM, TEM, and FTIR. Leaf explants of E. purpurea were cultured on MS medium supplemented with 2.0 mg/l 2,4-D for callus induction. The established calli were then treated with various concentrations (0-150 mg/l) of biosynthesized ZnO NPs and commercial ZnO microparticles (MPs). The total flavonoid content (TFC) of the callus extracts was quantified. The cytotoxic activity of the extracts against Michigan Cancer Foundation-7 (MCF-7) breast cancer cells, non-tumorigenic MCF-10A breast cells, and Peripheral Blood Mononuclear Cells (PBMCs) was assessed using the MTT assay. The immunomodulatory effect was evaluated by analyzing CD4 expression on PBMCs via flow cytometry. Results: Characterization confirmed the successful synthesis of spherical ZnO NPs with an average size of 40 nm. The optimum concentration for callus growth and flavonoid production was 75 mg/l ZnO NPs, yielding a significantly higher TFC (3.7 mg/g DW) compared to the control and MPs-treated groups. Callus extracts treated with ZnO NPs exhibited potent and selective dose-dependent cytotoxicity against MCF-7 cells (CC₅₀ = 625 μg/ml), significantly higher than those treated with MPs (CC₅₀ = 875 μg/ml). Crucially, these extracts showed no significant cytotoxicity against MCF-10A cells or PBMCs. Furthermore, the extracts did not alter the frequency or mean fluorescence intensity of CD4+ T cells, indicating no non-specific immune activation. Conclusion: Biosynthesized ZnO NPs act as a powerful elicitor in E. purpurea callus cultures, significantly enhancing the production of flavonoids with selective anticancer activity against breast cancer cells. This nanotechnology-based approach presents a sustainable and efficient strategy for boosting the yield of bioactive compounds from medicinal plants for potential chemotherapeutic or adjuvant applications. Keywords: Green Synthesis, Zinc Oxide Nanoparticles, Echinacea purpurea, Callus Culture, Flavonoids, Anticancer Activity, MCF-7 Cells. 1. Introduction Cancer remains one of the leading causes of mortality worldwide, driving the relentless search for novel and more effective therapeutic agents. Natural products, especially those derived from medicinal plants, have served as a cornerstone for anticancer drug discovery due to their structural diversity and biological activity [1]. Echinacea purpurea, a member of the Asteraceae family, is widely recognized for its immunostimulatory properties [2]. Modern pharmacological studies have revealed that its bioactive compounds, including caffeic acid derivatives, alkylamides, polysaccharides, and notably, flavonoids, are responsible for its anti-inflammatory, antioxidant, and anticancer activities [3, 4]. A significant challenge in utilizing plant-derived compounds is their low natural abundance, which makes direct extraction from wild plants economically and environmentally unsustainable. Plant tissue culture technology offers a controlled and scalable alternative for the continuous production of these high-value secondary metabolites independent of geographical and seasonal constraints [5]. Callus culture, in particular, is a potent system for enhancing the production of specific phytochemicals through the application of elicitors—substances that trigger stress responses and activate defense-related biosynthetic pathways in plants [6]. In recent years, nanotechnology has emerged as a revolutionary tool in agriculture and biotechnology. Nanoparticles (NPs), due to their unique physicochemical properties, can penetrate plant cells and act as effective elicitors [7]. Among them, Zinc Oxide Nanoparticles (ZnO NPs) are particularly interesting. Zinc is an essential micronutrient that acts as a cofactor for numerous enzymes, and ZnO NPs have been shown to influence plant growth, development, and metabolic activity more effectively than their bulk counterparts [8, 9]. The "green synthesis" of NPs using plant extracts is favored over chemical methods due to its eco-friendliness, cost-effectiveness, and production of biocompatible NPs capped with organic compounds that may enhance their biological activity [10]. While previous studies have explored the effects of metals on plants, the use of green-synthesized ZnO NPs to specifically modulate the anticancer properties of medicinal plant callus cultures remains largely unexplored. We hypothesize that biosynthesized ZnO NPs will act as superior elicitors compared to ZnO microparticles (MPs), leading to enhanced flavonoid production in E. purpurea callus and consequently, a more potent and selective extract against cancer cells. This study aims to: (1) synthesize and characterize ZnO NPs using Allium jesdianum extract, (2) establish an optimal callus culture system for E. purpurea, (3) investigate the eliciting effect of ZnO NPs and MPs on callus biomass and flavonoid content, and (4) evaluate the selective cytotoxicity and immunomodulatory potential of the elicited callus extracts against breast cancer cells.
  • Methods: 2.1. Green Synthesis and Characterization of ZnO NPs ZnO NPs were synthesized according to a modified method of Karnan and Selvakumar [11]. Briefly, 10 ml of aqueous Allium jesdianum extract was added to 50 ml of 0.1 M zinc nitrate hexahydrate solution and stirred at 75°C for 2 hours. The resulting precipitate was centrifuged, washed, dried, and calcined at 350°C for 2 hours. The synthesized NPs were characterized using X-ray Diffraction (XRD; Philips PW1730), Scanning Electron Microscopy (SEM; TESCAN VEGA3), Transmission Electron Microscopy (TEM; Philips EM208S), Atomic Force Microscopy (AFM; Bruker), and Fourier-Transform Infrared Spectroscopy (FTIR; PerkinElmer Spectrum RX I). 2.2. Plant Material and Callus Induction Leaf explants from 3-month-old E. purpurea plants were surface-sterilized and cultured on solid MS (Murashige and Skoog) medium supplemented with 30 g/l sucrose, 7 g/l agar, and different concentrations of 2,4-D (1, 2, 5 mg/l) or NAA (1, 2, 5 mg/l). The pH was adjusted to 5.8. Cultures were maintained at 25 ± 1°C under dark and light (16/8 h photoperiod) conditions. After four weeks, the optimum hormone concentration for callus induction was determined based on induction frequency and biomass. 2.3. Elicitor Treatment The established calli were subcultured onto the optimized MS medium (containing 2 mg/l 2,4-D) supplemented with different concentrations (0, 10, 25, 50, 75, 100, 150 mg/l) of sterile biosynthesized ZnO NPs or commercial ZnO MPs (Sigma-Aldrich). The cultures were maintained for 30 days, after which the callus biomass (Dry Weight) and morphology were recorded. 2.4. Preparation of Callus Extracts Dry calli from each treatment group were powdered and extracted with methanol using the maceration method at room temperature for 72 hours. The extracts were filtered, concentrated using a rotary evaporator, and freeze-dried. 2.5. Quantification of Total Flavonoid Content (TFC) The TFC was determined using the aluminum chloride colorimetric method [12]. Briefly, 1 mg/ml of each extract was mixed with 1 ml of 2% AlCl₃ methanolic solution. After 30 minutes of incubation, the absorbance was measured at 415 nm. A standard curve was prepared using quercetin, and the results were expressed as mg of quercetin equivalent per gram of dry weight (mg QE/g DW). 2.6. Cell Culture and Cytotoxicity Assay (MTT) The human breast adenocarcinoma cell line (MCF-7), the non-tumorigenic breast cell line (MCF-10A), and Peripheral Blood Mononuclear Cells (PBMCs) isolated from healthy donors were used. Cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/well and treated with various concentrations (0–1000 μg/ml) of the callus extracts for 48 hours. Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The concentration causing 50% cell death (CC₅₀) was calculated. 2.7. Flow Cytometric Analysis of CD4 Expression PBMCs were cultured with or without the callus extracts for 72 hours. The cells were then harvested, washed, and stained with PE-conjugated anti-human CD4 monoclonal antibody. Analysis was performed using a BD FACS flow cytometer, and the frequency and mean fluorescence intensity (MFI) of CD4+ T cells were determined. 2.8. Statistical Analysis All experiments were performed in triplicate. Data were analyzed using two-way ANOVA followed by Tukey's post-hoc test in SPSS software (v. 16.0). A p-value of less than 0.05 was considered statistically significant.
  • Results: 3.1. Characterization of Biosynthesized ZnO NPs The XRD pattern confirmed the crystalline nature of the synthesized NPs, showing characteristic peaks for zincite (JCPDS card no. 36-1451). The broadening of peaks indicated their nanoscale size. SEM and TEM analysis revealed that the NPs were predominantly spherical with an average size of 40 nm (Figures 1A & 1B). AFM further confirmed the size and surface topography. FTIR spectra showed a peak at ~536 cm⁻¹, corresponding to Zn-O stretching vibration, and peaks around 3429 cm⁻¹ and 1631 cm⁻¹ indicated the presence of O-H and C=O groups from the plant extract capping the NPs, which contributes to their stability. 3.2. Callus Induction and Elicitor Treatment The highest callus induction frequency (96%) and biomass yield (3.37 g DW) were achieved on MS medium supplemented with 2 mg/l 2,4-D under dark conditions (Table 1). The addition of ZnO NPs significantly influenced callus growth in a dose-dependent manner. The optimal concentration was 75 mg/l ZnO NPs, which produced friable, pale-yellow callus with the highest biomass. Concentrations above 100 mg/l proved inhibitory, likely due to Zn²⁺ ion-induced oxidative stress. ZnO MPs also enhanced growth but were consistently less effective than NPs at equivalent concentrations, highlighting the superior efficacy of the nano form (Table 2). 3.3. Enhancement of Flavonoid Production The TFC analysis revealed a direct correlation between ZnO NP treatment and flavonoid biosynthesis. The highest TFC of 3.7 mg QE/g DW was recorded in calli treated with 75 mg/l ZnO NPs, representing a more than four-fold increase compared to the control (0.89 mg QE/g DW) (Table 3). Calli treated with ZnO MPs also showed an increase, but it was significantly lower (max 3.09 mg QE/g DW at 100 mg/l). This dramatic enhancement can be attributed to the NPs acting as abiotic elicitors. Their small size allows for easier uptake into plant cells, where they may induce mild stress, activating the plant's defense mechanisms and thereby upregulating the phenylpropanoid pathway responsible for flavonoid biosynthesis. 3.4. Selective Cytotoxicity Against Cancer Cells The MTT assay demonstrated the potent and selective anticancer activity of the extracts (Figure 2). The extract from callus treated with 75 mg/l ZnO NPs exhibited the highest cytotoxicity against MCF-7 breast cancer cells, with a CC₅₀ value of 625 μg/ml. The extract from the ZnO MPs-treated group was less potent (CC₅₀ = 875 μg/ml). Most importantly, both extracts showed significantly lower toxicity towards the non-tumorigenic MCF-10A cell line and PBMCs (Figure 3). This selectivity is a crucial finding, suggesting that the elicited flavonoids target cancer cell mechanisms without harming normal cells. 3.5. Effect on Immune Cell Phenotype Flow cytometric analysis confirmed that none of the callus extracts, including the most potent one, induced a non-specific activation of the immune system. There was no significant change in the percentage of CD4+ T cells or the MFI of the CD4 marker on PBMCs after 72 hours of treatment (Table 4). This indicates that the observed anti-proliferative effect on PBMCs in the MTT assay was due to cytotoxicity at high concentrations and not an immunomodulatory event, and that the extracts are not mitogenic to T-cells.
  • Conclusion: This study successfully demonstrates a novel and efficient strategy for enhancing the anticancer potential of Echinacea purpurea through the integration of plant tissue culture and nanotechnology. Biosynthesized ZnO NPs served as effective elicitors, significantly boosting the production of flavonoids in callus cultures. The resulting extracts displayed potent and selective cytotoxicity against MCF-7 breast cancer cells while showing minimal effects on normal cells and no non-specific immune activation. These findings position green-synthesized ZnO NPs as a powerful tool in phyto-biotechnology for the sustainable production of high-value anticancer compounds. Future work will focus on identifying and quantifying the specific flavonoid compounds upregulated by this treatment and elucidating their precise mechanism of action against cancer cells.
  • Keywords: Green Synthesis, Zinc Oxide Nanoparticles, Echinacea purpurea, Callus Culture, Flavonoids, Anticance

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