Bioactive macromolecules are proposed as an alternative option to synthetic drugs due to their health benefits and minimal adverse effects. A wide range of bioactive macromolecules derived from natural sources are used in foods for their functionality and as nutraceuticals [1]. These bioactive macromolecules can be sourced from microorganisms [2], animals [3], and plants [4], with plants being the common source due to their biodiversity. Proteins (peptides) [[4], [5], [6]], lipids [[7], [8], [9]], and dietary fibers [10] are examples of plant derived macromolecules that possess biological activity such as antioxidation [4,7,10,11], anti-inflammation [5,8,12], and anticancer [6,9,13]. Various extraction methods can be utilized to obtain these plant macromolecules by cell disruption, including ultrasonication, enzymatic reactions, and microwave [1,11]. In contrast to cell disruption, bioactive macromolecules can also be released from the plant cells through packing into the releasing vesicles. Extracellular vesicles (EVs) are nanosized lipid bilayer membrane-enclosed vesicles that are naturally released from plant cells for cellular waste disposal, intercellular communication, and defense response against pathogens [14,15].

Regardless of explicit isolation and characterization [16], plant derived EVs showed promising potential to be drug delivery systems [17] and significant bioactivity in different pathological models (e.g., skin wounds [18,19], inflammation [20,21], myocardial infarction [22], and cancer [[23], [24], [25]]) due to their low immunogenicity, biocompatibility, and stability under stimulated gastrointestinal conditions [[26], [27], [28]]. The plant derived EVs exhibit the possibility for clinical uses as evidenced by the phase I clinical study of EVs derived from ginger (NCT04879810), grape (NCT01668849), and other fruits (NCT01294072) [15,29]. These clinical studies assure their potential to be developed as novel pharmaceutical products. Different plant derived EVs consist of different bioactive components, e.g., small molecules (e.g., sulforaphane [30], shogaol [26], and ginsenoside Rg3 [31]) and biological macromolecules (e.g., proteins [32,33], lipids [32,34], and nucleic acids [31,32,35]). In our previous study, plant derived bioactive macromolecules were successfully prepared in the form of nanosized EVs from Raphanus sativus L. var. caudatus microgreens (RSM) by a simple differential centrifugation technique [32]. Fourier-transform infrared (FTIR) microspectroscopy analysis revealed that RSM derived EVs contained proteins as the major biological macromolecules over lipids and nucleic acids. β-Pleated sheets were found to be the major secondary structure proteins. None of the small molecules were detected in RSM derived EVs. Our previous study demonstrated a simple isolation method to obtain stable nanosized EVs that consisted of active biological macromolecules [32].

Among potential bioactivities, anticancer is one of the major bioactivities that were investigated in plant derived bioactive macromolecules and EVs. Our screening study on anticancer activity demonstrated that RSM derived EVs possessed selective antiproliferative activity against HCT116 colon cancer cells over Vero normal cells [32]. The anticancer activity, thus, was aimed to be an interesting biological activity. Lifestyle changes with decreased physical activity and increased consumption of a Westernized diet are the important causes of colitis and delayed recovery of the gut injury. These behavioral changes promote colon cancer, the third most common cancer worldwide [36]. Oral administration is a high-patient compliance, convenient, and non-invasive route of administration [37]. Oral administration, thus, is a desired route of administration for plant derived EVs. Tissues and organs in the gastrointestinal tract (i.e., mouth, esophagus, stomach, small intestine, large intestine, and anus) have the chance to be directly exposed to the EVs without first-pass metabolism by the liver. The colon or large intestine is an organ among them, which might be beneficial from the direct contact effect. This study, thus, aimed to study the preferable mechanism of action of RSM derived EVs in HCT116 colon cancer cells for their anticancer potential uses and possibility to use as by the oral administration.

Apoptosis is a preferable death mechanism of cancer cells and a pharmacodynamic endpoint of anticancer agents. Apoptosis, therefore, is focused on this study. A neutral red uptake assay was performed to screen the antiproliferative activity. The morphological changes were also observed in apoptotic cells, including cell blebbing, chromatin condensation, DNA fragmentation, and apoptotic body formation. Moreover, the FTIR microspectroscopy analysis was performed to determine the altered cellular biochemical components. Circular dichroism spectroscopy was performed to determine the effect of EVs on DNA structure. The comet assay was performed to determine the DNA damage effect. The nuclear morphological change observed by DAPI staining was performed to determine the changing of nuclear morphology related to apoptosis. Flow cytometry with annexin V-FITC-propidium iodide double staining was performed to determine the mode of cell death. An increase in caspase activity was determined to investigate the apoptotic-inducing pathways. By using different techniques to confirm each undergoing process, this study provided comprehensive information after the colon cancer cells were treated with EVs in terms of mechanism.