# Plant Extracellular Vesicles: Investigating Their Utilization as Beneficial Nutrients in Diet
## 3 Conventional Methods for P-EV Isolation and Characterization  
To date, a major challenge associated with P-EV research is the lack of standardized protocols for good-quality purification [4]. Despite this inconvenience, EVs can be isolated in large amounts from many parts of the plant including the fruits and apoplastic fluid from the leaves, seeds, and roots [7]. Although different methods have recently been developed for plant vesicle isolation, including size exclusion chromatography, immunoprecipitation, and polymer-based precipitation [13], ultracentrifugation remains the preferred one, due to the greater amount of material that can be recovered [22]. Differential ultracentrifugation \begin{table}
\begin{tabular}{c c c} \hline \hline
**Potential Therapeutic Activities** & **microRNA** & **P-EVs Source** & **Ref** \\ \hline Reduced risk of & & Green leafy vegetables (i.e., cabbage, \\ cardiovascular disease & & spinach, lettuce) & [28] \\ Immuno-modulation & miR-168 & Strawberry & [30] \\ Modulation of inflammatory response & miR-164a & Hami melon & \\ by targeting cytokine genes (IL-16, & miR-398-b, miR-1078 & Orange & [31] \\ IL-1A, IL-6 and IL-5 respectively) & miR-4995 & Tomato & \\  & gma-miR159a-3p, & & \\ Antiproliferative effects on human & gma-miR159e-3p & Soybean & [32] \\ colon Caco-2 cancer cells & gma-miR-6300, mtr-miR-156a & & \\ Inhibition of SARS-CoV-2 replication & zma-miR-3980-5p & Blueberry & \\ by targeting viral genes & bdi-miR-5059, ns-miR-8077 & Graprefruit & [33] \\ Suppression of SARS-CoV-2-induced & gma-miR-4995 & Coconut & \\ cytopathic effect by inhibition of & gma-miR-6300, aqc-miR-159 & Ginger & [29] \\ Nsp12 and spike genes expression & aryl-miR396a-5p & & \\ Anti-viral effect against & miR-2911 & Honeysuckle & [34] \\ influenza virus & miR-156 & miR-162, miR-166 5p, & \\ Anti-inflammatory effects by & miR-167, miR-172, miR-390, & & \\ downregulation of NLRP3 & miR-394, miR-396 3p, miR-399, & & \\ inflammasome expression, enhancing & miR-529, miR-2111 5p & & \\  & mol-miR160h, mol-mir482b, & & \\  & mol-mir166, mol-mir159c, & & \\ Antiproliferative and proapoptotic & mol-mir1218a, mol-mir167f-3p, & & \\ mechanisms in cancer cells & mol-mir156e, mol-mir395d, & & \\  & mol-mir393a, mol-mir397a, & & \\  & mol-mir858b, mol-mir396a & & \\  & miR160 & Broccoli & \\ Inhibition of breast cancer growth in & mir-159 & Arabidopsis thaliana & [37] \\ mouse model & mir-156 & Wheat and corn & [38] \\ Intestinal epithelium homeostasis & mir-156 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Potential therapeutic activities and plant sources of miRNAs packaged in P-EVs. is a separating technique that involves centrifugation cycles of different centrifugal forces and duration to separate particles in the homogenate or the juice of plants, proportionally to their molecular weight and density [42]. During the EV isolation process, high-density particles such as dead cells and apoptotic bodies are first removed by low-speed centrifugation, followed by higher speed spins to remove larger vesicles and debris. In the final purification step, a supernatant containing EVs is pelleted and concentrated by high-speed centrifugation (40,000-200,000\(\times\)_g_) [22]. Ultracentrifugation is often supplemented by additional purifying methods [11], such as ultrafiltration, which consists in the use of ultrafine nano-membranes with different pore sizes, to trap particles above a certain dimension threshold, while allowing the smaller ones to flow through [7]. Moreover, since differential ultracentrifugation sediments contain contaminants such as proteins and other vesicles [13], it can be combined with density gradient ultracentrifugation to isolate P-EVs with higher purity [42]. Briefly, gradient ultracentrifugation uses a previously developed medium with a gradually decreasing density gradient, from the bottom to the top [22]. Under the application of the centrifugal force, solutes in the sample, including vesicles, separate into different zones according to the specific sedimentation rate [13].