Isolation of ginger ENPs by differential ultra-centrifugation and PEG method

ENPs from fresh ginger rhizomes were isolated as per previously published protocols with minor modifications⁵. About 250 g of fresh ginger rhizomes were procured from local market and washed thoroughly with water. After peeling, the rhizomes were ground using a mixer grinder (750-watt power, maximum rpm 18,500 under no load conditions and 11,000 rpm with load) at medium speed for 3 mins with 30 sec on/off cycles. Excess fiber was removed from the juice by filtering through a nylon mesh (pore size 125 µm). The fresh juice thus obtained was centrifuged at 2000g for 10 min, 6000 g for 20 min and 10,000 g for 45 min to remove large fibers/cells, large particles/cell debris and microparticles, respectively^19,39. The supernatant obtained after the 10,000 g step is referred as S10 extract. The yield of S10 extract varied between 70–80% of the initial ginger weight (e.g. 180 to 200 ml of S10 extract per 250 g of ginger) due to the removal of peel and insoluble debris. To purify ginger ENPs using conventional method, the S10 extract was subjected to ultracentrifugation in Beckman SW32.1 Ti swinging bucket rotor at 1,25,000 g for 2 hours at 4 °C. For isolation of ENPs using PEG, the S10 supernatant was mixed with PEG6000/4000/8000 (Sigma Aldrich) to reach a final concentration of 8%, 10%, 12% and 15% (weight by volume) and were incubated overnight at 4 °C with gentle rocking. After centrifugation at 8000 g for 30 min at 4 °C in a fixed angle rotor (Eppendorf), the excess PEG/liquid was removed by inverting the tube on a piece of tissue paper for 5 min. Both ultra and PEG-ENP pellets were suspended in sterile water and dialyzed overnight against sterile milli Q grade water using Dialysis membrane (Himedia) with a 10 kDa pore size. ENPs were lyophilized, weighed and resuspended in milli Q grade water to achieve a final concentration of 0.5 mg/µl of ENPs. The methodology developed here has been submitted to the EV-TRACK knowledgebase and this can be accessed with the EV-TRACK ID: {"type":"entrez-nucleotide","attrs":{"text":"EV190070","term_id":"151279710"}}EV190070⁴¹.