Plant-Derived Nanovesicles from Soaked Rice Water: A Novel and Sustainable Platform for the Delivery of Natural Anti-Oxidant γ-Oryzanol

Abstract

Gamma oryzanol (GO) is a natural anti-oxidant found in rice bran with potential health benefits. Conventional isolation of GO from rice bran requires the use of non-eco-friendly solvents such as acetone, ethyl acetate and hexane due to its low aqueous solubility. Further, nanoencapsulation of GO is required for the enhancement of stability and bioavailability. Plant-derived nanovesicles (PDNVs) are natural/intrinsic exosome-mimetic vesicles isolated from edible plants using green methods. Washed/soaked rice water (SRW) is often discarded as waste prior to cooking rice. However, traditional knowledge indicates its health-promoting anti-oxidant benefit, probably contributed by the presence of GO. Herein, for the first time, we isolated PDNVs from SRW by the cost-effective Polyethylene glycol 6000(PEG) precipitation method and demonstrated the presence of GO in PDNVs. In our initial screen, PDNVs were isolated from both rice grains (RGs) as well as the SRW of four different rice varieties, in which we identified the copious presence of GO in black RGs and brown SRW PDNVs. Both RG and SRW PDNVs were non-toxic to keratinocytes. SRW PDNVs displayed distinct cellular uptake mechanisms compared to RG PDNVs in human keratinocytes. Compared to native GO, brown SRW PDNVs containing GO displayed superior anti-oxidant activity in HaCaT keratinocytes, likely due to its enhanced cellular uptake. Overall, we describe here a waste-to-wealth green approach using an economical PEG method for the extraction of GO in bioavailable form. Given that oxidative stress is a driving factor for inflammation and related diseases, SRW PDNVs provide an affordable natural formulation for the treatment of diseases with underlying oxidative stress and inflammation.

Keywords: anti-oxidant activity; gamma oryzanol; plant-derived exosome-like nanovesicles; rice water.

Figure 1

Isolation of PDNVs from rice grain (RG) and soaked rice water (SRW) from indicated rice varieties. (A) Schematic depicting the methodology used for the isolation of rice grain (RG) and soaked rice water (SRW) PDNVs (S2, S6, S10 refer to supernatant obtained after centrifugation at 2000× g, 6000× g and 10,000× g, respectively). (B) Representative image of the average size distribution of PDNVs isolated from RG and SRW. (C) Representative image of average zeta potential of PDNVs isolated from RG and SRW. (D) Size and zeta potential values from three independent batches of RG or SRW PDNVs. (E) Scanning electron microscopic images of the RG and SRW PDNVs. Scale bar—2 μm. n.s. means nonsignificant. * p < 0.05, ** p < 0.01 *** p < 0.001 and **** p < 0.0001.

Figure 2

Biochemical characterization of RG and SRW PDNVs. (A) Total lipid content of isolated PDNVs assessed by phospho-vanillin method. (B) Total lipids were extracted from indicated PDNVs and resolved using a Silica Gel 60 F₂₅₄ plate. (C) Total protein content of isolated PDNVs estimated by BCA method. (D) Protein to lipid ratio was calculated based on the concentrations of PDNVs obtained with BCA and phospho-vanillin assay methods. All figures represent data from an average of three independent PDNV batches isolated from different rice varieties. All figures represent data from three independent PDNV batches isolated from different rice varieties. * p < 0.05.

Figure 3

Both RG and SRW PDNVs contain GO. (A) HPLC chromatograms of RG and SRW PDNV ethyl acetate extracts showing the presence of all four isomers of GO, namely cycloartenylferulate at 17.9 min, 24-methylenecyclo-artenyl ferulate at 19.3 min, campesteryl ferylate at 19.9 min and β-sitosteryl-ferulate at 22.3 min. (B) Total yield of GO isomers in RG and SRW PDNVs per mg of lipid, (a) cycloartenylferulate, (b) 24-methylenecyclo-artanyl ferulate, (c) campesteryl ferylate and (d) β-sitosteryl-ferulate. (C) Total GO content in RG and SRW PDNVs. (D) GO isomer composition in both RG and SRW PDNVs represented as the relative percentage. All figures represent data from three independent PDNV batches isolated from different rice varieties. n.s. means nonsignificant. * p < 0.05, ** p < 0.01 *** p < 0.001, and **** p < 0.0001.

Figure 4

RG and SRW PDNVs are non-toxic to HaCaT cells. HaCaT cells were incubated with either black or brown RGSRW PDNVs or only media (mock) at indicated concentrations for 24 h and relative cell viability was measured by MTT assay as indicated in Materials and Methods. All figures represent data from an average of three independent PDNVs batches; n.s. means nonsignificant. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 5

SRW PDNVs display distinct uptake kinetics compared to RG PDNVs. (A) HaCaT cells were incubated with 160 μg/mL (lipid equivalent) of DIL-labeled brown or black RG and SRW PDNVs. Cells were washed and fixed at indicated time points, counterstained with DAPI and observed under fluorescent microscope. PDNVs taken up are shown in red and nuclei are shown in blue. (B) To dissect out the uptake mechanism, cells were pre-incubated with specific inhibitors of endocytic pathways such as MβCD, AML, CPZ and IND, followed by the addition of indicated PDNVs. Mock refers to cells incubated with PDNV alone.

Figure 6

Brown SRW PDNV has potent anti-oxidant activity. (A) HaCaT cells were pretreated with 300 ng/mL of γ-Oryzanol or 480 μg/mL of PDNVs for 12 h followed by treatment with H₂O₂ to induce oxidative stress. ROS production was determined by treating cells with the ROS-sensitive fluorescent dye, DCFH2-DA, as described in the Materials and Methods section. Representative pictures of ROS production in cells (in green) and corresponding DIC images are shown. Bar graph indicates the relative ROS production measured by quantifying fluorescent intensity taken from multiple random areas within the wells. Data representative of three independent experiments; n.s. means nonsignificant, * p < 0.05, ** p < 0.01 and *** p < 0.001. (B) Table depicting the relative oryzanol concentration of PDNVs used in this experiment.