(M1530-04-24) Isolation and Biological Activity Evaluations of Exosome-Like Nanovesicles Derived from Dendrobium officinale

Purpose: Due to their biocompatibility, low toxicity, and resistance to gastrointestinal degradation, plant-derived exosome-like nanoparticles (PDENs) of a medicinal herb could serve as a carrier for the active ingredients in the herb to demonstrate treatment potential for various diseases ^[1]. Dendrobium officinale has been extensively reported by us and others to exhibit antioxidative, hypoglycemic, and mucosal barrier repair effects ^{[2, 3]}. It is hypothesized that the Dendrobium officinale-derived exosome-like nanoparticles will exhibit similar or even better activities than its aqueous extract. The current study aims to isolate Dendrobium officinale stem-derived exosome-like nanovesicles (DOS-ELNs) and Dendrobium officinale leaf-derived exosome-like nanovesicles (DOL-ELNs) to evaluate their antioxidative and hypoglycemic activities.

Methods: About 100 g of fresh Dendrobium officinale stems or leaves was homogenized in a blender for 30 min followed by serial centrifugations (3000×g for 20 min, 10000×g for 60 min and 150,000 ×g for 2 h) to obtain pellet. The pellet was then resuspended and subjected to purification using a sucrose gradient ultracentrifugation (8, 30, 45 and 60% sucrose in 20 mM Tris-Cl, pH 7.2) at 120,000×g for 2 h to collect DOS-ELNs and DOL-ELNs from the 30/45% sucrose interface. For characterization of DOS-ELNs and DOL-ELNs, their shape and particle size of were conducted via transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), respectively. In addition, the content of polysaccharides, flavonoids, and polyphenols in DOS-ELNs and DOL-ELNs were determined by H₂SO₄-anthrone method, plant flavonoids content assay kit and Folin-Ciocalteu assay, respectively. Moreover, levels of protein, RNA and lipids in DOS-ELNs and DOL-ELNs were detected by polyacrylamide gel electrophoresis, agarose gel electrophoresis, and thin-layer chromatography, respectively. Serial in-vitro tests were adopted to evaluate the antioxidative (scavenging capacities for ABTS radical, DPPH radical and superoxide anion), hypoglycemic (inhibition of α-glucosidase) potentials and intestinal integrity protection effect in LPS-induced inflammatory Caco-2 cells model of DOS-ELNs and DOL-ELNs.

Results: DOS-ELNs and DOL-ELNs were successfully obtained with average particle size of 181.9±51.8 nm and 224.8±83.3 nm, respectively (Fig. 1A) and a “double-layer shell” structure (Fig. 1B). It was found that DOS-ELNs and DOL-ELNs contained various types of lipids (Fig. 1C), RNAs with molecular weight range of 100-2500 bp (Fig. 1E) and proteins with molecular weight range of 16-95 kDa (Fig. 1F). DOS-ELNs and DOL-ELNs contained similar monosaccharide of mainly mannose and glucose (Fig. 1G) and slightly different contents of polysaccharide, flavonoid, and polyphenol contents (4.44±0.231, 2.06±0.277, and 2.45±0.063 mg/mL in DOS-ELNs, and 1.83±0.106, 3.12±0.318, and 4.56±0.105 mg/mL in DOL-ELNs). The IC₅₀ values of DOS-ELNs for α-glucosidase inhibitory activity (Fig. 2A), DPPH (Fig. 2B), ABTS (Fig. 2C) and superoxide anion radical scavenging activity (Fig. 2D) were 0.75, 1.28, 17.3, 43.34 mg/mL and those of DOL-ELNs were 0.93, 1.12, 13.2, 40.39 mg/mL, respectively. Moreover, both DOS-ELNs and DOL-ELNs could effectively improve intestinal integrity by elevating the expression of tight junction proteins (ZO-1 and Occludin) (Fig. 3A) and reduced the structural damage of cell membrane in Caco-2 cells (Fig. 3B).

Conclusion: Our current study for the first time isolated and characterized exosome-like nanoparticles from Dendrobium officinale stem and leaves. Despite slight difference in average particle size and contents of polysaccharide, flavonoid, and polyphenol between DOS-ELNs and DOL-ELNs, no significant difference was observed for their antioxidative activity, α-glucosidase inhibitory potential and intestinal integrity protection effect.

References: [1]. Munir J, Lee M, Ryu S. Exosomes in Food: Health Benefits and Clinical Relevance in Diseases. Advances in Nutrition. 2020, 11(3):687-696.
[2]. Zou J, Song Q, Shaw PC, Zuo Z. Dendrobium officinale regulate lipid metabolism in diabetic mouse liver via PPAR-RXR signaling pathway: Evidence from an integrated multi-omics analysis. Biomed Pharmacother. 2024, 173:116395.
[3]. Song Q, Cheng SW, Zou J, Li KSL, Cheng H, Wai Lau DT, Han Q, Yang X, Shaw PC, Zuo Z. Role of gut microbiota on regulation potential of Dendrobium officinale Kimura & Migo in metabolic syndrome: In-vitro fermentation screening and in-vivo verification in db/db mice. Journal of Ethnopharmacology. 2024, 321:117437.

Acknowledgements: Hong Kong Scholar program and China Postdoctoral Science Foundation; Li Dak Sum Yip Yio Chin R & D Center for Chinese Medicine at The Chinese University of Hong Kong.


Fig. 1 Isolation (A) and characterization of DOS-ELNs and DOL-ELNs for their size (B) and shape (C), contents of lipids (D)/nucleic acids (E)/proteins (F) and monosaccharide composition (G).


Fig. 2 Comparison of the hypoglycemic (A: α-glucosidase inhibitory activity) and antioxidative activities (B: DPPH radical scavenging activity; C: ABTS radical scavenging activity; D: Superoxide anion scavenging activity) of DOS-ELNs and DOL-ELNs.


Fig. 3 DOS-ELNs and DOL-ELNs at 500 μg/mL alleviated tight junction proteins (A) (ZO-1 and Occludin) and membrane damage (B) injury in LPS-induced Caco-2 cells.