Summary An efficient and cost‐effective enzymatic production method for preparation of a high‐valued natural sweetener (dihydrochalcone glucoside trilobatin) was developed by the combination of hydrogenation and enzymatic hydrolysis reactions with α‐l‐rhamnosidase as the catalyst in aqueous medium. This technology is adopting the cheap and largely available citrus flavanone naringin as the starting material for trilobatin synthesis, and the present enzymatic technology is possibly utilised by commercial for scale‐up production. The production is a straightforward two‐step process, in which naringin was hydrogenated into naringin dihydrochalcone and followed by removal of the rhamnosyl group of naringin dihydrochalcone by enzymatic hydrolysis using immobilised α‐l‐rhamnosidase as the catalyst. Under optimised conditions, an overall yield of 96% was achieved with a very low loading of α‐l‐rhamnosidase catalyst at 60 °C in a neutral aqueous buffer solution within 2 h. The immobilised α‐l‐rhamnosidase catalyst can be recycled for 10 reactions (90% yield retained).

The presence of blood-brain barrier (BBB) that limits effective penetration of therapeutics is the main reason for poor outcomes of glioblastoma (GBM) treatment. Ultrasound (US) combined with microbubbles (MBs) can precisely disrupt the tight junctions of brain endothelial cells, thus creating "acoustic pores" and non-invasive opening the BBB. Here, chitosan oligosaccharide (COS) is conjugated with a sonosensitizer protoporphyrin IX (PpIX) and an immune-enhancing adjuvant Poly(I:C) via electrostatic adsorption, and crosslinked with a tumor-targeting molecule hyaluronic acid (HA) affording nanosonosensitizers HA-Poly(I:C)/COS-PpIX (abbreviated as "HP/CP" NSs). HP/ CP NSs can target and penetrate GBMs, and trigger reactive oxygen species production upon US, simultaneously causing mitochondrial dysfunction and DNA damage. Tumor-associated antigens released by sonodynamic therapyinduced immunogenic cell death and loaded Poly(I:C) form an in situ vaccine together to potentiate antitumor immune responses. In orthotopic GBM mice models, the HP/CP+US treatments prolong mice survival, enhance cytotoxic T-lymphocytes infiltration, and activate peripheral immune circulation. Besides, HP/CP NSs possess favorable biosafety profiles. Collectively, this study sheds light on the application of HP/CP NSs for synergistic sonoimmunotherapy of GBMs after non-invasive opening of the BBB.

Summary The rhamnosyl group of naringin dihydrochalcone, neohesperidin dihydrochalcone, naringin and hesperidin was selectively removed by enzymatic hydrolysis using an immobilised α‐L‐rhamnosidase. Monoglycosylated products, including trilobatin, hesperetin dihydrochalcone‐7‐O‐glucoside, prunin and hesperetin‐7‐O‐glucoside, were isolated and characterised by 1H and 13C NMR and ESI‐MS. To optimise the enzymatic reaction conditions and its process costs, the hydrolysis of neohesperidin dihydrochalcone to produce trilobatin was selected as a model reaction. Using a ratio of neohesperidin dihydrochalcone: immobilised α‐L‐rhamnosidase equal to 1:0.6, the trilobatin yields was over 98%. The recycle of enzyme was also investigated, obtaining trilobatin with a yields of 80% even when the twentieth reaction cycle was conducted. Moreover, antiradical and antimicrobial activities of the obtained flavonoid monoglucosides were examined by DPPH, FRAP and ORAC methods, and compared with the efficacy of parental flavonoid glycosides and their aglycone. The results highlight that some of the obtained flavonoid monoglucosides show significant improvement in the antioxidant activity.

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