N. benthamiana leaves were collected 6 days after infiltration
and immediately frozen in liquid nitrogen. Samples were lyophilized
(CHRIST Alpha 1-4 LD Plus, Martin Christ, Osterode am Harz, Germany)
and powdered with a ball mill grinder (MM301, RETSCH, Haan, Germany)
for 2 × 1 min at 29 Hz. Lyophilized samples were weighed with
a precision scale, and 100 mg of each was suspended in 5 mL of 80%
(v/v) aqueous methanol and sonicated for 20 min, followed by centrifugation
for 15 min at 3000 rpm. Two methanol extractions were performed for
each sample, and supernatants were combined into 50 mL Falcon tubes
and evaporated to dryness (SpeedVac SPD 300DDA, Thermo Scientific,
Bellefonte, USA).
Crude plant extract samples were mixed with
500 μL of 10 mM (MES)-KOH (2-(N-morpholino)
ethanesulfonic acid) and 150 μL of 5 mg/mL almond β-glycosidase
(Sigma-Aldrich, St. Louis, USA) and incubated at 37 °C for 15
h with gentle shaking. Samples were then transferred into glass KIMAX
tubes, and raspberry ketone was extracted with 4 mL of methyl tert-butyl ether (MTBE), transferred into 2 mL Agilent vials,
and evaporated to dryness under nitrogen flow.
and immediately frozen in liquid nitrogen. Samples were lyophilized
(CHRIST Alpha 1-4 LD Plus, Martin Christ, Osterode am Harz, Germany)
and powdered with a ball mill grinder (MM301, RETSCH, Haan, Germany)
for 2 × 1 min at 29 Hz. Lyophilized samples were weighed with
a precision scale, and 100 mg of each was suspended in 5 mL of 80%
(v/v) aqueous methanol and sonicated for 20 min, followed by centrifugation
for 15 min at 3000 rpm. Two methanol extractions were performed for
each sample, and supernatants were combined into 50 mL Falcon tubes
and evaporated to dryness (SpeedVac SPD 300DDA, Thermo Scientific,
Bellefonte, USA).
Crude plant extract samples were mixed with
500 μL of 10 mM (MES)-KOH (2-(N-morpholino)
ethanesulfonic acid) and 150 μL of 5 mg/mL almond β-glycosidase
(Sigma-Aldrich, St. Louis, USA) and incubated at 37 °C for 15
h with gentle shaking. Samples were then transferred into glass KIMAX
tubes, and raspberry ketone was extracted with 4 mL of methyl tert-butyl ether (MTBE), transferred into 2 mL Agilent vials,
and evaporated to dryness under nitrogen flow.