Amygdalin

Reference standards of amygdalin, coumarin, and cinnamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin, cinnamaldehyde, paeoniflorin, and paeonol were obtained from Wako (Osaka, Japan). The purity of the seven reference standards was ≥98.0%. The chemical structures of the seven marker components are shown in Fig. 1a. High-performance liquid chromatography (HPLC)-grade reagents methanol, acetonitrile, and water to obtain the aqueous extract of GJBRHE were obtained from J. T. Baker (Phillipsburg, NJ, USA). Acetic acid was purchased from Merck (Darmstadt, Germany).Fig. 1

HPLC chromatograms of (a) the reference standard mixture and (b) GJBRHE: 230 nm (I), 254 nm (II), and 280 nm (III)

Water, acetonitrile (ACN), methanol, ethanol and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ, USA, HPLC grade). β-glucosidase enzymes (250 mg lyophilized powder ≥ 6U/mg), amygdalin (1 g, ≥99%), dhurrin (1 mg, ≥98%), prunasin (1 mg, ≥95%) and linamarin standards (1 mg, ≥95%), together with picric acid (100 g moisten with water ≥ 98%) and potassium cyanide (≥98%) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Whatman no.1 filter paper, sodium carbonate, sodium hydroxide and pH 8 phosphate buffer (500 mL) were also purchased from Fisher Scientific (Fair Lawn, NJ, USA). Plastic backing and hobby glue (adhesive neutral pH) were purchased from the Mizzou Store (Columbia, MO, USA).

Titanium oxide, acridine orange, MTT, rhodamine 123, amygdalin, copper II nitrate hexahydrate ethidium bromide, and antibiotic penicillin-streptomycin were purchased from Sigma-Aldrich chemical company (Merck & Co., Inc. USA). Gibco Dulbecco's Modified Eagle (DMEM) Medium and Fetal Bovine Serum (FBS) were obtained from Thermo Scientific, USA. We purchased a ROS detection kit from Nanjing KeyGen Biotech Co., Ltd.

Amygdalin (CAS number: 29883-15-6) was purchased from Sigma-Aldrich (St. Louis, MO). Prunasin (CAS number: 99-18-3) and sambunigrin (CAS number: 99-19-4) were supplied by LGC standards (Teddington, Middlesex, UK). Methanol HPLC grade was purchased from JT Baker (Deventer, Holland). Water was purified using a Milli-Q system from Millipore (Bedford, MA, USA).

Prior to chemical analyses, the nuts were shelled manually and screened to remove bad seeds. All kernel samples were triturated in liquid nitrogen. The extraction of amygdalin used methanol (20 ml) as the solute, as well as 0.2–0.4 g of almond powder under an ultrasonic generator (500 W, 40 kHz) for 30 min. The supernatant was filtered through filter paper. Then, 1 ml was diluted with an equal volume of 20% methanol and filtered through a 0.22-μm microfiltration membrane for clarification.
High-performance liquid chromatography (HPLC) assays were carried out on a Waters 2650, 2487 UV detector, and a 4.6 × 250 mm C18 column (Waters Sunfire, MA, United States). The mobile phase used was CH₃OH:H₂O (20:80, v/v) under isocratic conditions at a flow rate of 1.0 ml/min. A 10-μl sample was injected for analysis. Detection was recorded at 210 nm. The amygdalin used as a standard was purchased from Sigma-Aldrich (MO, United States). The method produced a good separation chromatogram with excellent linearity (correlation r² = 0.9999, Supplementary Figure 1) between the peak area and the concentration of amygdalin.

Cinnamaldehyde (≥98.0%), cinnamic acid (≥98.0%), liquiritin (≥99.0%), 6-gingerol (≥98.0%), and glycyrrhizin (≥99.0%) were purchased from Wako (Osaka, Japan). Amygdalin and coumarin (both ≥99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). liquiritin apioside (≥98.0%) was purchased from Shanghai Sunny Biotech (Shanghai, China) and ephedrine HCl (≥95.0%) was provided from Ministry of Food and Drug Safety. High-performance liquid chromatography (HPLC) grade, methanol, acetonitrile, and water were purchased from J. T. Baker (Phillipsburg, NJ, USA). Reagent grade, trifluoroacetic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA).