Series 1100

High-performance liquid chromatography (HPLC) equipment was used to identify and quantify the sugars and organic acids as previously described by Lipan et al. [2 (link)] with some modification. Almond finely ground (1 g) in a Moulinex grinder (AR110830) for 10 s was homogenized with 5 mL of phosphate buffer 50 mM (pH = 7.8) with an homogenizer (Ultra Turrax T18 Basic) during 2 min at 11,300 rpm, while the tube was maintained in an ice bath, then was centrifuged (Sigma 3–18 K; Sigma Laborzentrifugen, Osterode and Harz, Germany) for 20 min at 15,000 rpm and 4 °C and was filtered (0.45 μm Millipore membrane filter). The filtered supernatant (10 μL) was injected into a Hewlett Packard (Wilmington DE) series 1100 (HPLC) using 0.1% ortophosphoric acid elution buffer. Sugars were measured using a Supelcogel TM C-610H column (30 cm × 7.8 mm) with a pre-column (Supelguard 5 cm × 4.6 mm; Supelco, Bellefonte, PA) and the detection was carried out with a refractive index detector (RID). Organic acids were separated in the same HPLC condition as sugars and absorbance was measured at 210 nm with a diode-array detector (DAD). Calibration curves were run in triplicate injection using standards of different organic acids and sugars provided by Sigma (Poole, UK). Analyses were run in triplicate and results were expressed as g kg⁻¹ dw.

All chemical reagents and solvents were purchased from commercial suppliers and used without further purification. ¹H and ¹³C NMR spectra were obtained on a Varian Inova 400 MHz spectrometer. Chemical shifts (δ) were expressed in ppm relative to residual: CHCl₃ (δH 7.26 ppm), CDCl₃ (δC 77.0 ppm). Abbreviations are: s, singlet; t, triplet; m, multiplet. Reactions were monitored by LC-MS analysis (Hewlett-Packard Series 1100, ESI MS) eluting with 0.1% TFA in H₂O and 0.05% TFA in CH₃CN and/or TLC chromatography using Merck TLC Silica Gel 60 F254 plates and visualized by staining with cerium molybdate (Hanessian’s Stain) or by absorbance of UV light at 254 nm. Crude reaction mixtures were purified by column chromatography using Merck Silica Gel 60 as stationary phase.

HPLC was used to quantify Pro, Glu, and Orn in leaf extracts after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as reported by Monteoliva et al. (2014 (link)). Amino acids separation was carried out using a Zorbax C18 Plus column (100 × 4.6 mm, 3.5 μm) on a quaternary HPLC system (Hewlett Packard series 1100) with: (A) sodium acetate buffer 140 mM pH 5.8 and 7 mM triethylamine; (B) acetonitrile and (C) water. The gradient elution was 0.01 min 100% A; 0.50 min 99% A + 1% B; 27.5 min 91% A + 9% B; 28.50 min 89% A + 11% B; 44.50 min 82% A + 18% B; 47.5 min 60% B + 40% C; 50.5 min 100% A. The equipment was coupled to a fluorescent detector (excitation at 300 nm and emission at 400 nm). Retention times and quantification were determined using external standards. Values are expressed as nmol/g FW.

The phospholipid content of Bupisomes was measured by a modified Bartlett procedure as described earlier [19 ,20 ]. The bupivacaine content of the formulation (free and encapsulated) was quantified using a Hewlett-Packard series 1100 high-performance liquid chromatography (HPLC) with UV detection. The samples were injected into a 150 × 4.6 mm column (Luna, Phenomenex OOF-4252-EO). A mobile phase of acetonitrile:phosphate buffer 25 mM pH 6.8 (70:30) was used, and absorption was measured at a wavelength of 200 nm. The retention time of the bupivacaine was approximately 5.5 min. The lipid content and encapsulated bupivacaine concentration were used to calculate the drug to lipid ratio. The Bupisomes size was determined using a laser diffraction particle size analyzer (Beckman Coulter LS 13 320). The trapped LMVV aqueous volume of 17.4 mL/mmole HSPC was determined from the intraliposome ammonium concentration [21 (link)]. A more detailed physicochemical characterization of Bupigel is described in Table 3 of [7 (link)].

Culture filtrates were extracted with 1 vol. ethyl acetate, and concentrated 1:100 in vacuo. Aliquots of 400 µg of the extracts were fractionated by HPLC (Series 1100, Hewlett–Packard, Waldbronn, Germany; equipped with a LiChrospher RP18 column; 5 µm, 125 mm×4 mm, Merck, Darmstadt, Germany) with a 0.1% v:v formic acid: MeCN gradient (1% to 100% MeCN in 20 min; flow: 1 ml min⁻¹) for bioactivity-guided fractionation as described previously (Buckel et al., 2013 (link)). The fractionated extracts and pure compounds obtained from these small-scale separation processes were re-analysed for their biological activity. Subsequently, the fractions from five runs were pooled (corresponding to 2 mg of extract) for those extracts that had been prioritized by their bioactivity into 96-well plates and dried in vacuo. To identify the bioactive compounds, a HPLC-MS (Series 1200, Agilent, Waldbronn, Germany) equipped with an UV-DAD, and a coupled LC/MSD trap atmospheric pressure chemical ionization mass spectrometer with positive and negative polarization were used. Methods were applied as described previously (Buckel et al., 2017 (link)). The MS spectrum information for molecules and HPLC-MS analysis results are respectively given in Supplementary Fig. S1 and Supplementary Table S1.