M combined from leader stem (LS), bark and xylem combined from
M combined from leader stem (LS), bark and xylem combined from interwhorl stem (IS), and roots (R). All collected tissues have been immediately frozen in liquid nitrogen and stored at -80 C until analysis. 3.2. Extraction and GC/MS Analysis of Diterpene Metabolites Right after thawing, tissue samples had been dried (482 h inside the dark) at area temperature and after that cut into fragments of about 1 mm by signifies of a scalpel. For all the tissue sorts, the extraction from the diterpenoid fraction was performed following the process described by L ez-Goldar et al. [28] with minor modifications. Briefly, approximately 250 mg of every with the 5 diverse tissue kinds were extracted twice with 2 mL of a nhexane/dichloromethane mixture (1:1; v/v). During each extraction cycle, the extracts have been kept in an ultrasonic bath at 25 C for 20 min. Immediately after pooling collectively the two aliquots obtained inside a recovery glass vial, residual water was removed by passing the extracts onto a column containing 2 g of anhydrous Na2 SO4 , along with the obtained eluates were kept in the dark and stored at -20 C. For derivatisation, first 200 of each extract had been passed onto a column containing 15 mg of graphitized carbon, to remove non-terpenic impurities, after which 50 of every Aryl Hydrocarbon Receptor Compound eluate were transferred into a conical vial and dried under a gentle stream of N2 . Right after drying, 100 of a 1:1 (v/v) mix of N,O-bis (trimethylsilyl) trifluoroacetamide, containing 1 (v/v) trimethylchlorosilane, plus pyridine were added to every sample, as well as the derivatization was allowed to proceed for 30 min at 65 C. Finally, the option was brought to dryness beneath a gentle stream of N2 , the residue was resuspended with 50 of n-hexane and finally stored in darkness at -20 C till GC-MS evaluation. For each with the aforementioned tissue types, 3 biological replicates have been processed and analysed, every of them in triplicate. Qualitative and quantitative analysis of diterpenes from Calabrian pine tissues were carried out by means of a high ast GC-MS method an Agilent Technologies GC (model 7890A, Santa Clara, CA, USA), equipped using a VF-5ms capillary column (Agilent Technologies; 15 m 0.15 mm of inner diameter along with a 0.15 film thickness) beneath the following P2Y Receptor Antagonist Formulation thermal situations: from 90 C (two min) to 350 C using a ramp of 44.7 C min-1 , then isothermal for 5 min. The He carrier gas constant flow was 1.two mL min-1 . The samplePlants 2021, ten,13 ofinjection (0.five ) was performed under the pulsed splitless technique (43 psi) at 300 C. The coupled detector consisted of an Agilent mass selective detector (VL MSD-Triple-Axis Detector), mod. 5975C. The transfer line, the ion supply plus the analyser were kept at 300 C, 230 C and 150 C, respectively. The acquisition was carried out below full scan mode (variety m/z: 5050). The identification with the various diterpene metabolites was carried out by comparison of experimental mass spectra both with those in NIST08 and Wiley02 Libraries and those on the accessible reference literature [22,31,39], as well as of their related retention indices [28]. As far because the Wiley and NIST mass spectra libraries are concerned, the spectral match scores obtained for the diterpenes analysed in the present work had been invariably greater than 850, regularly returning the right identification of every single metabolite as the “first hit”. As outlined by the NIST library suggestions, the above score value of mass spectra match is deemed to become satisfactory and dependable for the right identifi.