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 straight away frozen in liquid nitrogen and stored at -80 C until evaluation. three.2. Extraction and GC/MS Analysis of Diterpene Metabolites After thawing, tissue samples have been dried (482 h within the dark) at room temperature after which cut into fragments of about 1 mm by indicates of a scalpel. For each of the tissue kinds, the extraction from the diterpenoid fraction was performed following the procedure described by L ez-Goldar et al. [28] with minor modifications. Briefly, roughly 250 mg of every of the five distinctive tissue sorts were extracted twice with two mL of a nhexane/dichloromethane mixture (1:1; v/v). Through each extraction cycle, the extracts have been kept in an ultrasonic bath at 25 C for 20 min. After pooling collectively the two aliquots obtained within a recovery glass vial, residual water was removed by passing the extracts onto a column containing 2 g of anhydrous Na2 SO4 , and the obtained eluates were kept in the dark and stored at -20 C. For derivatisation, initial 200 of every extract have been passed onto a column containing 15 mg of graphitized carbon, to remove non-terpenic impurities, after which 50 of each and every eluate have been transferred into a conical vial and dried beneath a gentle stream of N2 . Just after drying, 100 of a 1:1 (v/v) mix of N,O-bis (trimethylsilyl) trifluoroacetamide, containing 1 (v/v) trimethylchlorosilane, plus pyridine had been added to each and every sample, plus the derivatization was allowed to proceed for 30 min at 65 C. Ultimately, the remedy was brought to dryness beneath a gentle stream of N2 , the SHP2 Inhibitor MedChemExpress residue was resuspended with 50 of n-hexane and CDK16 Synonyms finally stored in darkness at -20 C till GC-MS evaluation. For every single on the aforementioned tissue kinds, three biological replicates had 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 strategy an Agilent Technologies GC (model 7890A, Santa Clara, CA, USA), equipped with a VF-5ms capillary column (Agilent Technologies; 15 m 0.15 mm of inner diameter and a 0.15 film thickness) under the following thermal circumstances: from 90 C (2 min) to 350 C with a ramp of 44.7 C min-1 , then isothermal for five min. The He carrier gas constant flow was 1.two mL min-1 . The samplePlants 2021, ten,13 ofinjection (0.5 ) was performed under the pulsed splitless approach (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 source and also the analyser had been kept at 300 C, 230 C and 150 C, respectively. The acquisition was carried out beneath full scan mode (range m/z: 5050). The identification from the distinctive diterpene metabolites was carried out by comparison of experimental mass spectra both with these in NIST08 and Wiley02 Libraries and these in the available reference literature [22,31,39], also as of their related retention indices [28]. As far as the Wiley and NIST mass spectra libraries are concerned, the spectral match scores obtained for the diterpenes analysed inside the present work had been invariably higher than 850, regularly returning the right identification of each metabolite as the “first hit”. As outlined by the NIST library suggestions, the above score worth of mass spectra match is thought of to be satisfactory and trustworthy for the appropriate identifi.
