Inventa, a computational tool that highlights the structural novelty potential within extracts, considering untargeted mass spectrometry data, spectral annotation, and literature reports. Based on this information, Inventa calculates multiple scores that inform their structural potential. Thus, Inventa has the potential to accelerate new natural products discovery. Inventa was applied to a set of plants from the Celastraceae family as a proof of concept. The Pristimera indica (Willd.) A.C.Sm roots extract was highlighted as a promising source of potentially novel compounds. Its phytochemical investigation resulted in the isolation and de novo characterization of thirteen new dihydro-β-agarofuran sesquiterpenes, five of them presenting a new 9-oxodihydro-β-agarofuran base scaffold.Collections of natural extracts hold potential for the discovery of novel natural products with original modes of action. The prioritization of extracts from collections remains challenging due to the lack of a workflow that combines multiple-source information to facilitate the data interpretation. Results from different analytical techniques and literature reports need to be organized, processed, and interpreted to enable optimal decision-making for extracts prioritization. Here, we introduce  Natural products (NPs), specialized metabolites from different biological sources like plants, fungi, bacteria, and marine organisms, have enormously contributed to and inspired the development of drugs . These bvia compound dereplication, metabolomics, or genome mining ] within an extract by novelty or biological activity potential  annotation rates against databases of NPs. This study was centered on the discovery of novel marine NPs. It classified the extracts based on the presence and proportion of features in the chromatogram with a particular set of scores based on their area and intensity. The scores tried to reflect each extract’s chemical complexity and structural novelty. The application of this workflow in a small set of marine sponges and tunicates extracts resulted in the isolation of two new eudistomin analogs and two new nucleosides  within natural extracts based on chromatographic characteristics, bioactivity, and dereplication results. This tool aims to explore and suggest peaks of interest in a particular extract for isolation .Other published studies proposed mass-spectrometry-based workflows selecting extracts to accelerate the discovery of novel NPs, for example, utilizing liquid chromatography-mass spectrometry profiling and MSleosides . Anotherleosides  to priorleosides . FinallyWith the increasing capacities of the analytical profiling techniques, and the broad applications of bioinformatics tools in the field of NPs chemistry, the quantity of analytical information obtained increased proportionally. The clear and concise analysis of the resulting massive datasets is challenging and reduces the efficiency of data-driven prioritization . This isInventa, a computational tool that highlights the structural novelty potential of novel NPs within extracts, considering untargeted mass spectrometry data and literature reports for the organism’s taxa of interest. It was designed to accelerate mining data sets in a scalable manner. As a proof of concept, we applied it to a collection of taxonomically related extracts of the Celastraceae family. Plants from this family are characterized for producing a wide range of specialized bioactive metabolites from different chemical classes, like macrolide sesquiterpene pyridine alkaloids  and ethyl acetate (EtOAc) were purchased from Fisher Chemicals, Reinach, Switzerland, LC-MS grade water, acetonitrile (ACN), and formic acid were purchased from Fisher Chemicals, Reinach, Switzerland, Dimethyl Sulfoxide (DMSO) molecular biology grade was purchased from Sigma, St Louis, United States.J). The residual CD3OD signals  were used as internal standards for 1H and 13C, respectively. Complete assignments were based on 2D-NMR spectroscopy: COSY, edited-HSQC, HMBC, and ROESY. The Electronic Circular Dichroism (ECD) was recorded on a JASCO J-815 spectrometer  in acetonitrile using a 1 cm cell. The scan speed was 200 nm/min in continuous mode between 600 nm and 150 nm. The optical rotations were measured in acetonitrile on a JASCO P-1030 polarimeter  in a 1 ml, 10 cm tube.NMR spectroscopic data were recorded on a Bruker Avance Neo 600 MHz spectrometer equipped with a QCI 5mm Cryoprobe and a sampleJet automated extract changer . Chemical shifts are reported in parts per million , and coupling constants are reported in Hz . The PFL supplied all the vegetal material . The collected samples have photographs, herbarium vouchers, and leaf extracts preserved in dry silica gel. Precise localization of the initial collection, unique ID and barcode, and GPS data are stored in the dedicated data management system. The plant material was dried for 3 days at 55°C in an oven; then the material was grounded and stored in plastic pots at a controlled temperature and humidity in the Pierre-Fabre Laboratories facilities.The set comprises seventy-six extracts from different plant parts  of thirty-six species belonging to fourteen different genera. These plants belong to the Pierre-Fabre Laboratories (PFL) collection with over 17,000 unique samples collected worldwide. The PFL collection was registered at the European Commission under the accession number 03-FR-2020.This registration certifies that the collection meets the criteria set out in the EU ABS Regulation which implements at EU level the requirements of the Nagoya Protocol regarding access to genetic resources and the fair and equitable sharing of benefits arising from their utilization (Open Tree of Life (OTL v13.4) (The taxonomic names were searched in the L v13.4)  to most Analyses were performed with a Waters Acquity UPLC system equipped with a PDA detector coupled to a Q-Exactive Focus mass spectrometer , employing a heated electrospray ionization source (HESI-II) with the following parameters: spray voltage: + 3.5 kV; heater temperature: 220°C; capillary temperature: 350.00°C; S-lens RF: 45 (arb. units); sheath gas flow rate: 55 (arb. units) and auxiliary gas flow rate: 15.00 (arb. units). The mass analyzer was calibrated using a mixture of caffeine, methionine–arginine–phenylalanine–alanine–acetate (MRFA), sodium dodecyl sulfate, and sodium taurocholate, and Ultramark 1621 in an acetonitrile/methanol/water solution containing 1% formic acid by direct injection. The system was coupled to a Charged aerosol detector  kept at 40°C. The PDA wavelength range was from 210 nm to 400 nm with a resolution of 1.2 nm. Control of the instruments was done using Thermo Scientific Xcalibur 3.1 software.2 (dd-MS2) experiments in positive ionization mode, full scans were acquired at a resolution of 35,000 FWHM (at m/z 200) and MS2 scans at 17,500 FWHM in the range 100–1500 m/z. The dd-MS2 scan acquisition events were performed in discovery mode with an isolation window of 1.5 Da and stepped normalized collision energy (NCE) of 15, 30, and 45 units. Additional parameters were set as follows: default mass charge: 1; Automatic gain control (AGC) target 2E5; Maximum IT: 119 ms; Loop count: 3; Min AGC target: 2.6E4; Intensity threshold: 1. Up to three dd-MS2 scans (Top 3) were acquired for the most abundant ions per scan in MS1, using the Apex trigger mode (2–7 s), dynamic exclusion (9.0 s), and automatic isotope exclusion. A specific exclusion list was created for the measurement using the solvent as a background extract with an IODA Mass Spec notebook  through a linear gradient of 5–100% B over 7 min and an isocratic step at 100% B for 1 min. The mobile phases were: (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The flow rate was set to 600 µl/min, the injection volume was 2 μl, and the column was kept at 40°C. The set of extracts was randomized before injection, including pooled QC extracts and blanks, repeated once every ten extracts.MZmine3 software ; Minimum highest intensity, 1.0E6 (1.0E5 negative) and Scan to scan accuracy (m/z) of 0.0020 or 10.0 ppm. The ADAP feature resolver algorithm was used for chromatogram deconvolution with the following parameters: S/N threshold, 30; minimum feature height, 1.0E6 (1.0E5 negative); coefficient area threshold, 110; peak duration range, 0.01–1.0 min; RT wavelet range, 0.01–0.08 min. Isotopes were detected using the 13C isotope filter with an m/z tolerance of 0.0050 or 8.0 ppm, an Retention Time tolerance of 0.03 min (absolute), the maximum charge set at 2, and the representative isotope used was the lowest m/z. Each file was filtered by RT , and only the ions with an associated MS2 spectrum were kept. Alignment was done with the join-aligner , and the align list was filtered to remove any duplicate .The data were converted from.RAW (Thermo) standard data format to an open.mzXML format employing the MS Convert software, part of the ProteoWizard package . The consoftware . For mas5; Intensity correlation threshold 1.0E5 and the Correlation Grouping with the default parameters). Followed by the Ion identity networking , Modifications ], Annotation refinement , Add ion identities networks ) and Check all ion identities by MS/MS (m/z tolerance (MS2), 10 ppm; min-height (in MS2), 1.0E3; Check for multimers, yes; Check neutral losses (MS1—> MS2), yes) modules. The resulting aligned peak list was exported as an .mgf file for further analysis.The resulting filtered list was subjected to Ion Identity Networking  startingmgf file exported from MZmine, using the feature-based molecular networking workflow (https://ccms-ucsd.github.io/GNPSDocumentation/) on the GNPS website (https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=df71854c6e644b979228d96b521a490b (positive), https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=d477f360ddb344a593b935624782d8eb (negative).A molecular network was constructed from the . website . The premgf file exported from MZmine was also annotated by spectral matching against an in-silico database to obtain putative annotations  and re-ranking from the chemotaxonomical information available on LOTUS  and the LOTUS Initiative outputs (The .otations . The resotations  (https:/on LOTUS .The in-s outputs .mgf file exported from MZmine (using the SIRIUS export module) that contains MS1 and MS2 information was processed with SIRIUS (v 5.5.5) command-line tools on a Linux server , maximum m/z to compute: 1000. ZODIAC was used to improve molecular formula prediction using a threshold filter of 0.99 (The SIRIUS .x server . The molx server  and the  of 0.99 . Metabol of 0.99  and sign of 0.99 . The che of 0.99  using th of 0.99 .2 spectra were processed with the memo_ms package (0.1.3). The parameters were set as follows: min_rel_intensity: 0.01, max_relative_intensity: 1, min_peaks_required: 10, losses_from: 10, losses_to: 00, n_decimal: 2. All the Peak/loss present in the blanks were removed before the computation of the distance matrix (The MSe matrix .Inventa (https://luigiquiros.github.io/inventa/v1.0.0). These scripts are made available as a Jupyter notebook that can be deployed directly on the cloud using a Binder link A.C.Sm. (19.8 g) were extracted successively with hexane (3 × 200 ml), EtOAc (3 × 200 ml), and MeOH (3 × 200 ml), with constant agitation at room temperature for a 12 h period each. The organic solvents were filtered and evaporated under reduced pressure to give 61.5 mg of hexane extract, 100.4 mg of ethyl acetate extract, and 728.3 mg of methanolic extract.The dried ground roots of ® valve, and an FRC-10A fraction collector . The HPLC conditions were as follows: X-Bridge C18 column  equipped with a Waters C18 precolumn cartridge holder ; solvent system ACN (B) and H2O (A), both containing 0.1% FA. The separation was performed in gradient mode as follows: 5–40% B in 5 min, 40–55% B in 52 min, and 55–100% B in 25 min. The flow rate was fixed to 17.0 ml/min. The extract was injected by dry load according to a protocol developed in our laboratory , 2 , 3 , 6 , 7 , 8 , 12 , 5 , 13 , 9 , 4 . The fraction collected at RT 34.5 min (0.8 mg), was separated in a X-Bridge C18 column  equipped with a Waters C18 precolumn cartridge holder ; solvent system ACN (B) and H2O (A), both containing 0.1% FA, in an isocratic run 50% ACN, to give 8  and 10 .From the ethyl acetate extract (59.2 mg) 13 fractions (corresponding to the HPLC-UV peaks) were collected to give pure compounds 1 , 2 , 3 , 6 , 7 , 8 , 5 , 4 , 10  and 11 . Fractions collected at RT 41.0 min (0.9 mg) and RT 48.5 min (0.4 mg), were re-purified in a X-Bridge C18 column  equipped with a Waters C18 pre-column cartridge holder ; solvent system ACN (B) and H2O (A), both containing 0.1% FA, in an isocratic run 50% ACN, to give 13 .The methanolic extract (276.8 mg) was fractionated, in the same conditions as the ethyl acetate extract, to give compounds 1 -1α,6β-diacetoxy-15-iso-butanoyloxy-2α,8β-di-(5-carboxy-N-methyl-3-pyridoxy)-9-oxodihydro-β-agarofuran, Silviatine A). Amorphous white powder; max 193, 270 nm.Compound 1H NMR  δ 1.22 , 1.24 , 1.27 , 1.42 , 1.49 , 1.95 , 1.97 , 2.19 , 2.40 , 2.80 , 2.97 , 3.62 , 3.70 , 4.70 , 5.10 , 5.54 , 5.74 , 6.03 , 6.31 , 6.56 , 6.58 , 7.95 , 8.01 , 8.44 , 8.48 ; 13C NMR  δ 17.5 (CH3-14), 19.2 (CH3-15c), 19.3 (CH3-15d), 20.9 (CH3-6b), 21.0 (CH3-1b), 27.2 (CH3-13), 30.2 (CH3-12), 31.6 (CH2-3), 34.1 (CH-4), 35.2 (CH-15b), 38.7 , 54.1 (CH-7), 60.2 (C-10), 63.9 (CH2-15), 71.1 (CH-1), 72.5 (CH-2), 77.6 (CH-6), 78.8 (CH-8), 85.7 (C-11), 91.9 (C-5), 110.7 (C-8b), 111.1 (C-2b), 119.6 (CH-8e), 119.9 (CH-2e), 140.5 (CH-8f), 140.6 (CH-2f), 145.7 (CH-2c), 146.3 (CH-8c), 165.2 , 171.4 , 177.8 (C-15a), 203.1 (C-9).For NMR spectra see m/z 741.2864 [M + H]+ . MS/MS spectrum: CCMSLIB00009919267 (Q114866936).SMILES:CC1(C)[C@H]([C@@H]2OC(C3=CN(C)C(C=C3)=O)=O)[C@@H](OC(C)=O)[C@]4(O1)[C@H](C)C[C@H](OC(C5=CN(C)C(C=C5)=O)=O)[C@H](OC(C)=O)[C@@]4(COC(C(C)C)=O)C2=O. InChIKey=HRTFUSPNNUOJAM-AIVQPSNCSA-N.2: -6β-acetoxy-2α,8β-di-(5-carboxy-N-methyl-3-pyridoxy)-1α-hydroxy-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran, Silviatine B. Amorphous white powder, max 200, 268 nm.Compound 1H NMR  δ 0.93 , 1.15 , 1.27 , 1.43 , 1.48 , 1.58 , 1.77 , 1.98 , 2.18 , 2.31 , 2.36 , 2.67 , 2.97 , 3.63 , 3.71 , 4.61 , 4.77 , 5.05 , 5.44 , 6.10 , 6.24 , 6.57 , 7.97 , 8.04 , 8.47 , 8.48 ; 13C NMR  δ 11.6 (CH3-15d), 16.5 (CH3-15e), 17.4 (CH3-14), 20.6 (CH3-6b), 27.1 (CH3-13), 27.4 (CH2-15c), 29.9 (CH3-12), 31.3 (CH2-3), 34.1 (CH-4), 38.3 (CH3-2g), 38.4 (CH3-8g), 42.0 (CH-15b), 54.0 (CH-7), 61.4 (C-10), 63.6 (CH2-15), 70.0 (CH-1), 74.7 (CH-2), 77.4 (CH-6), 78.4 (CH-8), 85.0 (C-11), 91.3 (C-5), 110.5 (C-8b), 111.3 (C-2b), 119.4 , 140.1 (CH-8f), 140.5 (CH-2f), 145.1 (CH-2c), 145.9 (CH-8c), 163.6 (C-8a), 164.1 (C-2a), 165.0 (C-2d), 164.8 (C-8d), 171.1 (C-6a), 177.8 (C-15a), 206.8 (C-9). For NMR spectra see m/z 713.2323 [M + H]+ ;MS/MS spectrum: CCMSLIB00009919268 (Q114866937) .SMILES: O=C1[C@]([H])([C@]([H])([C@]([H])([C@@]2([C@@]([H])(C([H])([C@@]([H])([C@@]([H])([C@]21C([H])(OC([C@@]([H])(C([H])(C([H])([H])[H])[H])C([H])([H])[H])=O)[H])O[H])OC(C(C([H])=C3[H])=C(N(C3=O)C([H])([H])[H])[H])=O)[H])C([H])([H])[H])O4)OC(C([H])([H])[H])=O)C4(C([H])([H])[H])C([H])([H])[H])OC(C5=C(N(C(C([H])=C5[H])=O)C([H])([H])[H])[H])=O. InChIKey=HPZNCFSLZGFDST-QIADLSSESA-N.3: -1α,6β-diacetoxy-2α,8β-di-(5-carboxy-N-methyl-3-pyridoxy)-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran, Silviatine C. Amorphous white powder, max 194, 267 nmCompound 1H NMR  δ 0.92 , 1.22 , 1.25 , 1.41 , 1.49 , 1.57 , 1.76 , 1.95 , 1.97 , 2.19 , 2.41 , 2.65 , 2.97 , 3.62 , 3.70 , 4.65 , 5.12 , 5.55 , 5.75 , 6.01 , 6.30 , 6.56 , 6.58 , 7.95 , 8.01 , 8.44 , 8.47 ; 13C NMR  δ 11.9 (CH3-15d), 16.7 (CH3-15e), 17.5 (CH3-14), 21.0 , 27.2 (CH3-13), 27.8 (CH2-15c), 30.2 (CH3-12), 31.6 (CH2-3), 34.1 (CH-4), 38.7 , 42.3 (CH-15b), 54.1 (CH-7), 60.1 (C-10), 63.9 (CH2-15), 71.1 (CH-1), 72.5 (CH-2), 77.7 (CH-6), 78.8 (CH-8), 85.6 (C-11), 91.9 (C-5), 110.7 (C-8b), 111.1 (C-2b), 119.6 (CH-8e), 119.9 (CH-2e), 140.5 (CH-8f), 140.6 (CH-2f), 145.6 (CH-2c), 146.3 (CH-8c), 163.9 (C-8a), 164.8 (C-2a), 165.2 , 171.2 (C-1a), 171.3 (C-6a), 177.6 (C-15a), 203.1 (C-9). For NMR spectra see m/z 755.3017 [M + H]+ ;MS/MS spectrum: CCMSLIB00009919270 (Q114866938).SMILES: O=C(OC([H])([H])[C@@]1(C2=O)[C@@]([H])(OC(C([H])([H])[H])=O)[C@]([H])(C([H])([H])[C@@]([H])(C([H])([H])[H])[C@]13OC([C@@]([H])([C@]3([H])OC(C([H])([H])[H])=O)[C@]2([H])OC(C4=C([H])N(C(C([H])=C4[H])=O)C([H])([H])[H])=O)(C([H])([H])[H])C([H])([H])[H])OC(C5=C([H])N(C(C([H])=C5[H])=O)C([H])([H])[H])=O)[C@@]([H])(C([H])(C([H])([H])[H])[H])C([H])([H])[H]. InChIKey=ATASRQYZQYFECN-FWMXANSESA-N.4: -6β-acetoxy-8β-benzoyloxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-1α-hydroxy-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran, Silviatine D. Amorphous white powder, max 196, 229, 273 nm.Compound 1H NMR  δ 0.95 , 1.15 , 1.30 , 1.48 , 1.49 , 1.60 , 1.81 , 1.99 , 2.18 , 2.32 , 2.37 , 2.70 , 3.00 , 3.71 , 4.62 , 4.79 , 5.06 , 5.45 , 6.17 , 6.28 , 6.58 , 7.53 , 7.66 , 8.04 , 8.07 , 8.49 ; 13C NMR  δ 11.9 (CH3-15d), 16.8 (CH3-15e), 17.7 (CH3-14), 21.0 (CH3-6b), 27.5 (CH3-13), 27.8 (CH2-15c), 30.2 (CH3-12), 31.7 (CH3-3), 34.4 (CH-4), 38.7 (CH3-2g), 42.4 (CH-15b), 54.4 (CH-7), 61.7 (C-10), 64.0 (CH2-15), 70.4 (CH-1), 75.1 (CH-2), 77.8 (CH-6), 78.8 (CH-8), 85.2 (C-11), 91.6 (C-5), 111.7 (C-2b), 119.8 (CH-2e), 129.8 , 130.6 (C-8b), 130.8 , 134.7 (CH-8e), 140.8 (CH-2f), 145.4 (CH-2c), 165.2 (C-2d), 166.3 (C-8a), 171.3 (C-6a), 178.1 (C-15a), 207.0 (C-9).. For NMR spectra see m/z 682.2850 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919278 (Q114866944).SMILES: O=C1[C@](OC(C2=C([H])C([H])=C([H])C([H])=C2[H])=O)([H])[C@](C3(C([H])([H])[H])C([H])([H])[H])([H])[C@](OC(C([H])([H])[H])=O)([H])[C@]4(O3)[C@@](C([H])([H])[H])([H])C([H])([H])[C@@](OC(C(C([H])=C5[H])=C([H])N(C([H])([H])[H])C5=O)=O)([H])[C@@](O[H])([H])[C@]41C([H])([H])OC([C@@](C([H])([H])[H])([H])C([H])([H])C([H])([H])[H])=O. InChIKey=TXTJGSCAWSRSFC-GCFOXSEASA-N.5: -6β-acetoxy-8β-benzoyloxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-1α,3β-dihydroxy-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran, Silviatine E. Amorphous white powder. max 200, 226, 267 nm.Compound 1H NMR  δ 0.96 , 1.16 , 1.30 , 1.53 , 1.54 , 1.61 , 1.81 , 2.20 , 2.51 , 2.71 , 3.00 , 3.70 , 3.92 , 4.75 , 4.88 , 4.99 , 5.42 , 6.18 , 6.33 , 6.58 , 7.53 , 7.66 , 8.04 , 8.07 , 8.48 ; 13C NMR  δ 11.9 (CH3-15d), 15.4 (CH3-14), 16.8 (CH3-15e), 21.0 (CH3-6b), 27.4 (CH3-13), 27.8 (CH2-15c), 30.1 (CH3-12), 38.7 (CH3-2g), 40.9 (CH-4), 42.4 (CH-15b), 53.2 (CH-7), 61.6 (C-10), 63.8 (CH2-15), 67.0 (CH-1), 73.1 (CH-3), 77.6 (CH-2), 78.3 (CH-6), 78.7 (CH-8), 87.1 (C-11), 92.9 (C-5), 111.2 (C-2b), 119.8 (CH-2e), 129.8 , 130.6 (C-8b), 130.8 , 134.8 (CH-8e), 140.8 (CH-2f), 145.6 (CH-2c), 164.6 (C-2a), 165.3 (C-2d), 166.3 (C-8a), 171.2 (C-6a), 178.0 (C-15a), 206.3 (C-9). For NMR spectra see m/z 698.2802 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919275 (Q114866948).SMILES: O=C1[C@@](OC(C2=C(C([H])=C(C([H])=C2[H])[H])[H])=O)([H])[C@]3([H])[C@@](OC(C([H])([H])[H])=O)([H])[C@]([C@]1([C@]4(O[H])[H])C([H])([H])OC([C@@](C([H])([H])[H])([H])C([H])([H])C([H])([H])[H])=O)(OC3(C([H])([H])[H])C([H])([H])[H])[C@@](C([H])([H])[H])([H])[C@](O[H])([H])[C@@]4(OC(C(C([H])=C5[H])=C(N(C5=O)C([H])([H])[H])[H])=O)[H]. InChIKey=ZSYJSJVZJMUAMB-WPJZQHFNSA-N.6: -6β-acetoxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)- 9α,15-di-(2-methylbutanoyloxy)-dihydro-β-agarofuran. Amorphous white powder, max 194, 267 nm.Compound 1H NMR  δ 0.55 , 0.88 , 1.03 , 1.11 , 1.13 , 1.24 , 1.37 , 1.46 , 1.48 , 1.54 , 1.68 , 1.90 , 2.17 , 2.24 , 2.35 , 2.37 , 2.41 , 2.62 , 3.70 , 3.71 , 4.37 , 4.39 , 5.37 , 5.41 , 5.64 , 5.66 , 6.56 , 6.60 , 6.74 , 8.03 , 8.07 , 8.61 , 8.89 ; 13C NMR  δ 11.6 (CH3-15d), 12.0 (CH3-9d), 16.4 (CH3-9e), 17.5 (CH3-14), 17.8 (CH3-15e), 21.3 (CH3-6b), 24.9 (CH3-12), 27.3 (CH2-9c), 27.7 (CH2-15c), 30.5 (CH3-13), 32.4 (CH2-3), 33.9 (CH-4), 38.5 (CH3-2g), 38.8 (CH3-8g), 42.1 (CH-15b), 42.5 (CH-9b), 53.1 (C-10), 55.7 (CH-7), 63.0 (CH2-15), 72.8 (CH-8), 73.3 (CH-9), 75.1 (CH-2), 76.0 (CH-1), 76.9 (CH-6), 81.6 (C-11), 91.1 (C-5), 119.6 (CH-2e), 119.8 (CH-8e), 140.9 (CH-8f), 141.1 (CH-2f), 145.5 (CH-2c), 146.5 (CH-8c), 165.2 , 172.1 (C-6a), 176.6 (C-9a), 179.0 (C-15a). For NMR spectra see m/z 799.3649 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919271 (Q114866941).SMILES: O=C(C([H])([H])[H])O[C@]1([C@]2(OC(C([H])([H])[H])(C([H])([H])[H])[C@@]1([C@@](OC(C3=C([H])N(C([H])([H])[H])C(C([H])=C3[H])=O)=O)([C@@]4([H])OC([C@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)[H])[H])[C@@](C([C@]([C@@]([C@]24C(OC([C@@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)([H])[H])(O[H])[H])(OC(C(C([H])=C5[H])=C([H])N(C([H])([H])[H])C5=O)=O)[H])([H])[H])(C([H])([H])[H])[H])[H]. InChIKey=LVFIUDAMNWXFMK-OPURYRTMSA-N.7: - 1α,6β-diacetoxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-15-iso-butanoyloxy-9α-(2-methylbutanoyloxy)-dihydro-β-agarofuran. Amorphous white powder, max 195, 266 nm.Compound 1H NMR  δ 0.85 , 0.87 , 1.06 , 1.15 , 1.21 , 1.31 , 1.49 , 1.53 , 1.69 , 1.85 , 1.91 , 2.19 , 2.26 , 2.43 , 2.52 , 2.58 , 2.67 , 3.68 , 3.70 , 4.24 , 5.46 , 5.51 , 5.58 , 5.61 , 5.70 , 6.56 , 6.59 , 6.74 , 8.01 , 8.04 , 8.52 , 8.83 ; 13C NMR  δ 12.1 (CH3-9d), 16.2 (CH3-9e), 17.6 (CH3-14), 19.3 (CH3-15d), 19.4 (CH3-15c), 21.1 (CH3-1b), 21.2 (CH3-6b), 24.8 (CH3-12), 26.9 (CH2-9c), 30.4 (CH3-13), 32.3 (CH2-3), 33.8 (CH-4), 35.1 (CH-15b), 38.6 (CH3-2g), 38.7 (CH3-8g), 42.0 (CH-9b), 52.7 (C-10), 55.3 (CH-7), 62.8 (CH2-15), 70.9 (CH-2), 72.2 (CH-9), 72.4 (CH-8), 76.5 (CH-6), 77.9 (CH-1), 82.0 (C-11), 91.2 (C-5), 111.0 (CH-2b), 111.9 (CH-8b), 119.7 (CH-2e), 119.8 (CH-8e), 140.7 (CH-2f), 140.8 (CH-8f), 145.7 (CH-2c), 146.4 (CH-8c), 164.8 (C-2a), 165.0 (C-8a), 165.1 (C-2d), 165.2 (C-8d), 171.3 (C-1a), 172.0 (C-6a), 175.9 (C-9a), 179.0 (C-15a). For NMR spectra see m/z 827.3598 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919272 (Q114866942).SMILES: O=C(O[C@@]([H])([C@@]1([C@@]([H])(C([H])([C@@]([H])([C@@]([H])([C@]12C([H])(OC(C([H])(C([H])([H])[H])C([H])([H])[H])=O)[H])OC(C([H])([H])[H])=O)OC(C(C([H])=C3[H])=C(N(C3=O)C([H])([H])[H])[H])=O)[H])C([H])([H])[H])O4)[C@]([H])(C4(C([H])([H])[H])C([H])([H])[H])[C@@]([H])(OC(C5=C(N(C(C([H])=C5[H])=O)C([H])([H])[H])[H])=O)[C@]2(OC([C@]([H])(C([H])(C([H])([H])[H])[H])C([H])([H])[H])=O)[H])C([H])([H])[H]. InChIKey=HDNFKWOIOAMTET-VFPOJSPNSA-N.8: - 1α,6β-diacetoxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-9α,15-di-(2-methylbutanoyloxy)-dihydro-β-agarofuran. Amorphous white powder, max 204, 266 nm.Compound 1H NMR  δ 0.55 , 0.88 , 1.07 , 1.16 , 1.21 , 1.25 , 1.29 , 1.49 , 1.50 , 1.53 , 1.71 , 1.85 , 1.91 , 2.20 , 2.24 , 2.39 , 2.44 , 2.52 , 2.66 , 3.68 , 3.71 , 4.19 , 5.49 , 5.52 , 5.58 , 5.61 , 5.71 , 6.57 , 6.60 , 6.76 , 8.02 , 8.03 , 8.54 , 8.88 ; 13C NMR  δ 11.6 (CH3-15d), 12.2 (CH3-9d), 16.2 (CH3-9e), 17.6 (CH3-14), 17.7 (CH3-15e), 21.1 (CH3-1b), 21.2 (CH3-6b), 24.9 (CH3-12), 26.8 (CH2-9c), 27.7 (CH2-15c), 30.4 (CH3-13), 32.3 (CH2-3), 33.8 (CH-4), 38.6 (CH3-2g), 38.8 (CH3-8g), 42.0 (CH-9b), 42.1 (CH-15b), 52.7 (C-10), 55.5 (CH-7), 62.6 (CH2-15), 70.9 (CH-2), 72.0 (CH-9), 72.4 (CH-8), 76.6 (CH-6), 77.8 (CH-1), 82.0 (C-11), 91.1 (C-5), 111.0 (C-2b), 112.1 (C-8b), 119.8 , 140.8 (CH-2f), 140.9 (CH-8f), 145.7 (CH-2c), 146.6 (CH-8c), 164.8 (C-2a), 165.0 (C-8d), 165.2 (C-8a), 165.2 (C-2d), 171.3 (C-1a), 172.0 (C-6a), 175.9 (C-9a), 178.6 (C-15a). For NMR spectra see m/z 841.3736 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919274 (Q114866947).SMILES: O=C(C([H])([H])[H])O[C@]1([C@]2(OC(C([H])([H])[H])(C([H])([H])[H])[C@@]1([C@@](OC(C3=C([H])N(C([H])([H])[H])C(C([H])=C3[H])=O)=O)([C@@]4([H])OC([C@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)[H])[H])[C@@](C([C@@]([C@@]([C@]24C(OC([C@@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)([H])[H])(OC(C([H])([H])[H])=O)[H])(OC(C(C([H])=C5[H])=C([H])N(C([H])([H])[H])C5=O)=O)[H])([H])[H])(C([H])([H])[H])[H])[H]. InChIKey=IJMXFBHJNXUVLI-ZPLOSWSHSA-N.9: - 1α,6β-diacetoxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-9α,15-di-(2-methylbutanoyloxy)-8α-nicotinoyloxydihydro-β-agarofuran. Amorphous white powder, max 194, 267 nm.Compound 1H NMR  δ 0.35 , 0.85 , 1.06 , 1.10 , 1.12 , 1.21 , 1.29 , 1.35 , 1.51 , 1.56 , 1.68 , 1.85 , 1.91 , 2.18 , 2.24 , 2.29 , 2.45 , 2.52 , 2.75 , 3.68 , 4.15 , 5.51 , 5.57 , 5.62 , 5.72 , 5.76 , 6.56 , 6.78 , 7.65 , 8.02 , 8.55 , 8.57 , 8.82 , 9.40 ; 13C NMR  δ 11.1 (CH3-15d), 11.8 (CH3-9d), 15.8 (CH3-9e), 17.2 , 20.7 , 24.6 (CH3-12), 26.5 (CH2-9c), 27.2 (CH2-15c), 30.2 (CH3-13), 31.9 (CH2-3), 33.4 (CH-4), 38.2 (CH3-2g), 41.4 (CH-15b), 41.8 (CH-9b), 54.8 (CH-7), 62.1 (CH2-15), 70.5 (CH-2), 71.7 (CH-9), 72.3 (CH-8), 76.1 (CH-6), 77.5 (CH-1), 81.8 (C-11), 91.0 (C-5), 119.4 (CH-2e), 125.0 (CH-8e), 139.1 (CH-8f), 140.5 (CH-2f), 145.2 (CH-2c), 151.4 (CH-8c), 154.2 (CH-8d), 164.9 (C-2d), 171.0 (C-1a), 171.2 (C-6a), 175.5 (C-9a), 178.6 (C-15a). For NMR spectra see m/z 811.3668 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919277 (Q114866949).SMILES: O=C(C([H])([H])[H])O[C@]1([C@]2(OC(C([H])([H])[H])(C([H])([H])[H])[C@@]1([C@@](OC(C3=C([H])N=C([H])C([H])=C3[H])=O)([C@@]4([H])OC([C@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)[H])[H])[C@@](C([C@@]([C@@]([C@]24C(OC([C@@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)([H])[H])(OC(C([H])([H])[H])=O)[H])(OC(C(C([H])=C5[H])=C([H])N(C([H])([H])[H])C5=O)=O)[H])([H])[H])(C([H])([H])[H])[H])[H]. InChIKey=UDRYEQNSFSPBNV-HKZKQFCWSA-N.10: -1α,6β-diacetoxy-9α-iso-butanoyloxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-15-methylbutanoyloxydihydro-β-agarofuran. Amorphous white powder, max 206, 269 nm.Compound 1H NMR  δ 0.54 , 1.07 , 1.09 , 1.15 , 1.21 , 1.25 , 1.47 , 1.49 , 1.52 , 1.85 , 1.92 , 2.21 , 2.38 , 2.44 , 2.45 , 2.52 , 2.67 , 3.68 , 3.70 , 4.18 , 5.49 , 5.52 , 5.56 , 5.61 , 5.70 , 6.56 , 6.60 , 6.77 , 8.02 , 8.04 , 8.54 , 8.88 ; 13C NMR  δ 11.6 (CH3-15d), 17.5 (CH3-14), 17.7 (CH3-15e), 18.8 (CH3-9d), 19.0 (CH3-9c), 21.0 (CH3-1b), 21.2 (CH3-6b), 24.9 (CH3-12), 27.7 (CH2-15c), 30.4 (CH3-13), 32.3 (CH2-3), 33.8 (CH-4), 35.3 (CH-9b), 38.6 (CH3-2g), 38.8 (CH3-8g), 42.1 (CH-15b), 52.7 (C-10), 55.5 (CH-7), 62.6 (CH2-15), 70.9 (CH-2), 71.9 (CH-9), 72.4 (CH-8), 76.6 (CH-6), 77.8 (CH-1), 81.9 (C-11), 91.1 (C-5), 111.0 (C-2b), 112.0 (C-8b), 119.8 , 140.8 (CH-2f), 140.9 (CH-8f), 145.7 (CH-2c), 146.6 (CH-8c), 164.8 , 171.3 (C-1a), 172.0 (C-6a), 176.3 (C-9a), 178.7 (C-15a). For NMR spectra see m/z 827.3595 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919279 (Q114866939).SMILES: O=C(C([H])([H])[H])O[C@]1([C@]2(OC(C([H])([H])[H])(C([H])([H])[H])[C@@]1([C@@](OC(C(C([H])=C3[H])=C([H])N(C([H])([H])[H])C3=O)=O)([C@@]4([H])OC(C(C([H])([H])[H])(C([H])([H])[H])[H])=O)[H])[H])[C@@](C([C@@]([C@@]([C@]24C(OC([C@@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)([H])[H])(OC(C([H])([H])[H])=O)[H])(OC(C(C([H])=C5[H])=C([H])N(C([H])([H])[H])C5=O)=O)[H])([H])[H])(C([H])([H])[H])[H])[H]. InChIKey=VKILZIVFMPURPQ-KAGCMFHOSA-N.11: -6β-diacetoxy-9α-iso-butanoyloxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-1α-hydroxy-15-methylbutanoyloxydihydro-β-agarofuran. Amorphous white powder, max 195, 266 nm.Compound 1H NMR  δ 0.54 , 1.07 , 1.09 , 1.11 , 1.13 , 1.22 , 1.46 , 1.46 , 1.54 , 1.90 , 2.18 , 2.36 , 2.40 , 2.43 , 2.63 , 3.70 , 4.36 , 4.38 , 5.37 , 5.41 , 5.62 , 5.65 , 6.56 , 6.60 , 6.75 , 8.05 , 8.07 , 8.61 , 8.90 ; 13C NMR  δ 11.3 (CH3-15d), 17.2 (CH3-14), 17.4 (CH3-15e), 18.6 , 20.9 (CH3-6b), 24.6 (CH3-12), 27.5 (CH2-15c), 30.2 (CH3-13), 32.2 (CH2-3), 33.5 (CH-4), 35.3 (CH-9b), 38.6 (CH3-2g), 38.3 , 41.9 (CH-15b), 55.4 (CH-7), 67.9 (CH2-15), 72.6 (CH-8), 73.0 (CH-9), 74.7 (CH-2), 75.6 (CH-1), 76.7 (CH-6), 81.2 (C-11), 90.8 (C-5), 111.5 (C-2b), 112.0 (C-8b), 119.2 (CH-2e), 119.4 (CH-8e), 140.7 , 145.2 (CH-2c), 146.3 (CH-8c), 165.0 , 171.7 (C-6a), 176.7 (C-9a), 178.7 (C-15a). For NMR spectra see m/z 785.3511 [M + H]+ ;MS/MS spectrum: CCMSLIB00009919269 (Q114866946).SMILES: O=C(C([H])([H])[H])O[C@]1([C@]2(OC(C([H])([H])[H])(C([H])([H])[H])[C@@]1([C@@](OC(C(C([H])=C3[H])=C([H])N(C([H])([H])[H])C3=O)=O)([C@@]4([H])OC(C(C([H])([H])[H])(C([H])([H])[H])[H])=O)[H])[H])[C@@](C([C@@]([C@@]([C@]24C(OC([C@@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)([H])[H])(O[H])[H])(OC(C(C([H])=C5[H])=C([H])N(C([H])([H])[H])C5=O)=O)[H])([H])[H])(C([H])([H])[H])[H])[H]. InChIKey=KXKFNEWNZKWNFD-MRBOHXSOSA-N.12: - 1α,3β,6β,8α-tetraacetoxy-15-iso-butanoyloxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-9α-tigloyloxydihydro-β-agarofuran. Amorphous white powder, max 207, 268 nm.Compound 1H NMR  δ 1.21 , 1.24 , 1.26 , 1.44 , 1.53 , 1.75 , 1.77 , 1.78 , 2.11 , 2.12 , 2.12 , 2.50 , 2.56 , 2.90 , 3.71 , 4.28 , 4.87 , 5.36 , 5.49 , 5.52 , 5.91 , 6.58 , 6.58 , 6.83 , 8.00 , 8.55 ; 13C NMR  δ 11.6 (CH3-9e), 14.1 (CH3-9d), 14.9 (CH3-14), 19.3 , 20.2 (CH3-1b), 20.7 , 24.6 (CH3-12), 30.2 (CH3-13), 34.8 (CH-15b), 38.0 (CH-4), 38.3 (CH3-2g), 52.0 (C-10), 53.3 (CH-7), 61.9 (CH2-15), 70.5 (CH-8), 71.3 (CH-2), 71.9 (CH-9), 74.9 (CH-1), 75.8 (CH-3), 76.5 (CH-6), 82.5 (C-11), 90.3 (C-5), 109.9 (C-2b), 119.6 (CH-2e), 128.9 (C-9b), 140.0 (CH-9c), 140.4 (CH-2f), 145.8 (CH-2c), 163.9 (C-2a), 165.0 (C-2d), 167.0 (C-9a), 170.8 (C-1a), 171.1 (C-6a), 171.3 (C-3a), 171.5 (C-8a), 178.9 (C-15a). For NMR spectra see m/z 790.3289 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919273 (Q114866943). SMILES: O=C(O[C@@]([H])([C@](O1)([C@@]([H])([C@]([H])([C@@]2(OC(C(C([H])=C3[H])=C(N(C3=O)C([H])([H])[H])[H])=O)[H])OC(C([H])([H])[H])=O)C([H])([H])[H])[C@]4([C@]2(OC(C([H])([H])[H])=O)[H])C([H])(OC(C([H])(C([H])([H])[H])C([H])([H])[H])=O)[H])[C@]([H])(C1(C([H])([H])[H])C([H])([H])[H])[C@@]([H])(OC(C([H])([H])[H])=O)[C@]4(OC(/C(C([H])([H])[H])=C(C([H])([H])[H])\[H])=O)[H])C([H])([H])[H]. InChIKey=WUSPTHMFTBWJDO-KOPSDWRJSA-N.13: -1α,3β,6β,8α-tetraacetoxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-15-(2-methylbutanoyloxy)-9α-tigloyloxydihydro-β-agarofuran. Amorphous white powder, max 194, 270 nm.Compound 1H NMR  δ 0.97 , 1.20 , 1.25 , 1.43 , 1.53 , 1.58 , 1.75 , 1.78 , 1.80 , 2.12 , 2.12 , 2.13 , 2.50 , 2.56 , 2.74 , 3.71 , 4.22 , 4.87 , 5.44 , 5.49 , 5.53 , 5.92 , 6.55 , 6.58 , 6.83 , 8.02 , 8.58 ; 13C NMR  δ 12.1 , 14.4 (CH3-9d), 15.2 (CH3-14), 17.4 (CH3-15e), 20.5 (CH3-1b), 21.1 (CH3-6b), 21.2 (CH3-3b), 21.4 (CH3-8b), 24.9 (CH3-12), 27.9 (CH2-15c), 30.6 (CH3-13), 38.2 (CH-4), 38.6 (CH3-2g), 42.3 (CH-15b), 52.1 (C-10), 53.6 (CH-7), 62.1 (CH2-15), 70.8 (CH-8), 71.6 (CH-2), 72.1 (CH-9), 75.2 (CH-1), 76.1 (CH-3), 76.9 (CH-6), 82.7 (C-11), 90.6 (C-5), 110.3 (C-2b), 119.9 (CH-2e), 129.2 (C-9b), 140.2 (CH-9c), 140.7 (CH-2f), 146.1 (CH-2c), 164.1 (C-2a), 165.3 (C-2d), 167.3 (C-9a), 171.1 (C-1a), 171.2 (C-6a), 171.5 (C-3a), 171.7 (C-8a), 178.9 (C-15a). For NMR spectra see m/z 804.3445 [M + H]+ ; MS/MS spectrum: CCMSLIB00009919276 (Q114866940).SMILES: O=C(C([H])([H])[H])O[C@]1([C@]([C@@]([C@]([C@]2([H])OC(C(C([H])=C3[H])=C([H])N(C([H])([H])[H])C3=O)=O)(OC(C([H])([H])[H])=O)[H])(C([H])([H])[H])[H])([C@@]4(C(OC([C@@](C(C([H])([H])[H])([H])[H])(C([H])([H])[H])[H])=O)([H])[H])[C@@]2([H])OC(C([H])([H])[H])=O)OC(C([H])([H])[H])(C([H])([H])[H])[C@@]1([C@@](OC(C([H])([H])[H])=O)([C@@]4([H])OC(/C(C([H])([H])[H])=C([H])\C([H])([H])[H])=O)[H])[H])[H]. InChIKey=IEENCNNCPOLOQP-CYNYGQNTSA-N. The Q-codes shown for each compound correspond to WikiData Q-identifiers.© 2015–2022, Gaussian Inc., Wallingford, CT, United States) using the CPCM model in acetonitrile  were performed at the University of Geneva on the HPC Baobab cluster.The absolute configuration of all compounds was assigned according to the comparison of the calculated and experimental ECD. Based on their relative configuration proposed by NMR 2D ROESY experiments, the structures were employed for the random conformational search using MMFF94s force field by Spartan Student v7 . From the results, the 20 isomers with lower energy were subjected to further successive PM3 and B3LYP/6-31G optimizations in Gaussian 16 software  job as minimum input. This workflow is preferred over the classical MN since it incorporates mass spectrometry (MS1 and MS2) and semi quantitative chromatographic information  specific for each feature . Other software can be used, if compatible with GNPS, the user can recover the table from the MN results. Their annotation status is based on the GNPS annotation results. To guarantee a minimum quality of the putative identities, the GNPS annotations are automatically cleaned and filtered  before the calculations (https://github.com/lfnothias/gnps_postprocessing). Additional feature dereplications results using in-silico databases and reponderation strategies to improve the putative annotation ( feature . FBMN wanotation  can be iMZmine supporting IIN (custom 2.53 version or MZmine 3), the user can leverage the grouping of multiple ion forms identified for a given molecule and reduce the total number of features. The species generated from the same molecules  are collapsed into a single feature group  through an MS1 feature chromatographic shape correlation. Inventa will perform the calculations related to FC based on the new MS1-based group features and MS2 spectral similarity cosine comparison  are pondered at the features level to express each extract’s measurable unknown structural richness. At the extract level, each extract’s available spectral space is compared to each other to spot dissimilarities using a dissimilarity matrix based on the MEMO vectors , Literature component (LC), Class component (CC), and Similarity component (SC) . Each cown in  to give Feature Component . Then, the annotation status (annotated or unannotated) is checked based on the dereplication results used as input. Finally, the total number of specific unannotated features in each extract is calculated and divided by the total number of detected features in the same extract. The evaluation of the specificity of the features (without information on their annotation status) a given extract within the set can be done based on the “Feature Specificity” (FS) value (is computed similarly to FC without considering the annotations). The nent FC,  is the rminimum specificity” defined by the user. When multiple extracts with the same species are present, even if a feature is specific at the species level, its relative intensity may be spread over its various extracts. Consequently, that feature will not be considered specific and will be ignored in the calculations. To address this limitation, the user can define the maximum occurrence of the species allowing the script to consider a feature as “specific” based on a shared specificity within multiple extracts ” is 1, many features will be not considered specific because they are shared between the plant parts. If for the data set the “maximum occurrence (N)” is set to 4, the number of specific features increased. This immediately raised the FS and FC in general, and the common tissue parts  gained up to 4 fold the FC’s original value.Usually, collections of natural extracts include extracts of the same species but with distinct characteristics, such as organs , collection sites, culture media (in the case of micro-organisms) or extraction solvents, among others. As explained above, the FS and FC consider a feature specific if its relative intensity is higher than the “Literature Component  is retrieved from the metadata table and used to query the NPs occurrences reported in the LOTUS initiative  is set to 0.5. The same calculation is performed for comparing the reports at the genus level, and similarly, a value of CCg  is set to 0.5 if at least one unreported class is found. Both values are added to give the final CC value. To avoid inconsistent proposed chemical classes throughout a given extract, a ‘minimum recurrence filter’ is used to verify that at least more than ‘n’ features are annotated with a given NPClassifier class . .The ent CC,  indicatenotation . The chenotation . This chSimilarity Component . An SC value of “1” implies the extract is classified as an outlier within the extract set studied. This score highlights spectrally dissimilar extracts. Such information may be linked to spectral fingerprints that are likely related to singular chemistry. This score can be compared to the FC, and since it is independent of alignment and annotation might help to evaluate the specificity of the extract from an orthogonal perspective. For its calculation, the dissimilarity matrix created is subjected to three different unsupervised algorithms: Local outlier factor   , One-Cla method) , and Iso method) . In gene method) . If an eInventa’s results table contains all the components scoring and overall priority score , involving around 38 genera and 168 species. These NPs present 130 different chemical classes (NPClassifier (According to LOTUS  and the assifier ), coveriset of plants from the Celastraceae family considered in this study consists of 36 species and 14 different genera. Several plant parts were considered, depending on the availability, yielding 76 extracts in total. To improve the detection of medium polarity specialized metabolites, only ethyl acetate extracts were prepared. Extensive metabolite profiling of all extracts was performed by UHPLC-HRMS/MS operating in Data Dependent Acquisition mode. A careful comparison of the Base Peak Intensity (BPI) traces for both positive and negative ionization modes with the semiquantitative Charged Aerosol Detector trace (CAD) indicated that the positive mode was the most representative of the composition of the extracts. Thus, for this study, only the positive ionization data was considered. The data were processed with MZmine3 , less than fifty in the genus (crg), and less than five hundred in the family (crf); these correspond to user-defined parameters. In the case of this extract, only two compounds were reported in the species and 8 in the genus . Applicaβ-agarofuran sesquiterpenoids for the major peaks in the extract according to the BPI  and two friedelane triterpenoids, pristimerin, and maytenin (GNPS matches), both previously reported in the Celastraceae family  is displayed. In this map, a color coding represents the category for the features: specific unannotated , specific annotated (green), and not interesting . Such visualization helps localize inside the extract of interest the TIC peaks and their features, potentially corresponding to novel NPs.The CC value of 1, addition of CCs 0.5 and CCg 0.5, implied that at least one chemical class proposed by SIRIUS-CANOPUS had not been reported in species or the genus. CANOPUS proposed the chemical class dihydro-mily See . ConsideInventa’s score and the above considerations, the Pristimera indica roots extract was prioritized and subjected to an in-depth phytochemical investigation for de novo structural identification of the potentially new NPs.Based on Pristimera indica roots extract, shown in Inventa. The aim of such filters is to enable the user to explore how filtering-out the least abundant features affects the Inventa scoring results. For this, the original aligned feature table is normalized sample-wise (each row corresponding to an extract). The filters are applied to each sample. These filtered data are then treated by Inventa as the input for all the computations, as described above. The first filter minimizes to zero all the features with a normalized area of less than 2% in each extract  A.C.Sm roots was reduced by 85% (from 727 to 104). The second filter uses the quantile distribution for the features normalized area intensity. With this quantile filter only features with a normalized area intensity above the defined quantile value are considered ; the features that have their normalized areas below this quantile value are minimized to zero  were used. A comparison of their UHPLC-HRMS profiling with the original ethyl acetate extract showed that the main NPs of interest were present in the ethyl acetate and methanolic extracts.Inspection of the . The isolation was done using a dry-load-based injection, keeping a high resolution, and maximizing the sample load  and a mixture containing two compounds  were obtained. The mixture was separated under optimized conditions to purify both compounds, giving twelve pure compounds in total. To obtain compound 11, the methanolic extract was separated in the same conditions. de novo structural elucidation and absolute configuration are described below.This methodology efficiently allowed to yield thirteen compounds with enough material for tion see , only wiβ-agarofuran as proposed by the CC chemical classes of Inventa. They all presented the characteristic 2 methyl singlets at δH between 1.41 and 1.56 (H3-12 and H3-13), a methyl doublet at δH between 1.11 and 1.22 (H3-14), an oxymethylene at δH between 4.99–5.51 and 4.18–4.79 , an acetate in C-6 at δH between 2.12 and 2.21 (H3-6b), a particularly deshielded H-6 proton (δH between 6.24 and 6.78) and several oxygenated methines (δH between 3.92 and 6.18). These compounds could be divided into 3 series.Analysis of the NMR data confirmed that all the isolated compounds were dihydro-1-5, see C between 203 and 207 on the 13C and HMBC spectra. The purest compound and the one isolated in the greatest quantity is compound 3, it will be described first.The first one (compounds 3 was isolated as an amorphous powder with a [M + H]+ of m/z 755.3017 and a molecular formula of C38H47N2O14. The 1H-NMR and HSQC spectra indicated the presence of 3 oxymethine (in addition to H-6) at δH/δC 5.75/71.1 (H/C-1), 5.55/72.5 (H/C-2), and 6.01/78.8 (H/C-8). These methines were substituted by an acetate at δH 1.95, and two 5-carboxy-N-methyl-pyridone at δH 3.70 , 6.58 , 8.01 , and 8.47  for the first one and 3.62 , 6.56 , 7.95 , and 8.44  for the second one. The acetate at δH 1.95 was positioned thanks to its HMBC correlation with the carbonyl C-1a at δC 171.2 and this latter correlating with H-1. The position of both 5-carboxy-N-methyl-pyridones was confirmed by the HMBC correlations from H-8, H-8c, and H-8f to C-8a, from H-2c and H-2f to C-2a, and the weak correlation from H-2 to C-2a. The ROESY correlations from the aromatic protons of the pyridone in C-2 with H-15 agreed with that. The C-3 position was not substituted as indicated by the presence of two methylene protons at δH 1.97 and 2.41. Finally a 2-methyl-butanoyl group at δH 0.92 , 1.25 , 1.57 , 1.76 , and 2.65  was linked to C-15 due to the HMBC correlations from H2-15, H-15b, H2-15c and H3-15e to the ester carbonyl C-15a at δC 177.6. The HMBC correlations from H-1, H-8, and H2-15 to the ketone C-9 at δC 203.1 confirmed the presence of the carbonyl in C-9 , and losses of 2-methyl-butanoyl (m/z 85) and acetyl groups (m/z 59) in agreement with the literature (Compound l in C-9 . Oxidatis in C-8 . All theterature . This tr2-15 to the aromatic protons of the 5-carboxy-N-methyl-pyridone in C-2 (H-2c/H-2f) and H-6, from H-6 to H-8 and H3-14 indicated that these protons were on the same side of the molecule. The correlation from H3-12 to the acetate in C-6 confirmed that the C5-O-C11-C7 bridge is on the opposite side. The weak correlation between H3-13 and H-1 indicated that H-1 is in the same orientation as the bridge and that H-1 should be axial . Thus, the relative configuration of the substituents in 3 was proposed as 1α, 2α, 4α, 5β, 6β, 7β, 8β and 10α.The ROESY correlations from H3, the ECD spectrum was calculated based on the relative configuration proposed by NMR and compared to the experimental data  agrees with the reports in the literature for the type of chemical structure proposed.To establish the absolute configuration of compound data See . The abs3 was assigned as -1α,6β-diacetoxy-2α,8β-di-(5-carboxy-N-methyl-3-pyridoxy)-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran and named Silviatine C.After the comparison, compound 1 was isolated as a white amorphous powder. The molecular formula, C37H45N2O14, was calculated based on the HRMS-ESI-MS for [M + H]+ of m/z 741.2864. The NMR data of 1 were very similar to that of 3, except that an iso-butanoyl group at C-15 replaced the 2-methylbutanoyl group present at the same position in 3. The HMBC correlations from H-15′ at δH 5.10 , H-15b at δH 2.80 , H3-15c at δH 1.27  and H3-15d at δH 1.24  to the ester group C-15a at δC 177.8 confirmed the position of the iso-butanoyl group. Same cross-peaks in the ROESY spectrum as 3 were observed, suggesting the same relative configuration as 3. After calculation and comparison of the ECD spectra compound 3 was assigned as -1α,6β-diacetoxy-15-iso-butanoyloxy-2α,8β-di-(5-carboxy-N-methyl-3-pyridoxy)-9-oxodihydro-β-agarofuran, and named Silviatine A.Compound 2 was a white amorphous powder with a molecular formula of C36H45N2O13, calculated for [M + H]+ of m/z 713.2323. The 1H-NMR signals were closely related to those of 3, indicating that they shared the same core. The major differences were observed for the substituents. Only one acetyl group was found and positioned at C-6 due to the HMBC correlation fromH-6 (δH 6.24) and the acetate at δH 2.18 to the carbonyl at δc 171.1. Position one was suggested to bear a hydroxyl group due to the higher field signal of H-1 (δH 4.61) and no other HMBC correlations. Two 5-carboxy-N-methyl-3-pyrodone substituents were found in positions C-2 (δc 74.7) and C-8 (δc 78.4). The substituent in position C-15 (δC 63.6) was as in 3 a 2-methyl-butanoate moiety consisting of a carbonyl (δC 177.8), one methine , one methylene as a diastereotopic system , two methyl groups . Analysis of the ROESY correlations indicates that the relative configuration is the same as those of compounds 1 and 3. After comparison of the experimental and calculated ECD spectra, compound 2 was assigned as -6β-acetoxy-2α,8β-di-(5-carboxy-N-methyl-3-pyridoxy)-1α-hydroxy-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran, and named Silviatine B.Compound 4 had a molecular formula of C36H44NO12 for a [M + H]+ of m/z 682.2850. The core structure agrees with the one proposed for 2, due to the same patterns and correlations observed in the NMR data. The molecular formula suggested the presence of just one nitrogen. According to the 1H-NMR signals, two different aromatic groups were identified: a 5-carboxy-N-methyl-3-pyridone, as in the previous compounds, at δH 6.58 , 8.04 , and 8.49 , and a benzene at δH 7.53 , 7.66 , and 8.07 . The 5-carboxy-N-methyl-3-pyridone was positioned in C-2 thanks to the ROESY correlation from H2-15 to H-2c and H-2f. . As for compounds 2 and 3, the position C-15 was functionalized with a 2-methylbutanoate moiety according to the HMBC correlations from H-15a/H-15b (δH 4.79 and 5.06), H-15b (δH 2.70), H2-15c (δH 1.60 and 1.80) and H3-15e (δH 1.30) to the carbonyl at δC 178.1. As in 2, H-2 (δH 5.45) is in a higher field, suggesting this position carries an OH group.Compound 4 was assigned based on the ROESY data as 1α, 2α, 6β, and 8β. After comparison of the experimental and calculated ECD spectra, compound 4 was assigned as -6β-acetoxy-8β-benzoyloxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-1α-hydroxy-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran, and named Silviatine D.As explained above, the relative configuration of 5 was obtained as an amorphous white powder, giving a [M + H]+ of m/z 698.2802 with a molecular formula of C36H44NO13, one oxygen more than compound 4. NMR data was closely related to 4; the major difference was the absence of the diastereotopic methylene in C-3; instead, a signal in a lower field was found at δH 3.92, integrating for one proton as a doublet of doublets . The chemical shift for C-3 (δC 73.1) suggested the presence of an OH group, and the COSY correlations fromH-2 (δH 5.42) and H-4 (δH 2.51) to H-3 corroborated its position. 5 was thus a hydroxyl-derivative of compound 4. The key ROESY correlations were the same as for previous compounds: from H-15 to H-6, H3-14, H-2c, and H-2f, from H-6 to H-8, and from H3-12/13 to H-4 and H-1. The ROESY correlation from H-3 to H3-14 indicated their trans configuration. The absolute configuration of compound 5 was assigned as -6β-acetoxy-8β-benzoyloxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-1α,3β-dihydroxy-15-(2-methylbutanoyloxy)-9-oxodihydro-β-agarofuran, after comparison with the calculated ECD spectra, and named Silviatine E.Compound β-agarofuran structures was composed of 6 new alatol-type structures -6β-acetoxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)- 9α,15-di-(2-methylbutanoyloxy)-dihydro-β-agarofuran. It presented typical 1H-NMR signals of a dihydro-β-agarofuran scaffold, with a molecular formula of C41H55N2O14 for [M + H]+ of m/z 799.3649. Position one carried a hydroxyl group as indicated by the chemical shift of H-1 at δH 4.39. The other positions  were esterified by two 5-carboxy-N-methyl-3-pyridone and two 2-methylbutanoate. These latter were positioned in C-9 and C-15 due to the HMBC correlations from H-9b at δH 2.24, H2-9c at δH 1.37 and 1.68, H3-9e at δH 1.03, and H-9 at δH 5.64 to C-9a δC 176.6 and from H-15b at δH 2.37, H2-15c at δH 1.24 and 1.48, H3-15e at δH 1.13, and H-15′ at δH 5.41 to C-15a δC 179.0. The 5-carboxy-N-methyl-3-pyridones were thus in C-2 and C-8. The ROESY correlation from the aromatic protons H-2c (δH 8.61) and H-2f (δH 8.07) to H2-15 (δH 5.41 and 4.37) placed this 5-carboxy-N-methyl-3-pyridone in C-2 while the correlations from H-8c (δH 8.89) to H-6 (δH 6.74) placed the second one in C-8. The ROESY correlation from H-15 to H-6 and H3-14 indicated that H2-15, the two 5-carboxy-N-methyl-3-pyridone in C-2 and C-8, H3-14, and H-6 were on the same side of the molecule. On the other side, the acetate in C-6 (H3-6b) correlated with H3-13, H3-12 with H-1, and H-1 with H-9. Altogether these data indicated that the relative configuration should be 1α, 2α, 4α, 5β, 6β, 7β, 8α, 9α, 15α.Compound 7 was obtained as an amorphous white powder with a molecular formula of C42H55N2O15 for [M + H]+ of m/z 827.3598. The 1D and 2D NMR data displayed a significant resemblance with 6. One extra quaternary carbon at δC 171.4 was observed, belonging to an acetyl group fixed in position C-1 (δC 77.9), corroborated by the HMBC correlation with H-1 (δH 5.70). Positions C-2, C-8, and C-9 were substituted with the same groups as 6. However, protons in C-15 (δH 4.24 and 5.46) correlated in the HMBC spectrum with a carbonyl group at δC 179.0, coupled to a methine (δH 2.58), and two methyl doublets (δH 0.85 and 1.15), corresponding to a methylpropanoate system. The ROESY spectrum presented the same correlations as 6. After calculation of the ECD spectrum and comparison with the experimental, the compound 7 was assigned as - 1α,6β-diacetoxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-15-iso-butanoyloxy-9α-(2-methylbutanoyloxy)-dihydro-β-agarofuran.Compound 8 was obtained as a white amorphous powder with a molecular formula of C43H57N2O15 for [M + H]+ of m/z 841.3736. The NMR data showed to be closely related to 7. However, in position C-15 (δC 62.6), the substituent corresponds to a 2-methylbutanoate as in 6. The absolute configuration was assigned by comparison of the calculated ECD based on the relative configuration proposed as 1β, 2β, 8β, and 9β due to the observed ROESY correlations. Compound 8 was assigned as - 1α,6β-diacetoxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-9α,15-di-(2-methylbutanoyloxy)-dihydro-β-agarofuran.Compound 9 was assigned as - 1α,6β-diacetoxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-9α,15-di-(2-methylbutanoyloxy)-8α-nicotinoyloxydihydro-β-agarofuran, with a molecular formula C42H53N2O11 for [M + H]+ of m/z 811.3668. The major difference with 8 was the presence of a nicotinate moiety at δH 9.40 , 8.82 , 8.57 , and 7.65  instead of one of the 5-carboxy-N-methyl-3-pyridone. The 5-carboxy-N-methyl-3-pyridone was positioned in C-2 due to the ROESY correlation of H-15′ with H-2c. The nicotinate was thus placed in C-8. The ROESY correlations remained the same as other alatol-type compounds. Calculations of the ECD spectrum were done to define the absolute configuration.Compound 10, C42H55N2O15, calculated for [M + H]+ of m/z 827.3595, presented the same formula and mass as 7. The same core structure was proposed, but substituents in C-9 (δC 71.9) and C-15 (δC 62.6) were inverted. H-9 (δH 5.52) had an HMBC correlation with carbon at δC 176.3 which was connected to an iso-propyl system ). The relative configuration was the same as 7 . Compound 10 was assigned as -1α,6β-diacetoxy-9α-iso-butanoyloxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-15-methylbutanoyloxydihydro-β-agarofuran.Compound 11 was assigned as -6β-diacetoxy-9α-iso-butanoyloxy-2α,8α-di-(5-carboxy-N-methyl-3-pyridoxy)-1α-hydroxy-15-methylbutanoyloxydihydro-β-agarofuran, with a molecular formula of C40H53N2O14, calculated for [M + H]+ of m/z 785.3511. It presented the same substitution pattern as 10, but position C-1 (δC 75.6) had a proton signal at a higher field (δH 4.38), suggesting the presence of a free hydroxyl group. The relative configuration was the same as the rest of the molecules from this group and the absolute assigned configuration was checked by ECD comparison between the calculated and experimental spectra.Compound β-agarofuran structures was composed of 2 new euonymol-type structures -1α,3β,6β,8α-tetraacetoxy-15-iso-butanoyloxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-9α-tigloyloxydihydro-β-agarofuran, based on the NMR data. It presented a molecular formula of C39H52NO16, calculated for [M + H]+ of m/z 790.3289. In the HMBC spectrum, six carbonyls, presumably esters, were observed at δC 178.9, 171.5, 171.3, 170.8, 167.0, and 163.9, in addition to the acetyl fixed in C-6 (δC 171.1). The HMBC correlations from H-1 (δH 5.91) and H3-1b (δH 1.75) to C-1a (δC 170.8) positioned an acetate in C-1, from H-2 (δH 5.52), H-2c (δH 8.55), and H-2f (δH 8.00) to C-2a (δC 163.9) positioned a 5-carboxy-N-methyl-pyridone in C-2, from H-3 (δH 4.87) and H3-3b (δH 2.12) to C-3a (δC 171.3) positioned an acetate in C-3, from H-8 (δH 5.52) and H3-8b (δH 2.11) to C-8a (δC 171.5) positioned an acetate in C-8, from H-9 (δH 5.49), H-9c (δH 6.83) and H3-9e (δH 1.77) to C-9a (δC 167.0) positioned a tiggeloyl in C-1, and from H2-15 (δH 4.28 and 5.36), H-15b (δH 2.90), H3-15c (δH 1.26) and H3-15d (δH 1.24) to C-15a (δC 178.9) positioned an iso-butanoyl in C-15. The ROESY correlations showed that the configuration of the ester groups in C-1, C-2, C-8, and C-9 was the same as the alatol-type structures (6–11). The ROESY between H-3 and H3-14 indicated that the acetate in C-3 and methyl 14 were in a trans configuration. This relative configuration was corroborated after a comparison of the experimental and calculated ECD spectra.Compound 13, was obtained as an amorphous powder, giving a [M + H]+ of m/z 804.3445 with a molecular formula of C40H54NO16. The mass difference of 14 observed between itself and 12, suggested the presence of an extra CH2. This was corroborated due to the close resemblance of all the 1D and 2D NMR, except for the substituent in position C-15 (δC 62.1), which fitted with a 2-methylbutanoate moiety. The absolute configuration was corroborated by ECD calculation, using the relative configuration proposed by the ROESY spectrum. Thus, compound 13 was assigned as -1α,3β,6β,8α-tetraacetoxy-2α-(5-carboxy-N-methyl-3-pyridoxy)-15-(2-methylbutanoyloxy)-9α-tigloyloxydihydro-β-agarofuran.Compound de novo structural identifications, the chemical class proposed by Sirius-Canopus was confirmed, as well as the potential that Inventa holds to speed the discovery of novel NPs.Based on the FC values and highlighted ions, the filtering results, and the Inventa, an innovative computational tool capable of combining various level of information  from state-of-the-art bioinformatics programs, to highlight and prioritize extracts based on the possibility of finding structurally novel NPs. Inventa can be modulated according to the study parameters, and run locally or remotely via a web-based Binder notebook. The application of Inventa on a set of plant extracts showed how it can identify extracts where new compounds have high probability to be discovered. As a proof of concept, Inventa succeeded in the prioritization of the Pristimera indica roots extract among a set of seventy-six extracts from the Celastraceae family. An in-depth phytochemical investigation of this extract led to the isolation and de novo structural identification of thirteen new β-agarofuran sesquiterpene compounds. Five of them presented a new 9-oxodihydro-β-agarofuran base scaffold. This example illustrates how Inventa can speed up the discovery of original NPs.As explained throughout the article, prioritization of library extracts is difficult, multifactorial and time consuming. For this reason, the development of comprehensive prioritization pipelines combining the results of several bioinformatics tools is necessary to speed up and streamline extract selection for further in-depth phytochemical study. In this context, we propose Inventa, which allows prioritization of extract from large collections can be complemented by other tools, such as FERMO, under development , which will allow in-detail analysis and visualization for a particular extract. Collaboration efforts are in place to make them compatible and enhance their applicability.It is expected that in a near future


Compared with C70(OCH3)10 containing a single kind of functional group, both the C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 exhibited enhanced fluorescence properties with blue fluorescence emission. The fluorescence quantum yields of the C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 were 1.94% and 2.30%, respectively, which were about ten times higher than that of C70(OCH3)10. The theoretical calculations revealed that the multi-functionalization of the C70 increased the S1–T1 energy gap, reducing the intersystem crossing efficiency, resulting in the higher fluorescence quantum yield of the C70 derivatives. The results indicate that multi-functionalization is a viable strategy to improve the fluorescence of fullerene derivatives.Due to the partially reduced π-conjugation of the fullerene cage, multi-functionalized fullerene derivatives exhibit remarkable fluorescent properties compared to pristine fullerenes, which have high potential for application in organic light-emitting diodes (OLEDs). In this study two multi-functionalized C By carefully controlling the molar ratio of the reactants, C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 can be readily synthesized from C70(OCH3)10 by using the Bingel–Hirsch reaction with high selectivity. Due to the reduced π-conjugated system of C70, the fluorescence quantum yield of C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 was about ten times higher than that of C70(OCH3)10. The results provide a method for synthesizing fullerene derivatives with excellent fluorescence, offering valuable materials for organic light-emitting diodes.Herein, we report the synthesis and fluorescence properties of two multi-functionalized C70, iodine monochloride (ICl), silver perchlorate, diethyl bromomalonate, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were purchased from commercial suppliers and used as received without further purification. Solvents were distilled and dried by standard procedures.C70Cl10 and C70(OCH3)10 were prepared according to the procedure in the literature : Diethyl bromomalonate  and DBU  were added to a solution of C70(OCH3)10  in anhydrous toluene (50 mL). The mixture was stirred overnight under atmosphere at room temperature. The solvent was removed under reduced pressure and the crude product was purified by column chromatography over silica gel with toluene/acetate (10:1) as the eluents to produce C70(OCH3)10[C(COOEt)2] as a pale-yellow solid . 1H NMR : δ 4.21 , 3.98 , 3.93 , 3.86 , 3.75 , and 1.22 . 13C NMR : δ 163.56, 153.40, 153.07, 151.78, 151.29, 150.78, 150.73, 149.91, 149.40, 148.90, 148.54, 148.42, 148.24, 148.22, 148.11, 147.88, 147.79, 146.61, 146.49, 146.13, 145.41, 145.12, 143.71, 142.96, 139.00, 138.83, 138.58, 137.46, 135.93, 134.20, 129.04, 128.23, 86.18, 81.21, 81.03, 80.81, 80.69, 67.79, 65.89, 63.10, 56.18, 56.11, 55.94, 55.91, 55.85, 43.47, and 14.03. ESI-FT-ICR-HRMS C87H40O14 [M+Na]+m/z calculated 1331.2310 found 1331.2311.C70(OCH3)10[C(COOEt)2]2: Diethyl bromomalonate  and DBU  were added to a solution of C70(OCH3)10  in anhydrous toluene (50 mL). The mixture was stirred overnight under atmosphere at room temperature. The solvent was removed under reduced pressure and the crude product was purified by column chromatography over silica gel with toluene/acetate (5:1) as the eluents to afford C70(OCH3)10[C(COOEt)2]2 as a light-yellow solid . 1H NMR : δ 4.33 , 4.00–3.77 , and 1.38–1.30 . 13C NMR : δ 163.70, 163.67, 163.64, 163.21, 154.21, 153.22, 151.89, 151.80, 151.39, 151.34, 150.58, 150.33, 150.00, 149.62, 149.02, 148.90, 148.59, 148.31, 147.83, 147.53, 147.24, 146.91, 146.62, 146.52, 146.44, 146.30, 145.85, 145.60, 145.30, 145.26, 145.01, 144.60, 144.47, 143.67, 142.71, 139.95, 139.66, 139.12, 138.89, 138.14, 137.77, 137.52, 136.59, 136.24, 135.58, 134.38, 133.50, 130.01, 85.49, 84.51, 81.06, 80.94, 80.86, 80.80, 80.76, 80.70, 67.89, 67.80, 63.10, 63.07, 62.94, 62.88, 55.97, 55.93, 55.87, 55.79, 55.71, 55.68, 55.20, 43.44, 41.00, and 14.06. ESI-FT-ICR-HRMS C94H50O18 [M+Na]+m/z calculated 1490.3847 found 1490.2916.The C1H NMR, 13C NMR, and 2D NMR spectra were recorded using Bruker AVⅢ500 spectrometers . High-resolution mass spectra (HRMS) were recorded on Agilent G6545XT mass spectrometers . UV-vis absorption spectra in solution were recorded using an Agilent Cary 5000 spectrophotometer . The spectra were measured in quartz glass cuvettes using spectroscopic grade solvents. Fluorescence spectroscopy in solution was carried out with an FLS980 spectrometer . Time-resolved measurements were performed with a PS laser diode and a TCSPC detection unit. Single-crystal X-ray data were collected on a Rigaku Xtalab Synergy diffractometer . Using Olex2  and C70(OCH3)10[C(COOEt)2]2 were synthesized from C70(OCH3)10 by Bingel–Hirsch reaction. The deca-adduct C70 derivative C70(OCH3)10 was readily prepared by treating the C70Cl10 with anhydrous methanol in the presence of silver perchlorate. C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 can be readily synthesized with high selectivity by carefully controlling the molar ratio of the reactants. Their molecular structures were confirmed by 1H, 13C NMR spectroscopy and high-resolution mass spectrometry (70(OCH3)10[C(COOEt)2] and the C70(OCH3)10[C(COOEt)2]2 molecules (As shown in trometry . The twoolecules .70(OCH3)10[C(COOEt)2] was unambiguously determined by X-ray crystallographic analysis (70(OCH3)10[C(COOEt)2]. As shown in 70 cage. The malonate group was added to the pole of the C70 cage, and the ester groups were pointed in different directions to minimize the steric hindrance. In the crystalline state, the C70(OCH3)10[C(COOEt)2] molecules displayed ordered packing in all the directions of the a-, b- and c-axes. Although the single crystal of the C70(OCH3)10[C(COOEt)2]2 was not obtained, the most favorable structure of C70(OCH3)10[C(COOEt)2]2 was determined through a series of theoretical calculations 10, C70(OCH3)10[C(COOEt)2], and C70(OCH3)10[C(COOEt)2]2 were measured at room temperature. As shown in 70(OCH3)10 exhibited two absorption peaks at 435 and 480 nm in the visible region, and one absorption peak at 315 nm in the ultraviolet region. By contrast, there was no absorption peak in the visible region, but there was one absorption peak in the ultraviolet region (313 nm) for C70(OCH3)10[C(COOEt)2], which was slightly blue-shifted with respect to the C70(OCH3)10. Similarly, the C70(OCH3)10[C(COOEt)2]2 showed an absorption peak at 305 nm, which was further blue-shifted compared with that of the C70(OCH3)10[C(COOEt)2]. Moreover, a broad shoulder peak around 370 nm was observed for the C70(OCH3)10[C(COOEt)2]. The blue-shifting of the absorption peaks of both C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 was caused by the decrease in the π-conjugated system of the C70 cage, indicating that the energy gap between the S1 and S0 became lager.The UV-vis absorption spectra of the C70(OCH3)10, C70(OCH3)10[C(COOEt)2], and C70(OCH3)10[C(COOEt)2]2, we measured the steady-state fluorescence spectra of these C70 derivatives. As shown in 70(OCH3)10 was 498 nm, with a shoulder peak at 521 nm. The major emission peak at 498 nm was ascribed to the S1→S0 transition, and the shoulder peak was ascribed to the transition involving the vibronic interactions [70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 were rather similar. The major peaks appeared at 451 and 454 nm for the C70(OCH3)10[C(COOEt)2] and the C70(OCH3)10[C(COOEt)2]2, respectively, while the shoulder peaks were shown at 480, and 481 nm. Obviously, the fluorescence emission peaks of the C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 were blue-shifted compared to those of the C70(OCH3)10, indicating that the Bingel–Hirsch reaction can effectively reduce the π-conjugated system of the C70 cage [70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 were 1.94, and 2.30%, respectively, which were about ten times higher than that of the C70(OCH3)10 (0.25%). However, the fluorescence quantum yields of both the C70(OCH3)10[C(COOEt)2] and the C70(OCH3)10[C(COOEt)2]2 were not particularly high, which made them difficult to use as fluorescent labels.To obtain information about the photophysical properties of the C70(OCH3)10, C70(OCH3)10[C(COOEt)2], and C70(OCH3)10[C(COOEt)2]2 in chloroform were recorded using the time-correlated single-photon counting (TCSPC) method. The fluorescence lifetime of the C70(OCH3)10 was described by a single-exponential component with τ = 1.16 ns. However, the fluorescence lifetime of the C70(OCH3)10[C(COOEt)2] (τ = 1.99 ns) was described by double-exponential components with τ1 = 1.18 ns (70.9%) and τ2 = 3.95 ns (29.1%). Similarly, the fluorescence lifetime of the C70(OCH3)10[C(COOEt)2]2 (τ = 1.82 ns) was also described by bi-exponential components with τ1 = 1.18 ns (72.0%) and τ2 = 3.44 ns (28.0%) (70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 were slightly longer than those of the C70(OCH3)10, which implies that the number of adducts on fullerene can influence the fluorescence lifetime of fullerene derivatives , and C70(OCH3)10[C(COOEt)2]2 were computed through TD-DFT. The excitations of the C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 were similar and spatially localized in the same fragment of the molecule, which meant a large S1–T1 energy gap. However, the large spatial extension led to a small S1–T1 energy gap, as with the C70(OCH3)10. Therefore, the further functionalization of the C70(OCH3)10 increased the S1–T1 energy gap, reducing the intersystem crossing efficiency, resulting in the higher fluorescence quantum yield of the C70 derivatives.To gain insight into the mechanisms of the fluorescence enhancements, we carried out theoretical calculations. Generally, the compounds with high fluorescence quantum yields had large S70 derivatives, C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2, were synthesized from C70(OCH3)10 by Bingel–Hirsch reaction with high selectivity. Compared with the C70(OCH3)10, the UV-vis absorption and fluorescence of both the C70(OCH3)10[C(COOEt)2] and the C70(OCH3)10[C(COOEt)2]2 were blue-shifted due to the decrease in the π-conjugated system of the C70. Moreover, the C70(OCH3)10[C(COOEt)2] and C70(OCH3)10[C(COOEt)2]2 showed blue fluorescence, and their fluorescence quantum yield was about ten times higher than that of the C70(OCH3)10. The TD-DFT calculations indicated that the multi-functionalization of the C70 increased the S1–T1 energy gap, reducing the intersystem crossing efficiency, resulting in the higher fluorescence quantum yield of the C70 derivatives. The results reveal that multi-functionalization is an effective strategy to improve the fluorescence of fullerene derivatives, providing novel organic electronic materials for organic light-emitting diodes.In summary, two multi-functionalized C


Premna szemaoensis resulted in the isolation of twelve new abietane diterpenoids, szemaoenoids A–L (1–12), together with four known abietane diterpenoids (13–16). The structures involved two rearranged-abietane skeletons: 17(15 → 16)-abeo-abietane  and 17(15 → 16),18(4 → 3)-diabeo-abietane . The structures of the new compounds were established mainly by analyzing NMR and HRESIMS data. The absolute configurations of 1, 3 and 10 were confirmed by single crystal X-ray diffraction analysis. In bioactivity assays, compounds 11, 12, 14 and 15 were active against two human colon cancer cell lines (HCT-116 and HT-29) with IC50 values ranging from 8.8 to 34.3 μM, and compounds 10, 13 and 14 exhibited effective free radical scavenging activity with IC50 values ranging from 35.6 to 41.5 μM by DPPH experiment.Investigation of the leaves and stems of  Premna szemaoensis.Active abietane diterpenoids with two rearranged-abietane skeletons: 17(15 → 16)-abeo-abietane and 17(15 → 16),18(4 → 3)-diabeo-abietane were isolated from  Premna (family: Verbenaceae) comprises approximately 200 species, which are mainly distributed in the tropical zone of Asia and Africa.1 There are about 44 species and 5 varieties grown in the south of China, especially in Southwest China. The dried aerial parts of some Premna species have been used in traditional folk medicine for the treatment of pyogenic infections, trauma, fracture, dysentery, haemorrhoids, and rheumatic arthritis.2 Previous phytochemical investigations of Premna have indicated the presence of diterpenoids,3 flavonoids,3–5 iridoid glycosides,4–7 xanthones,8 phenylethanoid glycosides,9 triterpenoids,10,11 and lignins.12 Their pharmacological effects, including neuroprotective,13 analgesic,14,15 antioxidative, cytotoxic,16,17 anti-inflammatory,18 and α-glucosidase inhibition,19 have been reported for crude extracts and pure compounds from Premna plants.The genus Premna szemaoensis Pei, locally called “simao dofu chai”, is mainly distributed in the south of Yunnan province of China.20 This plant has drawn the attention of local farmers and has been cultivated to be an important commercial crop due to its various applications. Its fresh leaves can be rubbed and squeezed in water to yield a mucilaginous juice, which was used to prepare a food named “green tofu” by local people through addition of materials containing Ca2+. In addition, the local villagers also used the leaves of this plant to cure injuries and fracture.20 However, the phytochemical investigation of this species was extremely rare, except a small number of flavonoids. In this investigation, we firstly afforded 12 new abietane diterpenoids (1–12) and four known abietane diterpenoids (13–16) from the aerial parts of P. szemaoensis. Most of the diterpenoids were with rearranged-abietane skeleton: 17(15 → 16)-abeo-abietane framework or 17(15 → 16),18(4 → 3)-diabeo-abietane framework, which were mainly isolated from the plants of genus Clerodendron and reported bioactivities including cytotoxic, angiotensin converting enzyme (ACE) inhibitory, antiviral actives.21–24 Herein, we describe the isolation and structural elucidation of these diterpenoids and the biological activities of selected compounds.P. szemaoensis were extracted three times with 70% acetone aqueous. After recycling acetone, the rest of portion was partitioned by liquid–liquid extraction between n-butanol and H2O. The n-butanol-soluble portion was repeatedly subjected to silica gel, Sephadex LH-20, and RP-C18 gel column chromatography (CC) and semi-preparative HPLC to afford 16 abietane diterpenoids, including 12 new compounds. The structures and stereochemistry of these isolates were elucidated mainly using spectroscopic analysis, X-ray diffraction analysis, and compared to data in the literature. Ultimately, the new compounds were named as szemaoenoids A–L (1–12), and known compounds were identified as teuvincenone F (13),25, (16R)-12,16-epoxy-11,14,17-trihydroxy-17(15 → 16)-abeo-8,11,13-abieta-triene-7-one (villosin B) (14),26,27 12,16-epoxy-11,14,17-trihydroxy-6-methoxy-17(15 → 16)-abeo-5,8,11,13-abietatetraene-7-one (15),28 11,16-dihydroxy-12-O-β-d-glucopyranosyl-17(15 → 16),18(4 → 3)-diabeo-4-carboxy-3,8,11,13-abietatetraene-7-one (16),21 respectively.The aerial parts of α]25D +36.3 . Its HRESIMS data showed a sodium adduct ion [M + Na]+ at m/z 531.2205 , which together with 13C NMR , 1.41 (s) and 1.20 (s), respectively; three olefinic proton signals at δH 7.46 (s), 5.20 (s), and 4.74 (s), and a double peak at δH 4.57 . The 13C NMR and DEPT spectra of 1 , five methylenes (one olefinic), three methines , and eight quaternary carbons , along with signals for a hexose unit. These data suggested that 1 is a diterpene glycoside and in accordance with the characteristics of a 17(15 → 16),18(4 → 3)-diabeo-8,11,13-abietatriene.Compound 1 was isolated as an optically active, white monoclinic crystals (MeOH);  and Hooft parameter [0.10(6)] for 1225 Bijvoet pairs obtained by low-temperature [100(2) K] Cu Kα radiation X-ray crystallography -12-O-β-d-glucopyranosyl-3,11,16-trihydroxy-17(15 → 16),18(4 → 3)-diabeo-4(19),8(9),11(12),13(14)-abietatetraene-7-one, named szemaoenoid A.The β-3-20/H-2 , but α- lography .29,30 Thα]25D −8.8 . The HRESIMS gave a quasi-molecular ion at m/z 531.2203 [M + Na]+ . Thus, in conjunction with 13C NMR and DEPT data, the molecular formula was established as C26H36O10, representing nine indices of hydrogen deficiency. The 1H and 13C NMR data of 2 , 4.27 , δC 59.2] and two olefinic quaternary carbons  in 2vs. an oxygenated quaternary carbon and an exocyclic double bond group in 1. Through HMBC experiment, observed correlations from H2-19  to C-3/C-4/C-5, from CH3-18 (δH 1.26) to C-2/C-3/C-4 indicated that an OH group at C-19 and a double bond at C-3 and C-4. There was another hydroxyl group at C-16 (assigned the configuration as R) via comparing chemical shift and coupling constant of 2 with 1 , and the connecting correlations of 1H–1H COSY and HMBC data.Compound 2 was isolated as a white amorphous powder with −). The NMR data for this compound were highly close to those of 2 (δC 157.4) in 3, correspondingly absence of an olefinic methine  vs.2. In the HMBC spectrum, the correlations of H2-15  to C-12 (δC 153.3), C-13 (δC 121.0), C-14 (δC 157.4) indicated that an OH group was at C-14, which made the chemical shifts of aromatic fields and ketone group (δC 207.1) obviously change in 13C NMR spectrum vs.2. Ultimately, the absolute configuration was confirmed by single crystal X-ray diffraction analysis, which assigned C-16 as R configuration (R)-12-O-β-d-glucopyranosyl-11,14,16,19-tetrahydroxy-17(15 → 16),18(4 → 3)-diabeo-3(4),8(9),11(12),13(14)-abietatetraene-7-one, named szemaoenoid C.Compound 3 was obtained as white monoclinic crystals (MeOH). Its molecular formula assigned was determined to be Cose of 2  and 2, eguration . Therefo26H32O10 according to HRESIMS (m/z 527.1888 [M + Na]+). The 1H NMR and 13C NMR data were closely related to those of 3 , one quaternary carbon at C-16 (δC 156.5). The HMBC correlations from CH3-17  to C-15/C-16 and from H-15 to C-12 (δC 153.4)/C-13 (δC 119.8)/C-14 (δC 156.8) powerfully confirmed the locations of the double bond between C-15 and C-16, and an OH group at C-16. In consideration of its molecular formula as C26H32O10, and chemical stability, this structure must be absence of one H2O, and linked from C-12 to C-16 through a vinyl ether bond, likewise the known compound 13 with a furan ring. The sugar moieties were determined to be located at C-11, by the HMBC correlation of the anomeric proton H-1′  to C-11 (δC 133.7). Thus, compound 4 was established as 11-O-β-d-glucopyranosyl-12,16-epoxy-14,19-dihydroxy-17(15 → 16),18(4 → 3)-diabeo-3(4),8(9),11(12),13(14),15(16)-abietapentaene-7-one, named szemaoenoid D.Compound 4, a yellowish amorphous powder, had a molecular formula of Cose of 3  and 2. T26H28O11 by HRESIMS data, indicating 12 degrees of unsaturation (two more than that of compound 4). The 1H and 13C NMR resonances of 5 closely resembled those of 4  and CH3-18  to C-2 (δC 201.1) and from H-6  to C-4 (δC 149.6)/C-8 (δC 110.1)/C-10 (δC 44.2) demonstrated above inference. Therefore, the structure of 5 was elucidated as 11-O-β-d-glucopyranosyl-12,16-epoxy-14,19-dihydroxy-17(15 → 16),18(4 → 3)-diabeo-3(4),5(6),8(9),11(12),13(14),15(16)-abietahexaene-2,7-dione, named szemaoenoid E.Compound 5 was obtained as a brown amorphous powder. Its molecular formula was determined to be Cose of 4  and 2, e26H32O10 . The NMR data of 6 (δC 71.3) and an exocyclic double bond  in 6 (in accord with compound 1) vs. an oxygenated methylene and two olefinic quaternary carbons in 4. In the HMBC spectrum, the cross-connection signals of CH3-18 (δH 1.40) to C-2 (δC 38.1), C-3 (δC 71.3) and C-4 (δC 153.6), and of H2-19  to C-3, C-4 and C-5 (δC 43.7), supported the presence of an OH group at C-3 and a double bond at C-4/C-19. In consideration of identical NMR data and biogenesis, absolute configuration of C-3 in 6 was identical with 1 (assigned as R configuration). Consequently, compound 6 was established as (3R)-11-O-β-d-glucopyranosyl-12,16-epoxy-3,14-dihydroxy-17(15 → 16),18(4 → 3)-diabeo-4(19),8(9),11(12), 13(14),15(16)-abietapentaene-7-one, named szemaoenoid F.Compound 6 was isolated as a yellowish amorphous powder. The HRESIMS gave its molecular formula as Cata of 6  and 2 we26H36O11 on the basis of the HRESIMS. From the NMR data, it was similar to those of 2  and an oxygenated methylene (δC 59.2) vs.2, correspondingly presence of an obvious carboxyl (δC 185.1), a quaternary carbon (δC 45.7) and a methylene (δC 39.9) in 7. The HMBC correlations of CH3-19  to C-3 (δC 39.9)/C-4 (δC 45.7)/C-5 (δC 53.6) and the carboxyl C-18 (δC 185.1), of H-5 to C-4/C-18/CH3-19 (δC 30.1), and the 1H–1H COSY correlations of H2-1/H2-2/H2-3, all indicated that CH3-19 and the carboxyl C-18 were both linked to C-4. CH3-19 was assigned as β-orientation from the NOE effect of CH3-19/CH3-20. In view of identical NMR data and biogenesis, absolute configuration of C-16 in 7 was also identical with 1 (assigned as R configuration). Therefore, the structure of 7 was determined as (16R)-12-O-β-d-glucopyranosyl-11,16-dihydroxy-17(15 → 16)-abeo-8(9),11(12),13(14)-abietatriene-7-one-18-acid, named szemaoenoid G.Compound 7, a white amorphous powder, had a molecular formula of Cose of 2  and 2, e26H38O10 according to HRESIMS. The 1H and 13C NMR spectra of 8  in 8, but the known compound was absent of this group. The HMBC correlations of H-5 (δH 1.73)/H2-6 /H-14 (δH 7.44) with C-7 (δC 201.3), of CH3-18 (δH 1.03)/CH3-19 (δH 0.93) with C-3 (δC 78.7), and 1H–1H COSY connections of H2-1/H2-2/H-3, suggested the carbonyl group at C-7 and an OH group at C-3, respectively. 1H–1H COSY correlations from CH3-17  through H-15  to CH2-16 , and HMBC correlations from CH3-17 to C-13 (δC 138.2) and C-15 (δC 35.1), from H-15 to C-16 (δC 68.8), C-17 (δC 18.2), C-12 and C-14, were suggestive of an 1-hydroxy-isopropyl moiety linked to C-13 in this structure. Configurations of C-15 was established as S, by comparing the chemical shifts at C-13, C-15, C-16 with that of two known compounds (S)-12-O-d-glucopy-ranosyl-3,11,16-trihydroxy-8,11,13-abie-tatriene21 and (15R)-cyrtophyllone B,32 whose structures were undoubtedly established by Mosher method and X-ray crystallography respectively. The β orientation of OH-3 was confirmed by the NOE effect of H-3/H-5. Therefore, this structure was established as (15S)-12-O-β-d-glucopyranosyl-3β,11,16-trihydroxy-8(9),11(12),13(14)-abietatriene-7-one, named szemaoenoid H.Compound 8, a white amorphous powder, exhibited a molecular formula of Ctra of 8  and 2 weompounds : (15S)-126H40O10. Its 1H and 13C NMR spectra  in 9 instead of a methyl group (δC 17.0) in the known structure. Moreover, the oxygenated methylene signals at δH 3.45 and 4.18 showed the HMBC correlations with the signals at C-3 (δC 80.9), C-4 (δC 44.3) and C-5 (δC 55.0), and the NOE effect with H-5/H-3, which evidently inferred that the OH was located at C-18, in combination with NOE effect of CH3-19/CH3-20. The β orientation of OH-3 and S configuration of C-15 were confirmed like that of compound 8. Consequently, compound 9 was assigned as (15S)-12-O-β-d-glucopyranosyl-3β,11,16,18-tetrahydroxy-8(9),11(12),13(14)-abietatriene, named szemaoenoid I.The HREIMS and NMR data of compound 9 were consistent with the molecular formula of C spectra  and 2 we20H26O5 was determined by HRESIMS, indicating 8 degrees of unsaturation in the structure. From the 1H and 13C NMR spectrum  indicated the β-orientation of OH-3. The absolute configuration of this compound was established as 3S, 5R, 10S, 16S by single crystal X-ray diffraction analysis -12,16-epoxy-3,11,14-trihydroxy-17(15 → 16)-abeo-8(9),11(12),13(14)-abietatriene-7-one, named szemaoenoid J.Compound 10, yellowish monoclinic crystals (MeOH), its chemical formula as Cspectrum  and 2, ianalyses  suggestespectrum  between analysis . Accordi20H24O5 by HRESIMS data, with one more degree of unsaturation then 10. As seen from 1H NMR and 13C NMR spectrum -abeo-8(9),11(12),13(14),15(16)-abietatetraene-7-one (11), named szemaoenoid K; and 12,16-epoxy-11,14,17-trihydroxy-17(15 → 16)-abeo-8(9),11(12), 13(14),15(16)-abietatetraene-7-one (12), named szemaoenoid L.Compound 11 and 12 were assigned to an identical molecular formula of Cspectrum  and 2, tPremna genus,33–36 but this was also the first to report diterpenoids isolated from the Premna plants distributed in China, and the described rearranged-abietane skeletons were firstly isolated from Premna genus. Abietane diterpenoids represented a large group of secondary metabolites that have shown interesting biological activities.31 But the rearranged abietane diterpenoids with 17(15 → 16)-abeo-abietane or 17(15 → 16),18(4 → 3)-diabeo-abietane were not common in nature. Structurally, the rearranged diterpenoids contain abundant hydroxyl groups and aromatic carbons, as well as trans-fused rings A and B according to biosynthetic pathway. However, the stereocenter of C-16 replaced with OH had never been established previously. Within this work, we firstly employed X-ray crystallography to assign absolute configuration of C-16 for a small series of 16-hydroxy-17(15 → 16)-abeo-abietane diterpenoids, which might assist future unambiguous identification of structurally related compounds.In our cognition, many diterpenoides had been reported from plants of in vitro against two human colon carcinoma cell lines (HCT-116 and HT-29). As a result (50 8.7 ± 1.4 μM) with positive control   . Almost 37–39 some selective compounds of the diterpenosides were executed free radical scavenging activity assay in the DPPH experiment . Compound 12 exhibited slightly weaken antioxidant activity with IC50 values of 74.9 ± 6.9 μM. None of the tested diterpene glucosides  showed potent free radical scavenging activity. Concerning the structure–activity relationship, a reasonable conclusion was reasoned out that the more the structure possessed phenolic hydroxyl groups, the more its scavenging activity was strong.As most of natural products possessing phenolic hydroxy exhibited antioxidant activity,periment . Among tδ) were expressed in parts per million with reference to the solvent signals. HRESIMS was performed on an Agilent G6230 TOF MS. Semi-preparative HPLC was performed on an Agilent 1260 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm) column. Column chromatography (CC) was performed on silica gel , Lichroprep RP-18 gel , MCI gel , and Sephadex LH-20 (Pharmacia). Fractions were monitored by TLC, and spots were visualized by UV light (254 nm) and sprayed with 8% H2SO4 in ethanol, followed by heating.X-ray data were collected using a Bruker APEX DUO instrument. Optical rotations were measured with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained on a Tenor 27 spectrophotometer with KBr pellets. One-dimensional (1D) and two-dimensional (2D) NMR spectra were recorded on Bruker DRX-600 spectrometers with TMS as the internal standard. Chemical shifts  has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences.Aerial parts of P. szemaoensis (10 kg) were extracted with 70% aqueous acetone (40 L) four times (two days each time) at room temperature and then filtered. The filtrate was evaporated under reduced pressure at 40 °C and then partitioned between n-butanol and H2O. The n-butyl alcohol soluble portion (600 g) was subjected to silica gel CC , eluted with a CHCl3–Me2CO gradient system (1:0–0:1) that afforded fractions A–E. The fractions were then decolorized using MCI gel and eluted with 95% MeOH–H2O.The air-dried and powdered aerial parts of 3–MeOH gradient (150:1–1:1), to yield fractions B1–B5. Fraction B1 was purified by repeated silica gel CC  to yield compound 14 (10.2 mg). Fraction B3 was purified by Sephadex LH-20 (MeOH) to yield fractions B31–B34. Then compound 13 (75.0 mg) was crystallized from fraction B34, and compound 15 (8.4 mg) was isolated by HPLC . B4 was purified by RP-18 CC  to yield fractions B41–B45, then B43 was isolated by semi-preparative HPLC  to obtain compound 10 (10.1 mg). B5 was subjected to Sephadex LH-20 CC (MeOH) to give B51–B55, then compounds 11 (8.7 mg) and 12 (15.4 mg) were isolated from fraction B52 by semi-preparative HPLC .Fraction B (33 g) was subjected to silica gel CC (200–300 mesh), eluted with a CHCl3–MeOH (gradient system: 120:1–1:1) to yield fractions C21–C27. C23 was isolated by semi-preparative HPLC  to afford compounds 1 (8.5 mg) and 2 (11.7 mg). C25 was purified by HPLC  to yield compounds 4 (5.2 mg), 5 (4.6 mg), and 6 (2.3 mg).Fraction C (120 g) was separated by Sephadex LH-20 (MeOH) to give fractions C1–C5. Fraction C2 was subjected to repeated silica gel CC (200–300 mesh), eluted with CHCl2O, 10–100%) to give fractions D1–D6. Compound 16 (20.1 mg) was crystallized from fractions D2. Fraction D4 was separated to by Sephadex LH-20 eluted with MeOH to give D41–D47, then D43 was isolated by semi-preparative HPLC  to yield compounds 3 (6.1 mg) and 7 (4.3 mg). Fraction D5 was separated to silica gel CC (200–300 mesh) eluted with CHCl3–MeOH (50:1–1:2) to yield D51–D56, then D54 was followed by HPLC  to afford compounds 8 (4.6 mg) and 9 (2.3 mg).Fraction D (100 g) was subjected to RP-18 CC ; UV (MeOH) λmax (log ε) 213 (4.23), 269 (3.91), 318 (3.49) nm; IR (KBr) νmax 3441, 1632, 1049 cm−1; 1H and 13C NMR data, see m/z 531 [M + Na]+; positive-ion HRESIMS [M + Na]+m/z 531.2205 .White monoclinic crystals (MeOH); mp 257–259 °C (MeOH); [α]22D +28.8 ; UV (MeOH) λmax (log ε) 204 (4.17), 270 (3.96), 318 (3.32) nm; IR (KBr) νmax 3441, 1632, 1071 cm−1; 1H and 13C NMR data, see m/z 531 [M + Na]+; positive-ion HRESIMS [M + Na]+m/z 531.2203 .White amorphous powder; [α]22D +17.8 ; UV (MeOH) λmax (log ε) 203 (4.55), 239 (1.55), 283 (1.51) nm; IR (KBr) νmax 3414, 1616, 1424, 1072 cm−1; 1H and 13C NMR data, see m/z 523 [M − H]−; negative-ion HRESIMS [M − H]−m/z 523.2177 .White monoclinic crystals (MeOH); mp 275–278 °C (MeOH); [α]22D −54.1 ; UV (MeOH) λmax (log ε) 204 (4.47), 239(4.41), 254 (4.33), 355 (3.61) nm; IR (KBr) νmax 3425, 2924, 1631, 1384, 1069, 586 cm−1; 1H and 13C NMR data, see m/z 527 [M + Na]+; positive-ion HRESIMS [M + Na]+m/z 527.1888 .Yellowish amorphous powder; [α]22D −53.0 ; UV (MeOH) λmax (log ε) 202 (4.13), 295 (4.06) nm; IR (KBr) νmax 3426, 1629, 1466, 1216, 580 cm−1; 1H and 13C NMR data, see m/z 539 [M + Na]+; positive-ion HRESIMS [M + Na]+m/z 539.1519 .Brown amorphous powder; [α]22D +78.8 ; UV (MeOH) λmax (log ε) 202 (4.27), 240 (4.34), 254 (4.27), 356 (3.52) nm; IR (KBr) νmax 3422, 2924, 1635, 1357, 1201, 1077 cm−1; 1H and 13C NMR data, see m/z 503 [M − H]−; negative-ion HREIMS [M − H]−m/z 503.1912 .Yellowish amorphous powder; [α]22D +15.9 ; UV (MeOH) λmax (log ε) 215 (4.25), 269 (3.84), 316 (3.44) nm; IR (KBr) νmax 3441, 1632, 1068 cm−1; 1H and 13C NMR data, see m/z 523 [M − H]−; negative-ion HRESIMS [M − H]−m/z 523.2176 .White amorphous powder; [α]22D −8.57 ; UV (MeOH) λmax (log ε) 215 (4.11), 269 (3.76), 316 (3.33) nm; IR (KBr) νmax 3428, 1632, 1069 cm−1; 1H and 13C NMR data, see m/z 509 [M − H]−; negative-ion HRESIMS [M − H]−m/z 509.2388 .White amorphous powder; [α]22D +9.25 ; UV (MeOH) λmax (log ε) 204 (4.65), 275 (3.27) nm; IR (KBr) νmax 3424, 1632, 1422, 1063, 596 cm−1; 1H and 13C NMR data, see m/z 511 [M − H]−; negative-ion HRESIMS [M − H]−m/z 511.2540 .White amorphous powder; [α]22D +30.56 ; UV (MeOH) λmax (log ε) 203 (4.84), 212 (4.70), 298 (3.10), 353 (1.67) nm; IR (KBr) νmax 3427, 1632, 1462, 1351, 1015 cm−1; 1H and 13C NMR data, see m/z 345 [M − H]−; negative-ion HRESIMS [M − H]−m/z 345.1708 .Yellowish monoclinic crystals (MeOH); mp 213–215 °C (MeOH); [α]22D +61.3 ; UV (MeOH) λmax (log ε) 202 (4.23), 237 (4.25), 258 (4.18), 369 (3.52) nm; IR (KBr) νmax 3441, 1631, 1458, 1372, 1277, 1179, 1069, 602 cm−1; 1H and 13C NMR data, see m/z 343 [M − H]−; negative-ion HRESIMS [M − H]−m/z 343.1551 .Yellowish amorphous powder; [α]22D +43.3 ; UV (MeOH) λmax (log ε) 202 (3.99), 237 (4.06), 260 (4.02), 366 (3.36) nm; IR (KBr) νmax 3429, 2926, 1631, 1456, 1370 cm−1; 1H and 13C NMR data, see m/z 343 [M − H]−; negative-ion HRESIMS [M − H]−m/z 343.1548 .Yellowish amorphous powder;  × 100%, where S, B and C are the absorbencies of the sample, the blank and the control, respectively.44Trolox  and vitamin C  were used as positive control. A 0.1 mM solution of DPPH radical in ethanol was prepared, and 100 μL of this solution was mixed with 100 μL of sample solution. The mixture was incubated for 5 min in a dark room at room temperature. Scavenging capacity was read spectropho-tometrically by monitoring the decrease in absorbance at 517 nm. DPPH scavenging activity (%) = [1 − . Structurally, these compounds involved two rearranged-abietane skeletons: 17(15 → 16)-abeo-abietane and 17(15 → 16),18(4 → 3)-diabeo-abietane. Their structures with absolute configurations were characterized by a series of spectroscopic methods and X-ray diffraction. In bioactivity assays, compounds 11, 12, 14 and 15 were active against two human colon cancer cell lines (HCT-116 and HT-29) with IC50 values ranging from 8.8 to 34.3 μM, and compounds 10, 13 and 14 exhibited effective free radical scavenging activity with IC50 values ranging from 35.6 to 41.5 μM by DPPH experiment. In short, the current study adds to understanding of the chemical composition and biological effects of this plant prepared for green food and ethnodrugs.In summary, we have firstly reported twelve new abietane diterpenoids (1–12) isolated from There are no conflicts to declare.RA-008-C7RA13309J-s001RA-008-C7RA13309J-s002