Pseudoamaolides A-O, anti-inflammatory triterpene spiroketal lactones from seeds of Pseudolarix amabilis.

Fifteen new triterpenoids (1-15), along with twenty known ones (16-35), were isolated from Pseudolarix amabilis. The triterpenoid structures include multiple skeleton types, such as 2,3-seco-cycloartane, 3,4-seco-cycloartane, 3,4:9,10-diseco-cycloartane, and 3,4:8,9:9,10-triseco-cycloartane, as elucidated by extensive spectroscopy (1D NMR, 2D NMR, HRESIMS, and IR) and single-crystal X-ray diffraction. The anti-inflammatory activities of compounds 1-35 were evaluated. Compounds 3, 11, 16, 24, 25, and 26 suppressed the transcription of the NF-κB-dependent reporter gene in LPS-induced 293T/NF-κB-Luc cells with IC50 values of 0.12, 0.10, 0.30, 0.09, 0.49, and 0.35 µM, respectively. In addition, compound 16 showed anti-inflammatory activity against xylene-induced ear swelling in vivo with an inhibition rate of 44.7 % (30 mg/kg). Compound 16 significantly improved the disease activity index (DAI) of ulcerative colitis at a dose of 400 mg/kg in a dextran sodium sulfate (DSS)-induced mouse model of experimental ulcerative colitis (P < 0.01).

Disclosing the composition of unknown historical drug formulations: an emblematic case from the Spezieria of St. Maria della Scala in Rome.

This paper reports a pioneering study of an unknown historical drug formulation preserved in the Spezieria of Santa Maria della Scala in Rome, founded at the end of the seventeenth century by the Discalced Carmelites. Due to limited literature related to pharmaceutical remedies and drugs of the Early Modern Era (between the XV and XVIII centuries) and the complexity in their formulations, the study of these drugs represents a great challenge. The untargeted nature of the selected drug required a multi-analytical approach with complementary techniques to formulate a compositional hypothesis: FT-IR spectroscopy, gas chromatography-associated/mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) were successfully employed to identify different organic compounds. Systematic archaeobotanical research was performed as well, allowing us to acquire data related to the possible genus of plants from which these natural compounds derive and their geographical origin. The unknown drug formulation turned out to be a complex mixture used as an ointment with an anti-inflammatory purpose. It mainly contains a mixture of Venetian turpentine; a Pine resin (colophony) from the Pinaceae family; an exudate of a plant from South America, whose identified components are triterpenic compounds such as alpha- and beta-amyrins, betulin and lupeol; and saturated fatty acids which act as carriers and/or to reduce the viscosity of abovementioned exudates and resins. The study of historical drugs is important not only in order to know the practices handed down by the speziali in the past but also to reconstruct historical recipes, which can inspire new dermatological, cosmetic, hygienic and current healing products.Graphical abstract.

Chemical characterization and identification of Pinaceae pollen by infrared microspectroscopy.

MAIN CONCLUSION: FTIR microspectroscopy, in combination with spectral averaging procedure, enables precise analysis of pollen grains for chemical characterization and identification studies of fresh and fossilised pollen in botany, ecology and palaeosciences. Infrared microspectroscopy (µFTIR) of Pinaceae pollen can provide valuable information on plant phenology, ecophysiology and paleoecology, but measurements are challenging, resulting in unreproducible spectra. The comparative analysis of µFTIR spectra belonging to morphologically different Pinaceae pollen, namely bisaccate Pinus and monosaccate Tsuga pollen, was conducted. The study shows that the main cause of spectral variability is non-radial symmetry of bisaccate pollen grains, while additional variation is caused by Mie scattering. Averaging over relatively small number of single pollen grain spectra (approx. 5-10) results with reproducible data on pollen chemical composition. The practical applicability of the µFTIR spectral averaging method has been demonstrated by the partial least-squares regression-based differentiation of the two closely related Pinus species with morphologically indistinguishable pollen: Pinus mugo (mountain pine) and Pinus sylvestris (Scots pine). The study has demonstrated that the µFTIR approach can be used for identification, differentiation and chemical characterization of pollen with complex morphology. The methodology enables analysis of fresh pollen, as well as fossil pollen from sediment core samples, and can be used in botany, ecology and paleoecology for study of biotic and abiotic effects on plants.

Evaluating climate signal recorded in tree-ring δ13 C and δ18 O values from bulk wood and α-cellulose for six species across four sites in the northeastern US.

RATIONALE: We evaluated the applicability of tree-ring δ13 C and δ18 O values in bulk wood - instead of the more time and lab-consuming α-cellulose δ13 C and δ18 O values, to assess climate and physiological signals across multiple sites and for six tree species along a latitudinal gradient (35°97'N to 45°20'N) of the northeastern United States. METHODS: Wood cores (n = 4 per tree) were sampled from ten trees per species. Cores were cross-dated within and across trees at each site, and for the last 30 years. Seven years, including the driest on record, were selected for this study. The δ13 C and δ18 O values were measured on two of the ten trees from the bulk wood and the α-cellulose. The offsets between materials in δ13 C and δ18 O values were assessed. Correlation and multiple regression analyses were used to evaluate the strength of the climate signal across sites. Finally the relationship between δ13 C and δ18 O values in bulk wood vs α-cellulose was analyzed to assess the consistency of the interpretation, in terms of CO2 assimilation and stomatal conductance, from both materials. RESULTS: We found offsets of 1.1‰ and 5.6‰ between bulk and α-cellulose for δ13 C and δ18 O values, respectively, consistent with offset values reported in the literature. Bulk wood showed similar or stronger correlations to climate parameters than α-cellulose for the investigated sites. In particular, temperature and vapor pressure deficit and standard precipitation-evaporation index (SPEI) were the most visible climate signals recorded in δ13 C and δ18 O values, respectively. For most of the species, there was no relationship between δ13 C and δ18 O values, regardless of the wood material considered. CONCLUSIONS: Extraction of α-cellulose was not necessary to detect climate signals in tree rings across the four investigated sites. Furthermore, the physiological information inferred from the dual isotope approach was similar for most of the species regardless of the material considered.

Inhibition of HL-60 cell growth via cell cycle arrest and apoptosis induction by a cycloartane-labdane heterodimer from Pseudolarix amabilis.

Pseudolaridimer C (), a rarely encountered cycloartane-labdane Diels-Alder adduct was isolated from the cones of Pseudolarix amabilis. The structure and absolute configuration of were established by comprehensive NMR and CD spectral analysis. The WST-8 assay indicated that time and dose dependently inhibited the proliferation of human leukemia cells HL-60 at 1-10 µM. DAPI and Annexin V-FITC/PI double staining method, and DNA ladder experiments all proved that had significant dose-dependent effects on HL-60 cell apoptosis. A further mechanism study indicated that the apoptosis was associated with the cell cycle arrest during the G2/M phase, and the activation of caspase-9, -3, -7, and poly-(ADP-ribose)-polymerase (PARP).

Methyl Jasmonate-Induced Monoterpenes in Scots Pine and Norway Spruce Tissues Affect Pine Weevil Orientation.

In large parts of Europe, insecticide-free measures for protecting conifer plants are desired to suppress damage by the pine weevil Hylobius abietis (L.). Treatment with methyl jasmonate (MeJA), a chemical elicitor already used in crop production, may enhance expression of chemical defenses in seedlings in conifer regenerations. However, in a previous experiment, MeJA treatment resulted in substantially better field protection for Scots pine (Pinus sylvestris L.) than for Norway spruce (Picea abies (L.) Karst.). Hypothesizing that the variations may be at least due partly to volatiles released by MeJA-treated seedlings and their effects on pine weevil orientation, we examined tissue extracts of seedlings (from the same batches as previously used) by two-dimensional GC-MS. We found that the MeJA treatment increased contents of the monoterpene (-)-ß-pinene in phloem (the weevil's main target tissue) of both tree species, however, the (-)-ß-pinene/(-)-α-pinene ratio increased more in the phloem of P. sylvestris. We also tested the attractiveness of individual monoterpenes found in conifer tissues (needles and phloem) for pine weevils using an arena with traps baited with single-substance dispensers and pine twigs. Trap catches were reduced when the pine material was combined with a dispenser releasing (-)-ß-pinene, (+)-3-carene, (-)-bornyl acetate or 1,8-cineole. However, (-)-α-pinene did not have this effect. Thus, the greater field protection of MeJA-treated P. sylvestris seedlings may be due to the selective induction of increases in contents of the deterrent (-)-ß-pinene, in contrast to strong increases in both non-deterrent (-)-α-pinene and the deterrent (-)-ß-pinene in P. abies seedlings.

Antiproliferative Constituents of the Roots of Ethiopian Podocarpus falcatus and Structure Revision of 2α-Hydroxynagilactone F and Nagilactone I.

Bioassay-guided fractionation using the human colorectal adenocarcinoma (HT-29) cell line of the methanol extract of dried roots of Podocarpus falcatus led to the isolation of two new type C nagilactones, 16-hydroxynagilactone F (1) and 2ß,16-dihydroxynagilactone F (2), and the new totarane-type bisditerpenoid 7ß-hydroxymacrophyllic acid (4), along with the seven known compounds 2ß-hydroxynagilactone F (3), macrophyllic acid (5), nagilactone D (6), 15-hydroxynagilactone D (7), nagilactone I (8), inumakiol D (9), and ponasterone A (10). The structures of the new compounds were determined by 1D and 2D NMR, HRESIMS, UV, and IR and by comparison with the reported spectroscopic data of their congeners. The orientation of the C-2 hydroxy group of 3 and 8 was revised to be ß based on evidence from detailed analysis of 1D and 2D NMR data and single-crystal X-ray diffraction studies. Among the isolated compounds, the nagilactones, including the new dilactones 16-hydroxynagilactone F (1) and 2ß,16-dihydroxynagilactone F (2), were the most active (IC50 0.3-5.1 µM range) against the HT-29 cell line, whereas the bisditerpenoids (4 and 5) and the other known compounds 9 and 10 were inactive. The presence of the bioactive nagilactones in P. falcatus supports its traditional use.

New eudesmane-type sesquiterpenoids from the root bark of Pseudolarix kaempferi.

The phytochemical investigation of the root bark of Pseudolarix kaempferi yielded eight eudesmane-type sesquiterpenoids, including three new ones, 1α-hydroxyl-4(14)-en-ß-dihydroagarafuran (1), 1α, 2α-diacetoxy-8ß-isobutanoyloxy-9α-benzoyl oxy-15-ß-(ß-furancarbonyloxy)-4ß, 6ß-dihydroxy-ß-dihydroagarofuran (7), and 1α-acetoxy-2 α-furancarbonyloxy-8ß-isobutanoyloxy-9α-benzoyloxy-15ß-(ß-acetoxy)-4ß, 6ß-dihydroxy-ß-dihydroagarofuran (8). Herein the new compounds 7 and 8 were reported as a mixture. The molecular structures of the isolated compounds were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, NMR, and MS, and comparison with the literature data.

Trade-offs between constitutive and induced defences drive geographical and climatic clines in pine chemical defences.

There is increasing evidence that geographic and climatic clines drive the patterns of plant defence allocation and defensive strategies. We quantified early growth rate and both constitutive and inducible chemical defences of 18 Pinaceae species in a common greenhouse environment and assessed their defensive allocation with respect to each species' range across climatic gradients spanning 31° latitude and 2300 m elevation. Constitutive defences traded-off with induced defences, and these defensive strategies were associated with growth rate such that slow-growing species invested more in constitutive defence, whereas fast-growing species invested more in inducible defence. The position of each pine species along this trade-off axis was in turn associated with geography; moving poleward and to higher elevations, growth rate and inducible defences decreased, while constitutive defence increased. These geographic patterns in plant defence were most strongly associated with variation in temperature. Climatic and geographical clines thus act as drivers of defence profiles by mediating the constraints imposed by trade-offs, and this dynamic underlays global patterns of defence allocation.

Flavonoids from Podocarpus macrophyllus and their cardioprotective activities.

One new 8-aryl flavone, podocarflavone A (1), together with 15 previously reported flavonoids were isolated from the twigs and leaves of Podocarpus macrophyllus. Their structures were established on the basis of extensive spectroscopic analysis and by the comparison with spectroscopic data reported in the literature. Antioxidant capacities of the isolated substances were determined using the 1,1-diphenyl-2-picrylhydrazyl, ferrous ions, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical in vitro assays, and their cytoprotective activities were also tested on H2O2-induced apoptosis in H9c2 cardiomyocytes. The results showed that those flavonoids exhibited significant cardioprotective effects by decreasing the H2O2-induced death of H9c2 cell, and the levels of lactate dehydrogenase and creatine kinase, and by inhibiting the elevated intracellular concentration of reactive oxygen species.

[Metabolic pathway and metabolites of total diterpene acid isolated from Pseudolarix kaempferi].

The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.

Coupling of sterically hindered trisubstituted olefins and benzocyclobutenones by C-C activation: total synthesis and structural revision of cycloinumakiol.

The first total syntheses of the proposed structure of cycloinumakiol (1) and its C5 epimer (18) are achieved in a concise and efficient fashion. Starting from the known 3-hydroxybenzocyclobutenone, 1 and 18 are obtained in nine and five steps with overall yields of 15% and 33%, respectively. The key for the success of this approach is the use of a catalytic C-C activation strategy for constructing the tetracyclic core of 1 through carboacylation of a sterically hindered trisubstituted olefin with benzocyclobutenone. In addition, the structure of the natural cycloinumakiol was reassigned to 19-hydroxytotarol (7) through X-ray diffraction analysis. This work demonstrates the potential of C-C activation for streamlining complex natural product synthesis.

Evaluation of a liquid chromatography method for compound-specific δ13C analysis of plant carbohydrates in alkaline media.

RATIONALE: Isotope analysis of carbohydrates is important for improved understanding of plant carbon metabolism and plant physiological response to the environment. High-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) for direct compound-specific δ(13)C measurements of soluble carbohydrates has recently been developed, but the still challenging sample preparation and the fact that no single method is capable of separating all compounds of interest hinder its wide-spread application. Here we tested in detail a chromatography method in alkaline media. METHODS: We examined the most suitable chromatographic conditions for HPLC/IRMS analysis of carbohydrates in aqueous conifer needle extracts using a CarboPac PA20 anion-exchange column with NaOH eluent, paying specific attention to compound yields, carbon isotope fractionation processes and the reproducibility of the method. Furthermore, we adapted and calibrated sample preparation methods for HPLC/IRMS analysis. OnGuard II cartridges were used for sample purification. RESULTS: Good peak separation and highly linear and reproducible concentration and δ(13)C measurements were obtained. The alkaline eluent was observed to induce isomerization of hexoses, detected as reduced yields and (13)C fractionation of the affected compounds. A reproducible pre-purification method providing ~100% yield for the carbohydrate compounds of interest was calibrated. CONCLUSIONS: The good level of peak separation obtained in this study is reflected in the good precision and linearity of concentration and δ(13)C results. The data provided crucial information on the behaviour of sugars in LC analysis with alkaline media. The observations highlight the importance for the application of compound-matched standard solution for the detection and correction of instrumental biases in concentration and δ(13)C analysis performed under identical chromatographic conditions. The calibrated pre-purification method is well suited for studies with complex matrices that disable the use of a spiked internal standard for the detection of procedural losses.

Natural resins and balsams from an eighteenth-century pharmaceutical collection analysed by gas chromatography/mass spectrometry.

Historical nomenclature has not always been unequivocally associated with the botanical origin of natural resins. The availability of natural resins has changed throughout the centuries and so have their trade names. Furthermore, adulterations and lack of knowledge have led to variations in the composition of the products traded under the same name. This investigation aims at a new understanding of the interrelation between the historical and modern terms for natural resins. Different Pinaceae and Pistacia resins, mastic, sandarac, copaiba balm and burgundy pitch from Vigani's Cabinet, a 300-year-old pharmaceutical collection owned by Queens' College, Cambridge (UK) were investigated. Related reference materials from modern collections were analysed together with natural resins derived from reliable botanical sources. The analytical method was gas chromatography/mass spectrometry (GC-MS) with and without derivatisation with trimethylsulfonium hydroxide. This technique provided detailed molecular compositions of the studied materials, which in turn led to particular data profiles of the materials. Marker molecules of Copaifera, Pinaceae, Cupressaceae and Pistacia resins were identified. By comparing the GC-MS data profiles to the reference samples, a clearer picture of the connection between nomenclature and botanical origin was obtained. With the aid of the marker molecules and data profiles, it was then possible to clarify the nomenclature of the aged resins sampled from Vigani's Cabinet.

Inhibitors of the oncogenic transcription factor AP-1 from Podocarpus latifolius.

An activator protein-1 (AP-1) based bioassay-guided phytochemical investigation on Podocarpus latifolius led to the isolation of three new sempervirol-type diterpenes, cycloinumakiol (1), inumakal (2), and inumakoic acid (3), along with three known norditerpenes (4-6). Compounds 4 and 6 were responsible for the observed bioactivity.

Antimicrobial, cytotoxic lignans and terpenoids from the twigs of Pseudolarix kaempferi.

Seven new compounds, including four lignans, (+)-(8S,8'S)-9,9'-dibenzoylsecoisolariciresinol (1), (+)-(8S*,8'R*)-4,4'-dimethyloxomatairesinol (2), (+)-(7S*,8R*,8'R*,9'S*)-9'-n-butoxytsugacetal (3), and pseudolarkaemin A (4), a pyronane glycoside, pseudolarkaemin B (5), an ent-beyerene glycoside, pseudolarkaemin C (6), and a triterpene, 25-epi-pseudolarolide Q (7), along with 25 known compounds (8­32) were isolated from the twigs of Pseudolarix kaempferi. Their structures were elucidated mainly by the analysis of their NMR and MS data. Pseudolarolide C acid (24) was isolated for the first time as a natural product. All compounds were evaluated for antimicrobial activity against Candida albicans and Staphylococcus aureus, and cytotoxic activity against K562, HT-29, B16, BGC-823, BEL-7402, SGC-7901, U251, and A549 cancer cell lines were assayed. Results indicated that the new compounds 3, 7, and some known compounds showed antimicrobial and cytotoxic activities.

Characterization of diterpenoids in the bark of Pseudolarix kaempferi by HPLC-ESI/MSn.

Fragmentation behavior of diterpenoids was investigated by ESI/MSn and the qualitative analysis of diterpenoids in the bark of Pseudolarix kaempferi was performed using high-performance liquid chromatography/ multi-stage mass spectrometry (HPLC-ESI/MSn). The characteristic fragmentation behaviors of the diterpenoids are the cleavages of the lactone ring and C4-O bond. Furthermore, the eliminations of substituent groups at C-18, C-7 and C-8 can also be observed in the MS" (n = 3-4) spectra. For C-4 acetoxy subsititued diterpenoids, [M+Na-60]+ and [M-H-104] are the base peaks of MS2 spectra in the positive and negative ionization modes, respectively. For C-4 hydroxyl subsititued diterpenoids, [M+Na-44]+ and [M-H-62] are the base peaks of MS2 in the positive and negative ionization modes, respectively. For C-18 glucosylated or esterized diterpenoids, [M+Na-44]+ is the base peak of MS2 spectra in positive ionization mode. These fragmentation rules were successfully exploited in the identification of diterpenoids in methanol/water (6:4) extract of P. kaempferi by LC-MS in positive ionization mode. A total of 9 diterpenoids were identified or tentatively characterized, and one of them is reported here for the first time. The described method could be utilized for the sensitive and rapid qualitative analysis of P. kaempferi.

[Metabolic pathway and metabolites of pseudolaric acid B].

The metabolic profile of pseudolaric acid B (PB) was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the specific metabolite of PB in plasma, urine, bile and feces using HPLC and HPLC-ESI/MS(n) after both oral and intravenous administration to rats, and almost no prototype was detected in all kinds of samples. The metabolic behaviors of PB orally administered in rats treated with antibiotics to eliminate intestinal microflora were identical with those in untreated rats, demonstrating that the metabolism of PB is independent of intestinal microflora. PB was stable in 48 h respective incubation with artificial gastric juice and artificial intestinal juice, suggesting that neither pepsin nor trypsin is in charge of metabolism of PB, and also demonstrating that PB is stable in both pH environments of gastric tract and intestinal tract. In vitro research on metabolism of PB in rat liver microsomes incubation revealed that little PB was metabolized and that the proposed metabolites were the demethoxy and demethoxydecarboxy products of the prototype. The amount of metabolites was extremely low compared with the prototype, indicating that liver microsomes are not responsible for the metabolism of PB either. PB was gradually metabolized into PC2 during 1 h in whole blood incubation in vitro, and the metabolic process showed dynamically dependent manner with incubation time. Once absorbed into blood, PB was quickly metabolized into PC2, accordingly, little prototype was detected in all kinds of samples. The metabolism was attributed to the rapid hydrolysis of C-19 ester bond by plasma esterase. These results clarified the metabolic pathway of PB for the first time, which was of great significance to identify the in vivo active form and interpret acting mechanism of the active compounds of P. kaempferi.

Analysis of the functional group composition of the spruce and birch phloem lignin.

In this article, the functional group composition of the spruce (Pícea ábies) and birch (Bétula péndula) phloem lignin is described. The features of the chemical structure were studied by analyzing dioxane lignin using the elemental analysis, UV-Vis, FT-IR, and 1D NMR spectroscopy. For comparison, samples of xylem dioxane lignin isolated from the corresponding wood species were also analyzed. FT-IR spectroscopy data suggest that the lignins of birch phloem and xylem are similar in chemical structure. However, there are differences in absorption bands in the spectra of spruce dioxane lignin, which indicate the opposite. Quantitative analysis of the functional group composition was performed using 13C and 31P NMR data. It was found that free phenolic hydroxyl groups of catechol and p-hydroxyphenyl types are dominated in the composition of spruce phloem lignin. Birch phloem lignin has a qualitative and quantitative composition of functional groups characteristic of hardwood lignins. However, the content of G-units is greater than S-units, in contrast to the birch xylem lignin, where S-units predominate. The revealed differences are relevant from the point of view of plant physiology. The practical significance of the study is connected with understanding the reactivity of lignins when considering the chemical processing of tree bark.

Characterization of plant exudates by principal-component and cluster analyses with nuclear magnetic resonance variables.

Principal-component and cluster analyses have been applied to nuclear magnetic resonance data for exudates derived from both conifers and angiosperms in order to classify these materials on the basis of molecular structure. The method succeeds in distinguishing resins produced by the conifer families Araucariaceae, Cupressaceae, and Pinaceae from each other and from resins produced by the angiosperm family Fabaceae. Other exudate types, including gums, gum resins, and kinos, also are distinguished from each other and from the resins.