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Extracts of Eremophila phyllopodasubsp. phyllopodashowed α-glucosidase and PTP1B inhibitory activity with IC50values of 19.6 and 13.6 μg/mL, respectively. High-resolution α-glucosidase/PTP1B/radical scavenging profiling was performed to establish a triple high-resolution inhibition profile that allowed direct pinpointing of the constituents responsible for one or more of the observed bioactivities. Subsequent targeted isolation and purification by analytical-scale HPLC led to the identification of 21 previously undescribed serrulatane diterpenoids, eremophyllanes A–U, as well as two known serrulatane diterpenoids, 1β-trihydroxyserrulatane (8) and 1α-trihydroxyserrulatane (10d), and five known furofuran lignans, (+)-piperitol (6), horsfieldin (7e), (−)-sesamin (9), (+)-sesamin (10h), and asarinin (10i). Their structures were elucidated by extensive analysis of HRMS and 1D and 2D NMR spectroscopic data. The relative configurations of the previously undescribed compounds were established by analysis of ROESY spectra as well as by DFT-GIAO NMR calculations followed by DP4+ probability analysis. The absolute configurations were determined by comparison of experimental and calculated ECD spectra. Serrulatane diterpenoids 7band 14exhibited α-glucosidase inhibitory activity with IC50values of 28.4 and 64.2 μM, respectively, while 11, 12, 14, and 15exhibited PTP1B inhibitory activity with IC50values ranging from 16.6 to 104.6 μM. Hypothetical routes for formation of all identified serrulatane diterpenoids are proposed.

Ten new branched-chain fatty acid (BCFA) dimers with a substituted cyclohexene structure, five new monomers, and two known monomers, (2E,4Z,6E)-5-(acetoxymethyl)tetradeca-2,4,6-trienoic acid and its 5-hydroxymethyl analogue, were identified in the leaf extract of Eremophila oppositifoliasubsp. angustifoliausing a combination of HPLC-PDA-HRMS-SPE-NMR analysis and semipreparative-scale HPLC. The dimers could be classified as three types of Diels–Alder reaction products formed between monomers at two different sites of unsaturation of the dienophile. Two of the monomers represent potential biosynthetic intermediates of branched-chain fatty acids. Several compounds were found by high-resolution bioactivity profiling to inhibit PTP1B and were purified subsequently by semipreparative-scale HPLC. The dimers were generally more potent than the monomers with IC50values ranging from 2 to 66 μM, compared to 38–484 μM for the monomers. The ten fatty acid dimers represent both a novel class of compounds and a novel class of PTP1B inhibitors.

Type 2 diabetes is a chronic multifactorial disease affecting more than 425 million people worldwide, and new selective α-glucosidase inhibitors with fewer side effects are urgently needed. In this study, a crude ethyl acetate extract of Machilus litseifoliawas fractionated by solid-phase extraction using C18cartridges to give a fraction enriched in α-glucosidase inhibitors. Subsequent microfractionation and bioassaying of the eluate by high-performance liquid chromatography (HPLC) using a complementary pentafluorophenyl column allowed construction of a high-resolution α-glucosidase inhibition profile (biochromatogram). This was used to target high-performance liquid chromatography–photodiode array detection–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HPLC-PDA-HRMS-SPE-NMR) analysis toward α-glucosidase inhibitors. This led to the identification of 13 dicoumaroylated flavonol rhamnosides, of which seven (8, 10, 12a, 12b, 16, 17, and 18) are reported for the first time, and two lignans, of which one (5) is reported for the first time. IC50values of isolated compounds toward α-glucosidase range from 5.9 to 35.3 μM, which is 8 to 91 times lower than the IC50value of 266 μM measured for the reference compound acarbose.

A hyphenated procedure involving high-performance liquid chromatography, photodiode array detection, high-resolution mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-PDA-HRMS-SPE-NMR, has proven an effective technique for the identification of compounds in complex matrices. Most HPLC-PDA-HRMS-SPE-NMR investigations reported so far have relied on analytical-scale reversed-phase C18columns for separation. Herein is reported the use of an analytical-scale pentafluorophenyl column as an orthogonal separation method following fractionation of a crude ethyl acetate extract of leaves of Coleonema albumon a preparative-scale C18column. This setup allowed the HPLC-PDA-HRMS-SPE-NMR analysis of 23 coumarins, including six new compounds, 8-O-β-d-glucopyranosyloxy-6-(2,3-dihydroxy-3-methylbut-1-yl)-7-methoxycoumarin (4), (Z)-6-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (6), 6-(4-β-d-glucopyranosyloxy-3-methylbut-1-yl)-7-hydroxycoumarin (8), (Z)-7-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yloxy)coumarin (13), (S)-8-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)-7-methoxycoumarin (19), and 7-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)coumarin (20). The use of the pentafluorophenyl column even allowed separation of several regioisomers that are usually difficult to separate using reversed-phase C18columns. The phytochemical investigation described for C. albumin this report demonstrates the potential and wide applicability of HPLC-PDA-HRMS-SPE-NMR for accelerated structural identification of natural products in complex mixtures.

Serrulatanes constitute a class of unique diterpenoids derived from all-Znerylneryl diphosphate rather than the conventional all-Editerpenoid precursor geranylgeranyl diphosphate and thus provide an intriguing expansion of the chemical space of plant specialized metabolites. Plants of the Australian Eremophilagenus are rich sources of structurally diverse serrulatanes. Here, we report the identification of 15 hitherto undescribed serrulatanes (eremoculatanes A–N), together with 16 previously reported compounds, from the EtOAc extract of Eremophila denticulataleaves. Isolation was performed by a combined use of systematic HPLC-PDA-HRMS-based phytochemical profiling and orthogonal reversed-phase C18and pentafluorophenyl separations. Among the new compounds isolated, eremoculatane A contains a C12backbone, for which the configuration was established by comparison of experimentally measured and theoretically calculated ECD spectra. The antihyperglycemic and antibacterial activities of the E. denticulataextract were investigated by high-resolution inhibition profiling, and they indicated that major constituents, mainly serrulatanes and flavonoids, contributed to the observed activity of the extract. One flavonoid, eupafolin (4), displayed moderate α-glucosidase inhibitory activity with an IC50value of 41.3 μM, and four serrulatanes (8, 9, 19g, and 19j) showed more than 50% PTP1B inhibition at 200 μM.

According to the International Diabetes Federation, type 2 diabetes (T2D) has reached epidemic proportions, affecting more than 382 million people worldwide. Inhibition of protein tyrosine phosphatase-1B (PTP1B) and α-glucosidase is a recognized therapeutic approach for management of T2D and its associated complications. The lack of clinical drugs targeting PTP1B and side effects of the existing α-glucosidase drugs, emphasize the need for new drug leads for these T2D targets. In the present work, dual high-resolution PTP1B and α-glucosidase inhibition profiles of Eremophila gibbosa, E. glabra, and E.aff. drummondii“Kalgoorlie” were used for pinpointing α-glucosidase and/or PTP1B inhibitory constituents directly from the crude extracts. A subsequent targeted high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) analysis and preparative-scale HPLC isolation led to identification of 21 metabolites from the three species, of which 16 were serrulatane-type diterpenoids (12 new) associated with either α-glucosidase and/or PTP1B inhibition. This is the first report of serrulatane-type diterpenoids as potential α-glucosidase and/or PTP1B inhibitors.

The endophytic fungus Pestalotiopsis virgatula, derived from the plant Terminalia chebulaand previously found to produce a large excess of a single metabolite when grown in the minimal M1D medium, was induced to produce a variety of unusual metabolites by growing in potato dextrose broth medium. Analysis of the fermentation medium extract was performed using an HPLC-PDA-MS-SPE-NMR hyphenated system, which led to the identification of a total of eight metabolites (1–8), six of which are new. Most of the metabolites are structurally related and are derivatives of benzo[c]oxepin, rare among natural products. This includes dispiro derivatives 7and 8(pestalospiranes A and B), having a novel 1,9,11,18-tetraoxadispiro[6.2.6.2]octadecane skeleton. Relative and absolute configurations of the latter were determined by a combination of NOESY spectroscopy and electronic circular dichroism spectroscopy supported by time-dependent density-functional theory calculations (B3LYP/TZVP level). This work demonstrates that a largely complete structure elucidation of numerous metabolites present in a raw fermentation medium extract can be performed by the HPLC-SPE-NMR technique using only a small amount of the extract, even with unstable metabolites that are difficult to isolate by traditional methods.

The hyphenated technique HPLC-PDA-MS-SPE-NMR was used to assist targeted preparative-scale isolation of constituents of Lobelia siphiliticaand Hippobroma longiflora(both Campanulaceae). This resulted in the isolation of two new alkaloids, (S)-2-[(2S,6R)-1-methyl-6-(2-oxo-2-phenylethyl)piperidin-2-yl]-1-phenylethyl acetate (3) and 6-[(E)-2-(3-methoxyphenyl)ethenyl]-2,3,4,5-tetrahydropyridine (4), the latter possessing a skeleton hitherto unseen among alkaloids of Lobeliaand related genera. Lobeline (1), (1S,1′S)-2,2′-[(2R,6S)-1-methylpiperidine-2,6-diyl]bis(1-phenylethanol) (2), and lobetyolin (5) were also isolated. The structures of 1−5were established using spectroscopic methods including homo- and heteronuclear two-dimensional NMR experiments and optical rotation data.

Despite recent demonstration of the power of HPLC-PDA-MS-SPE-NMR (high-performance liquid chromatography−photodiode-array detection−mass spectrometry−solid-phase extraction−nuclear magnetic resonance) in structure determination of natural products directly from minute amounts of crude extracts, this technique leaves chirality of the compounds uncharacterized. In this work we demonstrate that postcolumn SPE is a useful method of analyte concentration and accumulation not only for NMR but also for CD (circular dichroism) spectroscopy. Thus, use of HPLC-PDA-MS-SPE-NMR in combination with CD allowed rapid detection of ( R)-(−)-gossypol [( R)- 1] in Thespesia danis, providing a very rare example of the predominance of the levorotatory enantiomer of gossypol. Enantioselectivity of the in vitro antiplasmodial activity of gossypol was also demonstrated; the IC 50value of ( R)- 1was 4.5 ± 0.2 µM, with the eudismic ratio of about 2.5. No gossypol was detected in Gossypioides kirkii.

Phytochemical investigations of Dovyalis abyssinica, D. hebecarpa, and D. macrocalyxrevealed two new spermidine-type alkaloids, dovyalicin E (3) and dovyalicin F (4), along with the previously described dovyalicin A (1), dovyalicin B (2), and dovyalicin C (5). In addition, a new phenol glucoside, 4-hydroxytremulacin (7), and the new 1,2-cyclohexanediol glucoside 9, as well as the known compounds methyl 1-hydroxy-6-oxocyclohex-2-enecarboxylate (6) and tremulacin (8), were isolated. The structures were established using homo- and heteronuclear two-dimensional NMR experiments and chiroptical methods. At ambient temperature, the N-disubstituted amide 4exists as a mixture of cisand transconformers. Variable-temperature 1H NMR studies showed that time-averaged spectra are obtainable at 348 K, and the activation parameters determined for the rotation about the amide bond were H 89 ± 4.6 kJ/mol, S 65 ± 14 kJ/mol·K, and G(298K) 70 ± 4.5 kJ/mol.

The HPLC-SPE-NMR technique, supported by HPLC-MS measurements, was used to determine structures of major as well as some minor constituents of ethanol and petroleum ether extracts of Harpagophytum procumbens(Devil's claw) roots. This method was also shown to be applicable for rapid and precise on-line identification of secondary metabolites present in commercial herbal products of H. procumbens. A total of 15 compounds (1−14and 17) were identified from the ethanol and petroleum ether extracts, including a novel Diels−Alder dimer 14. Optimization of the HPLC-SPE-NMR experiments included quantitative 1H NMR measurements, determination of trapping and elution efficiency, effect of multiple trapping of analytes, use of various deuterated solvents for SPE cartridge elution, and effect of post-column dilution ratio of eluent with water. Linear accumulation of apolar and relatively polar analytes was demonstrated for at least 8−10 repeated trappings, resulting in greatly improved signal-to-noise ratios in NMR spectra and reduced acquisition times. Thus, the HPLC-SPE-NMR technique provides an efficient means of identification of multiple components of crude extracts. By allowing on-line generation of high-quality 2D NMR data without traditional purification of extract components, the HPLC-SPE-NMR methodology represents a paradigm shift in natural products research with respect to structure elucidation.

HPLC-SPE-NMR experiments with crude extracts of Harpagophytum procumbens allowed the rapid identification of novel, unstable chinane-type tricyclic diterpenes (1 and 2), along with numerous other constituents. Dramatic sensitivity gains achieved with this novel hyphenated technique establish a new paradigm in natural products research:  rapid and rigorous structure determination of multiple extract components, including minor components, without preparative-scale isolation.