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Here, we explore the mechanism of action of isoxylitone (ISOX), a molecule discovered in the plant Delphinium denudatum, which has been shown to have anticonvulsant properties. Patch-clamp electrophysiology assayed the activity of ISOX on voltage-gated sodium channels (VGSCs) in both cultured neurons and brain slices isolated from controls and rats with experimental epilepsy(kindling model). Quantitative transcription polymerase chain reaction (qRT-PCR) (QPCR) assessed brain-derived neurotrophic factor (BDNF) mRNA expression in kindled rats, and kindled rats treated with ISOX. ISOX suppressed sodium current (I(Na)) showing an IC50 value of 185 nM in cultured neurons. ISOX significantly slowed the recovery from inactivation (ISOX τ = 18.7 ms; Control τ = 9.4 ms; p < 0.001). ISOX also enhanced the development of inactivation by shifting the Boltzmann curve to more hyperpolarized potentials by -11.2 mV (p < 0.05). In naive and electrically kindled cortical neurons, the IC50 for sodium current block was identical to that found in cultured neurons. ISOX prevented kindled stage 5 seizures and decreased the enhanced BDNF mRNA expression that is normally associated with kindling (p < 0.05). Overall, our data show that ISOX is a potent inhibitor of VGSCs that stabilizes steady-state inactivation while slowing recovery and enhancing inactivation development. Like many other sodium channel blocker anti-epileptic drugs, the suppression of BDNF mRNA expression that usually occurs with kindling is likely a secondary outcome that nevertheless would suppress epileptogenesis. These data show a new class of anti-seizure compound that inhibits sodium channel function and prevents the development of epileptic seizures.

RATIONALE Tandem mass spectrometric studies of natural products revealed the identification of key fragments which can be helpful for their rapid dereplication in plant extracts utilizing a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach, particularly for the thermally labile compounds. The knowledge of the collision-induced dissociation (CID) fragmentation pattern of the molecule is essentially required prior to the analysis by LC/MS/MS. METHODS The fragmentation patterns of eleven Buxus steroidal alkaloids were studied by using a positive ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Chromatographic separation of a Buxus papillosa extract was achieved using a capillary HPLC system coupled with the mass instrument. RESULTS ESI-QqTOF-MS (positive ion mode) showed the presence of several characteristic fragments which can be used to rapidly identify various classes of Buxus steroidal alkaloids. The presence of a cyclopropane ring in the cycloartenol skeleton and the hydroxyl group at C-10 was found to effect on the fragmentation pattern and afford characteristic peaks. This study distinguishes between different types of Buxus steroidal alkaloids based on logical fragmentation pathways. This strategy was successfully applied in LC/ESI-QqTOF-MS/MS analysis of Buxus papillosa extract to investigate and characterize Buxus steroidal alkaloids and 14 compounds were identified as steroidal alkaloids. CONCLUSIONS The knowledge of the fragmentation pattern was used for the rapid identification of this bioactive group of biosynthetically unique steroidal alkaloids in complex plant extracts of Buxus species, especially in the absence of any reference material, by combining key fragments, exact mass measurements and relative abundances of diagnostic fragment ions.

Background Orange peel (Citrus sinensis) and green tea (Camellia sinensis) leaves, rich sources of food flavonoids, were analyzed for their trypsin inhibitory potential. Hesperetin, rutin and hesperidin from orange peel, and catechin from green tea leaves, were isolated and their chemical structures were analyzed. All four compounds were evaluated for their trypsin inhibitory potential. Results Among all the isolated compounds, rutin exhibited the highest protease inhibition activity (75.4 ± 0.9%) with IC50 = 16 ± 2 µmol L−1, followed by catechin (65.3 ± 1.4%; IC50 = 83 ± 9 µmol L−1), hesperetin (62.1 ± 1.3%; IC50 = 104 ± 12 µmol L−1) and hesperidin (59.7 ± 1.1%; IC50 = 127 ± 14 µmol L−1). Lineweaver–Burk and Dixon plots and their secondary replots indicated that all four compounds possessed non-competitive inhibition. The Ki values of hesperetin, rutin, hesperidin and catechin were calculated as 90.2 ± 1.1, 17.5 ± 0.6, 84.2 ± 1.5 and 65.1 ± 1.5 µmol L−1 respectively. Conclusion The present results suggest that the four isolated flavonoids can be used as a supplement in food for the treatment of pathologies associated with the degradation of a specific protein. © 2012 Society of Chemical Industry

The current study was aimed at investigating the potential antidepressant activity of Areca catechu nut ethanol extract and its various fractions using behavioral (acute and sub-chronic forced swim tests) and biochemical (monoamines and their metabolite levels using high performance liquid chromatography) tests. The areca nut ethanol extract and its aqueous fraction exhibited antidepressant activity in both acute and sub-chronic forced swim tests (IC50 ~ 50 and 20 mg/kg, respectively), which was further confirmed by unaltered locomotor (horizontal and vertical) activities of rats in the activity cage. Phytochemical analysis revealed that saponins of areca nut may be the active component in its antidepressant action. The rats treated sub-chronically with areca nut extract displayed toxic effects, whereas its active aqueous fraction was non-toxic, indicating the presence of different constituents for antidepressant and toxic effects. In the hippocampus of rats, the areca nut extract (50 mg/kg) and aqueous fraction (20 mg/kg) caused a significant elevation of serotonin (around 35%) and noradrenaline (around 30%) compared with the control (261 ± 25 and 512 ± 29 ng/g, respectively). In conclusion, the areca nut possesses potential antidepressant effect via the elevation of serotonin and noradrenaline. Copyright © 2012 John Wiley & Sons, Ltd.

Structural elucidation and gas-phase fragmentation of ten withanolides (steroidal lactones) were studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Withanolides form an important class of plant secondary metabolites, known to possess a variety of biological activities. Withanolides which possess hydroxyl groups at C-4, C-5, C-17, C-20, and C-27, and an epoxy group at C-5/C-6, were evaluated to determine the characteristic fragments and their possible pathways. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M + H]+. Low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis of the protonated molecule [M + H]+ indicated multiple losses of water and the removal of the C-17-substituted lactone moiety affording the [M + H–Lac]+ product ion as the predominant pathways. However, withanolides containing a hydroxyl group at C-24 of the lactone moiety showed a different fragmentation pathway, which include the loss of steroidal part as a neutral molecule, with highly diagnostic ions at m/z 95 and 67 being generated from the cleavage of lactone moiety. Our results also determined the influence of the presence and positions of hydroxyl and epoxy groups on product ion formation and stability. Moreover, the knowledge of the fragmentation pattern was utilized in rapid identification of withanolides by the LC/MS/MS analysis of a Withania somnifera extract. Copyright © 2010 John Wiley & Sons, Ltd.

Several secondary metabolites, artemetin (1), casticin (2), 3,3'-dihydroxy-5,6,7,4'-tetramethoxy flavon (3), penduletin (4), methyl 4-hydroxybenzoate (5), p-hydroxybenzoic acid (6), methyl 3,4-dihydroxybenzoate (7), 5-hydroxy-2-methoxybenzoic acid (8), vanillic acid (9) and 3,4-dihydroxybenzoic acid (10) were isolated from a folkloric medicinal plant, Vitex agnus-castus. The structures of compounds 1-10 were identified with the help of spectroscopic techniques. Compounds 3-10 were isolated for the first time from this plant. These compounds were screened for their antiinflammatory and lipoxygenase inhibitory activities. Compounds 6, 7 and 10 were found to have significant antiinflammatory activity in a cell-based contemporary assay, whereas compounds 1 and 2 exhibited a potent lipoxygenase inhibition.

Casticin (1), a flavonoid isolated from the aerial parts of Vitex agnus-castus, was found to be a potent immunomodulatory and cytotoxic compound. The activity was tested in vitro for chemiluminescence, chemotaxis, T-cell proliferation and cytotoxicity. Casticin (1) exhibited a significant inhibitory effect on monocyte oxidative burst in a dose dependent manner. It was found to have a significant suppressive effect on the chemotaxic action at higher concentrations on fMLP (10−8m) stimulated neutrophils. It also showed a potent suppressive effect on PHA stimulated T-cell (PMBC). Copyright © 2009 John Wiley & Sons, Ltd.

The biotransformation of the synthetic hormone dydrogesterone (1) by a number of fungal strains - including Cephalosporium aphidicola, Rhizopus stolonifer, Cunninghamella elegans, and Fusarium lini - afforded ten different metabolites, compounds 2-11. From a structural point of view, these transformations involved a combination of hydroxylation, oxidation, reduction, and/or epoxidation. The pregnane-based metabolites 10 and 11 are new compounds. All the known compounds were obtained for the first time from 1 by fungal transformation. The metabolites 3, 5, and 8 showed more-potent respiratory-burst inhibitory activity than the substrate 1 in a neutrophil-based cellular assay (Table 3).

This paper attempts to discover the ex post impact of the Chashma Right Bank Canal (CRBC) on the land use and cropping pattern of D.I.Khan district. The CRBC commands 250 000 ha and is spread over two provinces. The CRBC project was completed in three stages. Stage I was commissioned in 1987–88, and stages II and III in 1989 and 2001, respectively. The CRBC brought radical changes in the land use and cropping pattern of D.I.Khan district. For this study, three variables were selected including land use, cropping pattern and land values. To discover these changes at micro-level, five sample villages were randomly selected, four from within the CRBC command area and one outside. The analysis found that after commissioning of the CRBC, prime cultivable land was brought under non-agricultural use. The net sown area was also enhanced. Dry farming crops were replaced by water-intensive cash crops, which in effect caused the twin problem of waterlogging and salinity, particularly in stage I. Likewise, land values increased considerably. This study is a sort of ex post evaluation of the CRBC and provides policy guidelines for decision makers and for the proposed Chashma Right Bank 1st Lift Irrigation Project not to repeat the weaknesses of Chashma Right Bank Irrigation Project. Copyright © 2008 John Wiley & Sons, Ltd.

The current phytochemical investigation on Buxus hyrcana Pojark. has resulted in the isolation of the triterpenoid alkaloids 1-10. The structures of five new alkaloids, hyrcanone (1), hyrcanol (2), hyrcatrienine (3), N(b)-dimethylcycloxobuxoviricine (4), and hyrcamine (5), were elucidated by means of modern spectroscopic techniques, while the known alkaloids, buxidin (6), buxandrine (7), buxabenzacinine (8), buxippine-K (9) and E-buxenone (10), were identified by comparing their spectral data with those reported earlier. Compounds 1 and 3-9 were found to be acetyl- and butyrylcholinesterase inhibitors. The IC50 values were estimated to be in the range of 83.0-468.0 microM against AChE and 1.12-350.0 microM against BChE. The structure-activity relationship studies suggested that the presence of dimethylamino moieties at C(3) and C(20) is the most important factor influencing the activity of these compounds against the cholinesterase enzymes. All compounds were also evaluated for cytotoxicity on a fibroblast cell line with incubation of 24 h. No cytotoxic effects were exerted by any compound.

Three new acylphloroglucinols, myrtucommulone-D (Compound 1), myrtucommulone-E (Compound 2), myrtucommulone-C (Compound 3), and a known acyphloroglucinol myrtucommulone B (Compound 4) were isolated from a methanolic extract of Myrtus communis L. The structures of compounds 1, 2 and 4 were also unambiguously determined by single X-ray diffraction analysis. The compounds 1-4 were found to be more potent α-glucosidase inhibitors than the clinically used standards, acarbose and deoxynojirimycin. The compound 3 exhibited the highest activity among all the acylphloroglucinols, with an IC 50 =35.4±1.15 μM. The compounds 1 and 2 also exhibited strong antibacterial activities.

The transformation of the antibacterial diterpene sclareol (1) by two different fungal strains was investigated (Scheme). In the presence of Rhizopus stolonifer, (3beta)-3-hydroxysclareol (2), 18-hydroxysclareol (3), (6alpha)-6,18-dihydroxysclareol (4), and (11S)-11,18-dihydroxysclareol (5) were formed. Fermentation of 1 with Fusarium lini afforded (1beta)-1-hydroxysclareol (6) and (12S)-12-hydroxysclareol (7). Compounds 4-7 were identified as new compounds, and some of them were active against Bacillus subtilis (Table 3).

Microbial transformation of gelomulide G (3beta,6beta-diacetoxy-8beta,14beta-epoxyabiet-13(15)-en-16,12-olide, 1) was carried out. Incubation of 1 with Aspergillus niger afforded two new metabolites, 3beta,6beta-diacetoxy-8beta,14beta-dihydroxyabiet-13(15)-en-16,12-olide (2) and 3beta,6beta-diacetoxy-14beta-hydroxyabieta-8(9),13(15)-dien-16,12-olide (3). While Cunninghamella elegans afforded the 14-epimer of 2, i.e., 3beta,6beta-diacetoxy-8beta,14alpha-dihydroxyabiet-13(15)-en-16,12-olide (4), along with 3beta-acetoxy-6beta-hydroxy-8beta,14beta-epoxyabiet-13(15)-en-16,12-olide (5). The structures of the transformed products 2-5 were deduced to be new on the basis of MS and NMR data.

Three new physalins (1-3) and a new withanolide 7 have been isolated from the whole plant of Physalis minima, along with three known physalins: physalin H (4), isophysalin B (5), and 5beta,6beta-epoxyphysalin B (6). Their structures were deduced on the basis of in-depth spectroscopic analyses. Compounds 1-6 showed significant in vitro leishmanicidal activities (0.92-19.4 microg/ml) against promastigotes of Leishmania major.

Fungal transformation of (E)-guggulsterone (= (17E)-pregna-4,17-diene-3,16-dione; 1) by Rhizopus stolonifer, Fusarium lini, Cunninghamella elegans, or Gibberella fujikuroi afforded ten hydroxylated metabolites (2-11; Scheme), which were fully characterized. Compounds 4-11 have not been described yet. Some of these novel hydroxylated metabolites, as well as acetylated derivatives thereof, exhibited significant antibacterial and radical-scavenging activities (Table 3).

Bioassay-guided phytochemical investigation of Sarcococca hookeriana has resulted in the isolation and structure elucidation of five new pregnane-type steroidal alkaloids: (−)-hookerianamide A (=(2β,3β,4β,20S)-20-(dimethylamino)-3-[(3-methylbut-2-enoyl)amino]-5α-pregn-16-ene-2,4-diol; 1), (+)-hookerianamide B (=(2α,3β,4β,20S)-4-acetoxy-20-(dimethylamino)-3-[(3-methylbut-2-enoyl)amino]-5α-pregnan-2-ol; 2), (−)-hookerianamide C (=(2β,3β,20S)-2-acetoxy-20-(dimethylamino)-3-[(3-methylbut-2-enoyl)amino]-5α-pregnane; 3), (−)-hookerianamine A (=(3β,20S)-20-(dimethylamino)-3-(methylamino)-5α-pregn-14-ene; 4), and (+)-phulchowkiamide A (=(3β,20S)-20-(methylamino)-3-[(2-methylbut-2-enoyl)amino]-5α-pregn-2-en-4-one; 5). These compounds, as well as the two chemically derived acetyl derivatives 6 and 7, displayed cholinesterase inhibition in a concentration-dependent manner.

Seven new steroidal alkaloids, 2-hydroxysalignarine-E (=(2′E,20S)-20-(dimethylamino)-2β-hydroxy-3β-(tigloylamino)pregn-4-ene; 1), 5,6-dihydrosarconidine (=(20S)-20-(dimethylamino)-3β-(methylamino)-5α-pregn-16-ene; 2), salignamine (=(20S)-20-(methylamino)-3β-methoxypregna-5,16-diene; 3), 2-hydroxysalignamine (=(20S)-20-(dimethylamino)-2β-hydroxy-3β-methoxypregna-5,16-diene; 4), salignarine-F (=(2′E, 20S)-20-(dimethylamino)-4β-hydroxy-3β-(tigloylamino)pregn-5-ene; 5), salonine-C (=(2′E,20S)-20-(dimethylamino)-3β-(tigloylamino)pregna-4,14-diene; 6), and N-[formyl(methyl)amino]salonine-B (=(20S)-20-[formyl(methyl)amino]-3β-methoxypregna-5,16-diene; 7) have been isolated from the MeOH extract of Sarcococca saligna, along with the six known alkaloids dictyophlebine (8), epipachysamine-D (9), saracosine (10), iso-N-formylchonemorphine (11), sarcodinine (12), and alkaloid-C (13). The structures of 1–7 were deduced from spectral data. Compounds 1–13 demonstrated significant activity against acetyl- and butyrylcholinesterase.

Bioassay-guided extraction leads to the isolation of five new alkenyl phenol and salicylic acid derivatives (I) and (II), respectively.

The microbial transformation of (+)-isolongifolen-4-one (4) by a number of fungi by means of a standard two-stage fermentation technique afforded (7R)-12-hydroxyisolongifolen-4-one (5), (7S)-13-hydroxyisolongifolen-4-one (6), (11R)-11-hydroxyisolongifolen-4-one (7), (10R)-10-hydroxyisolongifolen-4-one (8), and (9R)-9-hydroxyisolongifolen-4-one (9) (Scheme). All five metabolites were found to be new, and metabolites 6 and 9 showed potent tyrosinase inhibitory activity (Table 1). The metabolites and their derivatives were characterized on the basis of spectroscopic and single-crystal X-ray-diffraction techniques.

A novel dimeric lignan, bispicropodophyllin glucoside (1) and a highly oxygenated new withanolide, coagulin S (2) were isolated from the ethanolic extract of Withania coagulans. The structures were established on the basis of the spectroscopic data and have been identified as (5S*,5aR*,8aR*,9S*,15S*,15aS*,18aS*,19S*)-9,19-di-β-D-glucopyranosyl-5,8a,9,15,15a,18,18a,19-octahydro-5,15-bis(3,4,5-trimethoxyphenyl)bis([1,3]dioxolo[4′,5′:6,7]naphtho)[2,3-c:2,3-h][1,6]dioxecin-6,16(5aH,8H)-dione (1) and (20S*,22R*)-5α,6β,14α,15α,17β,20,27-heptahydroxy-1-oxowith-24-enolide (2), respectively.

Phytochemical investigation on the whole plant of Salsola foetida resulted in the isolation of three new phenolic compounds 1, 2, and 3, which exhibit tyrosinase inhibition with moderate antioxidant activity. Compounds 1–3 inhibited tyrosinase with IC50 2.61, 1.85, and 0.40 μM, while exhibiting DPPH radical scavenging activity with IC50 383, 427 and 378 μM, respectively. The structures of 1, 2, and 3 were determined by modern spectroscopic techniques.

The effects were investigated of a partially purified subfraction (FS-1) isolated from Delphinium denudatum on sustained repetitive firing (SRF) of cultured neonatal rat hippocampal pyramidal neurons. The blockade of sustained repetitive firing is one of the basic mechanisms of antiepileptic drugs at the cellular level. Using the whole cell current-clamp technique, sustained repetitive firing was elicited in pyramidal neurons under study by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1-0.6 nA current strength. FS-1 (0.01-0.06 mg/mL) reduced the number of action potentials per pulse in a dose-dependent manner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitude and Vmax of action potentials. The Vmax of action potential 1 exhibited a dose-dependent reduction. At a dose of 0.06 mg/mL FS-1 reduced Vmax to 29%-38% and amplitude to 16%-20 % of the control values. The blockade of sustained repetitive firing by FS-1 was reversed by hyperpolarization of the membrane potential (-65 to -75 mV) while depolarization of the membrane potential (-53 mV to -48 mV) potentiated the block. The results suggest that FS-1 blocks sustained repetitive firing in hippocampal neurons in a use-dependent and voltage-dependent manner similar to the prototype anticonvulsant drug, phenytoin. However, unlike phenytoin, which binds preferably to the inactive state, the compounds present in FS-1 also interacted with the resting state of the Na+ channels by reducing Vmax of action potential 1. The results indicate that the partially purified FS-1 subfraction of Delphinium denudatum contains a potent anticonvulsant compound.

isolation, characterization, antiglycation and antioxidant activity of phenolic constituents from organic extracts of Viscum album including the new flavanone glycoside (I) and phenyl propanoid (II)