Four new alkaloids from the roots of Dactylicapnos scandens.

Four new alkaloids (1 - 4), together with five known ones (5 - 9), were isolated from the bulbs of Dactylicapnos scandens. The structures were determined by analysis of their spectroscopic data and quantum-chemical calculations. All the isolates were tested for their ability to modulate neuronal Ca2+ mobilization in primary cultured neocortical neurons. Compound 8 inhibited spontaneous Ca2+ oscillations at low micromolar concentrations.

A Drimane Meroterpenoid Borate as a Synchronous Ca+ Oscillation Inhibitor from the Coral-Associated Fungus Alternaria sp. ZH-15.

Territrem F (1), a drimane meroterpenoid bearing a unique borate ring system, was isolated together with its diol precursor territrem B (2) from the fungus Alternaria sp. ZH-15 associated with the soft coral Lobophytum crassum collected in the South China Sea. The structure of the new compound was elucidated by spectroscopic analysis and an X-ray single-crystal diffraction study, representing a new type of boron-containing natural product. Both compounds significantly inhibited spontaneous synchronous Ca2+ oscillations (SCOs) and epileptic discharges induced by 4-aminopyridine, showing the potential for antiepileptic drug research. The 5,9-boronic ester derivative of 2 did not change its SCO inhibitory activity.

Cadinane Sesquiterpenoids and Their Glycosides from Alangium chinense That Inhibit Spontaneous Calcium Oscillations.

Nine new cadinane sesquiterpenoids, alanenses A-I (1-9), were isolated from the leaves of Alangium chinense together with three previously reported analogues (10-12). The structures of these molecules were elucidated by interpretation of spectroscopic and spectrometric data. Absolute configurations were established by the comparison of experimental and calculated ECD data, chemical degradation studies for sugar moieties, and a single-crystal X-ray diffraction analysis. Compounds 1 and 2 were isolated as racemates, and enantiopurification was achieved by chiral HPLC. Compounds 3-5 are glycosylated cadinanes bearing a ß-d-glucose unit, while compounds 6-9 incorporate a hydroxymethyl group in either the free form or additional ring fusion. The structure of compound 11 was originally misassigned and later revised using additional NMR data. The corrected structure is here supported by X-ray single-crystal analysis. Compounds 1 and 2 inhibit spontaneous calcium channel oscillations at low micromolar concentrations.

Polysubstituted Cyclopentene Benzamides and Dianthramide Alkaloids from Delphinium anthriscifolium Hance.

Thirteen new benzamide alkaloids, delphiniumines A-M (1-13), together with one known analogue (14), were isolated from Delphinium anthriscifolium Hance. All of the structures were determined by spectroscopic and spectrometric analyses. Absolute configuration for 1 was established using experimental and calculated ECD data, as well as by X-ray crystallography analysis. Compound 1 possesses a previously undescribed polysubstituted cyclopentene carbon framework. Compound 2 was isolated as an artifact from 1 during the extraction process. Compound 7 is glycosylated with a ß-D-glucose unit. Compound 13 bears a chlorine substituent. At a concentration of 10 µM, compounds 6, 8, and 10-12 suppressed LPS-induced NO production in RAW264.7 cells with inhibition rates ranging from 40.3% to 78.8%.

Neuronal Modulators from the Coral-Associated Fungi Aspergillus candidus.

Three new p-terphenyl derivatives, named 4″-O-methyl-prenylterphenyllin B (1) and phenylcandilide A and B (17 and 18), and three new indole-diterpene alkaloids, asperindoles E-G (22-24), were isolated together with eighteen known analogues from the fungi Aspergillus candidus associated with the South China Sea gorgonian Junceela fragillis. The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic analysis, and DFT/NMR and TDDFT/ECD calculations. In a primary cultured cortical neuronal network, the compounds 6, 9, 14, 17, 18 and 24 modulated spontaneous Ca2+ oscillations and 4-aminopyridine hyperexcited neuronal activity. A preliminary structure-activity relationship was discussed.

Alkaloids from Corydalis decumbens modulate neuronal excitability.

Eight new alkaloids, including five isoquinoline alkaloids, a benzoazepine alkaloid, two isoindole alkaloids, and three synthetic alkaloids firstly obtained from the natural sources, together with three known ones were isolated from the bulbs of Corydalis decumbens. The structures were determined by analysis of their spectroscopic data and single-crystal X-ray diffraction. This is the first report of isoindole alkaloid and benzoazepine alkaloid from the genus Corydalis. Full NMR data for 9-11 are reported here for the first time. Moreover, the ability to modulate neuronal Ca2+ mobilization of the isolated alkaloids was tested in primary cultured neocortical neurons. Compound 7 inhibited spontaneous Ca2+ oscillations in primary neocortical neuron cultures at low micromolar concentrations.

Phthalideisoquinoline Hemiacetal Alkaloids from Corydalis decumbens That Inhibit Spontaneous Calcium Oscillations, Including Alkyl Derivatives of (+)-Egenine That Are Strikingly Levorotatory.

The new phthalideisoquinoline hemiacetal alkaloids (2-7) and the known analogues (1 and 8) were isolated from the bulbs of Corydalis decumbens. The new compounds were characterized by analysis of their NMR spectroscopic data, chemical degradation syntheses, X-ray crystallography, and comparison of experimental and calculated ECD data. All the isolates were screened in vitro for inhibitory activity of spontaneous calcium oscillations in primary cultured neocortical neurons. Compounds 1-3 and 5-7 were found to be active in the suppression of spontaneous calcium oscillations with IC50 values of 6.8, 5.6, 11.6, 10.2, 8.3, and 3.1 µM, respectively. It was also observed that the presence of hydroxy, methoxy, and ethoxy groups at the remote stereogenic center C-7' of some isolated phthalideisoquinoline hemiacetal alkaloids could alter the preferred conformation and invert the sign of optical rotation, rather than this resulting from configurational isomerism at C-1 or C-9, and that the 3J1,9 coupling constants of these analogues varied accordingly. For example, compounds 1 and 6 are levorotatory, despite these molecules having the same carbon skeleton and absolute configuration as (+)-egenine. This emphasizes the potential risk of incorrectly assigning absolute configuration based only on observed coupling constants or optical rotation when comparing the data of new compounds with literature values for known analogues, especially within this class of molecules.

Polycycloiridals with a Cyclopentane Ring from Iris tectorum.

Six new iridal-type triterpenoids containing an unprecedented cyclopentane ring, polycycloiridals E-J (1-6), were isolated from a large-scale re-extraction of Iris tectorum. A possible biosynthesis pathway is postulated. The known spirioiridotectal D (7) was also obtained in the current investigation, and its structure was unequivocally defined using X-ray diffraction data. Compound 7 suppressed LPS-activated NO production in the BV2 cell line with an IC50 value of 0.54 µM.

A unique homodimeric NAD⁺-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri.

In eukaryotes, NAD(+)-dependent isocitrate dehydrogenase (IDH) is strictly mitochondrial and is a key enzyme in the Krebs cycle. To date, all known NAD(+)-specific IDHs (NAD-IDHs) in the mitochondria are believed to be heteromeric in solution. Here, a unique homodimeric NAD-IDH from Ostreococcus tauri (OtIDH), the smallest autotrophic picoeukaryote, was unveiled. Active OtIDH has a molecular weight of ∼93 kDa with each subunit of 46.7 kDa. In the presence of Mn(2+) and Mg(2+), OtIDH displayed 42-fold and 51-fold preference for NAD(+) over NADP(+), respectively. Interestingly, OtIDH exhibited a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric homologs, and a remarkably high affinity for isocitrate (S0.5 < 10 µM) unlike other hetero-oligomeric homologs. Furthermore, its coenzyme specificity can be completely converted from NAD(+) (ancient trait) to NADP(+) (adaptive trait) by rational mutagenesis based on the evolutionary trace. Mutants D344R and D344R/M345H displayed a 15-fold and 72-fold preference for NADP(+) over NAD(+), respectively, indicating that D344 and M345 are the determinants of NAD(+) specificity. These findings also suggest that OtIDH may be an ancestral form of type II IDHs (all reported members are NADP(+)-linked enzymes) and may have evolved into NADP(+)-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation.

Hypeisoxazole A, a Racemic Pair of Tetrahydroisoxazole-Fused Benzylisoquinoline Alkaloids from Hypecoum erectum and Structural Revision of Hypecoleptopine.

(±)-Hypeisoxazole A (1), a racemic pair of rearranged benzylisoquinoline alkaloids possessing an unprecedented diindeno[2,1-c:2',1'-d] isoxazole scaffold, was isolated from the medicinal herb Hypecoum erectum, along with hypecoleptopine (2), whose structure is now revised as a novel spiro-benzylisoquinoline alkaloid with a 6/6/5/6/6 skeleton. Their structures were determined by comprehensive spectroscopic and spectrometric analyses, X-ray diffraction, and computational studies. Racemic mixture of 2 and its pure enantiomers modulated neuronal excitability activity.

Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680.

A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP+ as electron acceptor. Apparent Km values were 4.98 µM for NADP+ and 6,620 µM for NAD+, respectively, using Mn2+ as divalent cation. The enzyme performed a 33,000-fold greater specificity (kcat/Km) for NADP+ than NAD+. Moreover, SaIDH activity was entirely dependent on the presence of Mn2+ or Mg2+, but was strongly inhibited by Ca2+ and Zn2+. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP+.

New phenylpropanoid-substituted and benzyl-substituted flavonols from Alangium chinense.

Two previously undescribed flavonols with phenylpropanoid or benzyl substitution, named alangsine A (1), and alangsine B (2), together with four known compounds (3-6) were isolated from the leaves of Alangium chinense. Alangsine A was a racemic mixture, which was further separated into two enantiomers via high-performance liquid chromatography on a chiral column. The absolute configurations of the enantiomer pairs were deduced from the circular dichroism (CD) spectra. The activity of the isolated compounds towards neuronal excitability was examined.

Identification and biochemical characterization of a thermostable malate dehydrogenase from the mesophile Streptomyces coelicolor A3(2).

We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD(+)-specific and displayed about 400-fold (k(cat)) and 1,050-fold (k(cat)/K(m)) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (K(i)=5.8 mM) and excess L-malate (K(i)=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe(2+) and Zn(2+). Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.

Alternarin A, a Drimane Meroterpenoid, Suppresses Neuronal Excitability from the Coral-Associated Fungi Alternaria sp. ZH-15.

Alternarin A (1), a rearranged drimane meroterpenoid characterized by a thioglycerate moiety, was isolated together with two known analogues from the coral-associated fungi Alternaria sp. ZH-15. Its structure was determined based on spectroscopic analysis, modified Mosher's method, and TDDFT/ECD calculations. In a primary cultured cortical neuronal network, compound 1 effectively inhibited the activity of spontaneous synchronous Ca2+ oscillations and 4-aminopyridine induced epileptic discharges in the low micromolar concentration range.

New phthalideisoquinoline hemiacetal alkaloid derivatives from Corydalis decumbens.

Two new phthalideisoquinoline hemiacetal alkaloid derivatives, named corybensines A and B (1 and 2), and four known alkaloids (3-6) were isolated from the bulbs of Corydalis decumbens. Their structures were characterized by analysis of 1D/2D NMR and ECD data, quantum chemical ECD calculations, and X-ray diffraction analysis. Among them, compound 2 represents the first naturally occurring phthalideisoquinoline hemiacetal alkaloid derivative with a 2-pyrrolidinone moiety. The activity of the isolated compounds towards neuronal excitability was examined.

Huangkui Capsule Ameliorates Renal Fibrosis in a Unilateral Ureteral Obstruction Mouse Model Through TRPC6 Dependent Signaling Pathways.

Renal fibrosis is the final common pathological manifestation of almost all progressive chronic kidney diseases (CKD). Transient receptor potential canonical (TRPC) channels, especially TRPC3/6, were proposed to be essential therapeutic targets for kidney injury. Huangkui capsule (HKC), an important adjuvant therapy for CKD, showed superior efficacy for CKD at stages 1-2 in clinical practice. However, its anti-fibrotic effect and the underlying mechanisms remain to be investigated. In the present study, we evaluated the efficacy of HKC on renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and explored the potential underlying mechanism. Administration of HKC by intragastric gavage dose-dependently suppressed UUO-induced kidney injury and tubulointerstitial fibrosis. Similarly, HKC suppressed the expression level of α-smooth muscle actin (α-SMA), increased the expression of E-cadherin, and suppressed the mRNA expression of a plethora of proinflammatory mediators that are necessary for the progression of renal fibrosis. Mechanistically, HKC suppressed both canonical and non-canonical TGF-ß signaling pathways in UUO mice as well as the TRPC6/calcineurin A (CnA)/nuclear factor of activated T cells (NFAT) signaling axis. In addition, TRPC6 knockout mice and HKC treated wild type mice displayed comparable protection on UUO-triggered kidney tubulointerstitial injury, interstitial fibrosis, and α-SMA expression. More importantly, HKC had no additional protective effect on UUO-triggered kidney tubulointerstitial injury and interstitial fibrosis in TRPC6 knockout mouse. Further investigation demonstrated that HKC could directly suppress TRPC3/6 channel activities. Considered together, these data demonstrated that the protective effect of HKC on renal injury and interstitial fibrosis is dependent on TRPC6, possibly through direct inhibition of TRPC6 channel activity and indirect suppression of TRPC6 expression.

Chemical and biological study of aplysiatoxin derivatives showing inhibition of potassium channel Kv1.5.

Three new aplysiatoxins, neo-debromoaplysiatoxin D (1), oscillatoxin E (2) and oscillatoxin F (3), accompanied by four known analogues (4-7), were identified from the marine cyanobacterium Lyngbya sp. Structural frames differ amongst these metabolites, and therefore we classified compounds 1 and 4-6 as aplysiatoxins as they possess 6/12/6 and 6/10/6 tricyclic ring systems featuring a macrolactone ring, and compounds 2, 3 and 7 as oscillatoxins that feature a hexane-tetrahydropyran in a spirobicyclic system. Bioactivity experiments showed that compounds 1 and 4-6 presented significant expression of phosphor-PKCδ whereas compounds 2, 5 and 7 showed the most potent blocking activity against potassium channel Kv1.5 with IC50 values of 0.79 ± 0.032 µM, 1.28 ± 0.080 µM and 1.47 ± 0.138 µM, respectively. Molecular docking analysis supplementing the binding interaction of oscillatoxin E (2) and oscillatoxin F (3) with Kv1.5 showed oscillatoxin E (2) with a strong binding affinity of -37.645 kcal mol-1 and oscillatoxin F (3) with a weaker affinity of -32.217 kcal mol-1, further supporting the experimental data.

Rearranged iridal-type triterpenoids from Iris tectorum.

Three new iridal-type triterpenoids (1-3) featuring a rearranged homofarnesylside chain were isolated from the rhizomes of Iris tectorum. Compounds 2 and 3 were found to be a pair of epimers. Their structures were elucidated on the basis of comprehensive spectroscopic analysis. A possible biosynthetic pathway for them was postulated. Moreover, the mixture of compounds 2 and 3 exhibited moderate neuroprotective activity against serum deprivation-induced PC12 cell death.

Blockade of neuronal dopamine D2 receptor attenuates morphine tolerance in mice spinal cord.

Tolerance induced by morphine remains a major unresolved problem and significantly limits its clinical use. Recent evidences have indicated that dopamine D2 receptor (D2DR) is likely to be involved in morphine-induced antinociceptive tolerance. However, its exact effect and molecular mechanism remain unknown. In this study we examined the effect of D2DR on morphine antinociceptive tolerance in mice spinal cord. Chronic morphine treatment significantly increased levels of D2DR in mice spinal dorsal horn. And the immunoreactivity of D2DR was newly expressed in neurons rather than astrocytes or microglia both in vivo and in vitro. Blockade of D2DR with its antagonist (sulpiride and L-741,626, i.t.) attenuated morphine antinociceptive tolerance without affecting basal pain perception. Sulpiride (i.t.) also down-regulated the expression of phosphorylation of NR1, PKC, MAPKs and suppressed the activation of astrocytes and microglia induced by chronic morphine administration. Particularly, D2DR was found to interact with µ opioid receptor (MOR) in neurons, and chronic morphine treatment enhanced the MOR/D2DR interactions. Sulpiride (i.t.) could disrupt the MOR/D2DR interactions and attenuate morphine tolerance, indicating that neuronal D2DR in the spinal cord may be involved in morphine tolerance possibly by interacting with MOR. These results may present new opportunities for the treatment and management of morphine-induced antinociceptive tolerance which often observed in clinic.

Ge-Gen Decoction attenuates oxytocin-induced uterine contraction and writhing response: potential application in primary dysmenorrhea therapy.

The uterine tetanic contraction and uterine artery blood flow reduction are possible reasons for primary dysmenorrhea (PD). In the present study, we aimed to evaluate the uterine relaxant effect and the influence on uterine artery blood velocity of Ge-Gen Decoction (GGD), a well-known Chinese herbal formula. In female ICR mice, uterine contraction was induced by oxytocin exposure following estradiol benzoate pretreatment, and the uterine artery blood velocity was detected by Doppler ultrasound. Histopathological examination of the uterine tissue samples were performed by H&E staining. Ex vivo studies demonstrated that oxytocin, posterior pituitary, or acetylcholine induced contractions in isolated mouse uterus. GGD inhibited both spontaneous and stimulated contractions. In vivo study demonstrated that GGD significantly reduced oxytocin-induced writhing responses with a maximal inhibition of 87%. Further study demonstrated that GGD normalized oxytocin-induced abnormalities of prostaglandins F2 alpha (PGF2α) and Ca(2+) in mice. In addition, injection of oxytocin induced a decrease in uterine artery blood flow velocity. Pretreatment with GGD reversed the oxytocin response on blood flow velocity. Histopathological examination showed pretreatment with GGD alleviated inflammation and edema in the uterus when compared with the model group. Both ex vivo and in vivo results indicated that GGD possessed a significant spasmolytic effect on uterine tetanic contraction as well as improvement on uterine artery blood velocity which may involve PGF2α and Ca(2+) signaling, suggesting that GGD may have a clinic potential in PD therapy.