Diarylheptanoids from Myrica arborea

Investigations of the stem and root bark of Myrica arborea (Myricaceae) have yielded two novel diarylheptanoids, myricarborin and 11-O-beta-D-xylopyranosylmyricanol along with the known myricanol and 5-O-beta-D-glucopyranosylmyricanol. The structures of the novel compounds were determined by spectroscopic methods.     

Cyclic diarylheptanoids from Myrica nana inhibiting nitric oxide release

Investigation of the roots of Myrica nana afforded five new cyclic diarylheptanoids, myricananins A-E (1-5), two new artifacts of myricananins A and B (6-7), and four known compounds, 12-hydroxymyricanone (8), alnusonol (9), myricatomentogenin (10), and actinidione (11). The structures of these new compounds were established by detailed spectroscopic methods. The stereochemistry of compounds 1 and 2 were determined by single-crystal X-ray diffraction. In exception of compounds 2, 6 and 10, all the other compounds were examined for their inhibitory effects on nitric oxide production in lipopolysaccharides-activated macrophages. Compounds 1, 3, 7, 8 and 9 inhibited the release of nitric oxide with IC(50) values of 45.32, 63.51, 52.81, 30.19 and 46.18muM, respectively. Furthermore, compound 1 was found to inhibit the expression of inducible nitric oxide synthase.     

Chemopreventive potential of cyclic diarylheptanoids

Eleven cyclic diarylheptanoids and seven related compounds were screened as potential antitumor promoters by using the in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) activation assay. In addition, the cyclic diarylheptanoid myricanone (2) was examined for antitumor initiating activity in a two-stage carcinogenesis assay of mouse skin tumors induced by peroxynitrite as an initiator and TPA as a promoter. Myricanone (2) exhibited significant antitumor-initiating effect on mouse skin. These data suggest that cyclic, as well as linear, diarylheptanoids might be valuable chemopreventors.     

Absolute stereostructures of polypodane- and octanordammarane-type triterpenes with nitric oxide production inhibitory activity from guggul-gum resins

The methanolic extract from guggul-gum resin, the resin of Balsamodendron mukul, was found to inhibit nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages (IC(50) = 13 microg/mL). From the methanolic extract, three new polypodane-type triterpenes, myrrhanol B and myrrhanones B and A acetate, and a new octanordammarane-type triterpene, epimansumbinol, were isolated together with 17 known compounds including progesterone and the related steroids. The absolute stereostructures of new triterpenes were elucidated on the basis of chemical and physicochemical evidence. The several constituents showed inhibitory effects on nitric oxide production and induction of inducible nitric oxide synthase.     

Inhibitors from the rhizomes of Alpinia officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarylheptanoids for the activity

The 80% aqueous acetone extract from the rhizomes of Alpinia officinarum, a Chinese medicinal herb, were found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Through bioassay-guided separation, two diarylheptanoids [7-(4'-hydroxy-3'-methoxyphenyl-1-phenylhept-4-en-3-one and 3,5-dihydroxy-1,7-diphenylheptane] and a flavonol constituent (galangin) substantially inhibited LPS-induced NO production with IC50 values of 33-62 microM. To clarify structure-activity relationships of diarylheptanoids, related diarylheptanoids from Curcuma zedoaria were examined. Results indicate that the double bond or enone moiety at the 1-7 positions is important for the activity.     

Chemical constituents isolated from Polygala japonica leaves and their inhibitory effect on nitric oxide production in vitro

A methanolic extract of dried leaves of Polygala japonica Houtt (Polygalaceae) significantly attenuated nitric oxide production in lipopolysaccharide-simulated BV2 microglia. Five anthraquinones chrysophanol (1), emodin (2), aloe-emodin (3), emodin 8-O-beta-D-glucopyranoside (4) and trihydroxy anthraquinone (5), and four flavonoids kaempferol (6), chrysoeriol (7), kaempferol 3-gentiobioside (8) and isorhamnetin (9) were isolated from the methanolic extract using bioactivity-guided fractionation. Among them, compounds 1-4, 6 and 7 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglia at the concentrations ranging from 1.0 to 100.0 microM.     

Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1

The objective of the present study was to investigate the effect of leptin, alone or in combination with IL-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and IL-1. For this purpose, nitric oxide production was evaluated using the Griess colorimetric reaction in culture medium of cells stimulated over 48 hours with leptin (800 nmol/l) and IL-1 (0.025 ng/ml), alone or combined. Specific pharmacological inhibitors of NOS type II (aminoguanidine [1 mmol/l]), janus kinase (JAK)2 (tyrphostin AG490 and Tkip), phosphatidylinositol 3-kinase (PI3K; wortmannin [1, 2.5, 5 and 10 μmol/l] and LY294002 [1, 2.5, 5 and 10 μmol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, 20 and 30 μmol/l]) and p38 kinase (SB203580 [1, 5, 10, 20 and 30 μmol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by real-time PCR and NOS II protein expression was analyzed by western blot. Our results indicate that stimulation of chondrocytes with IL-1 results in dose-dependent nitric oxide production. In contrast, leptin alone was unable to induce nitric oxide production or expression of NOS type II mRNA or its protein. However, co-stimulation with leptin and IL-1 resulted in a net increase in nitric oxide concentration over IL-1 challenge that was eliminated by pretreatment with the NOS II specific inhibitor aminoguanidine. Pretreatment with tyrphostin AG490 and Tkip (a SOCS-1 mimetic peptide that inhibits JAK2) blocked nitric oxide production induced by leptin/IL-1. Finally, wortmannin, LY294002, PD098059 and SB203580 significantly decreased nitric oxide production. These findings were confirmed in mature and hypertrophic ATDC5 chondrocytes, and in human primary chondrocytes. This study indicates that leptin plays a proinflammatory role, in synergy with IL-1, by inducing NOS type II through a signalling pathway that involves JAK2, PI3K, MEK-1 and p38 kinase.     

Perivascular nerve fibres and endothelial cells of the rat basilar artery: immuno-gold labelling of antigenic sites for type I and type III nitric oxide synthase

Ultrastructural localisation of type I (neuronal) and type III (endothelial) isoforms of nitric oxide synthase in perivascular nerve fibres (axons) and endothelial cells was studied in the Wistar rat cerebral basilar artery, using monoclonal antibodies either to type I or type III nitric oxide synthase and post-embedding colloidal-gold immunocytochemistry. Labelling signal (gold particles) for type I and type III nitric oxide synthase was localised both in axons and endothelial cells. In the axon profiles, labelling for either type I or type III nitric oxide synthase was localised in the axoplasm and the lumen and/or membrane of small agranular synaptic vesicles. In the endothelial cells, labelling for either type-I or type-III nitric oxide synthase was predominantly in the cytoplasm. The present qualitative data extends our previous study of cerebrovascular nerve fibres and endothelial cells employing monoclonal antibodies; the localisation of nitric oxide synthase in a subpopulation of synaptic vesicles in nitric oxide synthase-positive cerebrovascular nerves suggests that vesicular mechanisms may be involved in the production/release of nitric oxide. © 1998 Chapman and Hall     

Angiotensin II stimulates nitric oxide production in pulmonary artery endothelium via the type 2 receptor

We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-dependent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone.     

Diarylheptanoids from Curcuma kwangsiensis and their inhibitory activity on nitric oxide production in lipopolysaccharide-activated macrophages

Eleven diarylheptanoids (1-11) were isolated from rhizomes of Curcuma kwangsiensis, together with seven known compounds. Their structures were elucidated by 1D and 2D NMR, circular dichroism (CD), and accurate mass measurements. Inhibitory effects of the isolated compounds on nitric oxide production in lipopolysaccaride-activated macrophages were evaluated. Compounds 1, 2, and 3 showed strong inhibitory activity on NO production with IC(50) values of 3.13, 2.81 and 2.41 μM, respectively.     

A new triterpene saponin from Chenopodium ficifolium

The new triterpene saponin 3-O-beta-D-glucopyranoside, 28-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosiduronic acid oleanolate was isolated from the roots of Chenopodium ficifolium. The known compounds stigmasterol-3-O-glucoside and 3-O-beta-D-glucopyranosiduronic acid, 28-beta-D-glucopyranosyl oleanolate were also isolated. The latter compound, oleanolic acid, beta-sitosterol and its glucoside were isolated from the aerial parts. The identity of these compounds was verified through different chemical and physico-chemical evidences including different 1D and 2D NMR experiments.     

Triterpenoid saponins and sapogenin lactones from Albizia gummifera

The structures of two new monodesmosidic and bisdesmosidic triterpenoid saponins (1 and 2) and the known compound delta 5-stigmasterol-3-O-beta-D-glucopyranoside (3) as well as two new oleanane type triterpene lactone glycosides 4, 5 and a new sapogenin lactone 6 isolated from the stem bark of Albizia gummifera C.A. Smith (Mimosaceae) have been elucidated as 3-O-?beta-D-glucopyranosyl(1-->2)-[alpha-L-arabinopyranosyl(1-->6) ]-beta-D- glucopyranosyl?-oleanolic acid (1), beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl 3-O-?beta-D-glucopyra-nosyl(1-->2)-[alpha-L-arabinopyrano syl(1-->6)]-beta-D- glucopyranosyl?-oleanolate (2), 3 beta-?O-D-glucopyranosyl-(1-->2)-[O-alpha-L-arabinopyranosyl(1-->6 )] beta-D-glucopyranosyloxy?-machaerinic acid gamma-lactone (4), 3 beta-O-beta-D-glucopyranosiduronic acid (1-->2)-beta-D-glucopyranosyloxy]-machaerinic acid gamma-lactone (5), and A-homo-3a-oxa-5 beta-olean-12-en-3-one-28-oic acid (6), respectively. The complete assignment of the 1H and 13C resonances of 1, 2, 4 and 6 and of the peracetate of 5 were achieved by means of 2D-NMR studies.     

Triterpenes from Mucuna birdwoodiana.

Methanolic extracts of the stalks of Mucuna birdwoodiana on acid hydrolysis and subsequent methylation with diazomethane provided four triterpene sapogenols. On the other hand, investigation of glycosides after methylation of the same extract led to the isolation of four triterpene glycosides. On the basis of chemical and spectral evidence, their structures were characterized as methyl asiatate, methyl maslinate, two new sapogenols, methyl 1 beta,2 alpha,3 beta,23-tetrahydroxyolean-12-en 28-oate (mucunagenin a), its urs-12-en isomer (mucunagenin b), 3-O-(6-O-methyl-beta-D-glucuronopyranosyl) methyl asiatate 3-O-[alpha-L-arabinopyranosyl(1----2)]-6-O-methyl-beta-D-glucur onopyranosyl methyl maslinate, 3-O-[alpha-L-arabinopyranosyl(1----2)]-6-O-methyl-beta-D-glucur onopyranosyl methyl asiatate and 3-O-(6-O-methyl-beta-D-glucuronopyranosyl) asiatic acid 28-O-beta-D-glucopyranoside.     

Triterpene saponins and iridoid glucosides from galium rivale

Three new glycosides of the oleanene-type triterpenes, rivalosides C-E (1-3), along with three known triterpene saponins, momordin IIb (4) and rivalosides A-B (5-6), and five known iridoid glucosides: monotropein, scandoside, deacetylasperulosidic acid, geniposidic acid and asperulosidic acid, were isolated from aerial parts of Galium rivale. The structures of the new compounds were elucidated by spectral methods and chemical means as 2alpha-acetoxy-3alpha, 19alpha-dihydroxy-olean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl-(1--> 6)-beta-D-glucopyranoside, 2alpha,3alpha, 19alpha-trihydroxy-olean- 12-en-28-oic acid 28-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside and 3-O-beta-D-glucuronopyranosyl-24-hydroxy-olean-12-en-28-oic acid 28-O-beta-D-glucopyranoside, for rivalosides C-E, respectively. The taxonomic significance of the rivalosides in G. rivale was discussed.     

Classical and slow-binding inhibitors of human type II arginase

Arginases catalyze the hydrolysis of L-arginine to yield L-ornithine and urea. Recent studies indicate that arginases, both the type I and type II isozymes, participate in the regulation of nitric oxide production by modulating the availability of arginine for nitric oxide synthase. Due to the reciprocal regulation between arginase and nitric oxide synthase, arginase inhibitors have therapeutic potential in treating nitric oxide-dependent smooth muscle disorders, such as erectile dysfunction. We demonstrate the competitive inhibition of the mitochondrial human type II arginase by N(omega)-hydroxy-L-arginine, the intermediate in the reaction catalyzed by nitric oxide synthase, and its analogue N(omega)-hydroxy-nor-L-arginine, with K(i) values of 1.6 microM and 51 nM at pH 7.5, respectively. We also demonstrate the inhibition of human type II arginase by the boronic acid-based transition-state analogues 2(S)-amino-6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-L-cysteine (BEC), which are known inhibitors of type I arginase. At pH 7.5, both ABH and BEC are classical, competitive inhibitors of human type II arginase with K(i) values of 0.25 and 0.31 microM, respectively. However, at pH 9.5, ABH and BEC are slow-binding inhibitors of the enzyme with K(i) values of 8.5 and 30 nM, respectively. The findings presented here indicate that the design of arginine analogues with uncharged, tetrahedral functional groups will lead to the development of more potent inhibitors of arginases at physiological pH.     

Impaired nitric oxide synthase-dependent dilatation of cerebral arterioles in type II diabetic rats

The goals of this study were to determine the effects of type II diabetes mellitus on nitric oxide synthase-dependent responses of cerebral arterioles and on endothelial nitric oxide synthase (eNOS) protein in cerebral arterioles. We examined dilatation of cerebral (pial) arterioles in 13-15 week old male lean and diabetic obese Zucker rats in response to nitric oxide synthase-dependent agonists (acetylcholine and adenosine diphosphate (ADP)) and a nitric oxide synthase-independent agonist (nitroglycerin). We found that acetylcholine (10 microM) increased cerebral arteriolar diameter by 10 +/- 3% (mean +/- SE) in lean Zucker rats, but by only 2 +/- 2% in diabetic obese Zucker rats (p<0.05). In addition, ADP (100 microM) increased cerebral arteriolar diameter by 20 +/- 2% in lean Zucker rats, but by only 8 +/- 2% in diabetic obese Zucker rats (p<0.05). In contrast, nitroglycerin produced similar vasodilatation in lean and diabetic obese Zucker rats. Thus, impaired dilatation of cerebral arterioles in diabetic obese Zucker rats is not related to non-specific impairment of vasodilatation. Following these functional studies, we harvested cerebral microvessels for Western blot analysis of eNOS protein. We found that eNOS protein was significantly higher in diabetic obese Zucker rats than in lean Zucker rats (p<0.05). Thus, type II diabetes mellitus impairs nitric oxide synthase-dependent responses of cerebral arterioles. In addition, eNOS protein from cerebral blood vessels is increased in diabetic obese Zucker rats.     

The nitric oxide pathway provides innate antiviral protection in conjunction with the type I interferon pathway in fibroblasts

The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.     

Sesquiterpenoids and diarylheptanoids from Nidus vespae and their inhibitory effects on nitric oxide production

Two coriamyrtin-type sesquiterpenes, fengfangin A (1) and tutin (2), and six diarylheptanoids, namely alnusone (3), centrolobol (4), muricarpone B (5), 1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-one (6), (3S)-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol (7), and (3S)-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)heptan-3-ol (8), were isolated from the 95% EtOH extract of nidus vespae, the nest of Polistes species. Their structures were identified by spectroscopic methods. Compounds 1 and 8 are new products. The absolute configuration of 1 was determined by single-crystal X-ray diffraction analysis using Flack parameter. The biological tests showed that compounds 5, 6, and 8 could inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells with IC(50) values in the range of 13-17 μM, whereas the sesquiterpenes were inactive in this assay (>25 μM). In addition, the ecological significance of the presence of neurotoxic sesquiterpene lactones in nidus vespae is briefly discussed.     

Activation of type II alveolar epithelial cells during acute endotoxemia

Lung injury induced by acute endotoxemia is associated with increased generation of inflammatory mediators such as nitric oxide and eicosanoids, which have been implicated in the pathophysiological process. Although production of these mediators by alveolar macrophages (AM) has been characterized, the response of type II cells is unknown and was assessed in the present studies. Acute endotoxemia caused a rapid (within 1 h) and prolonged (up to 48 h) induction of nitric oxide synthase-2 (NOS-2) in type II cells but a delayed response in AM (12-24 h). In both cell types, this was associated with increased nitric oxide production. Although type II cells, and to a lesser extent AM, constitutively expressed cyclooxygenase-2, acute endotoxemia did not alter this activity. Endotoxin administration had no effect on mitogen-activated protein kinase or protein kinase B-alpha (PKB-alpha) expression. However, increases in phosphoinositide 3-kinase and phospho-PKB-alpha were observed in type II cells. The finding that this was delayed for 12-24 h suggests that these proteins do not play a significant role in the regulation of NOS-2 in this model. After endotoxin administration to rats, a rapid (within 1-2 h) activation of nuclear factor-kappaB was observed. This response was transient in type II cells but was sustained in AM. Interferon regulatory factor-1 (IRF-1) was also activated rapidly in type II cells. In contrast, IRF-1 activation was delayed in AM. These data demonstrate that type II cells, like AM, are highly responsive during acute endotoxemia and may contribute to pulmonary inflammation.     

Diarylheptanoids from the fruits of Amomum kravanh and their inhibitory activities of nitric oxide production

Two new diarylheptanoids with a tetrahydropyran ring, kravanhol A (1) and kravanhol B (2), along with one known diarylheptanoid renealtin A (3) were isolated from the fruits of Amomum kravanh. The structures of compounds 1 and 2 were established by analysis of spectroscopic data and their absolute configurations were determined by Mosher's method and CD experiments. Compound 2 showed inhibitory effect on nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages with an IC₅₀ value of 38.9 ± 1.8 μM.