Synthesis and biological evaluation of Isosteviol derivatives as FXa inhibitors.

Firstly, a series of Isosteviol derivatives were synthesized and evaluated for FXa inhibitory activity. Among these compounds, the inhibitory activity of compounds 22, 35 and 38 on FXa was better than that of Isosteviol. Secondly, surface plasmon resonance (SPR) assays were performed for selected compounds. Compounds 22, 35, 38 have similar kinetic signatures, and affinity values were at µM level. Thirdly, compounds 22 and 35 displayed moderate-to-high anticoagulation activity and showed similar sensitivity to PT and aPTT. These findings will provide new insight into the exploration of FXa inhibition.

Synthesis and biological evaluation of Vinpocetine derivatives.

A new series of Vinpocetine derivatives were synthesized and evaluated for their inhibitory activity on PDE1A in vitro. Seven compounds with higher inhibitory activity were selected for surface plasmon resonance (SPR) binding experiments. Compared with Vinpocetine, these high potency compounds presented a higher binding affinity with PDE1A, which was consistent with inhibitory activity. After further screening, compounds 5, 7, 21, 34 and Vinpocetine were selected to examine the vasorelaxant effects on endothelium-intact rat thoracic aortic rings. The study suggested that the effects of compounds 7 and 21 were the most significant with the maximum value of 93.46 ±â€¯0.77% and 92.90 ±â€¯0.78% (n = 5) at a concentration of 100 µM respectively. Based on these studies, compounds 7 and 21 were considered for further development as hit compounds.

The synthesis and anti-metastatic effects of optical isomers of ionone alkaloid 9-(N,N-dimethyl)-4,7-megastigmedien-3-one.

Ionone alkaloid 9-(N,N-dimethyl)-4,7-megastigmedien-3-one (compound 1) is a new anti-metastatic natural product. However, it was previously reported as optical isomers mixture. Herein, the optical isomers (6a-6d) of compound 1 were synthesized. The absolute configurations of 6a-6d were determined by ECD experiments and calculated spectra with time-dependent density functional theory (TDDFT). The anti-metastatic effects of the optical isomers were examined by transwell assay. These results revealed that compound 6a had potential anti-metastatic activity with an IC50 value of 0.512 ±â€¯0.093 µM.

Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance.

Rheumatoid arthritis is characterized by the imbalance of T cells, which leads to increased pro-inflammatory and reduced anti-inflammatory cytokines. Modulating the balance among T cells is crucial for the treatment of RA. Kirenol is a major diterpenoid components of Herba Siegesbeckiae, which has been applied for arthritic therapy for centuries. Since prior research showed Kirenol exhibited anti-inflammatory effect in rats, in this study we have evaluated the effect and mechanism of bioactive Kirenol in a rat model of collagen-induced arthritis (CIA) on modulation of T cells. After immunization with bovine type II collagen (CII), Wistar rats were orally administered saline (CIA group), 2 mg/kg Kirenol or 2 mg/kg prednisolone daily for 30 days. The severity of arthritis was clinically and histologically assessed. The numbers of CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs) and IFNγ⁺CD4⁺ and IL4⁺CD4⁺ T cells were determined by flow cytometry, the mRNA expression level of Foxp3 was quantified by RT-PCR, cytokine levels were measured by ELISA and CII-induced cell proliferation was quantified in vitro. Kirenol significantly delayed the occurrence and reduced the disease severity of CIA. Histological analysis confirmed Kirenol suppressed joint inflammation and inhibited cartilage and bone destruction, compared to the CIA group. Kirenol also upregulated the mRNA expression of Foxp3, increased the numbers of CD4⁺CD25⁺Foxp3⁺ and IL4⁺CD4⁺ T cells, and reduced the number of IFNγ⁺CD4⁺ T cells. Kirenol reduced the levels of TNF-α, IL-17A and IL-6 in synovial fluid and TNF-α, IL-17A and IFN-γ in serum, and increased the serum levels of IL-4, IL-10 and TGF-ß1. In addition, Kirenol inhibited the ability of CII to induce splenocyte, PBMC and lymph node cell proliferation in vitro, compared to cells from CIA rats. In conclusion, these results suggest that Kirenol may be a potential immunosuppressant for the treatment for rheumatoid arthritis.

Pseudolaric acid B inhibits inducible cyclooxygenase-2 expression via downregulation of the NF-κB pathway in HT-29 cells.

PURPOSE: Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolaric kaempferi Gordon. Previous work has found that PAB has anti-inflammatory and anti-tumor effects in xenograft models of human hepatocellular carcinoma. The aim of this study is to evaluate the correlation between anti-cancer and anti-inflammatory effects of PAB and its molecular mechanisms on HT-29 cells. METHODS: Production of prostaglandin E2 (PGE2) in HT-29 cells was evaluated by ELISA. mRNA of cyclooxygenase-2 (COX-2) was analyzed by RT-PCR assay. High-content screening (HCS) method was adopted to detect the cytokine mixture (CM)-induced transcription activity of NF-κB and STAT3. Western blotting was used to evaluate the protein expression levels of inflammatory mediators induced by CM. After treatment with PAB in various concentrations, the inhibition rate of cell proliferation was measured with sulforhodamine B assays. For the in vivo studies, tumor-bearing models xenografted with HT-29 cells were developed in nude mice, and following oral administration with PAB, tumor inhibition rate was calculated. RESULTS: PAB inhibited the PGE2 production in HT-29 cells significantly (P < 0.05) with similar results detected at the COX-2 mRNA level. Furthermore, PAB suppressed the COX-2 protein expression and significant nuclear translocation of NF-κB and STAT3 induced by CM, which correlated with a concomitant degradation of I-κB and a decrease in constitutive STAT3 phosphorylation (P < 0.05). Moreover, various concentrations of PAB inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner. In vivo, after treatment with PAB for 17 days, the tumor weight of the 50 and 100 mg/kg treated groups was 0.62 ± 0.15 and 0.54 ± 0.06 g, respectively. When compared to the control group (0.82 ± 0.16 g), the inhibition rate of tumor weight was 24.2% at 50 mg/kg (P < 0.05) and 34.7% at 100 mg/kg (P < 0.001). CONCLUSIONS: PAB shows potential anti-cancer activity in HT-29 cells, and its molecular mechanisms are related to the anti-inflammatory action.

Structural determination of two new triterpenoid saponins acylated with monoterpenic acid from Gymnocladus chinensis Baill.

Two new triterpenoid saponins acylated with monoterpenic acid, 2ß,23-dihydroxy-3-O-α-L-rhamnopyranosyl-21-O-{(6S)-2-trans-2,6-dimethyl-6-O-[3-O-(ß-D-glucopyranosyl)-4-O-(2-methylbutanoyl)-ß-L-arabinopyranosyl]-2,7-octadienoyl)-acacic acid 28-O-ß-D-xylopyranosyl-(1 → 3)-ß-D-xylopyranosyl-(1 → 4)-[ß-D-glucopyranosyl-(1 → 3)]-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-ß-D-glucopyranosyl ester and 2ß,23-dihydroxy-3-O-α-L-rhamnopyranosyl-21-O-{(6S)-2-trans-2,6-dimethyl-6-O-[4-O-((6S)-2-trans-2,6-dimethyl-6-O-(ß-L-arabinopyranosyl)-2,7-octadienoyl)]-ß-L-arabinopyranosyl]-2,7-octadienoyl}-acacic acid 28-O-ß-D-xylopyranosyl-(1 → 3)-ß-D-xylopyranosyl-(1 → 4)-[ß-D-glucopyranosyl-(1 → 3)]-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-ß-D-glucopyranosyl ester were isolated from the fruit of Gymnocladus chinensis Baill. and the structural elucidation of both the compounds was accomplished by extensive studies of their spectroscopic (1D and 2D NMR, TOF-MS, QFT-MS) and chemical methods.

Three triterpenoid saponins acylated with monoterpenic acid from Gymnocladus chinensis.

A new triterpenoid saponin acylated with monoterpenic acid, together with two known triterpenoid saponins, has been isolated from the fruit of Gymnocladus chinensis Baill. Their structures were elucidated as 2ß,23-dihydroxy-3-O-α-L-rhamnopyranosyl-21-O-{(6S)-2-trans-2,6-dimethyl-6-O-[3-O-(ß-D-glucopyranosyl)-4-O-((6S)-2-trans-2,6-dimethyl-6-hydroxy-2,7-octadienoyl)-ß-L-arabinopyranosyl]-2,7-octadienoyl}-acacic acid 28-O-ß-D-xylopyranosyl-(1 → 3)-ß-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-ß-D-glucopyranosyl ester (1), gymnocladus saponin E (2), and gymnocladus saponin F(2) (3).

Kirenol upregulates nuclear annexin-1 which interacts with NF-κB to attenuate synovial inflammation of collagen-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Kirenol is a diterpenoid compound purified from the Chinese Herba Siegesbeckiae. Siegesbeckiae has been employed for the treatment of arthritis for centuries, its safety and efficacy are documented through a long history of human use. AIM OF THE STUDY: To investigate the effects on collagen-induced arthritis (CIA) and anti-inflammatory mechanism of kirenol. MATERIALS AND METHODS: Kirenol was administrated intragastrically in rats after the onset of CIA. Pathological changes were evaluated by paw swelling and histopathology. Concentration of IL-1ß in synovial fluid and adrenal corticotropin (ACTH) in plasma were determined by Elisa. Western blot was performed to detect the expression of annexin-1 and glucocorticoid receptor alpha (GRα) in synovium. NF-κB DNA binding activity was assessed by electrophoretic mobility shift assays (EMSA). RESULTS: Kirenol (1, 2, and 4 mg/kg) and prednisolone depressed paw swelling and reduced IL-1ß of synovial fluid in the CIA rats (p<0.05 or p<0.01). Kirenol and prednisolone upregulated nuclear annexin-1 and inhibited NF-κB activity in synovium of CIA. The inhibitory effect of kirenol and prednisolone on NF-κB activity was enhanced by anti-annexin-1 Ab. Prednisolone, but not kirenol, downregulated plasma ACTH and GRα expression significantly (p<0.01). CONCLUSION: Kirenol and prednisolone can upregulate nuclear annexin-1 which interacts with NF-κB to inhibit NF-κB activity, reduce cytokines expression and thereby attenuate inflammation of CIA joints. Kirenol does not lead to ACTH or GR downregulation, which is in contrast to classic glucocorticoid prednisolone. Kirenol shares with GCs similar anti-inflammatory mechanism but bypass the considerable limitation of GCs treatment.

A rapid and simple RP-HPLC method for quantification of kirenol in rat plasma after oral administration and its application to pharmacokinetic study.

A rapid and simple reverse-phase high-performance liquid chromatography (RP-HPLC) was developed and validated for the quantification of kirenol in rat plasma after oral administration. Kirenol and darutoside (internal standard, IS) were extracted from rat plasma using Cleanert™ C(18) solid-phase extraction (SPE) cartridge. Analysis of the extraction was performed on a Thermo ODS-2 Hypersil C(18) reversed-phase column with a gradient eluent composed of acetonitrile and 0.1% phosphoric acid. The flow rate was 1.0 mL/min and the detection wavelength was set at 215 nm. The calibration curve was linear over the range of 9.756-133.333 µg/mL (r(2) = 0.9991) in rat plasma. The lower limits of detection and quantification were 2.857 and 9.756 µg/mL, respectively. The intra- and inter-day precisions (relative standard deviation, RSD) were between 2.24 and 4.46%, with accuracies ranging from 91.80 to 102.74%. The extraction recovery ranged from 98.16 to 107.62% with RSD less than 4.81%. Stability studies showed that kirenol was stable in preparation and analytical process. The present method was successfully applied to the pharmacokinetic study of kirenol in male Sprague-Dawley rats after oral administration at a dose of 50 mg/kg.

[Studies on macrocyclic jatrophane diterpenes of Euphorbia kansui].

OBJECTIVE: To study macrocyclic jatrophane diterpenes of Euphorbia kansui. METHOD: The compounds were isolated and purified by various column chromatographic methods. Structures were elucidated by spectroscopic analyses. RESULT: Three macrocyclic jatrophane diterpenes were isolated from E. kansui and were characterized as kansuinin D1 (1), kansuinin D (2), kansuinin A (3). CONCLUSION: Kansuinin D1 (1) was a new compound.

[Chemical constituents in aerial part of Reineckea carnea].

OBJECTIVE: To study the chemical constituents in the aerial part of Reineckea carnea. METHOD: The compounds were isolated by extraction, silica gel, gel, and reversed-phase silica gel coloum chromatography, and high-performance liquid chromatography. The structures were identified by various spectroscopic methods including 1D and 2D NMR spectrum, MS, IR, etc. RESULT: Six compounds were isolated and identified as 1alpha, 3beta-dihydroxy-5beta-pregn-16-en-20-one-3-O-beta-D-glucopyranoside (1), syringaresinol-beta-D-glucoside (2), sophoraflavone B (3), stigmast-5, 22-dien-3-O-beta-D-glucopyranoside (4), daucosterol (5), a-D-glucose (6). CONCLUSION: Compound 1 was a new compound, coumpounds 2-6 were obtained from the plant for the first time.

Extract of Ganoderma lucidum potentiates pentobarbital-induced sleep via a GABAergic mechanism.

Ganoderma lucidum has been used for the treatment of a variety of diseases. For the first time here we report a detailed study on the mechanisms and effects of G. lucidum aqueous extract (GLE) on sleep and its sedative activity. GLE showed no effects on sleep architecture in normal rats at doses of 80 and 120 mg/kg. However, GLE significantly decreased sleep latency, increased sleeping time, non-REM sleep time and light sleep time in pentobarbital-treated rats. Suppression of locomotor activity in normal mice induced by GLE was also observed. Flumazenil, a benzodiazepine receptor antagonist, at a dose of 3.5 mg/kg showed a significant antagonistic effect on the shortening in sleep latency, increase in sleeping time, non-REM sleep time or light sleep time in pentobarbital-treated rat induced by GLE. Significant effect was also observed with GLE on delta activity during non-REM sleep and flumazenil did not block this effect. In conclusion, GLE may be a herb having benzodiazepine-like hypnotic activity at least in part.

An anticancer effect of a new saponin component from Gymnocladus chinensis Baillon through inactivation of nuclear factor-kappaB.

Gymnocladus chinensis Baillon is widely distributed in China, and its fruits have been used in the treatment of rheumatism, furunculosis, soreness and swelling in traditional Chinese medicine for a long time. Few biological components were, however, isolated. In this study, a new triterpenoid saponin (GC-1) was extracted from the fruit of Gymnocladus chinensis Baillon and its biological actions were investigated. The results showed that GC-1 inhibited growth of a panel of human cancer cell lines in vitro by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide and sulforhodamine B assays. Furthermore, GC-1 was demonstrated to induce apoptosis in HL-60 cells in a dose-dependent manner. By using a reporter gene assay, nuclear factor-kappaB activity induced by tumor necrosis factor-alpha was decreased gradually by addition of increasing concentration of GC-1 (1-40 micromol/l). In parallel, the blockage of nuclear factor-kappaB translocation from cytoplasm to nucleus was determined by Western blotting. This is the first study investigating the link of antiproliferative action of the compound with the inhibition of nuclear factor-kappaB activation. The mechanism of the actions of GC-1 might be due to the interruption of nuclear factor-kappaB translocation in the signaling pathway, which contributes to the chemotherapy potential.

[Chemical investingation of Chinese mangrove Sonneratia apetala II].

OBJECTIVE: Isolation and structural elucidation of the constituents of China mangrove Sonneratia apetala. METHOD: chromatography methods were used for isolation of compounds, spectroscopic methods were used for structural identifyication. RESULT: seven known compounds named (+/-) symgaresinol, betulinic acid, lupeol, lupeone, stigmast-5-ene-3beta, 7alpha-diol, beta-alpha myrin hexadecaneate, physcoion were isolated. CONCLUSION: these known compouns were unreported previously from this plant.

[Chemical constituents from the mangrove plant Ceriops tagal].

AIM: To investigate the secondary metabolites of the mangrove plant Ceriops tagal. METHODS: Column chromatography techniques including HPLC were used for the separation and purification, and extensive spectral analysis including various 2D NMR spectra were employed for structure elucidation. RESULTS: Nine compounds, namely, tagalsins A (1), ent-5alpha-dolabr-4 (18) -ene-15S,16-diol (2), squalene (3), betulinic acid (4), lup-20 (29) -en-3-on-28-oic acid (5), betulin (6), lup-20 (29) -en-3-on-28-ol (7), beta-sitosterol (8), n-hexacosanylferulate (9) were obtained. Of which 1 and 2 belong to dolabrane diterpene. CONCLUSION: Compound 1 is a new compound, and 2 to 9 are isolated from this species for the first time.

6-hydroxy-4-en-3-one sterols from the marine sponge Iotrochoto birotulata.

Five sterols with a nucleus skeleton of 6-hydroxy-4-en-3-one, namely cholesta-6beta-hydroxy-4-en-3-one (1), ergosta-6beta-hydroxy-4,24(28)-dien-3-one (2), ergosta-6alpha-hydroxy-4,24(28)-dien-3-one (3), ergosta-6alpha-hydroxy-4,22-E-dien-3-one (4), and ergosta-28-methyl-6beta-hydroxy-4,24(28)-dien-3-one (5) have been isolated from the marine sponge Iotrochoto birotulata, collected from the southern China sea. Sterols 2-4 are new compounds, and 1 has been isolated from marine organisms for the first time. The structures have been elucidated on the basis of extensive spectroscopic and chemical properties.

Cerebroside analogues from marine-derived fungus Aspergillus flavipes.

From the mycelium of the marine-derived fungus Aspergillus flavipes, isolated from the sea anemone Anthopleura xanthogrammica, two new cerebroside analogues, namely flavicerebrosides A (1): [(2S,2'R,3R,4E,8E)-N-2'-hydroxyoctadecanoyl-1-O-beta--galactopyranosyl-9-methyl-4,8-sphingadienine], and B (2): [(2S,2'R,3R,3'E,4E,8E)-N-2'-hydroxy-3'-octadecenoyl-1-O-beta--galactopyranosyl-9-methyl-4,8-sphingadienine], together with two known glycosphingolipids cerebrosides D (3) and C (4), were isolated. Their structures were identified by means of extensive spectroscopic analysis (IR, UV, 2D NMR, MS, CD) and chemical degradation. All four compounds showed cytotoxic activity against the KB cell line.

[Chemical constituents from Chinese marine sponge Cinachyrella australiensis].

OBJECTIVE: To investigate the secondary metabolites from Chinese marine Sponge Cinachyrella australiensis. METHODS: Column chromatography techniques including HPLC were used for the separation and purification of the compounds, and extensive spectral analyses including various 2D NMR spectra were employed for structure elucidation. RESULTS: Nineteen compounds were obtained,including 2-methyoxy-6,12,15-trien-8-yne-octadecanoic acid (1), 2-benzenedicarboxylic acid dibutyl ester(2), 1,2-benzenedicarboxylic acid bis(2-ethylhexyl)ester(3), (-) (3S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid(4), L-Tryptophan (5), p-hydroxylbenzaldehyde (6), p-hydroxyl-benzylethanol(7), p-hydroxyl-benzyl-propanol(8), cholesta-4-en-3-ol(9), 2-methyl-6-amino-9-(2-deoxy-beta-D- ribofuranosyl-purine(10), 2'-Deoxyadenosine (11), 6-amino-9-beta-D-ribofuranosyl-9H-purine (12),uracil(13), thymine(14), thymidine(15), 1-(2-deoxy-beta-D-Ribofuranosyl) uracil(16), 1-ethyl-alpha-(2-deoxy)-beta-D-ribofuranos(17),isolumichrome(18),and zarzissine(19). CONCLUSION: Compounds 1 and 18 are new natural products,and compounds 2 to 17 as well as 19 are isolated from this species for the first time.

Polyphenolic compounds from the leaves of Koelreuteria paniculata Laxm.

From the fresh leaves of Koelreuteria paniculata Laxm (Sapindaceae), four new compounds, named ethyl p-trigallate (1), 3''-O-galloyl-4'-O-galloyl-4-O-galloyl-gallic acid (2), ethyl p-heptagallate (3) and 3''-galloylquercitrin (4), together with 12 known compounds namely catechin (5), galloylepicatechin (6), isorhamnetin (7), kaempferol-3-O-arabinopyranoside (8), quercetin-3'-O-beta-D-arabinopyranoside (9), quercitrin (10), methyl p-digallate (11), methyl m-digallate (12), p-digalloyl acid (13), m-digalloyl acid (14), hyperin (15) and kaempferol-3-O-alpha-L-rhamnoside (16) were isolated by extensive column chromatographic separation. Their structures were elucidated on the basis of chemical and spectroscopic methods. Compound 9 was not reported previously with pyranoside of arabinose at C-3'. Compounds 4 and 9 possessed the activity for PTK inhibition.