Rapid screening for natural lipase inhibitors from Alisma orientale combining high-performance thin-layer chromatography-bioautography with mass spectrometry.

Lipase inhibitors are an attractive class of hypolipidemic compounds, which inhibit the activity of human pancreatic lipase, thereby preventing the absorption of triglycerides in vivo. As a library of promising lead compounds for drug development, traditional Chinese medicine (TCM) has gained growing attention in quick discovery and identification of enzyme inhibitors of natural-origin. The purpose of this work was to discover unknown lipase inhibitors from Alisma orientale by the activity oriented analysis method thin-layer chromatography-bioautography, then use electrospray ionization mass spectrometry technology via the elution based TLC-MS interface to identify their structures. As a result, eleven natural lipase inhibitors from Alisma orientale extracts were identified based on molecular mass and fragment ions obtained by HPTLC-MS, and further confirmed by a series of complementary means including UV spectra, 1H NMR characteristic proton signals and polarity of compounds, eleven lipase inhibitors were tentatively assigned as triterpenoids: alisol B (m/z 495.50 [M + Na]+), alisol B 23-acetate (m/z 537.58 [M + Na]+), 11-deoxy-alisol B (m/z 479.50 [M + Na]+), 11-deoxy-alisol B 23-acetate (m/z 521.50 [M + Na]+), alisol A/epialisol A (m/z 513.50 [M + Na]+), 16-oxo-11-deoxy-alisol A (m/z 511.50 [M + Na]+), 16-oxo-alisol A (527.50 [M + Na] +), alisol C (m/z 509.58 [M + Na]+), alisol C 23-acetate (m/z 551.50 [M + Na]+), alisol M 23-acetate (m/z 567.50 [M + Na]+), and alismanol Q/neoalisol (m/z 493.42 [M + Na]+). The integrated approach is an efficient method for rapid screening lipase inhibitors from complex plant extracts and provides a reasonable and favorable basis for the identification and separation of other enzymatic system and other important compounds with therapeutic values.

Isolation and Characterization of Chemical Constituents from the Poroid Medicinal Mushroom Porodaedalea chrysoloma (Agaricomycetes) and Their Antioxidant Activity.

The chemical analysis of the methanol extract of Porodaedalea chrysoloma (Fr.) Fiasson & Niemela afforded the isolation of five compounds (1-5). The first two are phenolic derivatives: methyl (E)-3-(4-methoxycar-bonylphenoxy)-acrylate (1) is a new natural product, while methyl 3-(4-methoxycarbonylphenoxy)-propionate (2) was isolated from a natural source for the first time. The triterpene steroids ergone (3), 3ß-hydroxyergosta-7,22-diene (4), and ergosterol (5) have not been previously identified in this species. The structures of the compounds were determined on the basis of NMR and MS spectroscopic analysis. The isolated fungal metabolites 1-5 were evaluated for their antioxidant activity. Compounds 1, 2, and 4 proved to possess considerable antioxidant effect in the ORAC assay with 2.21 ± 0.34, 1.58 ± 0.18, and 5.02 ± 0.47 mmol TE/g, respectively.

A bioactive new protostane-type triterpenoid from Alisma plantago-aquatica subsp. orientale (Sam.) Sam.

A new protostane-type triterpenoid, 5ß,29-dihydroxy alisol A (1) was isolated from Alisma plantago-aquatica subsp. orientale (Sam.) Sam. as well as 12-deoxyphorbol-13α-pentadecanoate (2). We first report the presence of compound 2 in the genus Alisma. Their structures were established on the basis of 1D and 2D NMR, and HRESIMS spectroscopic analyses. All the isolated compounds were assayed for their inhibitory effects against human carboxylesterase 2 (HCE-2). Compounds 1 and 2 displayed inhibitory activities against HCE-2 with IC50 values of 29.2 and 4.6 µM, respectively. The interaction mechanisms of HCE-2 with compounds 1 and 2 were investigated by molecular docking, respectively.

Alisol B-23-acetate, a tetracyclic triterpenoid isolated from Alisma orientale, induces apoptosis in human lung cancer cells via the mitochondrial pathway.

Alisol B-23-acetate (AB23A), a tetracyclic triterpenoid isolated from the rhizome of Alisma orientale, has been reported to exert anti-proliferative activities in human colon, ovarian and gastric cancer cells. However, the anti-cancer effect of this compound on human lung cancer cells has not yet been thoroughly elucidated. In the present study, we investigated the effects of AB23A on the cell viability and apoptosis in human lung cancer A549 and NCI-H292 cells. The results indicated that AB23A inhibited the growth of A549 and NCI-H292 cells in dose- and time-dependent manner, however, there was only weak cytotoxicity on normal bronchial epithelial cells. The induction of apoptosis by AB23A was demonstrated by DAPI and annexin-V-FITC/PI staining. Further investigation revealed that AB23A decreased mitochondrial membrane potential (MMP) and up regulated reactive oxygen species (ROS) level. Meanwhile, the increased Bax/Bcl-2 ratio, activated caspase-3, caspase-9 and PARP were observed. In addition, AB23A increased the release of cytochrome c from mitochondria and the translocation of apoptotic inducing factor (AIF) into nuclei. Taken together, these results indicated that AB23A induced apoptosis by activating the intrinsic pathway, and suggested that AB23A can be used as a potential modulating agent in lung cancer.

2-Ethoxycarbonyl-2-ß-hydroxy-a-nor-cholest-5-ene-4one: Extraction, structural characterization, antimicrobial, antioxidant, anticancer and acute toxicity studies.

Identification and characterization of marine natural products with antimicrobial, antioxidant activity with minimal toxicity has received much interest over the past few years. Among, Acropora formosa is one of the unexplored marine organism for the screening of natural products in marine resources. In this study, a novel steroid 2-ethoxycarbonyl-2-ß-hydroxy-A-nor-cholest-5-ene-4one (ECHC) was isolated from butanol extracts of A. formosa using vacuum liquid chromatography and sequentially purified by column chromatography. The chemical structure of the compound was elucidated based on spectroscopic analysis including GC-MS, 1H NMR and 13C NMR and identified as ECHC. Moreover, in vitro antioxidant activity showed that ECHC was highly scavenged the oxidative stress generative molecules. The in vitro cytotoxic activity of ECHC showed excellent activity against human breast cancer cells. Further, in vivo acute toxicity of ECHC on zebrafish Danio rerio was showed no toxicity as well as no morphological damage was observed after 21 days exposure. Histological analysis revealed that there is no apparent difference was observed between ECHC exposure and control group of D. rerio. Together, these results confirmed that ECHC has in vitro antioxidant and anticancer activity and could be developed as a potential drug against most contagious disease like cancer.

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins.

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Anti-inflammatory activity of 3ß-hydroxycholest-5-en-7-one isolated from Hippocampus trimaculatus leach via inhibiting iNOS, TNF-α, and 1L-1ß of LPS induced RAW 264.7 macrophage cells.

Hippocampus trimaculatus leach has been widely used in beverage and herbal medicine fabrication. However, the study of the molecular mechanism of its bioactivity especially the anti-inflammatory activity is still scanty. In the present study, the pure HEO isolated from the dried Hippocampus trimaculatus leach was firstly found to have anti-inflammatory activity in vitro. The molecular mechanism of the anti-inflammatory activity was detailed using the lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage cells, suggesting that the HEO can significantly suppress the inflammatory factors of nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin (1L)-1ß (1L-1ß). Cellular signaling analyses demonstrated that the HEO downregulated the NF-κB and extracellular signal-regulated kinase (ERK) of mitogen-activated protein kinase (MAPK) signaling pathways. All the results suggested that the HEO is a potential natural anti-inflammatory agent.

Alisol A 24-Acetate, a Triterpenoid Derived from Alisma orientale, Inhibits Ox-LDL-Induced Phenotypic Transformation and Migration of Rat Vascular Smooth Muscle Cells through Suppressing ERK1/2 Signaling.

Alisol A 24-acetate, a triterpenoid extracted from Alisma orientale, has shown antiatherosclerotic actions. The purpose of this study was to evaluate the inhibition of alisol A 24-acetate on oxidized low-density lipoprotein (Ox-LDL)-induced phenotypic transformation and migration of rat vascular smooth muscle cells (VSMCs), and to explore the underlying mechanisms. VSMCs were pretreated with alisol A 24-acetate and a specific extracellular signal-regulated kinase (ERK) inhibitor, U0126, and then stimulated with 50 mg/l Ox-LDL in vitro. The expression of VSMC phenotypic marker SM22α was determined using immunocytochemistry, and the migration of VSMCs was detected using a scratch-wound healing assay. The expression of matrix metalloproteinase (MMP)-9, MMP-2, phosphorylated ERK1/2 (pERK1/2) and total ERK was determined. Ox-LDL treatment caused a reduction in SM22α expression, VSMC transformation to the synthetic phenotype, increased MMP-2 and MMP-9 synthesis, the extension of VSMC migration distance and the upregulation of pERK1/2 expression, while the addition of alisol A 24-acetate or U0126 resulted in the elevation of SM22α expression, VSMC transformation to the contractile phenotype, a reduction in MMP-2 and MMP-9 expression, the shortening of cell migration distance and decreased pERK1/2 expression. The results of this study demonstrate that alisol A 24-acetate effectively reverses the phenotypic transformation and inhibits the migration of VSMCs, which may be associated with the suppression of the ERK1/2 signaling pathway.

The Protective Effects of Alisol A 24-Acetate from Alisma canaliculatum on Ovariectomy Induced Bone Loss in Vivo.

Alisma canaliculatum is a herb commonly used in traditional Korean medicine, and has been shown in scientific studies to have antitumor, diuretic hepatoprotective, and antibacterial effects. Recently, the anti-osteoclastogenesis of alisol A 24-acetate from Alisma canaliculatum was investigated in vitro. However, the influence of alisol A 24-acetate on osteoporosis in animals has not been investigated. The present study was undertaken to investigate the anti-osteoporotic effect of alisol A 24-acetate on bone mass in ovariectomized (OVX) mice and to identify the mechanism responsible for its effects. OVX mice were treated daily with 0.5 or 2 µg/g of alisol A 24-acetate for a period of six weeks. It was found that these administrations significantly suppressed osteoporosis in OVX mice and improved bone morphometric parameters. The serum estradiol, bone alkaline phosphatase levels, regulatory T/Th17 cell numbers were significantly increased by alisol A 24-acetate as compared with untreated OVX mice. In addition, TRAP activity was inhibited by alisol A 24-acetate in OVX mice. These results suggest alisol A 24-acetate effectively prevents bone loss in OVX mice, and that it can be considered a potential therapeutic for the treatment of postmenopausal osteoporosis.

Anti-inflammatory Steroid from Phragmitis rhizoma Modulates LPS-Mediated Signaling Through Inhibition of NF-κB Pathway.

Lipopolysaccharides (LPS) strongly stimulate immune cells, and unabated activation of immune system by LPS may lead to an exacerbation of sickness and depression. In this study, stigmasta-3,5-dien-7-one (ST) was isolated from Phragmitis rhizoma as a negative regulator of LPS-induced inflammation in macrophages. ST effectively reduced nitric oxide (NO), prostaglandin E2, and pro-inflammatory cytokine levels, which were markedly raised by LPS treatment. In addition, ST blocked the nuclear factor-kappa B (NF-κB) signaling pathway via down-regulation of phospho-p38 mitogen-activated protein kinase and phosphorylation and degradation of the inhibitor of NF-κB α. To our knowledge, this is the first study showing anti-inflammatory activities of ST isolated from Phragmitis rhizoma.

[Optimization of flash-type extraction technology of alisol B 23-acetate from Alismatis Rhizoma by response surface methodology].

Response surface methodology was used to optimize and obtain the optimal flash-type extraction technology of alisol B 23-acetate from Alismatis Rhizoma. With the extraction rate of alisol B 23-acetate as an indicator, single-factor test was used to investigate the effect of ethanol volume fraction, liquid-solid ratio, extraction times and extracting time on the extraction rate of alisol B 23-acetate.The results were combined with Box-Benhnken design and response surface analysis to optimize the technology parameters for extraction process of Alismatis Rhizoma and obtain the optimal flash-type extraction technology under the following conditions： ethanol volume fraction 80%, liquid-solid ratio 12∶1, extraction 4 times, 114 s/time. Flash-type extraction technology of alisol B 23-acetate by response surface methodology is stable, time-saving, efficient, and with the advantages of room temperature extraction and no component damage, so it can be used for massive production.

A new flavonone from seeds of Alpinia katsumadai and its neuroprotective effect on PC12 cells.

A new flavonone, named as (2R, 3S)-pinobanksin-3-cinnamate(1), together with six known compounds, pinocem-brin (2), pinobanksin (3), 3-O-acetylpinobanksin (4), galangin (5), kumatakenin(6), and 3-methylkaempferol (7), were isolated from a 95% ethanol extract of seeds of Alpinia katsumadai through a combination of various chromatographic techniques, including silica gel and Sephadex LH-20. The structure of compound 1 was elucidated by spectroscopic data analysis. Compound 1 exhibits a potent neuroprotective effect against the corticosterone-damaged PC12 cells, which may be underlying the effect by scavenging intracellular ROS.

Studies on chemical constituents of Ophiopogon japonicus.

Two new and six known steroidal glucosides were isolated from the tuber of Ophiopogon japonicus. The new steroidal glucosides were established as (20R,25R)-26-O-ß-d-glucopyranosyl-3ß,26-dihydroxycholest-5-en-16,22-dioxo-3-O-α-l-rhamnopyranosyl-(1 â†’ 2)-ß-d-glucopyranoside (1) and 26-O-ß-d-glucopyranosyl-(25R)-furost-5-en-3ß,14α,17α,22α,26-pentaol-3-O-α-l-rhamnopyranosyl-(1 â†’ 2)-ß-d-glucopyranoside (3) on the basis of spectroscopic data as well as chemical evidence.

The binding mechanisms of plasma protein to active compounds in Alismaorientale rhizomes (Alismatis Rhizoma).

Ultrafiltration and HPLC were employed to assess binding rates between rat plasma protein and two active compounds with lipid-regulating properties (alisol B 23-acetate and alisol A 24-acetate) from Alismaorientale rhizomes (Alismatis Rhizoma), a traditional Chinese medicine. SDS-PAGE was used for the evaluation of the binding between the alisol acetates and Hb in plasma. The fluorescence spectroscopy and circular dichroism spectroscopy were also combined with molecular modeling to explore binding mechanisms between Hb and the alisol acetates under imitative physiological condition. The ultrafiltration results show that alisol B 23-acetate bound more strongly than alisol A 24-acetate to plasma protein. SDS-PAGE results may suggest that alisols bind to Hb in plasma. The spectroscopy results are consisting with the molecular modeling results, and they indicate that the differences in plasma protein binding strength between the two compounds may be related to their side chains. A folded side chain/parent ring bound more strongly to Hb than an open side chain/parent ring.

Complete NMR assignments of undegraded asterosaponins from Asterias amurensis.

Four asterosaponins, thornasteroside A (1), versicoside A (2), anasteroside B (3), and asteronylpentaglycoside sulfate (4), were isolated from the predatory starfish Asterias amurensis Lütken. Unlike previous studies focusing on structure elucidation by degradation of the complex saponin molecules, complete nuclear magnetic resonance (NMR) assignment for the intact molecules was accomplished using 600 MHz high magnetic field NMR. The complete set of NMR assignments can help in the structure elucidation of asterosaponins isolated in low yields without resorting to chemical degradation. Furthermore, this approach can be extended to other complex steroidal saponins, which may accelerate the discovery of bioactive secondary metabolites from this invasive starfish species.

A new steroid with long cross-conjugation structure from the marine-derived fungus Aspergillus aculeatus.

A new steroid with a long cross-conjugation structure, 15a-hydroxy-(22E, 24R)-ergosta-3, 5, 8 (14), 22-tetraen-7-one (1), was isolated from the marine-derived fungus Aspergillus aculeatus. Its structure was established by the extensive spectroscopic analyses, and its cytotoxicities against P388, HL-60, and PC-3 cell lines were measured in vitro.

Molecular cloning and yeast expression of cinnamate 4-hydroxylase from Ornithogalum saundersiae baker.

OSW-1, isolated from the bulbs of Ornithogalum saundersiae Baker, is a steroidal saponin endowed with considerable antitumor properties. Biosynthesis of the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically via the phenylpropanoid biosynthetic pathway, but molecular biological characterization of the related genes has been insufficient. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11), catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1. Molecular isolation and functional characterization of the C4H genes, therefore, is an important step for pathway characterization of 4-methoxybenzoyl group biosynthesis. In this study, a gene coding for C4H, designated as OsaC4H, was isolated according to the transcriptome sequencing results of Ornithogalum saundersiae. The full-length OsaC4H cDNA is 1,608-bp long, with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 55-bp 5' non-coding region and a 35-bp 3'-untranslated region. OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD), HPLC-MS and nuclear magnetic resonance (NMR) analysis. The identification of the OsaC4H gene was expected to open the way to clarification of the biosynthetic pathway of OSW-1.

Cytotoxic cholesta-1,4-dien-3-one derivatives from soft coral Nephthea sp.

Five new steroids, (12ß, 22R)-12-acetoxy-22-hydroxy-cholesta-1,4-dien-3-one (1), (12ß, 22R)-12-hydroxy-22-acetoxy-cholesta-1, 4-dien-3-one (2), (12ß, 22R)-12, 22-diacetoxy-cholesta-1, 4-dien-3-one (3), (22R)-18, 22-diacetoxy-cholesta-1, 4-dien-3-one (4), (20R, 22R)-20-hydroxy-22-acetoxy-cholesta-1, 4-dien-3-one (5), and one known steroid astrogorgol N (6), were isolated from soft coral Nephthea sp. Their structures were established by spectroscopic analysis (1D, 2D NMR, HRMS) and comparisons of their spectral data with those of related steroids. The absolute configuration at C-22 of 1 was determined to be R by Mosher's analysis. All isolated compounds exhibited cytotoxic activity against HeLa cells with IC50 values ranged from 7.51±0.22 to 18.72±0.78µg/mL.

[Study on constituents of the aerial parts of Pogostemon cablin].

OBJECTIVE: To investigate the chemical constituents of the aerial parts of Pogostemon cablin. METHODS: The constituents were isolated by column chromatography over silica gel, Sephadex LH-20 and C8. Structures were identified by spectroscopic data analysis. RESULTS: Thirteen compounds were obtained and elucidated as patchouli alcohol (1), pogostone (2), friedelin (3), epifriedelinol (4), oleanolic acid (5), methyl oleanolate (6), 5alpha-stigmast-3,6-dione (7), stigmast-4-ene-3-one (8), beta-sitosterol (9), pachypodol (10), retusin (11), (-)-guaiacylglycerol (12) and dibutyl phthalate (13). CONCLUSION: Compounds 6, 7, 8, 12 and 13 are isolated from this genus for the first time.

Two new compounds from an endophytic fungus Alternaria solani.

Two new secondary metabolites, named 7-dehydroxyl-zinniol (1) and 20-hydroxyl-ergosta-4,6,8(14),22-tetraen-3-one (2), were isolated from the culture of Alternaria solani, an endophytic fungal strain residing in the roots of Aconitum transsectum. Their structures were elucidated on the basis of comprehensive spectroscopic analyses including IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR. Biological activity tests indicated that compound 1 showed moderate anti-HBV activity.