Improved chromatographic diastereoresolution of cyclopropyl dafachronic acid derivatives using chiral anion exchangers.

In the present paper we describe the optimization and the application of a chromatographic method suitable to get all four diastereoisomers of C24-C25 cyclopropyl dafachronic acid derivatives in sufficient amount for their biological appraisal towards the nuclear hormone receptor transcription factor DAF-12. A preliminary column screening of six anion exchange type Cinchona alkaloid-based chiral stationary phases (CSPs) allowed to identify the one with a quinine scaffold and carrying a O-9-(3,5-bis(trifluoromethyl)phenyl) moiety at C9 position as the best CSP. Few modifications of the experimental conditions revealed that a content of 18mM acetic acid used as counterion and displacer in acetonitrile and a column temperature fixed at 35°C were optimal for the simultaneous discrimination of all four diastereoisomers with a 1.0mL/min flow rate. With such conditions, transoid (S,S) and (R,R) diastereoisomers were resolved with RS>1.4. With non chiral reversed-phase columns, neither the cisoid nor the transoid diastereoisomers could be resolved. This way, ca. 1.0mg of each stereoisomer was isolated with a diastereomeric purity >98%, suitable for the following biological tests. The indirect stereochemical assignments of the four diastereoisomers, and hence the corresponding chromatographic elution order (24R,25R)<(24S,25S)<(24R,25S)<(24S,25R) were made in an analogy manner on the basis of the resolution of fully assigned and structurally very similar ursodeoxycholic acid derivatives. As support of this indirect way of assigning the absolute configuration of the C24 and C25 chiral centre a molecular modeling procedure based on dynamic simulation was successfully applied.

Bis-sesquiterpene from the Marine Sponge Dysidea fragilis.

Two new sesquiterpenes and one new bis-sesquiterpene, named dysinidins C-E (1-3) along with three known sterols, dysideasterol F, 9α,l lα-epoxycholest-7-en-3ß,5α,6α-triol, and 9α,11α-epoxycholest-7-en-3ß,5α,6α,19-tetrol 6-acetate (4-6) were isolated from the Vietnamese marine sponge Dysidea fragilis (Montagu, 1814). Their structures were determined by 1D- and 2D-NMR spectroscopies and HR-ESI-MS, as well as by comparison with reported literature data. Compounds 4-6 were found to inhibit eight human cancer cell lines (KB, LU-1, HL-60, LNCaP, SK-Mel-2, HepG-2, MCF-7, and PC-3), with IC50 values ranging from 7.3 to 31.5 µM.

Antcin K, an Active Triterpenoid from the Fruiting Bodies of Basswood-Cultivated Antrodia cinnamomea, Inhibits Metastasis via Suppression of Integrin-Mediated Adhesion, Migration, and Invasion in Human Hepatoma Cells.

Previous research demonstrated that the ethyl acetate extract from Antrodia cinnamomea suppresses the invasive potential of human breast and hepatoma cells, but the effective compounds are not identified. The main bioactive compounds of A. cinnamomea are ergostane-type triterpenoids, and the content of antcin K is the highest. The objective of this study was to evaluate the antimetastatic activity and mechanisms of antcin K purified from the fruiting body of basswood-cultivated A. cinnamomea on human liver cancer Hep 3B cells. The results showed that adhesion, migration, and invasion of Hep 3B cells were effectively inhibited by antcin K within 24 h of treatment. Antcin K not only reduced the protein expression and activity of MMP-2 and MMP-9 but also down-regulated vimentin and up-regulated E-cadherin in Hep 3B cells. In depth investigation for the molecular mechanism revealed that antcin K could reduce the protein expression of integrin ß1, ß3, α5, and αv and suppress phosphorylation of FAK, Src, PI3K, AKT, MEK, ERK, and JNK. These results suggested that antcin K was able to inhibit the metastasis of human hepatoma cells through suppression of integrin-mediated adhesion, migration, and invasion. Coupled with these findings, antcin K has a good potential to reduce the risk of liver cancer metastasis.

Cytotoxic 5α,8α-epidioxy sterols from the marine sponge Monanchora sp.

Three new sterols, 5α,8α-epidioxy-24-norcholesta-6,9(11),22-trien-3ß-ol (1), 5α,8α-epidioxy-cholesta-6,9(11),24-trien-3ß-ol (2), and 5α,8α-epidioxy-cholesta-6,23-dien-3ß,25-diol (3), with four known sterols (4-7) were isolated from a marine sponge Monanchora sp. Their chemical structures were elucidated by extensive spectroscopic analysis. Compounds 1 and 3-7 showed moderate cytotoxicity against several human carcinoma cell lines including renal (A-498), pancreatic (PANC-1 and MIA PaCa-2), and colorectal (HCT 116) cancer cell lines.

Fast separation and quantification of steroid hormones Δ4- and Δ7-dafachronic acid in Caenorhabditis elegans.

Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Δ4- and Δ7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Δ4- and Δ7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3 µm core-shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Δ4- and Δ7-DA in C. elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation.

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.

A new alkaloid from the fruit of Nandina domestica Thunb.

A new steroidal alkaloid, (20S,22R,24R)-24-ethyl-3-oxocholest-4-en-22-amino, named as nandsterine (1), together with 10 known alkaloids, palmatine (2), O-methylbulbocapnine (3), nantenine (4), dehydronantenine (5), glaucine (6), didehydroglaucine (7), dehydrocorydaline (8), jatrorrhizine (9), magnoflorine (10) and berberine (11), was isolated from the fruit of Nandina domestica Thunb. Their structures were elucidated by using spectroscopic methods as well as by comparing with the published data. Compound 1 was a new class of steroidal alkaloid isolated from the family Berberidaceae, meanwhile compounds 2, 3, 6-8 and 10 were obtained from N. domestica for the first time. Compound 1 exhibited cytotoxicity against HL-60 cells (human leukaemia) with IC50 values of 52.1 µM.

Chromatographic separation of free dafachronic acid epimers with a novel triazole click quinidine-based chiral stationary phase.

For the first time, a successful chromatographic method based on the use of a novel triazole click quinidine (QD) derivative anion-exchange chiral stationary phase (CSP) is applied for the epimer separation of dafachronic acids (Δ(4)- and Δ(7)-DAs). The use of a polar-ionic eluent system consisting of 18mM AcOH in ACN furnished an excellent separation of both Δ(4)- (α=1.19, RS=2.52) and Δ(7)-DA (α=1.14, RS=2.06) C-25 epimer couples. The pool of data collected during the chromatographic analyses revealed the prominent role of anion-exchange interactions in governing the analyte (SA) retention and also indicated the occurrence of stereoselective H-bond contacts with the chiral selector (SO) substructural motifs. In any case, molecular modelling studies corroborate the need for sufficient spatial freedom for optimum binding between the SO and the SAs which seems less the case for the immobilised SO unit of the commercialized QD-based CSP.

A steroidal molecule present in the egg wax of the tick Rhipicephalus (Boophilus) microplus inhibits bacterial biofilms.

The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization.

High performance liquid chromatography used for quality control of Achyranthis Radix.

To establish a standard of quality control and to identify reliable Achyranthis Radix, three phytoecdysones including ecdysterone (1), 25R-inokosterone (2) and 25S-inokosterone (3) were determined by quantitative HPLC/UV analysis. Three phytoecdysones were separated with an YMC J'sphere ODS C(18) column (250 mm × 4.6 mm, 4 µm) by isocratic elution using 0.1% formic acid in water and acetonitrile (85:15, v/v%) as the mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 245 nm. The standards were quantified by HPLC/UV from Achyranthes bidentata Blume and Achyranthes japonica Nakai, as well as Cyathula capitata Moq. and Cyathula officinalis Kuan, which are of a different genus but are comparative herbs. The method was successfully used in the analysis of Achyranthis Radix of different geographical origin or genera with relatively simple conditions and procedures, and the assay results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of eighteen A. bidentata Blume samples and ten A. japonica Nakai samples. The results indicate that the established HPLC/UV method is suitable for quantitation and pattern recognition analyses for quality evaluation of Achyranthis Radix.

Chemical characterization of a commercial Commiphora wightii resin sample and chemical profiling to assess for authenticity.

The gum resin of Commiphora wightii [(Hook. ex Stocks) Engl.] is an ayurvedic medicine for the treatment of arthritis, inflammation, obesity, lipid disorders, and cardiovascular diseases and is known as guggul. Morphologically, it is not easy to distinguish guggul from closely related gum resins of other plants. Reliability of the commercially available guggul is critical due to the high risk of adulteration. To check authenticity, a commercial guggul sample was investigated for its chemical markers and 17 metabolites were identified, including three new, 20(S),21-epoxy-3-oxocholest-4-ene (1), 8 ß-hydroxy-3,20-dioxopregn-4,6-diene (2), and 5-(13' Z-nonadecenyl)resorcinol (17) from the ethyl acetate soluble part. During the current study, compounds 14- 17 were identified as constituents of Mangifera indica gum, as an adulterant in the commercial guggul sample. This discovery highlighted the common malpractices in the trade of medicinal raw material in the developing world. The structures of the compounds were deduced by the spectroscopic technique and chemical methods, as well as by comparison with the reported data. The structure of 20(S),21-epoxy-3-oxocholest-4-ene (1) was also unambiguously deduced by single-crystal X-ray diffraction technique.

[Two new steroid glycosides from the Far East starfish Hippasteria kurilensis].

Two new steroid glycosides were isolated from the Far East starfish Hippasteria kurilensis collected in the Sea of Okhotsk. They were characterized as (22E,24R)-3-O-(2-O-methyl-beta-D-xylopyranosyl)-24-O-[2-O-methyl-beta-D-xylopyranosyl-(1-->5)-alpha-L-arabinofuranosyl]-5alpha-cholest-22-ene-3beta,4beta,6alpha,7alpha,8,15beta,24-heptaol (kurilensosid I) and (24S)-3-O-(2-O-methyl-beta-D-xylopyranosyl)-24-O-(alpha-L-arabinofuranosyl)-5alpha-cholestan-3beta,4beta,6beta,15alpha,24-pentaol (kurilensosid J). In addition, the earlier known glycosides linkosides F and L1, levisculoside G, forbeside L, desulfated echinasteroside A, and granulatoside A were isolated and identified. The structures of the new compounds were established with the help of bidimentional NMR spectroscopy and mass spectrometry.

(25S)-cholesten-26-oic acid derivatives from an Indonesian soft coral Minabea sp.

(25S)-3-Oxocholesta-1,4-dien-26-oic acid (1) and a new (25S)-18-acetoxy-3-oxocholesta-1,4-dien-26-oic acid (2) were isolated from a soft coral Minabea sp. (cf. aldersladei) collected in North Sulawesi, Indonesia, together with two known cholic-acid-type compounds, 3-oxochol-1,4-dien-24-oic acid (3) and 3-oxochol-4-en-24-oic acid (4). The structures of these compounds were determined on the basis of their spectroscopic data. The absolute stereochemistry at C-25 of 2 was determined by comparative (1)H NMR study using chiral anisotropic reagents [(S)- and (R)-phenylglycine methyl esters]. This is the first to report compound 1 as a natural product.

In vitro antioxidant activity of Oldenlandia herbacea and isolation of 9,9-dimethyl hexacosane and 23-ethyl-cholest-23-en-3beta-ol.

The petroleum ether, chloroform, ethyl acetate and methanol extracts of the whole plant of Oldenlandia herbacea were screened for in vitro antioxidant activity by using ABTS, DPPH, nitric oxide and hydrogen peroxide assay methods. The chloroform, ethyl acetate and methanol extracts showed potent antioxidant activity against ABTS free radicals. In the hydrogen peroxide method, the methanol and ethyl acetate extracts showed potent activity. Chemical investigation of the petroleum ether extract yielded a long chain hydrocarbon, 9,9-dimethyl hexacosane (1), and a steroid, 23-ethyl-cholest-23-en-3beta-ol (2).

Purification of dinosterol for hydrogen isotopic analysis using high-performance liquid chromatography-mass spectrometry.

A semi-preparative normal-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS) method is presented for the purification of various alcohol fractions from total lipid extracts derived from sediments, for the purpose of hydrogen isotopic measurement by gas chromatography-isotope ratio mass spectrometry (GC-IRMS). 4-methylsterols, including the dinoflagellate-specific marker dinosterol (4,23,24-trimethylcholestan-22-en-3beta-ol), were successfully separated from notoriously co-eluting plant-derived pentacyclic triterpenoid alcohols and alkyl alcohols. We find that substantial hydrogen isotope fractionation occurs during chromatographic separation, demonstrating the importance of recovering the entire peak when subsequent hydrogen isotope analyses are to be performed. This is the first report of such hydrogen isotopic fractionation for a natural unlabelled compound.

[Trofosides A and B and other cytostatic steroid-derived compounds from the Far East starfish Trofodiscus über].

Three new polar steroids identified as trofoside A, (20R,24S)-24-O-(3-O-methyl-beta-D-xylopyranosyl)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfoxy-(20R,24S)-5alpha-cholestane-3beta,6beta,8,15alpha,24-pentaol sodium salt, were isolated from Trofodiscus uber starfish extracts collected in the Sea of Okhotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3beta,6alpha,8,15beta-tetrahydroxy-5alpha-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius (C(min)) and terminating the cell division at the stage of the first division (C(min) embr.), as well as the concentrations causing 50% immobilization of sperm cells (ImC50) and inhibiting their ability to fertilize egg-cells by 50% (IC50) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono- and double chained glycosides with the monosaccharide fragment at C3 and C24 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.

[Steroid compounds from the Far East starfish Pteraster obscurus and the ophiura Asteronyx loveni].

Seven sulfated polyhydroxysteroids were isolated from the Far East starfish Pteraster obscurus and the ophiura (snake star) Asteronyx loveni (collected in the Sea of Okhotsk) and characterized: disodium and sodium salts of (20R)-24-methyl-2beta-hydroxycholesta-5,24(28)-diene-3alpha,21-diyl disulfate, (20R)-5alpha-cholestane-3beta,21-diyl disulfate, (20R)-3beta-hydroxy-5alpha-cholestan-21-yl sulfate, (20R)-cholest-5-ene-3beta,21-diyl disulfate, (20R)-2beta-hydroxycholest-5-ene-3alpha,21-diyl disulfate, (20R)-cholest-5-en-3beta-yl sulfate, and (20R)-5alpha-cholestan-3beta-yl sulfate. The first four compounds turned out to be new, whereas the others were identical to the known compounds. Structures of the isolated steroids were identified by two-dimensional NMR spectroscopy and other physicochemical methods. The compounds isolated from starfish are structurally similar to typical ophiuroid metabolites, which support the opinion of some taxonomists that starfish and ophiuroids are phylogenetically related classes.

[Studies on chemical constituents of Laurencia tristicha ( II )].

OBJECTIVE: To search for chemical constituents with structural diversity from Laurencia tristicha to supply for biological assay. METHOD: Compounds were isolated by means of column chromatography over normal phase silica gel and Sephadex LH-20, recrystallization and HPLC. Structures were identified by spectroscopic methods including 1D NMR, IR and MS. Cytotoxicities of the purified compounds were evaluated by MTT method. RESULT: Seven compounds were isolated from L. tristicha. Their structures were elucidated as cholesterol (1), cholesta- 5-en-3beta, 7alpha-diol (2), beta-stigmasterol (3), phytol (4), zeaxanthin (5), 4 -hydroxybenzaldehyde (6), indolyl-3-carbaldehyde (7). In the cytotoxic assay compound 2 was active against human cancer cell lines HCT-8, Bel-7402, BGc-823, A549 and HELA with IC50 values of 1.90, 2.02, 1.99, 6.52 and 1.20 microg x mL(-1), respectively. Compound 4 showed cytotoxicity against HCT-8 and HELA with IC50 value of 3.51 and 2.04 microg x mL(-1), and other compounds were inactive ( IC50 > 10 microg x mL(-1)). CONCLUSION: Compounds 1-7 were isolated from L. tristicha for the first time. In additon, compounds 2 and 4 were cytotoxic against several human cancer cell lines.

Structural identification of an anthrasteroid hydrocarbon from the sheep tick Ixodes ricinus.

A new anthrasteroid hydrocarbon was isolated from cuticular integument of engorged female Ixodes ricinus, and its structure was determined by interpretation of spectroscopic and nuclear magnetic resonance data as 14alpha(H)-1(10-->6)-abeo-cholesta-3,5,7,9(10)-tetraene (1).

New cytotoxic steroids from the Indian Ocean sponge Axinella cf. bidderi.

Four new sterols have been isolated from the marine sponge Axinella cf. bidderi, 17alpha-hydroxy-22,23-epoxy-24-methylcholest-5-en-3beta-ol (1) and 17alpha-hydroxy-22,23-epoxycholest-5-en-3beta-ol (2), together with 3 and 4, which possess respectively the cholestene and the cholestane skeleton with a cyclic enol ether linkage between C-18 and C-22. The structures were elucidated using spectroscopic data. The in vitro activity was evaluated against prostate, ovary, pancreas, colon, and lung cell lines.