Preparative method for isosilybin isolation based on enzymatic kinetic resolution of silymarin mixture

The flavonolignan isosilybin (1) is one of the silybin congeners contained in the silymarin complex, which is isolated from the seeds of the milk thistle (Silybum marianum). A number of recent studies have demonstrated that isosilybin is probably the most potent anticancer agent found in silymarin. It occurs as a mixture of two diastereoisomers, A and B. Lipase Novozym 435 was found to allow the preparative production of both optically pure isosilybin A and B in a diastereoisomeric purity of over 95%. A preparatory method based on the enzymatic resolution and other purification methods has been developed, enabling multigram amounts of both optically pure isosilybins A (1a) and B (1b) to be obtained.     

Preparation of silybin and isosilybin sulfates by sulfotransferase from Desulfitobacterium hafniense

Flavonolignans silybin and isosilybin are major components of silymarin complex isolated from seeds of the milk thistle (Silybum marianum) featuring strong antioxidant and hepatoprotective effects, and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Natural silybin and isosilybin are mixtures of diastereoisomers: silybin/isosilybin A (1a, 1b) and silybin/isosilybin B (2a, 2b). The metabolism of these compounds is supposed to be strongly linked to Phase II of biotransformation and the respective conjugates are rapidly excreted in bile and urine. The aim of this study was to obtain optically pure sulfated metabolites of both silybins and isosilybins. Aryl-sulfate sulfotransferase (EC 2.8.2.22) from Desulfitobacterium hafniense was found to be a highly effective tool for the regiospecific enzymatic synthesis of silybin A-20-O-sulfate, silybin B-20-O-sulfate, isosilybin A-20-O-sulfate and isosilybin B-20-O-sulfate providing nearly quantitative yields and employing cheap p-nitrophenyl sulfate as sulfate donor. The isolated sulfated products will be used as authentic standards in metabolic studies of both silybins and isosilybins.     

Enhanced bioactivity of silybin B methylation products

Flavonolignans from milk thistle (Silybum marianum) have been investigated for their cellular modulatory properties, including cancer chemoprevention and hepatoprotection, as an extract (silymarin), as partially purified mixtures (silibinin and isosilibinin), and as pure compounds (a series of seven isomers). One challenge with the use of these compounds in vivo is their relatively short half-life due to conjugation, particularly glucuronidation. In an attempt to generate analogues with improved in vivo properties, particularly reduced metabolic liability, a semi-synthetic series was prepared in which the hydroxy groups of silybin B were alkylated. A total of five methylated analogues of silybin B were synthesized using standard alkylation conditions (dimethyl sulfate and potassium carbonate in acetone), purified using preparative HPLC, and elucidated via spectroscopy and spectrometry. Of the five, one was monomethylated (3), one was dimethylated (4), two were trimethylated (2 and 6), and one was tetramethylated (5). The relative potency of all compounds was determined in a 72 h growth-inhibition assay against a panel of three prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a human hepatoma cell line (Huh7.5.1) and compared to natural silybin B. Compounds also were evaluated for inhibition of both cytochrome P450 2C9 (CYP2C9) activity in human liver microsomes and hepatitis C virus infection in Huh7.5.1 cells. The monomethyl and dimethyl analogues were shown to have enhanced activity in terms of cytotoxicity, CYP2C9 inhibitory potency, and antiviral activity (up to 6-fold increased potency) compared to the parent compound, silybin B. In total, these data suggested that methylation of flavonolignans can increase bioactivity.     

Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation—the Phase II conjugation reaction—of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3′-phosphoadenosine-5′-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.     

Characterization of tautomeric limonoids from the fruits of Melia toosendan

Four nimbolinin-type limonoids, 12α/β-1-O-tigloyl-1-O-deacetyl-nimbolinin B (1), 1-deacetylnimbolinin B (2), nimbolinin B (3) and nimbolinin A (4), were isolated from the fruits of Melia toosendan. 1 was a new compound and existed as a mixture of a pair of tautomers, 12α- (1a) and 12β- (1b). The structures of both tautomers were fully determined by extensive spectroscopic methods including UV, IR, NMR and ESI-MS. Tautomeric behaviors and their relative molar ratios in compounds 1–4 were further investigated using optical rotation, TLC, ¹H NMR and HPLC. Equilibrium equation of nimbolinin was proposed accordingly, with 12α- and 12β-isomers interchanging via a 12-hemiacetal intermediate.     

Tyrosinase inhibitory study of flavonolignans from the seeds of Silybum marianum (Milk thistle)

Anti-melanogenesis effects of silymarin from milk thistle have been reported recently, but detailed tyrosinase inhibition properties of individual components have not been investigated. This study purported to substantiate tyrosinase inhibition and its mechanism based on a single metabolite. The responsible components for tyrosinase inhibition of target source were found out as flavonolignans which consist of isosilybin A (1), isosilybin B (2), silydianin (3), 2,3-dihydrosilychristin (4), silychristin A (5), silychristin B (6) and silybin (7), respectively. The isolated flavonolignans (1–7) inhibited both monophenolase (IC₅₀ = 1.7–7.6 µM) and diphenolase (IC₅₀ = 12.1–44.9 µM) of tyrosinase significantly. Their inhibitions were 10-fold effective in comparison with their mother skeletons (8–10). Inhibitory functions were also proved by HPLC analysis using N-acetyl-l-tyrosine as substrate. The predominant formation of E_met·I was confirmed from a long prolongation of lag time and a decrease of the static state activity of the enzyme. All tested compounds had a significant binding affinity to tyrosinase with K_SV values of 0.06–0.27 × 10⁴ L·mol⁻¹, which are well correlated with IC₅₀s. In kinetic study, all flavonolignan (1–7) were mixed type I (K_I < K_IS) inhibitors, whereas their mother skeletons (8–10) were competitive ones. The UPLC-ESI-TOF/MS analysis showed that the isolated inhibitors are the most abundant metabolites in the target plant.     

Australian marine sponge alkaloids as a new class of glycine-gated chloride channel receptor modulator

Chemical analysis of a specimen of the sponge Ianthella cf. flabelliformis returned two new sesquiterpene glycinyl lactams, ianthellalactams A (1) and B (2), the known sponge sesquiterpene dictyodendrillin (3) and its ethanolysis artifact ethyl dictyodendrillin (4), and five known sponge indole alkaloids, aplysinopsin (5), 8E-3′-deimino-3′-oxoaplysinopsin (6), 8Z-3′-deimino-3′-oxoaplysinopsin (7), dihydroaplysinopsin (8) and tubastrindole B (9). The equilibrated mixture 6/7 exhibited glycine-gated chloride channel receptor (GlyR) antagonist activity with a bias towards α3 over α1 GlyR, while tubastrindole B (9) exhibited a bias towards α1 over α3 GlyR. At low- to sub-micromolar concentrations, 9 was also a selective potentiator of α1 GlyR, with no effect on α3 GlyR—a pharmacology that could prove useful in the treatment of movement disorders such as spasticity and hyperekplexia. Our investigations into the GlyR modulatory properties of 1–9 were further supported by the synthesis of a number of structurally related indole alkaloids.     

A pair of new tetrahydro-naphthalenone enantiomers from Eremurus altaicus (Pall.) Stev.

A new racemic mixture of a 4-hydroxytetralone derivative, altaicusin A (1), was isolated from the whole plant of Eremurus altaicus (Pall.) Stev., together with three anthraquinones (compounds 2–4) and two naphthalene derivatives (5–6). The racemic altaicusin A (1) was further purified by chiral HPLC to yield a pair of enantiomers, (+)-(4S)-altaicusin A (1a) and (−)-(4R)-altaicusin A (1b). Their structures were established on the basis of spectroscopic analysis, including IR, HR-TOF-MS, and NMR. The absolute configurations of compounds 1a and 1b were elucidated by quantum chemical ECD calculations. Compounds 3 and 6 exhibited inhibitory activity against protein tyrosine phosphatase 1B (PTP1B).     

Light induced isomerization of piperlonguminine to scutifoliamide A,isopiperlonguminine and hoffmannseggiamide A

Light induced isomerization of piperlonguminine (1) to scutifoliamide A (2), isopiperlonguminine (3) and hoffmannseggiamide A (4) is reported in this work. In vivo antihyperlipidemic study showed that a mixture of 1 and 2 (1:1) had significantly decreased serum total cholesterol (TC) and triglyceride (TG) in rats, which were similar to those of the pure 1 and simvastatin. Additionally, 2 was less toxic on HepG2 liver cell than the 1 and simvastatin.     

Phenolic acid glycosides from Parmentiera cereifera Seem. (Candle tree)

Two new phenolic acid glycosides, parmentins A (1) and B (2) were isolated from the methanolic extract of the leaves and stems of candle tree (Parmentiera cereifera Seem). These compounds were accompanied by a mixture of β-sitosterol and stigmasterol (3), β-sitosterol glucoside (4), isovanillic acid (5), vanillic acid (6), and p-hydroxybenzoic acid (7). The structures of the isolated compounds were determined on the basis of physical and spectroscopic analyses, including 1D and 2D NMR (¹H, ¹³C, COSY, HSQC and HMBC) and mass spectrometry (HR-ESI-MS).     

N-Formyllapatin A,a new N-formylspiroquinazoline derivative from the marine-derived fungus Penicillium adametzioides AS-53

N-Formyllapatin A (1), a new spiroquinazoline derivative, and four known quinazoline metabolites, lapatins A (2) and B (3), prelapatin B (4), and glyantrypine (5), along with two known indolediketopiperazine derivatives, fumitremorgin B (6) and verruculogen (7), were characterized from Penicillium adametzioides AS-53, a fungus obtained from the fresh tissue of an unidentified marine sponge. The structure of compound 1 was established by detailed interpretation of NMR and MS data, and its absolute configuration was established by a single-crystal X-ray diffraction analysis. N-Formyllapatin A (1) represents the first N-formylspiroquinazoline secondary metabolite. Compounds 3 and 5–7 showed moderate inhibitory activity against aqua-pathogenic bacterial Vibrio harveyi.     

Neo-clerodane diterpenes from Ajuga turkestanica

The ethyl acetate extract of the aerial parts of Ajuga turkestanica afforded 6 neo-clerodane diterpenes, including two novel compounds, 14,15-dihydroajugachin B (1) and 14-hydro-15-methoxyajugachin B (2), in addition to the known diterpenoids chamaepitin (3), ajugachin B (4), ajugapitin (5) and lupulin A (6). Structures were established through exhaustive NMR spectroscopic analysis and chemical transformation in the case of 1. The full ¹H and ¹³C NMR assignment of the C-15 R and S configurations of 14-hydro-15-methoxyajugachin B and chamaepitin were elucidated.     

Three new phthalide glycosides from the rhizomes of Ligusticum chuanxiong

Three new phthalide glycosides, named chuanxiongoside A (1), chuanxiongoside B (2) and ligusticoside B (3) together with two known glycosides, celephthalide A (4) and icariside F₂ (5), were isolated from the rhizomes of Ligusticum chuanxiong. This is the first report of compounds 4⿿5 from the title plant. The structures of compounds 1⿿3 were identified using various spectroscopic methods.     

The co-occurrence of bufadienolides and podophyllotoxins from Helleborus thibetanus

Phytochemical investigation of Helleborus thibetanus led to the isolation of two new bufadienolides with a trans A/B ring fusion mode (1–2) and two known ones (3–4), three podophyllotoxins (5–7), two flavones (8 and 9), one phytoecdysteroid (10), one spirostane (11) and one pseudo-spirostane (12). The structures of the new compounds were identified using extensive spectroscopic analyses and the known compounds were identified by comparing the spectral data with those previously reported. Isolation of bufadienolides with a trans A/B ring fusion mode could be chemotaxonomic marker for the differentiation of H. thibetanus from the toad venom and other plants of this genus, on the other hand, co-occurrence of podophyllotoxin from H. thibetanus supported the inclusion of Berberidaceae and Ranunculaceae in the same monophyletic order Ranunculales based on the outgroup comparison and parsimony analysis.     

Isopimarane diterpenes and flavan derivatives from the twigs of Caesalpinia furfuracea

The first phytochemical investigation of Caesalpinia furfuracea twigs led to the isolation and identification of four new compounds including two isopimarane diterpenes, caesalfurfuric acids A (1) and B (2), and two flavans, (2R)-caesalflavans A (5) and B (6), together with four known compounds, 4-epi-isopimaric acid (3), methyl (E)-3-(3,4-dihydroxyphenyl)acrylate (4), (E)-resveratrol (7) and oxyresveratrol (8). Their structures were elucidated by intensive spectroscopic analysis. Compound 1 was found to exhibit antibacterial activity against MRSA SK1 with an MIC value of 16 μg/mL.     

Triterpene saponins and megastigmane glucosides from Camellia bugiamapensis

Two new triterpene saponins, camelliosides I and J (1 and 2), two new megastigmane glycosides, camellistigosides A and B (3 and 4), and two known megastigmane glycosides, icariside B₁ (5) and (6S,9R)-roseoside (6), were isolated from a methanol extract of the Camellia bugiamapensis leaves using various chromatographic separation techniques. Their structures were elucidated based on spectroscopic analyses, including HR ESI MS, CD, 1D and 2D NMR. Their inhibitory effects on LPS-induced NO production in RAW264.7 cells were evaluated. This is the first report of the chemical constituents and biological activity of C. bugiamapensis.     

Biphenyl and xanthone derivatives from the twigs of a Garcinia sp. (Clusiaceae)

The genus Garcinia is a well known rich source of bioactive xanthones and benzophenones. Some species of this genus also produce flavonoids and biphenyls as minor constituents. In this study, two new biphenyls, doitungbiphenyls A (1) and B (2), along with two biphenyls, schomburgbiphenyl (3) and nigrolineabiphenyl B (4); and four xanthones, 1,3,6-trihydroxy-8-isoprenyl-7-methoxyxanthone (5), morusignin K (6), 1,5-dihydroxyxanthone (7), and 1,7-dihydroxyxanthone (8), were isolated from the acetone extract of the twigs of a Garcinia sp. Their structures were characterized extensively by 1D and 2D NMR spectroscopy and HR-EI-MS. The cytotoxicity of the two new biphenyls against the oral cavity cancer (KB) and the breast cancer (MCF7) cell lines was also evaluated.     

Acylphloroglucinol derivatives from Hypericum laricifolium Juss

A new dimeric acylphloroglucinol derivative, hyperlaricifolin (1), and two new monomeric acylphloroglucinols, laricifolin A (2) and laricifolin B (3), were isolated from an extract of the aerial parts of Hypericum laricifolium Juss., along with the four known dimeric acylphloroglucinols uliginosin B, isouliginosin B, hyperbrasilol B, and isohyperbrasilol B. Their structures were elucidated by interpretation of their 1D-, 2D-NMR and mass spectra.     

Two new flavonol glycosides from the leaves of Cleome viscosa L.

From the leaves of Cleome viscosa L., two new flavonol glycosides, named visconoside A (1) and visconoside B (2), together with six known flavonol glycosides, vincetoxicoside A (3), vincetoxicoside B (4), kaempferitrin (5), kaempferide 3-O-β-d-glucopyranoside 7-O-α-l-rhamnopyranoside (6), kaempferol 3-O-β-d-glucopyranoside 7-O-α-l-rhamnopyranoside (7), and isorhamnetin 3-O-β-d-glucopyranoside (8) were isolated by various chromatography methods. Its chemical structure was elucidated by IR, UV, HR-ESI-MS, NMR 1D and 2D experiments and compared with literatures.     

Anti-HBV active constituents from Piper longum

In the screening search for Hepatitis B virus inhibitory agents from medicinal plants, the ethanol extract of Piper longum Linn. was found to possess superior anti-HBV activity in vitro. Bioassay-guided fractionation coupled with repeated purification resulted in the isolation of four new compounds, involving two new glycosides longumosides A (1) and B (2) and two new amide alkaloids erythro-1-[1-oxo-9(3,4-methylenedioxyphenyl)-8,9-dihydroxy-2E-nonenyl]-piperidine (3), threo-1-[1-oxo-9(3,4-methylenedioxyphenyl)-8,9-dihydroxy-2E-nonenyl]-piperidine (4), as well as two compounds 3β,4α-dihydroxy-2-piperidinone (5), 5,6-dihydro-2(1H)-pyridinone (6) from natural source for the first time. The structures of the four new compounds were determined by extensive analyses of the MS, IR, 1D and 2D NMR data. Besides, the compounds 2–6, together with the known compounds 7–11 obtained previously, were assayed for their anti-HBV activity by using Hep G 2.2.15 cell line in vitro. Results suggested the compound piperine (7) possessed remarkable inhibitory HBV activity, against the secretion of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) with the Selectivity Index (SI) values of 15.7 and 16.8, respectively.