Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229

Ginsenoside Rb1 is the most abundant ginsenoside in Panax (ginseng). The hydrolysis of this ginsenoside produces compound K, the biologically active ginsenoside of ginseng. We previously identified a fungus Paecilomyces Bainier sp. 229 (sp. 229), which can efficiently convert ginsenoside Rb1 to compound K. In this report, the ginsenoside hydrolyzing β-glucosidases were isolated from sp. 229 and the pathway of the biotransformation of ginsenoside Rb1 to compound K by sp. 229 was investigated. Based on reverse-phase HPLC and TLC analysis, we found the main metabolic pathway is as follows: ginsenoside Rb1 → ginsenoside Rd → ginsenoside F2 → compound K. Moreover, the results showed that there were other metabolic pathways: ginsenoside Rb1 → ginsenoside XVII → ginsenoside F2 → compound K and ginsenoside Rb1 → ginsenoside Rg3 → ginsenoside Rh2. These processes would allow the specific bioconversion of ginsenoside Rb1 to various ginsenosides using an appropriate combination of specific microbial enzymes.     

Changes of ginsenosides in Korean red ginseng (Panax ginseng) fermented by Lactobacillus plantarum M1

To obtain microorganisms for the microbial conversion of ginsenosides in red ginseng powder (RGP), Lactobacillus species (M1–M4 and P1–P4) were isolated from commercial ginseng products. Strain M1 was determined to be L. plantarum by 16S rRNA sequencing. Red ginseng powder (RGP) fermented by L. plantarum M1 had a high total content of ginsenosides (142.4 mg/g) as compared to the control (121.8 mg/g). In particular, the ginsenoside metabolites Rg3, Rg5, Rk1, Compound K (CK), Rh1, and Rg2 showed a high level in the fermented RGP (65.5 mg/g) compared to the control (32.7 mg/g). During fermentation for 7 days, total sugar content decreased from 8.55 mg/g to 4 mg/g, uronic acid content reached its maximum (53.43 μg/g) at 3 days, and total ginsenoside content increased to 176.8 mg/g at 4 days. In addition, ginsenoside metabolites increased from 38.0 mg/g to 83.4 mg/g at 4 days of fermentation. Using everted instestinal sacs of rats, the fermented red ginseng showed a high transport level (10.3 mg of polyphenols/g sac) compared to non-fermented red ginseng (6.67 mg of polyphenols/g sac) after 1 h. These results confirm that fermentation with L. plantarum M1 is very useful for preparing minor ginsenoside metabolites while being safe for foods.     

Triterpene glycosides from Astragalus icmadophilus

Six cycloartane-type triterpene glycosides were isolated from Astragalus icmadophilus along with two known cycloartane-type glycosides, five known oleanane-type triterpene glycosides and one known flavonol glycoside. The structures of the six compounds were established as 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxy cycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3,4-diacetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane by the extensive use of 1D- and 2D-NMR experiments along with ESIMS and HRMS analysis.The first four compounds are cyclocanthogenin and cycloastragenol glycosides, whereas the last two are based on cyclocephalogenin as aglycone, more unusual in the plant kingdom, so far reported only from Astragalus spp.     

Excited state properties of dinaphthyl ditelluride

Di-1-naphthyl ditelluride (Te₂naphthyl₂) is characterized by two low-energy excited states. The corresponding electronic transitions nTe → σ¹ Te–Te and nTe → π¹ naphthyl CT give rise to absorptions at λ_max = 403 and 311 nm, respectively. In solution nTe → σ¹ excitation leads to the cleavage of the Te–Te bond. In contrast to Te₂naphthyl₂ in the dissolved state the solid compound shows a luminescence (λ_max = 576 nm) which originates from nTe → π¹ naphthyl CT triplet.     

Highly glycosylated flavonols with an O-linked branched pentasaccharide from Iberis saxatilis (Brassicaceae)

Four flavonol glycosides isolated from non-flowering leafy shoots of Iberis saxatilis (Brassicaceae) were characterised by spectroscopic and chemical methods as saxatilisins A–D, the 3-O-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside, 3-O-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside, 3-O-(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside, and 3-O-(6-O-E-feruloyl)-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside of isorhamnetin (3,5,7,4′-tetrahydroxy-3′-methoxyflavone), respectively. Analysis of ²J_HC correlations detected with the H2BC (heteronuclear two-bond correlation) pulse sequence aided the unambiguous assignment of glycosidic resonances in the ¹H and ¹³C NMR spectra of these compounds. Saxatilisins A, C, and D, are the first flavonol glycosides to be described with a pentasaccharide chain at a single glycosylation site. Several pentaglycosides of kaempferol and quercetin, tentatively assigned as saxatilisin analogues from LC–MS/MS analyses, were present as minor constituents of the extracts.     

New cytotoxic steroidal saponins from Cestrum parqui

Two new steroidal saponins, 25(R)-3β [(O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 2)-O-[β-d-xylopyranosyl-(1 → 3)-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranosyl)oxy]-5α, 15β, 22R, 25R-spirostan-3,15-diol (1, named parquispiroside) and 25R-26-[(β-d-glucopyranosyl)Oxy]-(3β [(O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 2)-O-[β-d-xylopyranosyl-(1 → 3)-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranosyl)oxy], 5α, 15β, 22R, 25R)-furostane-3,15,22-triol (2, named parquifuroside), along with the known saponins, capsicoside D (3) and 22-OMe-capsicoside D (4) and the known glycoside, benzyl primeveroside (5), were isolated from the leaves of Cestrum parqui. The structures of these compounds were elucidated by careful analysis of 1D and 2D NMR spectra and ESIMS data. Parquispiroside (1) exhibited moderate inhibition of Hela, HepG2, U87, and MCF7 cell lines with IC₅₀ values in the range of 3.3–14.1 μM.     

Calcium protection of PS2 complex of Phaseolus coccineus from cadmium toxicity: In vitro study

The action of 10 and 20 mM Ca against harmful Cd effect on PS2 complex isolated from leaves of Phaseolus coccineus L. cv. Pi?kny Ja? was studied. The changes in fast chlorophyll a fluorescence induction kinetics and protein composition of PS2 complex were the symptoms of Cd toxicity and Ca protection of PS2 complex. Calcium applied at 10 mM concentration prevented F₀ reduction caused by the presence of 250–1000 μM Cd in the incubation mixture, but that of (the variable chlorophyll a fluorescence) F_v, F_m, F_v/F₀, and F_v/F_m only at 250 μM Cd. Ca concentration doubling in the incubation mixture resulted in complete overcoming the toxicity of 250–1000 μM Cd to F_v and F_m. However, the protection of F_v/F₀ and the photochemical efficiency of PS2 (F_v/F_m) from 1000 μM Cd was only partial even at 20 mM Ca. A protective effect of 10 mM Ca on D1, D2 and 17 kDa proteins was found in PS2 complex exposed to 250 μM Cd, and on 43 kDa protein in the complex treated with 500 μM Cd. However, 20 mM Ca counteracted the toxicity of 500 μM Cd to the 43, 47 and 17 kDa proteins, as well as the harmful effect of 1000 μM Cd on 47 and 17 kDa ones.     

Triterpene saponins from Antonia ovata leaves

Six pentacyclic triterpenoid saponins, named antoniosides E–J along with two known alkaloids, were isolated from the leaves of Antonia ovata. Their structures were determined by the extensive use of 1D and 2D-NMR experiments along with HRESIMS analysis and acid hydrolysis. All isolated saponins contained the same pentasaccharide chain: 3-O-[β-d-glucopyranosyl-(1 → 2)]-[β-d-glucopyranosyl-(1 → 4)]-[β-d-glucopyranosyl-(1 → 3)-α-l-arabinopyranosyl(1 → 6)]-β-d-glucopyranoside, linked at C-3 of esterified derivatives of polyhydroxyoleanene triterpenoids (theasapogenol A and 15α-hydroxy-theasapogenol A). Isolated compounds were evaluated for their cytotoxic activity against KB cell line by a WST-1 assay, and the IC₅₀ values ranged from 3.3 to 5.3 μM.     

Three new flavonoid glycosides from the aerial parts of Graptophyllum grandulosum Turril (Acanthaceae)

Grandulosides A-C, three new flavonoid glycosides, were isolated from the aerial parts of Graptophyllum grandulosum Turill and identified as chrysoeriol-7-O-β-d-apiofuranosyl-(1 → 2)-β-d-xylopyranoside (1), chrysoeriol-7-O-[4′′′-O-acetyl-β-d-apiofuranosyl-(1 → 2)]-β-d-xylopyranoside (2) and 7-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-(4′′-Sodium hydrogeno sulfate) glucopyranoside (3). Four known compounds, chrysoeriol-7-O-β-d-xyloside (4), isorhamnetin-3-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside (5), luteolin-7-O-β-d-apiofuranosyl-(1 → 2)-β-d-xylopyranoside (6) and sucrose (7) were also obtained. The structures of these compounds were established by interpretation of their spectral data, mainly HR-TOFESIMS, 1D-NMR (¹H, ¹³C) and 2D-NMR (COSY, NOESY, HSQC and HMBC) and by comparison with the literature data.     

Genetic differentiation in Pyrenophora teres populations measured with AFLP markers

The genetic structure and occurrence of mating types and forms of Pyrenophora teres, the causal agent of net blotch on barley, was studied among 278 isolates collected from the northern hemisphere and from Australia. Genetic differentiation was high (F_CT 0.238, P = 0.002) between P. teres f. teres (PTT) isolates originating from Northern Europe, North America, Russia and Australia. The P. teres population in Australia was clearly divided into two subgroups (F_CT 0.793, P < 0.001) according to the form identity: PTT and P. teres f. maculata (PTM), with the PTT samples showing a greater degree of differentiation (F_ST 0.573, P < 0.001) among Australian states than the PTM samples (F_CT 0.219, P < 0.001). No differentiation was found among locations within Australian states. Both mating types (MAT1 and MAT2) were equally common (1:1) in several locations in Australia and in Finland. The only exception was Krasnodar, Russia, where only MAT2 was identified. Our results show that the prevalence of sexual reproduction, occurrence of forms of P. teres, and genetic differentiation between geographical regions are highly variable. The paper discusses the various effects and outcomes of population selection in Australia and in the northern barley growing regions.     

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs)

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC₅₀ = 292.17 ± 27.71 μM and 331.94 ± 21.21 μM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC₅₀ = 275.24 ± 13.15 μM). These values are significantly lower than those of ascorbic acid (EC₅₀ = 1129.32 ± 88.79 μM) and α-tocopherol (EC₅₀ = 944.62 ± 148.00 μM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68 ± 0.27; synthetic (S,S)-SDG-1: 2.09 ± 0.16; synthetic (R,R)-SDG-2: 1.96 ± 0.27], peroxyl [natural (S,S)-SDG-1: 2.55 ± 0.11; synthetic (S,S)-SDG-1: 2.20 ± 0.10; synthetic (R,R)-SDG-2: 3.03 ± 0.04] and DPPH [natural (S,S)-SDG-1: EC₅₀ = 83.94 ± 2.80 μM; synthetic (S,S)-SDG-1: EC₅₀ = 157.54 ± 21.30 μM; synthetic (R,R)-SDG-2: EC₅₀ = 123.63 ± 8.67 μM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.     

Triterpene glycosides from Agrostemma gracilis

Four triterpene saponins, agrostemmosides A–D were isolated from the methanol extract of Agrostemma gracilis. The structures of the compounds were determined as 3-O-β-d-xylopyranosyloleanolic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-xylopyranosyloleanolic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-β-d-xylopyranosylechinocystic acid 28-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-β-d-xylopyranosylechinocystic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester by a combination of one- and two-dimensional NMR techniques, and mass spectrometry. To the best of our knowledge this is the first phytochemical report on A. gracilis, and echinocystic acid saponins were encountered for the first time in Caryophyllaceae family.     

Distinct chemotypes of Tephrosia vogelii and implications for their use in pest control and soil enrichment

Tephrosia vogelii Hook. f. (Leguminosae) is being promoted as a pest control and soil enrichment agent for poorly-resourced small-scale farmers in southern and eastern Africa. This study examined plants being cultivated by farmers and found two chemotypes. Chemotype 1 (C1) contained rotenoids, including deguelin, rotenone, sarcolobine, tephrosin and α-toxicarol, required for pest control efficacy. Rotenoids were absent from chemotype 2 (C2), which was characterised by prenylated flavanones, including the previously unrecorded examples (2S)-5,7-dimethoxy-8-(3-hydroxy-3-methylbut-1Z-enyl)flavanone, (2S)-5,7-dimethoxy-8-(3-methylbut-1,3-dienyl)flavanone, (2S)-4′-hydroxy-5-methoxy-6″,6″-dimethylpyrano[2″,3″:7,8]flavanone, (2S)-5-methoxy-6″,6″-dimethyl-4″,5″-dihydrocyclopropa[4″,5″]furano[2″,3″:7,8]flavanone, (2S)-7-hydroxy-5-methoxy-8-prenylflavanone, and (2R,3R)-3-hydroxy-5-methoxy-6″,6″-dimethylpyrano[2″,3″:7,8]flavanone. The known compounds (2S)-5-methoxy-6″,6″-dimethylpyrano[2″,3″:7,8]flavanone (obovatin 5-methyl ether) and 5,7-dimethoxy-8-(3-hydroxy-3-methylbut-1Z-enyl)flavone (Z-tephrostachin) were also found in C2. This chemotype, although designated Tephrosia candida DC. in collections originating from the World Agroforestry Centre (ICRAF), was confirmed to be T. vogelii on the basis of morphological comparison with verified herbarium specimens and DNA sequence analysis. Sampling from 13 locations in Malawi where farmers cultivate Tephrosia species for insecticidal use indicated that almost 1 in 4 plants were T. vogelii C2, and so were unsuitable for this application. Leaf material sourced from a herbarium specimen of T. candida contained most of the flavanones found in T. vogelii C2, but no rotenoids. However, the profile of flavonol glycosides was different to that of T. vogelii C1 and C2, with 6-hydroxy-kaempferol 6-methyl ether as the predominant aglycone rather than kaempferol and quercetin. The structures of four unrecorded flavonol glycosides present in T. candida were determined using cryoprobe NMR spectroscopy and MS as the 3-O-α-rhamnopyranosyl(1 → 6)-β-galactopyranoside-7-O-α-rhamnopyranoside, 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside, 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside, and 3-O-α-rhamnopyranosyl(1 → 2)[(3-O-E-feruloyl)-α-rhamnopyranosyl(1 → 6)]-β-galactopyranosides of 6-hydroxykaempferol 6-methyl ether. Tentative structures for a further 37 flavonol glycosides of T. candida were assigned by LC–MS/MS. The correct chemotype of T. vogelii (i.e. C1) needs to be promoted for use by farmers in pest control applications.     

Simultaneous determination of tolbutamide,omeprazole, midazolam and dextromethorphan in human plasma by LC–MS/MS—A high throughput approach to evaluate drug–drug interactions

Drug–drug interactions involving cytochrome P450 (CYP450s) are an important factor for evaluation of a new chemical entity (NCE) in drug development. To evaluate the potential inhibitory effects of a NCE on the pharmacokinetics of a cocktail of representative probes of CYP enzymes (midazolam for CYP3A4, tolbutamide for CYP2C9, omeprazole for CYP2C19 and dextromethorphan for CYP2D6) and the safety and tolerability of the NCE in the presence of probe substrates, a high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tolbutamide, omeprazole, midazolam and dextromethorphan in human plasma using tolbutamide-d₉, midazolam-d₄, (±)-omeprazole-d₃, and dextromethorphan-d₃ as the internal standards (ISs). Human plasma samples of 50 μL were extracted by a simple protein-precipitation procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. Reversed-phase HPLC separation was achieved with a Hypersil GOLD AQ column (50 mm × 4.6 mm, 5 μm). MS/MS detection was set at mass transitions of 271 → 172 m/z for tolbutamide, 346 → 198 m/z for omeprazole, 326 → 291 m/z for midazolam, 272 → 171 m/z for dextromethorphan, 280 → 172 m/z for tolbutamide-d₉ (IS), 349 → 198 m/z for (±)-omeprazole-d₃ (IS), 330 → 295 m/z for midazolam-d₄ (IS), and 275 → 171 m/z for dextromethorphan-d₃ (IS) in positive mode. The high throughput LC–MS/MS method was validated for accuracy, precision, sensitivity, stability, recovery, matrix effects, and calibration range. Acceptable intra-run and inter-run assay precision (<10%) and accuracy (<10%) were achieved over a linear range of 50–50,000 ng/mL for tolbutamide, 1–1000 ng/mL for omeprazole, 0.1–100 ng/mL for midazolam and 0.05–50 ng/mL for dextromethorphan in human plasma. Method robustness was demonstrated by the 100% pass rate of 24 incurred sample analysis runs and all of the 50 clinical study samples used for incurred sample reproducibility (ISR) test having met the acceptance criterion (%Diff within 20%). The overall ISR results for all compounds showed that over 95% of the samples had a %Diff of less than 10%. The method is simple, rapid and rugged, and has been applied successfully to sample analysis in support of a drug–drug interaction study.     

Rheediinosides A and B,two antiproliferative and antioxidant triterpene saponins from Entada rheedii

Two triterpenoid saponins have been isolated from the seed kernels of Entada rheedii. Their structures have been established using 1D- and 2D-NMR and mass spectrometry as 3-O-β-d-xylopyranosyl-(1 → 3)-O-α-l-arabinopyranosyl-(1 → 6)-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside A, 1) and 3-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-xylopyranosyl-(1 → 3)-α-l-arabinopyranosyl-(1 → 6)]-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside B, 2). Compounds 1 and 2 were tested for their antiproliferative activity against T98G, A431, PC3 and B16-F1 cell lines, and further for their antioxidant properties. Moderate cytotoxic potency and antioxidant properties were found for these compounds whereas Rheediinoside B was in all assays more active than Rheediinoside A.     

Enzymatically derived aldouronic acids from Eucalyptus globulus glucuronoxylan

A glucuronoxylan was extracted from the holocellulose of Eucalyputus globulus wood with 10% KOH and subjected to hydrolysis by a commercial cellulase preparation “Meicelase”. Neutral xylooligosaccharides liberated were analyzed by size exclusion chromatography. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were studied by monosaccharide analysis, comparison of volume distribution coefficients (Dvs) in anion exchange chromatography with those of the authentic samples, and ¹H and ¹³C NMR spectroscopy, resulting in the characterization of seven aldouronic acids including a novel one containing galactose residue.O-β-d-Xylp-(1 → 4)-[O-(4-O-Me-α-d-GlcAp)-(1 → 2)]-O-β-d-Xylp-(1 → 4)-O-β-d-Xylp-(1 → 4)-d-XylO-(4-O-Me-α-d-GlcAp)-(1 → 2)-O-β-d-Xylp-(1 → 4)-O-β-d-Xylp-(1 → 4)-d-XylO-(4-O-Me-α-d-GlcAp)-(1 → 2)-O-β-d-Xylp-(1 → 4)-d-XylO-β-d-Xylp-(1 → 4)-O-β-d-Xylp-(1→3)-O-α-l-Rhap-(1 → 2)-O-α-l-GalAp-(1 → 4)-d-XylO-β-d-Xylp-(1 → 4)-O-β-d-Xylp-(1 → 3)-O-α-l-Rhap-(1 → 2)-d-GalAO-β-d-Xylp-(1 → 3)-O-α-l-Rhap-(1 → 2)-O-α-d-GalAp-(1 → 4)-d-XylO-β-d-Galp-(1 → 2)-O-(4-O-Me-α-d-GlcAp)-(1 → 2)-O-β-d-Xylp-(1 → 4)-O-β-d-Xylp-(1 → 4)-d-Xyl.The oligosaccharides liberated provide information on multiplicity of xylanases secreted by Trichoderma viride. The presence of the last aldouronic acid shows a structural feature of E. globulus xylan.     

New triterpene saponins from the seed cake of Camellia Oleifera and their cytotoxic activity

Two new oleanane-type triterpene saponins, identified as 16α-hydroxy-22-O-angeloyl-23-formyl-28,31-dihydroxymethylene-olean-12-ene-3β-O-{β-d-galactopyranosyl-(1 → 2)[β-d-xylopyranosyl-(1 → 2)-α-l-arabinopyranosyl(1 → 3)]-β-d-glucopyranosiduronic acid} (oleiferasaponin B₁, 1) and 22-O-hydrocinnamoyl-23-formyl-28-dihydroxymethylene-olean-12-ene-3β-O-{β-d-glucopyranosyl-(1 → 2)[β-d-xylopyranosyl-(1 → 2)-α-l-arabinopyranosyl(1 → 3)]-β-d-glucopyranosiduronic acid} (oleiferasaponin B₂, 2), were isolated from the seed cake of Camellia oleifera Abel. Their structures were established by extensive 1D- and 2D-NMR experiments along with TOF-MS analysis and acid hydrolysis. The cytotoxicity of the isolated compounds was evaluated in four human carcinoma cell lines: A 549, SK-OV-3, SK-MEL-2 and HCT15. Both compounds 1 and 2 exhibited significantly cytotoxic activity with IC₅₀ values of 18.5 μM (A549), 11.3 μM (SK-OV-3), 13.9 μM (SK-MEL-2) and 1.6 μM (HCT15) for 1 and IC₅₀ values of 8.4 μM (A549), 6.3 μM (SK-OV-3), 9.2 μM (SK-MEL-2) and 0.8 μM (HCT15) for 2. In addition, compound 2 showed more effective cytotoxic activity than compound 1.     

Synthesis and in vitro activities of a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via an octadecylglycerol residue

To prepare a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via a lipophilic octadecylglycerol residue we condensed 1-O-4-monomethoxytrityl-3-O-octadecyl-sn-glycerol-2-hydrogenphosphonate obtained from 3-O-octadecyl-sn-glycerol with AZT by the phosphonate method. The purified condensation product was de-tritylated resulting in 3′-azido-3′-deoxythymidylyl-(5′ → 2-O)-3-O-octadecyl-sn-glycerol, followed by treatment with (ethoxycarbonyl)phosphoric dichloride. The resulting 3′-azido-3′-deoxy-thymidylyl-(5′ → 2)-3-O-octadecyl-sn-glycerol-1-O-(ethoxycarbonyl)phosphonate was purified by preparative RP-18 column chromatography. The antiviral duplex drug 3′-azido-3′-deoxythymidylyl-(5′ → 2-O)-3-O-octadecyl-sn-glycerol-1-O-phosphonoformate trisodium salt (AZT–lipid–PFA) was obtained after alkaline cleavage of the phosphonoformate ethylester residue. The overall yield of the five step synthesis performed at gram scale was about 30%. According to a supposed pathway AZT–lipid–PFA could be cleaved to yield a mixture of different antiviral compounds such as AZT, AZT-5′-monophosphate, octadecylglycerol–AZT, PFA and octadecylglycerol–PFA, possibly producing additive and/or synergistic antiviral effects. In vitro studies showed that the duplex drug exhibits antiviral activities against HIV and especially against drug-resistant strains and clinical isolates of HSV and HCMV. The E₅₀ values of AZT–lipid–PFA against HIV ranged between 170 and 200 nM. The half-maximal inhibitory doses (IC₅₀) against highly acyclovir (ACV)-resistant HSV isolates determined by a plaque reduction assay ranged between 1.87 and 4.59 μM. Using ganciclovir (GCV)-sensitive, GCV resistant and drug cross-resistant HCMV strains the IC₅₀-values of AZT–lipid–PFA were between 2.78 and 1.18 μM. With regard to PFA, the IC₅₀-value of AZT–lipid–PFA determined on a multi-drug-resistant HCMV strain was about 90-fold lower than that of PFA, demonstrating the superior antiviral effect of the duplex-drug.     

Simultaneous HPLC–MS–MS quantification of 8-iso-PGF2α and 8,12-iso-iPF2α in CSF and brain tissue samples with on-line cleanup

Quantitation of isoprostanes such as 8-iso-PGF_2α and 8,12-iso-iPF_2α-VI in biological fluids has been proposed as a reliable test of oxidant stress and inflammation in a variety of disorders. This paper presents a liquid chromatography method with tandem mass spectrometry detection for the simultaneous analysis of these two isoprostanes in human CSF and brain tissue samples. An API 5000 triple quadrupole instrument (AB Sciex, Foster City, CA, USA) with an APCI ion source was used in this study. Aliquots of CSF samples (0.25 mL) were treated with a methanol:zinc sulfate mixture followed by on-line cleanup on an extraction column (Validated-C₁₈) with 0.1% formic acid. The brain tissue samples were homogenized and lipids were extracted using Folch solution. Solid-phase extraction columns (C₁₈) were used for the purification of the brain isoprostane fraction. Chromatographic separation was achieved using an analytical column (Synergi C₁₈ HydroRP) with 0.1% formic acid in water and a mixture of methanol:acetonitrile under isocratic conditions. The mass spectrometer was operated in the MRM scan and negative ion mode. The quadrupoles were set to detect the molecular ions [M?H]^? and high mass fragments of isoprostanes: m/z 353 → 193 amu (8-iso-PGF_2α) and m/z 353 → 115 amu (8,12-iso-iPF_2α-VI) and their deuterated internal standards: m/z 357 → 197 amu (8-iso-PGF_2α-d₄) and m/z 364 → 115 amu (8,12-iso-iPF_2α-VI-d₁₁). The lower limit of quantification was 2.5 pg/mL for 8-iso-PGF_2α and 5.0 pg/mL for 8,12-iso-PF_2α-VI for the CSF method and 10.0 pg/0.1 g of tissue and 30.0 pg/0.1 g of tissue for 8-iso-PGF_2α and 8,12-iso-iPF_2α-VI, respectively, for the brain tissue method. No ion suppression or enhancement of the detection of 8-isoPGF_2α, 8,12-isoPF_2α-VI or both internal standards was found.     

Antiplatelet aggregation triterpene saponins from the leaves of Ilex kudingcha

Two new ursane-type triterpene saponins, 3-O-β-d-glucopyranosyl(1 → 3)-[α-l-rhamnopyranosyl(1 → 2)]-α-l-arabinopyranosylurs-12,19(29)-dien-28-oic acid 28-O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranosyl ester (1) and 3-O-β-d-glucopyranosyl(1 → 3)-[α-l-rhamnopyranosyl(1 → 2)]-α-l-arabinopyranosyl-19α,20α-dihydroxyurs-12-en-28-oic acid 28-O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranosyl ester (2), along with thirteen known triterpene saponins were isolated from the n-BuOH part of the MeOH extraction of the leaves of Ilex kudingcha C.J. Tseng (also called “Ku-Ding-Cha”). The structures of new compounds were elucidated on the basis of detailed spectroscopic analysis, including HR-ESI-TOF-MS, 1D and 2D-NMR experiments, and by acid hydrolysis. All the compounds were screened for antiplatelet aggregation activity in vitro, and compounds 1, 2, 3, 7, 12 and 15 showed significant inhibition of platelet aggregation induced by ADP (5 μM) with IC₅₀ values of 14.7 ± 3.7, 11.3 ± 2.5, 17.4 ± 4.6, 20.5 ± 3.1, 8.1 ± 1.5 and 18.9 ± 4.2 μM, respectively.