Synthesis, Antigenicity Against Human Sera and Structure-Activity Relationships of Carbohydrate Moieties from <em>Toxocara</em> larvae and Their Analogues

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galβ1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite Toxocara and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meβ1-3GalNAcα1-OR (A), Fucα1-2Gal4Meβ1-3GalNAcα1-OR (B), Fuc2Meα1-2Galβ1-3GalNAcα1-OR (C), Fucα1-2Galβ1-3GalNAcα1-OR (D) and a disaccharide Fuc2Meα1-2Gal4Meβ1-OR (E) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1?3)-2-azide-4-O-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the O-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides A, B, C and E have high serodiagnostic potential to detect infections caused by Toxocara larvae.     

Activity-Guided Isolation of Antioxidant Compounds from <em>Rhizophora apiculata</em>

Rhizophora apiculata (R. apiculata) contains an abundance of biologically active compounds due its special salt-tolerant living surroundings. In this study, the total phenolic content and antioxidant activities of various extract and fractions of stem of R. apiculata were investigated. Results indicated that butanol fraction possesses the highest total phenolic content (181.84 mg/g GAE/g dry extract) with strongest antioxidant abilities. Following in vitro antioxidant activity-guided phytochemical separation procedures, lyoniresinol-3α-O-β-arabinopyranoside (1), lyoniresinol-3α-O-β-rhamnoside (2), and afzelechin-3-O-L-rhamno-pyranoside (3) were separated from the butanol fraction. These compounds showed more noticeable antioxidant activity than a BHT standard in the DPPH, ABTS and hydroxyl radical scavenging assays. HPLC analysis results showed that among different plant parts, the highest content of 1-3 was located in the bark (0.068%, 0.066% and 0.011%, respectively). The results imply that the R. apiculata might be a potential source of natural antioxidants and 1-3 are antioxidant ingredients in R. apiculata.     

Three New Myrsinol Diterpenes from <em>Euphorbia prolifera</em> and Their Neuroprotective Activities

Three new myrsinol diterpenes were isolated from the roots of Euphorbia prolifera. Their structures were elucidated as 2α-O-isobutyryl-3β,5α,7β,10,15β-penta-O-acetyl-14α-O-benzoyl-10,18-dihydromyrsinol (1), 2α-O-isobutyryl-3β-O-propion-yl-5α,7β,10,15β-tetra-O-acetyl-10,18-dihydromyrsinol (2), and 2α,14α-di-O-benzoyl-3β,5α,7β,10,15β-penta-O-acetyl-10,18-dihydromyrsinol (3) on the basis of spectroscopic data analyses (IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR). Their neuroprotective activities were evaluated and compounds 1 and 2 showed neuroprotective effects against MPP⁺-induced neuronal cell death in SH-SY5Y cells.     

Two New Daucane Sesquiterpenoids from <em>Daphne aurantiaca</em>

Two new daucane sesquiterpenoids 1β,2β-epoxy-10(H)α-dauca-11(12)-ene-7α,14-diol (1) and 1α,2α-epoxy-10(H)α-dauca-11(12)-ene-7α,14-diol (2) were isolated from the plateau medicinal plant Daphne aurantiaca Diels. (Thymelaeceae). Their structures were elucidated by 1D and 2D NMR spectroscopy, as well as HR-ESI-MS data.     

Platelet-Activating Factor (PAF) Antagonistic Activity of a New Biflavonoid from <em>Garcinia nervosa</em> var. <em>pubescens</em> King

The methanol extract of the leaves of Garcinia nervosa var. pubescens King, which showed strong inhibitory effects on platelet-activating factor (PAF) receptor binding, was subjected to bioassay-guided isolation to obtain a new biflavonoid, II-3,I-5, II-5,II-7,I-4',II-4'-hexahydroxy-(I-3,II-8)-flavonylflavanonol together with two known flavonoids, 6-methyl-4'-methoxyflavone and acacetin. The structures of the compounds were elucidated by spectroscopic methods. The compounds were evaluated for their ability to inhibit PAF receptor binding to rabbit platelets using ³H-PAF as a ligand. The biflavonoid and acacetin showed strong inhibition with IC₅₀ values of 28.0 and 20.4 μM, respectively. The results suggest that these compounds could be responsible for the strong PAF antagonistic activity of the plant.     

Constituents of Clinopodium Umbrosum

The isolation and identification of fifteen crystalline components from the whole herb of Clinopodium umbrosum (Bieb.) C. Koch (Labiatae) are described. Their structures were determined on the basis of spectral evidence and chemical transformation. These compounds include five steroids (α-spinasterone, β-sitosterol, stigmasterol, α-spinasterol, and α-spinasteryl-3-O-β-glucopyranoside), four triterpenoids (3β-hydroxyurs-11-en-28,13-olide, betulinic acid, oleanolic acid, ursolic acid), four flavonoids (luteolin, luteolin-7-O-β-glucopyranoside, apigenin-7-O-β-glucuronide, and apigenin-7-O-β-methylglucuronate), and two lignolic acids [3-(3,4-dihydroxyphenyl)- lactic acid and rosmarinic acid].     

Cycloascauloside b from <Emphasis Type="Italic">Astragalus caucasicus</Emphasis>

The structure of a new cycloartane glycoside isolated from leaves of Astragalus caucasicus Pall. (Leguminosae) was elucidated using chemical transformations and spectral data. Cycloascauloside B is 20R, 25-epoxy-24S-cycloartan-3β,6α,16β,24-tetraol 3-O-[α-L-rhamnopyranosyl-(1å2)]-β-D-glucopyranoside.     

Diterpenoids from the Buds of <em>Pinus banksiana</em> Lamb

Three new diterpenoids, namely 7α-hydroxyabieta-8,11,13,15-tetraen-18-oic acid, 7β,15,18-trihydroxyabieta-8,11,13-triene, 13,15-dihydroxypodocarpa-8,11,13-triene, and 12 other known compounds were isolated from buds of Pinus banksiana Lamb. All these compounds, except for 7-oxodehydroabietinol, were isolated for the first time from this plant. Their structures were elucidated by detailed spectroscopic studies and comparison with published data. All isolated compounds were tested for cytotoxic and antibacterial activities. Overall, two compounds, 7-oxodehydroabietinol and 18-nor-4,15-dihydroxyabieta-8,11,13-trien-7-one, showed moderate cytotoxicity against a human lung carcinoma cell line.     

Cytotoxic Podophyllotoxin Type-Lignans from the Steam Bark of <em>Bursera fagaroides </em>var. <em>fagaroides</em>

The hydroalcoholic extract of the steam bark of B. fagaroides var. fagaroides displayed potent cytotoxic activity against four cancer cell lines, namely KB (ED₅₀ = 9.6 × 10^-2 μg/mL), PC-3 (ED₅₀ = 2.5 × 10^-1 μg/mL), MCF-7 (ED₅₀ = 6.6 μg/mL), and HF-6 (ED₅₀ = 7.1 × 10^-3 μg/mL). This extract also showed anti-tumour activity when assayed on mice inoculated with L5178Y lymphoma cells. Bioactivity-directed isolation of this extract, afforded seven podophyllotoxin-type lignans identified as podophyllotoxin (1), β-peltatin-A-methylether (2), 5'-desmethoxy-β-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7) by 1D and 2DNMR and FAB-MS analyses, and comparison with reported values. All the isolated compounds showed potent cytotoxic activity in the cell lines tested, especially compound 3, which exhibited greater activity than camptothecin and podophyllotoxin against PC-3 (ED₅₀ = 1.0 × 10^-5 μg/mL), and KB (ED₅₀ = 1.0 × 10^-5 μg/mL). This is the first report of the isolation of podophyllotoxin and its acetate in a Bursera species.     

Combination of preparative HPLC and HSCCC methods to separate phosphodiesterase inhibitors from Eucommia ulmoides bark guided by ultrafiltration-based ligand screening

Phosphodiesterase (PDE) inhibitors are widely used because of their various pharmacological properties, and natural products are considered the most productive source of PDE inhibitors. In this work, a new ultrafiltration–high-performance liquid chromatography (HPLC)–diode-array detection–mass spectrometry based ligand screening was developed for the first screening of PDE inhibitors from Eucommia ulmoides bark, and then the target bioactive compounds were prepared by combination of stepwise preparative HPLC and high-speed countercurrent chromatography (HSCCC) methods. Experiments were conducted to optimize the parameters in ultrafiltration, stepwise preparative HPLC, and HSCCC to allow rapid and effective screening and isolation of active compounds from complex mixtures. Seven lignans with purity over 97 % were isolated and identified by their UV, electrospray ionization mass spectrometry, and NMR data as (+)-pinoresinol-4,4′-di-O-β-D-glucopyranoside (1), (+)-pinoresinol-4-O-β-D-glucopyranosyl(1?→?6)-β-D-glucopyranoside (2), (+)-medioresinol-4,4′-di-O-β-D-glucopyranoside (3), (+)-syringaresinol-4,4′-di-O- β-D-glucopyranoside (4), (?)-olivil-4′-O-β-D-glucopyranoside (5), (?)-olivil-4-O-β-D- glucopyranoside (6), and (+)-pinoresinol-4-O-β-D-glucopyranoside (7). Compound 2 was first isolated from the genus Eucommia. Lignan diglucopyranosides (compounds 1–4) shower a greater inhibitory effect than lignan monoglucopyranosides (compounds 5–7). The method developed could be widely applied for high-throughput screening and preparative isolation of PDE inhibitors from natural products.     

Identification and Determination of <em>Aconitum</em> Alkaloids in <em>Aconitum</em> Herbs and <em>Xiaohuoluo Pill</em> Using UPLC-ESI-MS

A rapid, specific, and sensitive ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) method to examine the chemical differences between Aconitum herbs and processed products has been developed and validated. Combined with chemometrics analysis of principal component analysis (PCA) and orthogonal projection to latent structural discriminate analysis, diester-diterpenoid and monoester-type alkaloids, especially the five alkaloids which contributed to the chemical distinction between Aconitum herbs and processed products, namely mesaconitine (MA), aconitine (AC), hypaconitine (HA), benzoylmesaconitine (BMA), and benzoylhypaconitine (BHA), were picked out. Further, the five alkaloids and benzoylaconitine (BAC) have been simultaneously determined in the Xiaohuoluo pill. Chromatographic separations were achieved on a C₁₈ column and peaks were detected by mass spectrometry in positive ion mode and selected ion recording (SIR) mode. In quantitative analysis, the six alkaloids showed good regression, (r) > 0.9984, within the test ranges. The lower limit quantifications (LLOQs) for MA, AC, HA, BMA, BAC, and BHA were 1.41, 1.20, 1.92, 4.28, 1.99 and 2.02 ng·mL^-1, respectively. Recoveries ranged from 99.7% to 101.7%. The validated method was applied successfully in the analysis of the six alkaloids from different samples, in which significant variations were revealed. Results indicated that the developed assay can be used as an appropriate quality control assay for Xiaohuoluo pill and other herbal preparations containing Aconitum roots.     

<em>In Vitro </em>and <em>in </em><em>Vivo</em> Antitumor Effect of Trachylobane-360, a Diterpene from<em> Xylopia langsdorffiana</em>

Trachylobane-360 (ent-7α-acetoxytrachyloban-18-oic acid) was isolated from Xylopia langsdorffiana. Studies have shown that it has weak cytotoxic activity against tumor and non-tumor cells. This study investigated the in vitro and in vivo antitumor effects of trachylobane-360, as well as its cytotoxicity in mouse erythrocytes. In order to evaluate the in vivo toxicological aspects related to trachylobane-360 administration, hematological, biochemical and histopathological analyses of the treated animals were performed. The compound exhibited a concentration-dependent effect in inducing hemolysis with HC₅₀ of 273.6 μM, and a moderate in vitro concentration-dependent inhibitory effect on the proliferation of sarcoma 180 cells with IC₅₀ values of 150.8 μM and 150.4 μM, evaluated by the trypan blue exclusion test and MTT reduction assay, respectively. The in vivo inhibition rates of sarcoma 180 tumor development were 45.60, 71.99 and 80.06% at doses of 12.5 and 25 mg/kg of trachylobane-360 and 25 mg/kg of 5-FU, respectively. Biochemical parameters were not altered. Leukopenia was observed after 5-FU treatment, but this effect was not seen with trachylobane-360 treatment. The histopathological analysis of liver and kidney showed that both organs were mildly affected by trachylobane-360 treatment. Trachylobane-360 showed no immunosuppressive effect. In conclusion, these data reinforce the anticancer potential of this natural diterpene.     

Two New Compounds Isolated from<em> Liriope muscari</em>

Two new compounds, (2S,3R)-methyl 7-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-3-(hydroxymethyl)-2,3-dihydrobenzofuran-5-carboxylate (1) and (4R,5S)-5-(3-hydroxy-2,6-dimethylphenyl)-4-isopropyldihydrofuran-2-one (2), tentatively named norcurlignan and limlactone, respectively, were isolated from Liriope muscari, together with the known compound (-)-pinoresinol (3). The structures of these compounds were elucidated and characterized on the basis of 1D NMR, 2D NMR, CD and MS data. The in vitro antioxidant activities of compounds 1-3 were assessed by the DPPH and ABTS scavenging methods.     

Fumigant Antifungal Activity of Myrtaceae Essential Oils and Constituents from <em>Leptospermum petersonii</em> against Three <em>Aspergillus</em> Species

Commercial plant essential oils obtained from 11 Myrtaceae plant species were tested for their fumigant antifungal activity against Aspergillus ochraceus, A. flavus, and A. niger. Essential oils extracted from Leptospermum petersonii at air concentrations of 56 × 10^-3 mg/mL and 28 × 10^-3 mg/mL completely inhibited the growth of the three Aspergillus species. However, at an air concentration of 14 × 10^-3 mg/mL, inhibition rates of L. petersonii essential oils were reduced to 20.2% and 18.8% in the case of A. flavus and A. niger, respectively. The other Myrtaceae essential oils (56 × 10^-3 mg/mL) only weakly inhibited the fungi or had no detectable affect. Gas chromatography-mass spectrometry analysis identified 16 compounds in L. petersonii essential oil. The antifungal activity of the identified compounds was tested individually by using standard or synthesized compounds. Of these, neral and geranial inhibited growth by 100%, at an air concentration of 56 × 10^-3 mg/mL, whereas the activity of citronellol was somewhat lover (80%). The other compounds exhibited only moderate or weak antifungal activity. The antifungal activities of blends of constituents identified in L. petersonii oil indicated that neral and geranial were the major contributors to the fumigant and antifungal activities.     

Steroidal and Phenolic Glycosides from the Bulbs of <em>Lilium pumilum</em> DC and Their Potential Na⁺/K⁺ ATPase Inhibitory Activity

A new steroidal saponin, named pumilum A (1), and a new phenolic glycoside, threo-1-(4'-hydroxy-2'-methoxyphenyl)-2-(2',4'-dihydroxyphenyl)-1,3-propanediol-4'-O-β-D-glucopyranoside (7) were isolated from the methanolic extract of the bulbs of Lilium pumilum DC, along with five known steroidal saponins. Their chemical structures were elucidated on the basis of detailed spectroscopic analysis, including 1D and 2D NMR techniques and chemical methods. In addition, the inhibitory activity of all the isolates on Na⁺/K⁺ ATPase was evaluated.     

Two new flavonoid diglycosides from Cephalotaxus sinensis

Two new acetylated flavonoid diglycosides, scutellarein-5-O-[a-L-rhamnopyranosyl-(1→ 4)-b-D-6-O-acetylglucopyranoside] (1), apigenin-5-O-[a-L-rhamnopyranosyl-(1→2)-b-D-3,6-di-O-acetylglucopyranoside] (2), were isolated from the leaves of Cephalotaxus sinensis. Their structures were determined by chemical and spectroscopic methods including HR-FAB-MS, 1H–1H COSY, HSQC and HMBC experiments.     

Concise synthesis of two natural steroidal glycosides isolated from <Emphasis Type="Italic">Allium schoenoprasum</Emphasis>

Two natural steroidal glycosides, diosgenin 3-O-α-l-rhamnopyranosyl-(1→2)-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (1) and laxogenin 3-O-α-l-rhamnopyranosyl-(1→2)-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (2) with important cytotoxic activity against the HCT 116 and HT-29 human colon cancer cell lines have been efficiently synthesized via straightforward sequential glycosylation reaction with the combined use of N-phenyltrifluoroacetimidates and trichloroacetimidates donors at room temperature. All structures of the synthesized new compounds were identified by ¹H NMR, ¹³C NMR and HRMS spectra.     

<em>N</em>-Substituted 5-Chloro-6-phenylpyridazin-3(2<em>H</em>)-ones: Synthesis, Insecticidal Activity Against <em>Plutella xylostella </em>(L.) and SAR Study

A series of N-substituted 5-chloro-6-phenylpyridazin-3(2H)-one derivatives were synthesized based on our previous work; all compounds were characterized by spectral data and tested for in vitro insecticidal activity against Plutella xylostella. The results showed that the synthesized pyridazin-3(2H)-one compounds possessed good insecticidal activities, especially the compounds 4b, 4d, and 4h which showed > 90% activity at 100 mg/L. The structure-activity relationships (SAR) for these compounds were also discussed.     

Negative-Pressure Cavitation Extraction of Four Main Vinca Alkaloids from <em>Catharanthus </em><em>r</em><em>oseus</em> Leaves

In the present study, an improved method termed negative-pressure cavitation extraction (NPCE) followed by reverse phase high-performance liquid chromatography (RP-HPLC) was developed for the extraction and quantification of vindoline (VDL), catharanthine (CTR), vincristine (VCR) and vinblastine (VLB) from Catharanthus roseus leaves. The optimized method employed 60-mesh particles, 80% ethanol, a negative pressure of -0.075 MPa, a solid to liquid ratio of 1:20, 30 min of extraction and three extraction cycles. Under these optimized conditions, the extraction yields of VDL, CTR, VCR and VLB are 0.5783, 0.2843, 0.018 and 0.126 mg/g DW, respectively. These extraction yields are equivalent to those from the well-known ultrasonic extraction method and higher than the yields from maceration extraction and heating reflux extraction. Our results suggest that NPCE-RP-HPLC represents an excellent alternative for the extraction and quantification of vinca alkaloids for pilot- and industrial-scale applications.     

Towards cyclic,conformationally constrained,fluorine-containing β-amino acid derivatives from d-glucose

Novel, potentially bioactive, fluorinated branched-chain monosaccharides were obtained by reaction of diethylaminosulphur trifluoride (DAST) with a series of methyl 3-C-cyano-3-ethoxycarbonyl-β-d-glucopyranoside derivatives, including the 4,6-O-benzylidene derivative and their 3-C-(N-protected aminomethyl) reduction products, as well as the phenyl 3-C-cyano-3-ethoxycarbonyl-1-thio-α-d-(and β-d-)glucopyranosides. The absolute configuration at C(3) was unambiguously assigned for all compounds on the basis of X-ray crystallographic analysis of methyl 4,6-O-benzylidene-3-C-cyano-3-deoxy-3-ethoxycarbonyl-β-d-glucopyranoside, corroborating the previous tentative assignment by other authors for the 4,6-unprotected compound. The course of the fluorination depended on the reaction temperature and the substitution pattern of the substrate. Thus, for methyl 3-C-cyano-3-ethoxycarbonyl-β-d-glucopyranoside, fluorination occurred exclusively at C(6), but for the phenylthio analogue, a 2-deoxy-2-phenylthio-α-d-manno-configured glycosyl fluoride and its 6-fluoro derivative were obtained, resulting from the expected rearrangement reaction, whilst starting from the phenylthio α anomer, only the unrearranged 6-fluoro compound was formed. Rearrangement was also observed in the fluorination of methyl 4,6-O-benzylidene-3-C-(N-protected aminomethyl)-β-d-glucopyranoside, which led to the 2-O-methyl-α-d-mannopyranosyl fluoride derivative as the sole product. This methodology may constitute a simple route to enantiopure conformationally constrained cyclic fluorinated β-amino acids having the α carbon atom shared with a pyranose ring, although only moderate yields were achieved, particularly in the fluorination step.