Determination of nine nucleosides in Rhizoma Paridis by quantitative analysis of multi-components via a single marker method.

In this work, a new quantitative analysis method of multi-components analysis via a single marker strategy coupled with high-performance liquid chromatography (HPLC) analysis, was proposed to analyze nine nucleosides (cytidine, uridine, 2'-deoxyuridine, inosine, guanosine, 2'-deoxyguanosine, thymidine, adenosine, and 2'-deoxyadenosine) as quality control markers in Rhizoma Paridis. Guanosine was set as the internal reference substance, whose content in Rhizoma Paridis was determined using conventional external standard method. Then, relative correction factors between guanosine and the other eight nucleosides were measured respectively. The amounts of the other eight components were calculated according to the relative correction factors by the quantitative analysis of multi-components via a single marker method. Finally, the result of vector angle cosine analysis showed that there was no significant difference of the contents between the external standard method and the quantitative analysis of multi-components via a single marker method, indicating that the quantitative analysis of multi-components via a single marker method can be applied for the quality control of Rhizoma Paridis. As far as we know, this is also the first report to analyze nucleosides by the quantitative analysis of multi-components via a single marker method, providing an efficient and promising quality assessment method for other traditional Chinese medicine containing nucleosides.

A new strategy for the preparation of antibody against natural glycoside: With astragaloside IV as an example.

A new strategy for the hapten design of natural glycoside and application for the preparation of antibody is reported in this work. With astragaloside IV (AGS-IV) as an example, C6"-CH2OH on a glucosyl group was selectively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation to C6"-COOH, which was subsequently condensed with -NH2 on bovine serum albumin to get artificial antigen. Then, the successful preparation of artificial antigen was verified by TCL, SDS-PAGE, UV, and MALDI-TOF-MS. Finally, rabbits were immunized with artificial antigen to obtain an antibody against AGS-IV. After tests of the titer, IC50, and cross-reactivity, the results showed that the antibody prepared by TEMPO oxidation in this work had higher specificity than that the antibody prepared by conventional sodium periodate (NaIO4) oxidation. The hapten, as a carboxylic acid derivative of AGS-IV, has better water solubility than AGS IV, which is more suitable for the synthesis of the hapten-carrier protein conjugate in aqueous phase, achieving another virtue of TEMPO oxidation over NaIO4 oxidation. This new strategy provides new ideas for the design of haptens of other natural glycosides, as well as the preparation of their antibodies.

Dendrobine-type alkaloids and bibenzyl derivatives from Dendrobium findlayanum.

Five previously undescribed compounds, including two dendrobine-type alkaloids (1 and 2), three bibenzyl derivatives (3-5), along with six known compounds were isolated from orchids Dendrobium findlayanum. The structures and absolute configurations of the undescribed compounds were elucidated on the basis of HR-ESIMS, NMR spectroscopy, optical rotation value, as well as electronic circular dichroism (ECD) calculations. The cytotoxic effects of the isolated compounds on three human tumour cell lines (A172, SHSY5Y, and Hela) were evaluated by the MTT assay. Compound 6 showed excellent inhibitory activities against three human tumour cell lines with IC50 ranging from 1.65 µM to 3.77 µM. All these compounds were assessed for their activity of promoting the gastrointestinal motility of zebrafish treated with Nile red. Compound 6 have excellent activity to promote the gastrointestinal motility of zebrafish at the concentration of 0.3 µM.

Determination of a astragaloside IV derivative LS-102 in plasma by ultra-performance liquid chromatography-tandem mass spectrometry in dog plasma and its application in a pharmacokinetic study.

BACKGROUND: Astragalosidic acid (LS-102) is a new water-soluble derivative of astragaloside IV - a major effective component isolated from the Chinese herb Astragali Radix. Our previous study showed that LS-102 exhibited potent cardiovascular activity. PURPOSE: The objective of this study was to investigate the pharmacokinetic properties of LS-102 after single-dose, oral administration in beagle dogs by developing and validating an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. METHOD AND RESULT: The chromatographic separation was performed on a Acquity HSS C18 column (100 mm × 2.1 mm, 1.8 µm) by a gradient elution using a mobile phase consisting of water and acetonitrile at a flow rate of 0.35 ml/min. The analytes were detected with a triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode. Method validation revealed a wide linearity over the range of 2.0-10,000 ng/ml together with satisfactory intra- and inter-day precision, accuracy, and recovery. Stability testing showed that LS-102 spiked into dog plasma was stable for 4 h at room temperature, for up to 2 weeks at -80 °C, and during three freeze-thaw cycles. The method was effectively and successfully applied to the pharmacokinetics of LS-102 after oral administration (5, 10 and 20 mg/kg) to beagle dogs. Peak plasma concentrations are attained within approximately 2 h after oral administration with a half-life ranging from 1.55 h to 4.49 h. The plasma concentration-time curve of LS-102 after oral administration presents the phenomenon of a double-peak absorption phase. The peak concentration and area under the concentration-time curve of LS-102 seemed to increase with the increasing doses proportionally, that suggesting linear pharmacokinetics in dogs. Meanwhile, the doxorubicin (Dox)-injured H9c2 cell model was prepared by incubating the cells in 1 µM Dox for 24 h. MTT assay and LDH release measurement showed that LS-102 protected against Dox-induced cardiomyocyte death. CONCLUSION: The obtained results may help to guide the further pre-clinical research of LS-102 as a potentially novel cardioprotective agent.

A efficient method to identify cardioprotective components of Astragali Radix using a combination of molecularly imprinted polymers-based knockout extract and activity evaluation.

Although herb medicines have become the major source for new drug discovery, many of them are largely under-explored due to the purity-activity relationship. Efficient identification of bioactive compounds in conventional stepwise separation and isolation has not yet been elucidated. Therefore, we proposed a new separation strategy for holism understanding of herb pharmacology using molecularly imprinted polymers (MIPs). Astragali Radix (AR), important in traditional Chinese medicine, was chosen in this study for multicomponent knockout followed by bioactivity evaluation. We prepared calycosin molecularly imprinted polymers (calycosin-MIPs) which could selectively recognize flavonoid aglycons in AR. The molecular selectivity of calycosin-MIPs as a critical parameter was evaluated using the template and other high content compounds in AR. Based on it, using the calycosin-MIPs material via solid-phase extraction procedures was applied for the knockout of flavonoid aglycons in AR. Finally, hypoxia/reoxygenation model in H9c2 cells was used to evaluate the activity of the AR extract before and after knockout. The results showed that calycosin-MIPs as recognition materials for flavonoid aglycons in AR are applied in one-step separation with high selectivity and tunability. The subextract in the absence of flavonoid aglycons has been demonstrated to clarify the cardio-protective components of AR. In conclusion, this proof-of-principle study is the first one showing that molecular imprinting technology coupled with a bioassay can be used to explore the bioactive variability from the perspective of multicomponent separation of herbal medicine.

Antiproliferative Sesquiterpenoids from Ligularia rumicifolia with Diverse Skeletons.

Twenty-two new sesquiterpenoids with four skeletal types and 15 known analogues were isolated from the whole plants of Ligularia rumicifolia. The structures of the isolates were elucidated based on comprehensive spectroscopic data analysis. Compound 1 is a C14 nor-sesquiterpenoid featuring a 6/6/6 tricyclic skeleton with a 9,13-ether bridge. The absolute configuration of 2 was established through single-crystal X-ray diffraction data. Compounds 13-16 exhibited in vitro antiproliferative activity against the four human tumor cell lines A-549, HGC-27, HeLa, and MV4-11. Specifically, compounds 13 and 16 showed antiproliferative activity against the MV4-11 cell line with IC50 values of 0.5 ± 0.2 and 1.1 ± 0.5 µM, respectively.

Cytotoxic triterpenoid saponins from Clematis tangutica.

Eight previously undescribed oleanane-type triterpenoid saponins, clematangoticosides A-H, together with eight known saponins, were isolated from the whole plants of Clematis tangutica (Maxim.) Korsh. Their structures were elucidated by extensive spectroscopic analysis, in combination with chemical methods (acid hydrolysis and mild alkaline hydrolysis). Clematangoticosides D-G were found to be unusual 23, 28-bidesmosidic glycosides. The cytotoxic activities of all of the isolated saponins were evaluated against the four human cancer cell lines SGC-7901, HepG2, HL-60 and U251MG. Clematoside S, sapindoside B, kalopanax saponin A, and koelreuteria saponin A exhibited cytotoxicity against all of the test cancer cell lines with IC50 values in the range of 1.88-27.20 µM, while clematangoticoside D and F showed selective cytotoxicity against SGC-7901 with IC50 values of 24.22 and 21.35 µM, respectively.

Polycyclic Spiro Lignans and Biphenyl Tetrahydrofuranone Lignans from Gymnotheca involucrata.

Four rare polycyclic spiro lignans (1-4) and four new biphenyl tetrahydrofuranone lignans (5-8) were isolated from the whole plant of Gymnotheca involucrata. The structures of the new compounds were elucidated on the basis of detailed spectroscopic analysis and the absolute configuration of 1 was confirmed by single crystal X-ray diffraction. Bioassay results showed that compounds 2 and 6 exhibited weak antifungal activity against Uromyces viciae-fabae at 100 ppm in leaf-disc assays, while compound 3 demonstrated moderate insecticidal activity against Diabrotica balteata at 500 ppm in an artificial diet assay.

Preparation of ds-DNA functionalized magnetic nanobaits for screening of bioactive compounds from medicinal plant.

A novel magnetic nanocomposite (MNPs@DNA) was synthesized by bonding double strand DNA (ds-DNA) onto Fe3O4 magnetic nanoparticles (MNPs) directly. MNPs@DNA was characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV), Fourier transform infrared spectroscopy (FT-IR) and thermo-gravimetric analysis (TGA), which indicated that ds-DNA was immobilized onto MNPs. Vibrating sample magnetometer (VSM) analysis indicated that the MNPs@DNA had a high saturation magnetization of 42.97emu/g. A novel method for screening of active compounds from natural sources was developed by employing MNPs@DNA as a nanobait and high-performance liquid chromatography-mass spectrometry as detecting system. Columbamine, palmatine, jateorhizine, epiberberine and berberine were identified as DNA binders from the extract of Rhizoma coptidis. In addition, a comparison of the binding abilities among MNPs with different DNA strand lengths (25, 200 and 1200bp) showed that the shortest one exhibited the highest binding ability. This is the first report on fast chemical characterization of active ingredients in medicinal plant using ds-DNA immobilized on magnetic nano-baits. This method can be used not only for screening of DNA binders from complex herbal matrices, but also for assessing the affinities between a specific ds-DNA and its potential binders.

Metabolic study of Angelica dahurica extracts using a reusable liver microsomal nanobioreactor by liquid chromatography-mass spectrometry.

Highly active and recoverable nanobioreactors prepared by immobilizing rat liver microsomes on magnetic nanoparticles (LMMNPs) were utilized in metabolic study of Angelica dahurica extracts. Five metabolites were detected in the incubation solution of the extracts and LMMNPs, which were identified by means of HPLC-MS as trans-imperatorin hydroxylate (M1), cis-imperatorin hydroxylate (M2), imperatorin epoxide (M3), trans-isoimperatorin hydroxylate (M1') and cis-isoimperatorin hydroxylate (speculated M2'). Compared with the metabolisms of imperatorin and isoimperatorin, it was found that the five metabolites were all transformed from these two major compounds present in the plant. Since no study on isoimperatorin metabolism by liver microsomal enzyme system has been reported so far, its metabolites (M1' and M3') were isolated by preparative HPLC for structure elucidation by (1) H-NMR and MS(2) analysis. M3' was identified as isoimperatorin epoxide, which is a new compound as far as its chemical structure is concerned. However, interestingly, M3' was not detected in the metabolism of the whole plant extract. In addition, a study with known chemical inhibitors on individual isozymes of the microsomal enzyme family revealed that CYP1A2 is involved in metabolisms of both isoimperatorin and imperatorin, and CYP3A4 only in that of isoimperatorin.

A new ursane-type triterpenoid saponin from the aerial parts of Clematoclethra scandens subsp. actinidioides.

A new ursane-type triterpenoid saponin, 2α,3α,24-trihydroxyurs-12,20(30)-dien-28-oic acid ß-D-glucopyranosyl ester (1), together with six known triterpenoid saponins, was isolated and characterized from the aerial parts of Clematoclethra scandens subsp. actinidioides.

12-Membered Resorcylic Acid Lactones Isolated from Saccharicola bicolor, an Endophytic Fungi from Bergenia purpurascens.

Two new resorcylic acid lactones, 13-hydroxyhidroresorcylide (1) and 12-hydroxyhidroresorcylide (2), along with four known congeners (3-6) were isolated from Saccharicola bicolor, an endophytic fungus from Bergenia purpurascens. Their structures were elucidated by interpretation of the spectroscopic evidence.

Cyclic Lipopeptides with Herbicidal and Insecticidal Activities Produced by Bacillus clausii DTM1.

Seven cyclic lipopeptide biosurfactants (1-7) were isolated for the first time from the fermentation broth of endophytic Bacillus clausii DTM1 and were identified as anteisoC13[Val7] surfactin-(L-Glu)-O-methyl-ester (1), anteisoC12[Val7] surfactin (2), anteisoC15[Val7] surfactin (3), isoC14[Leu7] surfactin (4), anteisoC12[Leu7] surfactin (5), nC13[Leu7] surfactin (6), and anteisoC14[Leu7] surfactin-(L-Glu)-O-methyl-ester (7); 1 has not been isolated before as a natural product from any source. Plate-based herbicide and insecticide bioassays showed that all compounds exhibited interesting insecticidal and herbicidal activities.

A Rapid Study of Botanical Drug-Drug Interaction with Protein by Re-ligand Fishing using Human Serum Albumin-Functionalized Magnetic Nanoparticles.

A great many active constituents of botanical drugs bind to human serum albumin (HSA) reversibly with a dynamic balance between the free- and bound-forms in blood. The curative or side effect of a drug depends on its free-form level, which is always influenced by other drugs, combined dosed or multi-constituents of botanical drugs. This paper presented a rapid and convenient methodology to investigate the drug-drug interactions with HSA. The interaction of two steroidal saponins, dioscin and pseudo-protodioscin, from a botanical drug was studied for their equilibrium time and equilibrium amount by re-ligand fishing using HSA functionalized magnetic nanoparticles. A clear competitive situation was obtained by this method. The equilibrium was reached soon about 15 s at a ratio of 0.44: 1. Furthermore, the interaction of pseudo-protodioscin to total steroidal saponins from DAXXK was also studied. The operation procedures of this method were faster and more convenient compared with other methods reported.

Lipase ligands in Nelumbo nucifera leaves and study of their binding mechanism.

Lotus (Nelumbo nucifera) leaves have been widely used in weight-loss foods to prevent obesity in China. In this work, a facile procedure based on ligand fishing was developed to isolate and identify lipase inhibitors present in lotus leaves. Highly stable and active lipase-Fe3O4 superparamagnetic nanoparticle conjugates (LMNPs) were prepared and used as baits. Two flavonoids in lotus leaf extract were found to bind to the baits and were identified as quercetin-3-O-ß-d-arabinopyranosyl-(1→2)- ß-d-galactopyranoside (1) and quercetin-3-O-ß-d-glucuronide (4) based on electrospray ionization-mass spectrometric analyses. Their 50% inhibitory concentrations on lipase (IC50) were 52.9 ± 3.2 and 17.1 ± 1.5 µg/mL, respectively. In addition, they were found to significantly quench the fluorescence of lipase, suggesting their strong affinities with this enzyme, which was further evidenced by molecular docking. Ligand fishing based on LMNPs shows great power for fast screening and identification of lipase inhibitors present in edible and medicinal plants.

[Triterpenes from aerial parts of Clematoclethra scandens subsp. actinidioides].

To study the chemical constituents of Clematoclethra scandens subsp. actinidioides, chromatographic methods such as silica gel and MCI column chromatographic technology, and preparative HPLC were used and sixteen compounds were isolated from the aerial parts of this plant. By using spectroscopic techniques including 1H, 13C-NMR, HMBC and ESI-MS, these compounds were identified as betulinic acid (1), ursolic acid (2), oleanic acid (3), corosolic acid (4), 3beta-(trans-p-coumaroyloxy)-2alpha, 23-dihydroxyurs-12-en-28-oic acid (5), 3beta-(trans-p-coumaroyloxy)-2alpha, 23-dihydroxyurs-12, 20 (30)-dien-28-oic acid (6), 2alpha, 3alpha, 23-trihydroxyurs-12, 20 (30)-dien-28-oic acid (7), 2alpha, 3alpha, 23-trihydroxyurs-12-en-28-oic acid (8), asiatic acid (9), 2alpha, 3alpha, 24-tri-hydroxyurs-12-en-28-oic acid (10), 2alpha, 3beta, 23-trihydroxyurs-12, 20 (30)-dien-28-oic acid (11), 2alpha, 3beta, 19alpha, 24-tetrahydroxyurs-12-en-28-oic acid (12), 2alpha, 3alpha, 19alpha, 24-tetrahydroxyurs-12-en-28-oic acid (13), 2alpha, 3beta, 23, 24-tetrahydroxyurs-12-en-28-oic acid (14), 2alpha, 3alpha, 19alpha, 23, 24-pentahydroxyurs-12-en-28-oic acid (15) and daucosterol (16). Among them, compounds 3-6, 11-12, 14 and 15 were isolated from this endemic plant for the first time.

Three new phenolic compounds from the leaves of Rosa sericea.

Two new flavonoids, quercetin-3-O-ß-d-xylopyranosyl-(1→2)-α-d-ribopyranoside (1) and kaempferol-3-O-ß-d-xylopyranosyl-(1→2)-α-d-ribopyranoside (2), and one new phenolic derivative, gallicin-p-O-(6'-O-caffeoyl)-ß-d-glucoside (3), together with twelve known compounds were isolated from the leaves of Rosa sericea (Rosaceae family). The structures of the new compounds were established by means of spectroscopic analysis including one- and two-dimensional NMR spectroscopy. Some of the isolated compounds were tested for the cytotoxicity of a breast cancer cell (MCF-7) line. The results showed that rubanthrone A (4) has moderate cytotoxicity against the MCF-7 cell line.

19-oxygenated ent-kaurane diterpenoids from Isodon pharicus.

Eight new 19-oxygenated ENT-kaurane diterpenoids were isolated from the aerial parts of Isodon pharicus. Their structures were determined by means of extensive spectroscopic techniques including interpretation of 1D and 2D NMR spectra. Selected compounds were evaluated for their cytotoxicity against NB4, A549, PC-3, MCF-7, and SH-SY5Y cell lines.

Using baicalin-functionalized magnetic nanoparticles for selectively extracting flavonoids from Rosa chinensis.

An extraction agent featuring a natural product, baicalin, anchored on the surface of nanomagnetic particles (BMNPs) is herein reported. A facile solid-phase extraction (SPE) procedure with high selectivity toward flavonoids using BMNPs has been established. BMNPs were proven very effective for enriching flavonoids from extracts of medicinal plants such as Rosa chinensis. The SPE protocol involving a convenient solid-liquid separation by using an external magnet field was easy to carry out. Further, the SPE sorbent (BMNPs) could be reused for many times reducing the operation cost. Importantly, flavonoids retained on the BMNPs were effectively recovered by eluting with methanol. Coupling the proposed SPE with ESI-MS/MS allowed a quick quantification of flavonoids in herbal extracts. Simultaneous determination of eight flavonoids extracted from R. chinensis was demonstrated in this work.

Three new flavone C-glycosides from the aerial parts of Paraquilegia microphylla.

Three new flavone C-glycosides, paraquinins A-C, were isolated from the aerial parts of Paraquilegia microphylla (Royle) Dromm. et Hutch, a Tibetan medicine distributed in the Qinghai-Tibet plateau. On the basis of 1D and 2D NMR evidence, their structures were elucidated as acacetin-6-C-ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranoside (1), acacetin-6-C-α-L-rhamnopyranosyl-(1 → 2)-ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranoside (2), and acacetin-6-C-α-L-rhamnopyranosyl-(1 → 2)-(6'''-O-E-feruloyl)-ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranoside (3).