Development of an HPLC-DAD method for the determination of five alkaloids in Stephania yunnanensis Lo and in rat plasma after oral dose of Stephania yunnanensis Lo extracts.

For the rational utilization and the quantitative quality control of the Stephania yunnanensis Lo, an HPLC-DAD method was developed for the quantitative and simultaneous determination of five alkaloids in rat plasma (stepharine, sinomenine, palmatine, isocorydine and tetrahydropalmatine), which were the main active chemical constituents of this plant and belong to four kinds of isoquinoline-type alkaloids (protoberberine, morphine, aporphine and protaporphine alkaloids). The contents of five alkaloids ranged from 0.09 to 2.32% (w/w). The method validation was tested for the linearity (r2 > 0.9975), precision (intra-day RSD < 4.8% and inter-day RSD < 4.9%), extraction recovery (85.49 ± 2.29% to 99.21 ± 1.48%) and stability (98.5 ± 5.3% to 101.2 ± 3.4%). We developed an HPLC-DAD method to simultaneously measure these alkaloids in rat plasma after oral administration of the extract of this plant to rats. The results supported the hypothesis that isoquinoline alkaloids were the compounds responsible for the main pharmacological activities for anti-inflammatory and analgesic.

Design, synthesis, and anticancer properties of isocorydine derivatives.

Isocorydine (ICD), an aporphine alkaloid, is widely distributed in nature. Its ability to target side population (SP) cells found in human hepatocellular carcinoma (HCC) makes it and its derivative 8-amino-isocorydine (NICD) promising chemotherapeutic agents for the treatment of HCC. To improve the anticancer activity of isocorydine derivatives, twenty derivatives of NICD were designed and synthesized through chemical structure modifications of the aromatic amino group at C-8. The anti-proliferative activities of all synthesized compounds against human hepatocellular (HepG2), cervical (HeLa), and gastric (MGC-803) cancer cell lines were evaluated using an MTT assay. The results showed that all the synthetic compounds had some tumor cell growth inhibitory activity. The compound COM33 (24) was the most active with IC50 values under 10 µM (IC50 for HepG2 = 7.51 µM; IC50 for HeLa = 6.32 µM). FICD (12) and COM33 (24) were selected for further investigation of their in vitro and in vivo activities due to their relatively good antiproliferative properties. These two compounds significantly downregulated the expression of four key proteins (C-Myc, ß-Catenin, CylinD1, and Ki67) in HepG2 cells. The tumor inhibition rate of COM33 (24) in vivo was 73.8% after a dose 100 mg/kg via intraperitoneal injection and the combined inhibition rate of COM33 (24) (50 mg/kg) with sorafenib (50 mg/kg) was 66.5%. The results indicated that these isocorydine derivatives could potentially be used as targeted chemotherapy agents or could be further developed in combination with conventional chemotherapy drugs to target cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT), the main therapeutic targets in HCC.

[Research on anticancer activity of isocorydine and its derivatives].

Isocorydine and its analogs were extracted from Dicranostigma leptopodum and Stephania yunnanensis through the method of natural products chemistry. Its derivatives were prepared by chemical structure modifications from isocorydine. MTT method was used to study the inhibitory effect of those compounds on the growth of HepG2, HeLa and MGC-803 cancer cell lines in vitro. The results showed that isocorydine and its analogs all have the growth inhibition for those cancer cell lines. This paper investigated the structure-activity relationship of isocorydine and its derivatives with anticancer activity in the aspect of stereochemical structure, functional groups positions of the compounds and the electron density of aromatic rings based on the single crystal diffraction structure and the molecular docking of EGFR and isocorydine.

Allelochemicals from the Rhizosphere Soil of Potato (Solanum tuberosum L.) and Their Interactions with the Soilborne Pathogens.

To reveal the allelopathic effects of potato, seven compounds were isolated from the rhizosphere soil: 7-methoxycoumarin (1), palmitic acid (2), caffeic acid (3), chlorogenic acid (4), quercetin dehydrate (5), quercitrin (6), and rutin (7). Bioassays showed that compounds 1, 2, 4, and 6 had inhibitory effects on the growth of L. sativa and tissue culture seedlings of potato. The existence of the allelochemicals was confirmed by HPLC, and their contents were quantified with a total concentration of 9.02 µg/g in the rhizosphere soil of replanted potato. Approaches on the interactions of the allelochemicals and pathogens of potato including A. solani, B. cinerea, F. solani, F. oxysporum, C. coccodes, and V. dahlia revealed that compound 1 had inhibitory effects but compounds 2-4 promoted the colony growth of the pathogens. These findings demonstrated that the autotoxic allelopathy and enhancement of the pathogens caused by the accumulation of the allelochemicals in the continuously cropped soil should be one of the main reasons for the replant problems of potato.

Anti-aging effects on Caenorhabditis elegans of a polysaccharide, O-acetyl glucomannan, from roots of Lilium davidii var. unicolor Cotton.

The anti-aging activities on Caenorhabditis elegans of a polysaccharide, O-acetyl glucomannan (LPR), purified from roots of Lilium davidii var. unicolor Cotton, were assessed by observing the mean lifespan, reproduction, pharyngeal pumping and stress response on nematodes. Additionally, the fluorescence intensity of lipofuscin and the level of reactive oxygen species (ROS) were detected. Also the activities of superoxide dismutase (SOD), catalase (CAT) and contents of malondialdehyde (MDA) were determined by the kit method. The results showed that LPR effectively delayed the aging of C. elegans in a dose-dependent manner. When the concentration reached 4 mg/mL, LPR extended the mean lifespan of C. elegans by up to 40%, 61% (P < 0.01) and 50% (P < 0.05) under normal, thermal and oxidative stress culture conditions, respectively. Moreover, LPR remarkably increased the reproduction duration of the nematodes at a concentration of 1 mg/L, and significantly decreased the ROS and lipofuscin level of C. elegans in three dosage groups. Further study illustrated that LPR at 4 mg/mL strongly increased the activity of SOD and CAT by 39.03% (P < 0.01) and 41.89% (P < 0.05), and decreased the lipid peroxidation of MDA level in C. elegans by 52.59% (P < 0.005) compared to a control. It was inferred that LPR provided stress resistance to heat and oxidation, and prolonged the lifespan of wild type N2 C. elegans mainly by elevating the function of nematode antioxidant defense systems and by scavenging free radicals. These findings provided evidence for the anti-aging properties of this polysaccharide from L. davidii.

Diversity and Functions of Endophytic Fungi Associated with Roots and Leaves of Stipa purpurea in an Alpine Steppe at Qinghai-Tibet Plateau.

Stipa purpurea is a unique and dominant herbaceous plant species in the alpine steppe and meadows on the Qinghai-Tibet Plateau (QTP). In this work, we analyzed the composition and diversity of the culturable endophytic fungi in S. purpurea according to morphological and molecular identification. Then, we investigated the bioactivities of these fungi against plant pathogenic fungi and 1- aminocyclopropane-1-carboxylate deaminase (ACCD) deaminase activities. A total of 323 fungal isolates were first isolated from S. purpurea, and 33 fungal taxa were identified by internal transcribed spacer primers and grouped into Ascomycota. The diversity of endophytic fungi in S. purpurea was significantly higher in roots as compared to leaves. In addition, more than 40% of the endophytic fungi carried the gene encoding for the ACCD gene. The antibiosis assay demonstrated that 29, 35, 28, 37 and 34 isolates (43.9, 53.1, 42.4, 56.1, and 51.5%) were antagonistic to five plant pathogenic fungi, respectively. Our study provided the first assessment of the diversity of culturedepending endophytic fungi of S. purpurea, demonstrated the potential application of ACCD activity and antifungal activities with potential benefits to the host plant, and contributed to high biomass production and adaptation of S. purpurea to an adverse environment.

[Optimization of the fermentation conditions of Bacillus nematodes and screening of macroporous adsorption resin]. / æ€çº¿è™«èŠ½å­¢æ†èŒå‘é µæ¡ä»¶ä¼˜åŒ–åŠå¤§å­”æ ‘è„‚ç­›é€‰.

Bacillus sp. SMrs28, metabolites from which had significant nematicidal activity, was isolated from the rhizosphere soil of Stellera chamaejasme. To determine the optimal fermentation conditions of the strain and the resin type of preliminary purified active ingredient, fermentation conditions were optimized by single factor experiment, while the macroporous resin types were screened in a static adsorption experiment. The results showed that the optimal fermentation conditions of SMrs28 strain were as follows: glucose and yeast powder were the best carbon source and nitrogen source, fermentation for 48 h, inoculum volume of 10%, temperature at 28 ℃,a rotation speed of 180 r·min-1, liquid volume of 30 mL in 150 mL triangular flask, and with an initial pH of 7.2. The static adsorption experiments showed that the adsorption and desorption of active ingre-dients in the fermentation broth by the macroporous adsorption resin D101 was significantly better than that of XAD-4, HP20 and AB-8, with the nematicidal activity of the desorption liquid being significantly improved. The nematicidal activity of fermentation broth was significantly improved by the optimization of fermentation conditions and the screening of optimal macroporous adsorption resins. These results laid a foundation for the further isolation and purification of active ingredients from SMrs28 strain, and provided theoretical basis for the development and utilization of microbial nematicides.

Pharmacokinetics, tissue distribution, and safety evaluation of a ligustilide derivative (LIGc).

Ligustilide (LIG) is the main active ingredient of Angelica sinensis (Oliv.) Diels and has a neuroprotective effect against ischemic stroke. However, owing to its multi-conjugated unstable structure, the compound has poor drug-forming properties. Therefore, we synthesized highly stable colorless needle crystals (known as ligusticum cycloprolactam, LIGc) through the structural modification of LIG. After a stability experiment was conducted at room temperature for four months, no impurity peaks were found by HPLC-DAD analysis, which indicated that LIGc resolved the stability issues of LIG. LIGc was absorbed and eliminated rapidly after intravenous administration (Cmax = 6.42 ±â€¯1.65 mg/L at a dose of 20 mg/kg) and oral administration (Tmax = 0.5 h, Cmax = 9.89 ±â€¯1.62 mg/L at a dose of 90 mg/kg, t1/2z approximately 2.5 h). The absolute oral bioavailability (F) of LIGc was clearly higher than the F of LIG reported in the literature (F, 83.97 % versus 2.6 %). Linear dose-dependent pharmacokinetics were observed after oral administration, with a higher area under the curve (AUC0-t, 22.31 ±â€¯2.88 mg/L*h) observed at 90 mg/kg than that at 45 mg/kg (AUC0-t, 13.673 ±â€¯0.666 mg/L*h). Tissue distribution results indicated that LIGc easily crossed the blood-brain barrier (BBB) and was distributed widely to the main tissues and organs of rats. We also conducted a preliminary safety assessment of LIGc by means of an acute toxicity test in KM mice. All mice had excellent health status (ig, dosage of 5.0 g/kg), with no histopathological changes observed in the main organs.

Structural characterization, antioxidant and antibacterial activities of two heteropolysaccharides purified from the bulbs of Lilium davidii var. unicolor Cotton.

Two heteropolysaccharides, BHP-1 and BHP-2, were separated and purified from the edible bulbs of Lilium davidii var. unicolor Cotton by anion-exchange and gel-permeation chromatography. The primary structural characteristics of BHP-1 and BHP-2 were investigated by Congo red test, X-ray diffractometry (XRD) analysis, IR, GC and NMR. The results showed that the weight-average molecular weight (MW) and number-average molecular weight (Mn) of BHP-1 and BHP-2 were nearly 1.93 × 105 g/moL, 8.86 × 104 g/moL and 3.52 × 104 g/moL, 2.95 × 104 g/moL, respectively. Glucose and mannose comprised BHP-1 in a relative molar ratio of 5.9:2.0, and BHP-2 was consisted of glucose, galactose, mannose and arabinose with approximate molar ratios of 8.3:1.5:1.0:1.1. Their backbone mainly contained α-(1 → 4)-linked d-glucopyranosyl. In addition, the C-6 of the glucose and C-2 of mannose residue of BHP-1 and BHP-2 were substituted. The two polysaccharides were semi-crystalline substance with multi-branching structure and triple helix conformation. Bioassays showed that BHP-1 and BHP-2 had effective antioxidant activities evaluated by DPPH, scavenging superoxide radical and OH radical in a dose-dependent manner. The two heteropolysaccharides also displayed distinct and selective antibacterial activity against B. cereus, P. putida, M. luteus and K. pneumonia with inhibition zones of 13 mm, 16 mm, 17 mm, 23 mm and 12 mm, 17 mm, 19 mm, 20 mm, respectively. These findings provided a reference for potential applications of the polysaccharides from L. davidii as natural antioxidants and antibacterial agents in food and pharmaceuticals.

Purification, characterization and antioxidant activities of a polysaccharide from the roots of Lilium davidii var. unicolor Cotton.

A polysaccharide (LPR) was separated from the roots of Lilium davidii var. unicolor Cotton with hot water extraction, ethanol precipitation, and purification by anion-exchange and gel-permeation chromatography. LPR was characterized. The weight-average molecular weight (MW) of LPR was 5.12 × 104 g/moL. Glucose and mannose comprised LPR with a molar ratio of 2.9:3.3. IR, NMR and methylation analysis showed that LPR was a natural O-acetyl glucomannan, the backbone mainly contained ß-(1 → 4)-linked d-glucopyranosyl and ß-(1 → 4)-linked D-mannopyanosyl, and the branches probably linked at O-2 and/or O-3 of the mannosyl and glucosyl residues. The acetyl groups mainly attached at O-2 or O-3 of mannosyl residues. X-ray diffractometric (XRD) analysis and scanning electron microscopy (SEM) analysis revealed that LPR was a semi-crystalline substance with porous lamellar structure. Bioassays in vitro indicated that LPR had distinct scavenging activities on hydroxyl radical and DPPH radical. These findings provided a reference for functional underutilization roots of L. davidii as natural antioxidant in food and pharmaceutical industry.