The reason and mechanism of propylene glycol alginate sodium sulfate (PSS) mediated allergic side effect.

Propylene glycol alginate sodium sulfate (PSS) is a heparinoid polysaccharide drug used in clinic for >30 years in China. But its allergy events happened from time to time and should not be ignored. Here, ammonium salt in PSS (PSS-NH4+), PSS fractions with high Mw (PSS-H-Mw) and low mannuronic acid (M) to guluronic acid (G) ratio (PSS-L-M/G) were found to induce allergic response by the structure-activity and impurity-activity relationships in vitro. Furthermore, we confirmed the reason and elucidated the mechanism accounted for allergic side effect of PSS in vivo. It was found that high IgE levels in PSS-NH4+ and PSS-H-Mw groups upregulate the cascade expression of Lyn-Syk-Akt or Erk and second messenger Ca2+, which accelerated mast cells (MCs) degranulation to release histamine, LTB4, TPS, and finally induced lung tissue injury. PSS-L-M/G caused a mild allergic symptom because it only enhanced the expression of p-Lyn and histamine release. In brief, PSS-NH4+ and PSS-H-Mw were main reasons to result in allergic response. Our results suggested that it is very necessary to control the range of Mw and the content of impurities (< 1 % ammonium salt) of PSS to guarantee its safety and effectiveness in clinical treatment.

Integrating Activity-Guided Strategy and Fingerprint Analysis to Target Potent Cytotoxic Brefeldin A from a Fungal Library of the Medicinal Mangrove Acanthus ilicifolius.

Mangrove-associated fungi are rich sources of novel and bioactive compounds. A total of 102 fungal strains were isolated from the medicinal mangrove Acanthus ilicifolius collected from the South China Sea. Eighty-four independent culturable isolates were identified using a combination of morphological characteristics and internal transcribed spacer (ITS) sequence analyses, of which thirty-seven strains were selected for phylogenetic analysis. The identified fungi belonged to 22 genera within seven taxonomic orders of one phyla, of which four genera Verticillium, Neocosmospora, Valsa, and Pyrenochaeta were first isolated from mangroves. The cytotoxic activity of organic extracts from 55 identified fungi was evaluated against human lung cancer cell lines (A-549), human cervical carcinoma cell lines (HeLa), human hepatoma cells (HepG2), and human acute lymphoblastic leukemia cell lines (Jurkat). The crude extracts of 31 fungi (56.4%) displayed strong cytotoxicity at the concentration of 50 µg/mL. Furthermore, the fungus Penicillium sp. (HS-N-27) still showed strong cytotoxic activity at the concentration of 25 µg/mL. Integrating cytotoxic activity-guided strategy and fingerprint analysis, a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, brefeldin A, was isolated from the target active strain HS-N-27. It displayed potential activity against A549, HeLa and HepG2 cell lines with the IC50 values of 101.2, 171.9 and 239.1 nM, respectively. Therefore, combining activity-guided strategy with fingerprint analysis as a discovery tool will be implemented as a systematic strategy for quick discovery of active compounds.

Pharmacokinetics of Nuciferine and N-Nornuciferine, Two Major Alkaloids From Nelumbo nucifera Leaves, in Rat Plasma and the Brain.

The leaf of the lotus (Nelumbo nucifera) is a natural plant resource used as both food and herbal medicine (He-Ye) in China. Alkaloids are considered the major bioactive compound of the herb and exhibit various biological activities, including anti-hyperlipidemia, anti-obesity, anti-inflammatory, and anti-hyperuricemic effects. Nuciferine (NF) and N-nuciferine (N-NF) are two major alkaloids found in the herb. In the present work, the plasma and brain pharmacokinetics of the two compounds were investigated after oral and intravenous (i.v.) administration of a lotus leaf alkaloid fraction to SD rats via ultra-performance liquid chromatography coupled with photodiode array detection and brain microdialysis. After oral administration (50 mg/kg), the two compounds NF and N-NF were rapidly absorbed into the blood and reached a mean maximum concentration (Cmax) of 1.71 µg/mL at 0.9 h and 0.57 µg/mL at 1.65 h, respectively. After i.v. administration (10 mg/kg), NF and N-NF were found to have a relatively wide volume of distribution (Vd, λz, 9.48 and 15.17 L/kg, respectively) and slow elimination half-life (t1/2, λz, 2.09 and 3.84 h, respectively). The oral bioavailability of NF and N-NF was estimated as 58.13% and 79.91%, respectively. After i.v. dosing (20 mg/kg), the two compounds rapidly crossed the blood-brain barrier and reached their Cmax (in unbound form): 0.32 and 0.16 µg/mL at 0.89 and 1.22 h, respectively. Both alkaloids had widespread distribution in the brain, with Vd, λz/F-values of 19.78 L/kg and 16.17 L/kg, respectively. The mean t1/2, λz values of NF and N-NF in the brain were 1.24 and 1.39 h, respectively. These results can help us to better understand the characteristics and neuro-pharmacological effects of the lotus alkaloid fraction.

The mechanisms of sulfated polysaccharide drug of propylene glycol alginate sodium sulfate (PSS) on bleeding side effect.

Propylene glycol alginate sodium sulfate (PSS), a sulfated polysaccharide derivative, has been used as a heparinoid drug to prevent and treat hyperlipidemia and ischemic cardio-cerebrovascular diseases in China for 30 years. But its bleeding risk should not be overlooked. Here we clarified the reasons and mechanism leading to bleeding side effect of PSS. It was found that PSS fractions with low mannuronic acid (M)/guluronic acid (G) ratio and high molecular weight (Mw) can excessively extend activated partial thromboplastin time (APTT) and thrombin time (TT), over-inhibit the thrombin (FIIa) activity mediated by anti-thrombin III (ATIII) to induce bleeding risk. In addition, the fraction of low M/G ratio can suppress platelet aggregation mediated by adenosine diphosphate (ADP) and induce platelet reduction by improving platelet antibody (PA)-IgA/G in serum and by inhibiting or damaging the bone marrow hematopoietic function. And the fraction of high Mw can restrain the reticulated platelet (RP) production, then reduce mean platelet volume (MPV) and platelet-large cell counts or ratio, and finally decrease platelet amount by inhibiting or damaging the bone marrow hematopoietic function. In brief, PSS fractions with low M/G ratio and high Mw were the main reasons to bring about bleeding by excessively suppressing coagulant factors activities and weakening platelet function. Our results suggested that it is very necessary to control the M/G ratio and the range of Mw of PSS to guarantee its safety and effectiveness in clinical.

Study on quality control of sulfated polysaccharide drug, propylene glycol alginate sodium sulfate (PSS).

The combination of biological and chemical analysis methods was developed to improve quality control of propylene glycol alginate sodium sulfate (PSS), a sulfated polysaccharide drug. The allergic and anticoagulant assays revealed that PSS fractions with higher Mw and lower M/G ratio may have allergic response and bleeding risks. HPLC with pre-column derivatization, HPGPC and IC methods were combined to analyze 10 batches of PSS samples from different manufacturers. The results showed that the quality of these PSSs varied greatly which in turn led to the unstable anticoagulant activity and side effects. The study indicated that PSS with high purity, M/G ratio above 1.5, Mw of ∼9kD and DS of 9.0-13.0% can ensure clinical efficacy and low incidence of adverse drug reactions. In conclusion, the combined methods would be in favor of guiding manufacture and quality control of PSS to guarantee its effectiveness and safety.

Anticoagulant and antithrombotic activities of low-molecular-weight propylene glycol alginate sodium sulfate (PSS).

Propylene glycol alginate sodium sulfate (PSS), a sulfated polysaccharide derivative, has been used as a heparinoid drug to prevent and treat hyperlipidemia and ischemic cardio-cerebrovascular diseases in China for nearly 30 years. To extend the applications of PSS, a series of low-molecular-weight PSSs (named FPs) were prepared by oxidative-reductive depolymerization, and the antithrombotic activities were investigated thoroughly in vitro and in vivo. The bioactivity evaluation demonstrated a positive correlation between the molecular weight and the anticoagulant and antithrombotic activities of FPs. FPs could prolong the APTT and clotting time and reduce platelet aggregation significantly. FPs could also effectively inhibit factor IIa in the presence of AT-III and HC-II. FPs decreased the wet weights and lengths of the thrombus and increased occlusion times in vivo. FP-6k, a PSS fragment with a molecular weight of 6 kDa, is an optimal antithrombotic candidate for further study and showed little chance for hemorrhagic action.

Low molecular weight guluronate prevents TNF-α-induced oxidative damage and mitochondrial dysfunction in C2C12 skeletal muscle cells.

Muscle wasting is associated with a variety of chronic or inflammatory disorders. Evidence suggests that inflammatory cytokines play a vital role in muscle inflammatory pathology and this may result in oxidative damage and mitochondrial dysfunction in skeletal muscle. In our study, we used microwave degradation to prepare a water-soluble low molecular weight guluronate (LMG) of 3000 Da from Fucus vesiculosus obtained from Canada, the Atlantic Ocean. We demonstrated the structural characteristics, using HPLC, FTIR and NMR of LMG and investigated its effects on oxidative damage and mitochondrial dysfunction in C2C12 skeletal muscle cells induced by tumor necrosis factor alpha (TNF-α), a cell inflammatory cytokine. The results indicated that LMG could alleviate mitochondrial reactive oxygen species (ROS) production, increase the activities of antioxidant enzymes (GSH and SOD), promote mitochondrial membrane potential (MMP) and upregulate the expression of mitochondrial respiratory chain protein in TNF-α-induced C2C12 cells. LMG supplement also increased the mitochondrial DNA copy number and mitochondrial biogenesis related genes in TNF-α-induced C2C12 cells. LMG may exert these protective effects through the nuclear factor kappa B (NF-κB) signaling pathway. These suggest that LMG is capable of protecting TNF-α-induced C2C12 cells against oxidative damage and mitochondrial dysfunction.

Activated AMPK explains hypolipidemic effects of sulfated low molecular weight guluronate on HepG2 cells.

Low molecular weight and sulfated low molecular weight guluronate (LMG and SLMG) were prepared and hypolipidemic effects were studied in a human hepatocellular carcinoma HepG2 cell line. Both compounds decreased total cholesterol (TC) and triglycerides (TG) and inhibited 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) activity in HepG2 cells. In general, SLMG had greater effects than LMG. Activation of sterol regulatory element-binding protein 2 (SREBP-2), low density lipoprotein receptor (LDLR), AMP-activated protein kinase (AMPK), and AMPK's downstream targets were evidenced by increased phosphorylation of AMPK, HMGCR, and acetyl-CoA-carboxylase (ACC), which decreased HMGRC and ACC activity. We further demonstrated that activated AMPK was linked to down-regulated SREBP-1 and up-regulated cholesterol 7α-hydroxylase (CYP7A1).

Identification and characterization of potent CYP2D6 inhibitors in lotus leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: The herb of lotus (Nelumbo nucifera) leaves is a commonly used traditional Chinese herbal medicine that is utilized for the treatment of sunstroke, to assuage thirst, and to cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the herb exhibits various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-HIV, anti-microbial, and anti-hypoglycemic activities. Currently, the herb is becoming more popular in China as a "tea drink" or as a main ingredient of some herbal formulations, which implies that the herb and/or its products are now more likely to be concurrently administered with conventional medicines for losing body weight and reducing blood lipids. However, its potential inhibitory effect on human cytochrome P450 (CYP) has not been systemically investigated to date. The present study was performed to assess the potential inhibitory effects of lotus leaf alcoholic extract (LAE), its major fractions, and its main compounds on five CYP isoenzymes (CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in vitro. MATERIAL AND METHODS: Five probe substrates were incubated with human liver microsomes in the presence or absence of the LAE, the alkaloid fraction (AF), the flavonoid fraction (FF), or the individual aporphine alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and 2-hydroxy-1-methoxyaporphine (HMA). After the incubation, the relative metabolites of the substrates were analyzed using LC-MS/MS. RESULTS: The results showed that the LAE strongly inhibited CYP2D6 with an IC50 value of 12.05µg/mL and weakly inhibited other isoenzymes. In addition, FF was found to weakly inhibit CYP2D6, whereas AF exerted a markedly higher inhibitory effect on CYP2D6 activity with an IC50 value of 0.96µg/mL. The three aporphine alkaloids isolated from the AF (NF, N-NF, and HMA) significantly inhibited CYP2D6 with IC50 values of 3.78, 3.76, and 3.15µM, respectively. Their Lineweaver-Burk plots and Dixon plots showed that NF, N-NF, and HMA competitively inhibited CYP2D6 activity with Ki values of 1.88, 2.34, and 1.56µM, respectively. CONCLUSION: The study revealed that the alkaloid compounds in lotus leaves exert a potent inhibitory effect on CYP2D6 isoenzyme. The possible drug interactions of the leaves and their preparations with conventional medicines should thus be taken into account.

An HPLC method for microanalysis and pharmacokinetics of marine sulfated polysaccharide PSS-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles in rat plasma.

This study was aimed at developing a sensitive and selective HPLC method with postcolumn fluorescence derivatization for the detection of propylene glycol alginate sodium sulfate (PSS) in rat plasma. Plasma samples were prepared by a simple and fast ultrafiltration method. PSS was extracted from rat plasma with D-glucuronic acid as internal standard. Isocratic chromatographic separation was performed on a TSKgel G2500 PWxL column with the mobile phase of 0.1 M sodium sulfate at a flow rate of 0.5 mL/min. Analyte detection was achieved by fluorescence detection (FLD) at 250 nm (excitation) and 435 nm (emission) using guanidine hydrochloride as postcolumn derivatizing reagent in an alkaline medium at 120 °C. The calibration curve was linear over a concentration range of 1-500 µg/mL, and the lower limit of detection (LLOD) was found to be 250 ng/mL. This validated method was applied successfully to the pharmacokinetic study of PSS and PSS-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (PSS-NP) in rat plasma after a single intravenous (PSS only) and oral administration (PSS and PSS-NP). Significant differences in the main pharmacokinetic parameters of PSS and PSS-NP were observed. The relative bioavailability of PSS-NP was 190.10% compared with PSS which shows that PSS-NP can improve oral bioavailability.

Different pharmacokinetics of the two structurally similar dammarane sapogenins, protopanaxatriol and protopanaxadiol, in rats.

Dammarane Sapogenins (DS), with main ingredients of protopanaxatriol (PPT, 33%) and protopanaxadiol (PPD, 16%), is an alkaline hydrolyzed product of ginsenosides and had significant activities in improving learning and memory and decreasing chemotherapy-induced myelosuppression. In the present study, the pharmacokinetics and oral bioavailabilities of PPT and PPD were investigated when a single dose of DS was administrated orally (75 mg/kg) and intravenously (i.v., 30 mg/kg) to rats. Their in vitro stabilities in the GI tract were also investigated. PPT and PPD concentrations were measured by LC-MS. The results showed that PPT was eliminated rapidly from the body with an average t1/2, λz value of 0.80 h and CL of 4.27 l/h/kg after i.v. administration, while PPD was eliminated relatively slowly with a t1/2, λz of 6.25 h and CL of 0.98l/h/kg. After oral administration, both PPD and PPT could be absorbed into the body, but their systemic exposures were quite different. PPT was absorbed into the body quickly, with a Tmax of 0.58 h and a Cmax of 0.13 µg/ml, while PPD was absorbed relatively slowly with a Tmax of 1.82 h and a Cmax of 1.04 µg/ml. The absolute bioavailabilities of PPT and PPD were estimated as 3.69% and 48.12%, respectively. The stability test found that PPT was instable in the stomach with 40% degradation after 4h incubation at 37°C, both in pH1.2 buffer and in the stomach content solution. The instability in the stomach might be one of the reasons for PPT's poor bioavailability.

[Genotype I Japanese encephalitis virus is the main genotype in mosquito in Fujian province].

OBJECTIVE: To grasp the infection rate and genotypes of Japanese encephalitis virus (JEV) in mosquito in Fujian province. METHODS: Mosquito specimens in Sanming city, Jianyang city and Fuzhou city in Fujian province were collected in 2010. RT-PCR was used to detect the JEV sequence from the mosquitoes by specific primers. The sequence splicing and the differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree were performed by the software of ATGC, Clustal X (1.83), MegAlign, GeneDoc 3.2 and Mega (4.0). RESULTS: Totally 6987 mosquitoes were collected and main species was Culex tritaeniorhynchus and Anopheles sinensis. The infection rate of JEV in mosquitoes in Sanming, Jianyang and Fuzhou were 1.25%, 1.76% and 0.65%, respectively. One full genome in the positive specimens was sequenced. And further study showed that the positive JEV sequences belonged to genotype I. CONCLUSION: Genotype I Japanese encephalitis virus is the main genotype in mosquitos in Fujian province.