Simultaneous quantification of six flavonoids of Rhus verniciflua Stokes using matrix solid-phase dispersion via high-performance liquid chromatography coupled with photodiode array detector.

A simple and efficient matrix solid-phase dispersion via high-performance liquid chromatography coupled with a photodiode array detector was developed to analyze the following flavonoids of Rhus verniciflua Stokes: fisetin, fustin, butein, sulfuretin, garbanzol, and quercetin. The optimum conditions for the procedure was the use of Zeolite Socony Mobil-twenty-two molecular sieves as the adsorbent, sample:adsorbent ratio of 2:5, grinding for 3 min, and use of 8 mL of 70% methanol:water as the elution solvent. The method was validated for linearity, precision, reproducibility, limit of detection, and limit of quantification. The method exhibited excellent linearity for all six flavonoids. The intra- and interday precisions over a range of concentrations were below 3.0% and limits of quantification for the six flavonoids were 0.16 and 0.50 µg/mL. Compared with other published methods, the proposed method was more effective, rapid, and required less reagents. Therefore, the combination of matrix solid-phase dispersion and high-performance liquid chromatography coupled with photodiode array detector showed excellent reproducibility and simplicity and could be suitable for the extraction and quantification of multiple flavonoids in R. verniciflua Stokes samples.

Separation of polyprenols from Ginkgo biloba leaves by a nano silica-based adsorbent containing silver ions.

Polyprenols extracted from Ginkgo biloba leaves is a kinds of unsaturated compound containing double bonds. Traditionally, the separation methods for the polyprenols are lack of selectivity and their separation efficiency are low. We synthesized two kinds of functional nano-silica containing silver ions materials (AgTCM and AgTCN) which have selectivity for unsaturated compounds to separate Ginkgo biloba leaves polyprenols for the first time. AgTCN displays exceptionally high selectivity for polyprenols and high stability under extended heat and light exposure, while silver is virtually immobile during solvent elution. Importantly, the exceptional stability of AgTCN gives rise to much higher polyprenols recovery than conventional silica gel during the chromatographic elution. In addition, we found that the adsorption of polyprenols onto the AgTCN conforms to pseudo-second-order kinetic model and AgTCN has strong affinity with polyprenols by analyzing Langmuir, Freundlich, Temkin-Pyzhev, and Dubinin-Radushkevich isotherms. The calculation results of thermodynamic parameters demonstrate that decrease of temperature in favor of increasing the adsorbing capacity of polyprenols onto the AgTCN, and the adsorption process of which is exothermic reaction. Our results pave the way for the novel separation methods of polyprenols from Ginkgo biloba leaves.

Antibacterial, cytotoxic and genotoxic activity of nitrogenated and haloid derivatives of C50-C60 and C70-C120 polyprenol homologs.

BACKGROUND: Polyprenol is an important lipid with many bioactive effects. The study on differences in bioactive effects of polyprenol derivatives having different isoprene units are seldom reported and it is helpful to find out which type of polyprenol derivatives are effective for treating A549/HepG2 cells and E. coli /S. aureus. METHODS: All tested polyprenol derivatives were measured with inhibition halos by Oxford cup assays. MIC values were assessed by the broth dilution method. Time-killing curve studies were conducted in duplicate on separate days. Cytotoxicity study was measured by the MTT assay and genotoxic study was evaluated by comet assay. RESULTS: With regard to antibacterial activity, the sensitivities to the quaternary polyprenyl ammonium salt derivatives GAS and MAS were 31.3 µg/mL and 15.6-31.3 µg/mL, respectively. GAS and MAS exhibited cytotoxic activity toward HepG2 cells (IC50 of 10.1-11.6 µg/mL), which was stronger than that exhibited toward A549 cells (IC50 of 13.8-13.9 µg/mL). The bactericidal activity of MAS was stronger than that of GAS at the same concentration at least 48 h. The DNA damage in A549 and HepG2 cells exposed to all 10, 20 and 40 µg/mL MAS was statistically significant in comparison to the control. Our results indicate a dose-dependent increment in DNA damage in A549 and HepG2 cells exposed to 10, 20 and 40 µg/mL MAS for both the percentage of DNA in the tail and tail moment. CONCLUSION: The quaternary ammonium salt derivatives GAS and MAS exhibited higher antibacterial (E. coli and S. aureus) and cytotoxic activity (A549 and HepG2 cells) than the other derivatives evaluated in this study. The DNA damage in HepG2 cells suggests that MAS induced A549 and HepG2 cells death via apoptotic pathway. Our results provide new evidence supporting the medical use of polyprenol derivatives against bacterial and tumor diseases.

Analysis on the Physicochemical Properties of Ginkgo biloba Leaves after Enzymolysis Based Ultrasound Extraction and Soxhlet Extraction.

In this study, high performance liquid chromatography (HPLC), ultraviolet (UV), thermagravimetric analyzer (TGA), pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), and scanning electron microscope (SEM) were used as measurement techniques, contents of chemical composition, pyrolytic products, thermal stability, morphological characterization of Ginkgo biloba leaves (GBL) acted as the index, and physicochemical properties of GBL after enzymolysis based ultrasound extraction (EBUE) and Soxhlet extraction were studied. The detection results of chemical composition revealed that contents of general flavone, soluble protein, soluble total sugar and protein in the GBL declined significantly after EBUE, and contents of polyprenols and crude fat obviously reduced as well after Soxhlet extraction. Py-GC-MS results indicated that total GC contents of micromolecules with carbon less than 12 from 54.0% before EBUE decline to 8.34% after EBUE. Total GC contents of long-chain fatty acids with carbon less than 20 from 43.0% before EBUE reduced to 27.0% after Soxhlet extraction. Thermal stability results showed that GBL after Soxhlet extraction was easier to decompose than GBL before EBUE. SEM results illustrated that surface structure of GBL was damaged severely after EBUE, compared with GBL before EBUE, while organic solvent extraction had little influence on the morphological characterization of GBL after Soxhlet extraction compared with GBL after EBUE.

In Vivo and In Vitro Toxicity Evaluation of Polyprenols Extracted from Ginkgo biloba L. Leaves.

Polyprenols of Ginkgo biloba L. leaves (GBP) are a new type of lipid with 14-24 isoprenyl units, which in humans have strong bioactivity like the dolichols. A large amount of work showed that GBP had good antibacterial activity and powerful protective effects against acute hepatic injury induced by carbon tetrachloride and alcohol, as well as antitumor activity, but the safety of GBP was not considered. The current study was designed to evaluate the toxicity of these polyprenols. Acute toxicity in mice was observed for 14 days after GBP oral dosing with 5, 7.5, 10, 15 and 21.5 g/kg body weight (b. wt.) Further, an Ames toxicity assessment was carried out by plate incorporation assay on spontaneous revertant colonies of TA97, TA98, TA100 and TA102, with GBP doses designed as 8, 40, 200, 1000 and 5000 µg/dish, and subchronic toxicity was evaluated in rats for 91 days at GBP doses of 500, 1000 and 2000 mg/kg b. wt./day. The weight, food intake, hematological and biochemical indexes, the ratio of viscera/body weight, and histopathological examinations of tissue slices of organs were all investigated. The results showed that no animal behavior and appearance changes and mortality were seen during the observation period with 21.5 g/kg GBP dose in the acute toxicity test. Also, no mutagenicity effects were produced by GBP (mutation rate < 2) on the four standard Salmonella strains (p > 0.05) in the Ames toxicity test. Furthermore, the no observed adverse effect level (NOAEL) of GBP was 2000 mg/kg for 91 days feeding of rats in the subchronic toxicity tests. Results also showed the hematological and biochemical indexes as well as histopathological examination changed within a small range, and all clinical observation indexes were normal. No other distinct impacts on cumulative growth of body weight, food intake and food utilization rate were discovered with GBP. No significant difference was discovered for the rats' organ weight and the ratio of viscera to body weight (p > 0.05). Reversible pathological changes in the histopathological examinations of tissue slices of organs were not observed. GBP could therefore be considered as a safe material with minor side effects.

Effects of alfalfa saponin extract on mRNA expression of Ldlr, LXRα, and FXR in BRL cells.

We studied the effects of alfalfa saponin extract (ASE) on low density lipoprotein receptor (Ldlr), liver X receptor α (LXRα), and farnesoid X receptor (FXR) in normal and hyperlipidemic Buffalo rat liver (BRL) cells. Normal and hyperlipidemic BRL cells were divided into eight groups: normal, or normal cells treated with 50, 100, and 150 mg/L ASE, hyperlipidemic, or hyperlipidemic cells treated with 50, 100, and 150 mg/L ASE. After treatment for 24 h, Ldlr, LXRα, and FXR mRNA expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Data showed that mRNA expression of Ldlr in normal BRL cells was significantly up-regulated by ASE treatment and mRNA expressions of LXRα and FXR were significantly down-regulated both in normal and hyperlipidemic BRL cells after ASE treatment. Thus, ASE might ameliorate hepatic steatosis by regulating genes involved in cholesterol metabolism, including up-regulation of Ldlr as well as down-regulation of LXRα and FXR.

Antiviral activity of a nanoemulsion of polyprenols from ginkgo leaves against influenza A H3N2 and hepatitis B virus in vitro.

UNLABELLED: In order to improve the bioavailability levels of polyprenols (derived from ginkgo leaves (GBP)) in the human body, a GBP nanoemulsion was prepared, and its antiviral activity was evaluated against influenza A H3N2 and hepatitis B virus in vitro. METHODS: A GBP nanoemulsion was prepared by inversed-phase emulsification (IPE). Next, we investigated the antiviral activity of the GBP nanoemulsion on influenza A H3N2 and hepatitis B virus in vitro by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenlytetrezolium bromide) method. ELISA and the fluorescent quantitative PCR method were used to measure the content of HBsAg, HBeAg and DNA virus in human samples. RESULTS: The GBP nanoemulsion exhibited uniformity at an average particle size 97 nm with a hydrophilic-lipophilic balance (HLB) of 9.5. GBP is non-toxic to normal cells, hepatitis B virus DNA, hepatitis B virus antigen and HepG2215. Furthermore, GBP could reach a 70% virucidal activity and a 74.9% protection rate (*** p < 0.001) on MDCK cells infected with H3N2 virus at a high concentration of 100 µg/mL. GBP had a good inhibition rate on HBsAg (52.11%, ** p < 0.01) at 50 µg/mL and Day 9 of incubation, and a 67.32% inhibition effect on HBeAg at a high concentration of 100 µg/mL and Day 9. GBP had good inhibition on HBV DNA with CT 18.6 and lower copies (** p < 0.01) at a middle concentration of 12.5 to 25 µg/mL. CONCLUSIONS: The GBP nanoemulsion was very stable and non-toxic and had very strong antiviral activity against influenza A H3N2 and hepatitis B virus in vitro. The inhibitory effects and reactive mechanisms were similar to the drug, 3TC; by lengthening the incubation time and increasing the drug concentration, GBP has promising potential as an antiviral drug.

Chemical constituents and structural characterization of polysaccharides from four typical bamboo species leaves.

In order to find bamboo leaves with high contents of bioactive polysaccharides, 32 samples were chosen to analyze their polysaccharide content by GC and sulfuric acid-anthrone colorimetric assays. Purified polysaccharides (BLPS) were separated from the four varieties P. nigra (Lodd.) Munro (PN), P. vivax McClure (PV), Chimonobambusa quadrangularis (Fenzi) Makino (CQ), and P. bambussoides cv. Tanakae (PB) by ultrasound extraction, solution precipitation, ion exchange resin, DEAE-52 and Sephadex G-100 chromatography. BLPS structural characterization was accomplished by HPLC-GPC, Fourier transform infra-red spectroscopy (FTIR) and NaIO4-HIO4 oxidation reactions. The results showed that the total polysaccharides of the bamboo leaves in samples 1-32 ranged between 1.4% and 5.4%, Samples No. 29-No. 32 (PN, PV, CQ, and PB) contained 2-3 fold more polysaccharides than No. 1~No. 28 among the 32 different species, particularly the content of galactose was in a range of 21.5%-34.1% for these four typical bamboo species leaves, which was also more than 2-3 fold higher than in No. 1-No. 28. Sugar analysis indicated that PN-PBLPS-1, PV-PBLPS-1, CQ-PBLPS-1 and PB-PBLPS-1 from the four varieties were homogeneous polysaccharides with molecular weights of 2.04 × 104, 1.15 × 104, 8.75 × 104 and 1.48 × 104 Da, respectively. PB-PBLPS-1 was a mixture of α-galactopyranose and ß-d-glucopyranose linkages with α-(1→6) or ß-(1→6)glycosidic bonds, while PN-PBLPS-1, PV-PBLPS-1, and CQ-PBLPS-1 had α galactopyranose linkages with α-(1→6) glycosidic bonds.

Enzymatic hydrolysis of oleuropein from Olea europea (olive) leaf extract and antioxidant activities.

Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 µg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

Quantitative analysis of pancreatic polypeptide cell distribution in the human pancreas.

The pancreatic islet is mainly composed of beta-, alpha- and delta-cells with small numbers of pancreatic polypeptide (PP) and epsilon cells. It is known that there is a region in the head of the pancreas that is rich in PP-cells. In the present study, we examined the distribution of PP-cells, and assessed the influence of the PP-cell rich region to quantify the total islet mass. Pancreatic tissues were collected from donors with no history of diabetes or pancreatic diseases (n = 12). A stereological approach with a computer-assisted large-scale analysis of whole pancreatic sections was applied to quantify the entire distribution of endocrine cells within a given section. The initial whole pancreas analysis showed that a PP-cell rich region was largely restricted to the uncinate process with a clear boundary. The distinct distribution of PP-cells includes irregularly shaped clusters composed solely of PP-cells. Furthermore, in the PP-cell rich region, beta- and alpha-cell mass is significantly reduced compared to surrounding PP-cell poor regions. The results suggest that the analysis of the head region should distinguish the PP-cell rich region, which is best examined separately. This study presents an important implication for the regional selection and interpretation of the results.

Antibacterial/antifungal activity and synergistic interactions between polyprenols and other lipids isolated from Ginkgo biloba L. leaves.

Polyprenols separated from lipids are promising new components from Ginkgo biloba L. leaves (GBL). In this paper, ginkgo lipids were isolated by extraction with petroleum ether, saponification, and molecular distillation. Eight known compounds: isophytol (1), nerolidol (2), linalool (3), ß-sitosterol acetate (4), ß-sitosterol (5), stigmasterol (6), ergosterol (7), ß-sitosterol-3-O-ß-D-glucopyranoside (8) and Ginkgo biloba polyprenols (GBP) were separated from GBL by chromatography and identified mainly by NMR. The separated and identified compounds 1, 2 and 3 are reported here for the first time in GBL. The 3D-DAD-HPLC-chromatogram (190-232 nm) of GBP was recorded. This study provides new evidence as there are no previous reports on antibacterial/antifungal activities and synergistic interactions between GBP and the compounds separated from GBL lipids against Salmonella enterica, Staphylocococus aureus and Aspergillus niger. Nerolidol (2) showed the highest activity among all the tested samples and of all mixture groups tested the GBP with isophytol (1) mixture had the strongest synergistic effect against Salmonella enterica among the three tested strains. A proportion of isophytol and GBP of 38.19%:61.81% (wt/wt) was determined by mixture design as the optimal proportion for the synergistic effect of GBP with isophytol against Salmonella enterica.

Hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves on CCl4-induced hepatotoxicity in rats.

The hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves were evaluated against carbon tetrachloride induced hepatic damage in Sprague-Dawley rats. The elevated levels of serum ALT, AST, ALP, ALB, TP, HA, LN, TG, and CHO were restored towards normalization significantly by GBP in a dose dependent manner. The biochemical observations were supplemented with histopathological examination of rat liver sections. Meanwhile, GBP also produced a significant and dose-dependent reversal of CCl(4)-diminished activity of the antioxidant enzymes and reduced CCl(4)-elevated level of MDA. In general, the effects of GBP were not significantly different from those of the standard drug Essentiale.

[Mechanism of linoleic acid on the expression of plasminogen activator inhibitor type-1 in HepG2 cells].

AIM: To investigate the molecular mechanism underlying the effect of linoleic acid on plasminogen activator inhibitor type-1 (PAI-1) expression in HepG2 cells. METHODS: HepG2 cells were exposed to different concentrations of linoleic acid and PAI-1 expression was determined by RT-PCR and colorimetric assay. Luciferase reporter gene plasmids containing four sequentially truncated fragments of the PAI-1 promoter region (-804 to +17) were constructed, and plasmids carrying constructs of Smad binding element (SBE)-site directed deletions in PAI-1 promoter were also generated using overlap extention PCR and transiently transfected into HepG2 cells, the transcriptional activity of PAI-1 was demonstrated by the luciferase activity.The effect of linoleic acid on Smad3 and Smad4 protein levels in cultured HepG2 cells was measured by Western blot analysis. RESULTS: (1) Linoleic acid remarkably increased PAI-1 mRNA expression and transcription in varying concentrations. (2) The level of PAI-1 transcription was gradually decreased induced by linoleic acid when transfected the SBE- site directed-deletions plasmids in PAI-1 promoter at -734/-731. (3) Protein levels of both Smad3 and 4 in HepG2 cells were increased by linoleic acid. CONCLUSION: Linoleic acid regulated the expression of PAI-1 from transcriptional level in HepG2 cells and SBE involved in the regulation, and both Smads protein and Smad signaling pathway acted main role in this procession.