Effect of patchouli alcohol on Helicobacter pylori-induced neutrophil recruitment and activation.

Neutrophil infiltration typically occurs in Helicobacter pylori (H. pylori)-induced acute gastritis; however, this immune response fails to eradicate H. pylori in vivo. Moreover, reactive oxygen species (ROS), which are generated by neutrophils, cause severe damage to gastric mucosa. Patchouli alcohol (PA) has been reported to have effective anti-oxidative and anti-H. pylori activities, and we investigated its effects on H. pylori-induced neutrophil recruitment and activation in this research. In neutrophil recruitment experiment, H. pylori was injected into rat air pouch to explore the effects of PA (10, 20 and 40 mg/kg) on acute inflammatory response. The results revealed that PA significantly reduced the weight of exudate and the number of neutrophils in the air pouch. Meanwhile, remarkable decrements in TNF-α and IL-8 levels in exudates were observed. In neutrophil activation experiment, rat neutrophils were isolated and activated by using 50 µg/mL H. pylori water-soluble surface protein with or without the treatment of PA (5, 10 or 20 µmol/L). Results indicated that PA not only significantly inhibited the production of ROS, but also reduced the gene and protein expressions of p22/p47-phoxes, and the binding of p22/p47-phoxes. Furthermore, the influence of PA on the neutrophil activation genes of H. pylori (h-nap and sabA) was investigated, and the results showed that expressions of h-nap and sabA were remarkably decreased after PA treatment. In conclusion, PA reduced the recruitment and activation of neutrophils induced by H. pylori, as shown by its inhibition of pro-inflammatory factor generation, p22/p47-phoxes function and H. pylori neutrophil activation-related gene expression.

Geniposide attenuates Aß25-35-induced neurotoxicity via the TLR4/NF-κB pathway in HT22 cells.

Alzheimer's disease (AD), a neurodegenerative disorder, is marked by the accumulation of amyloid-ß (Aß) and neuroinflammation which promote the development of AD. Geniposide, the main ingredient isolated from Chinese herbal medicine Gardenia jasminoides Ellis, has a variety of pharmacological functions such as anti-apoptosis and anti-inflammatory activity. Hence, we estimated the inflammatory cytotoxicity caused by Aß25-35 and the neuroprotective effects of geniposide in HT22 cells. In this research, following incubation with Aß25-35 (40 µM, 24 h) in HT22 cells, the methylthiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) release assays showed that the cell survival rate was significantly decreased. In contrast, the reactive oxygen species (ROS) assay indicated that Aß25-35 enhanced ROS accumulation and apoptosis showed in both hoechst 33342 staining and annexin V-FITC/PI double staining. And then, immunofluorescence test revealed that Aß25-35 promoted p65 to transfer into the nucleus indicating p65 was activated by Aß25-35. Moreover, western blot analysis proved that Aß25-35 increased the expression of nitric oxide species (iNOS), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-1ß (IL-1ß). Simultaneously, Aß25-35 also promoted the expression of toll-like receptor 4 (TLR4), p-p65 and p-IκB-α accompanied with the increase in the level of beta-secretase 1 (BACE1) and caspase-3 which further supported Aß25-35 induced apoptosis and inflammation. Fortunately, this up-regulation was reversed by geniposide. In conclusion, our data suggest that geniposide can alleviate Aß25-35-induced inflammatory response to protect neurons, which is possibly involved with the inhibition of the TLR4/NF-κB pathway in HT22 cells. Geniposide may be the latent treatment for AD induced by neuroinflammation and apoptosis.

[Pharmacokinetics of genistein in urine of healthy volunteers].

In order to study the excretion of genistein (GEN) capsule, an estrogen drugs, in human, 30 healthy volunteers were selected and orally administered 50, 100, and 300 mg genistein in an parallel study. Genistein were determined in urine by LC-MS/MS and glucuronidated genistein (GENG) were indirectly determined with enzymatic hydrolysis in urine by LC-MS/MS, and the pharmacokinetic parameters were analyzed by DAS software (ver 2.0). The result showed that the concentrations of genistein in human urine were less than 1% of the GENG, and the cumulative excretion of GEN in 48 h were 0.037, 0.134, and 0.142 mg, separately, and the urinary excretion percentage were only 0.07%, 0.13%, and 0.05%, separately. But the cumulative excretion of GENG in 48 h was 5.3, 13.8, and 15.4 mg, separately, and the urinary excretion percentage were 10.6%, 13.8%, and 5.1%, separately, and the max urinary excretive rate was 0.4, 1.0, and 1.4 mg x h(-1), separately (tmax were 6 h). Studies showed that part of drug excreted through kidney in a form of GENG in human, and the cumulative urinary excretion and the maximum excretion rate of GENG showed a proportional increase conditioned with the dose in the range of 50-100 mg, but showed non-linear increase feature in 300 mg.

Chemical profiling of bioactive constituents in Sarcandra glabra and its preparations using ultra-high-pressure liquid chromatography coupled with LTQ Orbitrap mass spectrometry.

In this study, a fast and reliable method based on an ultra-high-pressure liquid chromatography coupled with photodiode-array detection (PDA) and a linear ion trap high-resolution mass spectrometer (LTQ-Orbitrap XL) has been developed for the identification of bioactive constituents in the whole plant of Sarcandra glabra and its related four preparations. By accurate mass measurements within 5 ppm error for each molecular ion and subsequent fragment ions in routine analysis, 50 compounds, including organic acids, caffeoyl derivatives, flavonoids, coumarins and terpenoids, were identified or tentatively characterized. Among them, 6,7,8-trihydroxycoumarin-O-rhamnopyranoside (17), (2R)-naringenin-6-C-B-D-glucopyranosyl-(6→1)-apiose (25) and (2S)-naringenin-6-C-B-D-glucopyranosyl-(6→1)-apiose (27) were tentatively identified as new compounds. Besides, 21 of these compounds were coexisting in four preparations of Sarcandra glabra. Fragmentation behaviors of the four major categories of compounds were also investigated. This established UPLC-PDA/ESI-MS(n) method was reliable and effective for the separation and identification of the major constituents and would be the basis for quality control of Sarcandra glabra and its related preparations.

Application of a liquid chromatography/tandem mass spectrometry method to pharmacokinetic study of mangiferin in rats.

A simple, rapid and accurate liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated for quantification of mangiferin in rat plasma. After the addition of the internal standard (IS) paracetamol, plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on a C(18) column by isocratic elution with methanol-acetonitrile-1% acetic acid (40:3:57, v/v/v). The detection was performed on a Sciex API 3000 LC/MS/MS with TurboIonSpray ionization (ESI) inlet in the positive ion MRM mode. Good linearity was achieved over the concentration range of 3.01-601 ng/mL. Intra- and inter-day precisions were less than 9.1%, and accuracy ranged from 100.5% to 104.0%. The pharmacokinetic profiles of free mangiferin at three dose levels and mangiferin in Zhimu decoction and Zhimu-Huangbai decoction were studied for the first time in rats by this method. After single intragastric administration of free mangiferin 17.5, 35 and 70 mg/kg, C(max) and AUC increased but non-proportional to the doses. At the same dose level (35 mg/kg), C(max) and AUC of mangiferin in two decoctions were significantly higher than the corresponding values of free mangiferin.

Pharmacokinetics and safety of ginsenoside Rd following a single or multiple intravenous dose in healthy Chinese volunteers.

The pharmacokinetics and safety of ginsenoside Rd (Rd) were assessed in healthy Chinese volunteers. In the single-dose study, a randomized, open-label, 3-way crossover design was used. Participants were assigned to receive 10, 45, or 75 mg Rd by intravenous infusion, with a 2-week washout period between dosing periods. Plasma levels of Rd were found to be proportional to dose, with the mean C(max) and AUC(0-infinity) ranging from 2.8 to 19.3 mg/L and 27.9 to 212.5 mg x h/L over the dose range studied. Ginsenoside Rd was slowly cleared from plasma (t(1/2Z) = 17.7-19.3 hours). In the multiple-dose study, 10 mg Rd was administered once daily for 6 days. Slight drug accumulation was noted. The mean steady-state C(max), AUC(0-infinity), and AUC(ss) were 4.0 mg/L, 51.7 mg x h/L, and 26.4 mg x h/L, respectively. The t(1/2Z) was 20.5 hours, which was similar to the single-dose value. Ginsenoside Rd was well tolerated with no pattern of dose-related adverse events. It had a favorable pharmacokinetic and safety profile that enables the drug to be explored in future clinical studies that target patients with acute ischemic stroke.

Application of a liquid chromatography/tandem mass spectrometry method for the pharmacokinetic study of dihydroartemisinin injectable powder in healthy Chinese subjects.

A simple, rapid and accurate liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated for quantification of dihydroartemisinin (DHA) in human plasma. Following a simple single-step liquid-liquid extraction with ethyl acetate, the analyte was separated on a C(18) column by isocratic elution with methanol-water-10mM ammonium acetate (80:10:10, v/v/v), and analyzed by mass spectrometry in the positive ion MRM mode. Good linearity was achieved over a wide range of 1.01-2020ng/mL. Intra- and inter-day precisions were less than 9.0%, and accuracy ranged from 93.0 to 98.2%. The pharmacokinetics of DHA injectable powder was studied for the first time in healthy subjects by this method. After single intravenous infusion of DHA injectable powder 40, 80 and 160mg, the elimination half-life (t(1/2lambdaZ)) was 1.69, 1.88 and 1.92h, respectively; mean C(max) and AUC increased in proportion to the doses. The pharmacokinetics of DHA fit the linear dynamic feature over the DHA dose range studied.

[Isolation, purification and biological activities of Arnebia euchroma glycosaminoglycans].

OBJECTIVE: To study the methods of extraction, isolation, purification and biological activities of Arnebia euchroma glycosaminoglycans (AEG). METHODS: AEG was purified by distilled water extraction, ethanol fractionation, Sephadex column chromatography. The purity and molecular weight and concentration of AEG were measured by HPLC; We divided the experiment into Physiological Saline group and the other eight groups with different doses of AEG, established RT-PRC method to observe the anti-HPV effect after their actions on the verruca tissues. RESULTS: Using HPLC, the group of AEG was divided into two glycosaminoglycans with different molecular weight as 27336 and 1152. Bioassay results showed that AEG had anti-HPV-DNA activity, the lowest effective concentration was 0.781 mg/ml. CONCLUSION: AEG extracted by this method is a mixture with two molecular weight glycosaminoglycans, and has obvious anti-HPV effect.

Single- and multiple-dose pharmacokinetics of genistein capsules in healthy chinese subjects: A phase I, randomized, open-label study.

BACKGROUND: Genistein capsules are currently being developed to treat osteoporosis in China. Genistein is extracted from the fruit of Sophora japonica Leguminosae. OBJECTIVE: The objective of this study was to assess the pharmacokinetics of genistein capsules after single and multiple oral doses in healthy Chinese subjects. METHODS: This was a Phase I, randomized, open-label, single- and multiple- dose study in healthy Chinese adults (aged 19-40 years). In the single-dose study, subjects were randomly assigned in a 1:1:1 ratio to receive genistein 50, 100, or 300 mg (in 50-mg capsules). To assess the effect of food on the pharmacokinetics, subjects in the 50-mg group were equally randomized again into fasting and postprandial (genistein was administered after a high-fat breakfast) groups according to a 2-way cross-over design. A separate equal-sized group of subjects were administered genistein 50 mg on day 1 (single dose), received no treatment on days 2 and 3, and were administered genistein 50 mg QD for 6 days (days 4-9) to obtain a multiple-dose pharmacokinetic profile. Because genistein is converted so rapidly and completely to glucuronidated genistein after administration, plasma concentrations of glucuronidated genistein were determined using a validated high-performance liquid chromatography/ tandem mass spectrometry method. Drug tolerability was assessed by monitoring adverse events (AEs) and laboratory parameters. RESULTS: The study enrolled 40 healthy subjects (24 men, 16 women; 10 each in the 50-, 100-, and 300-mg single-dose groups and 10 in the multiple-dose group). Three subjects voluntarily withdrew (2 in the 100-mg group and 1 in the 300-mg group) before study drug administration. Thirty-seven subjects (24 men, 13 women) completed the study and were included in the analysis. The mean (SD) values of the single-dose genistein 50-, 100-, and 300-mg groups were as follows: Tmax, 6.0 (2.4), 7.4 (2.4), and 5.6 (1.2) hours, respectively; tl/2, 13.0 (4.0), 12.6 (5.8), and 9.4 (1.1) hours; AUC0-t, 3344 (1635), 8389 (5164), and 9361 (2428) ng/mL · h(-1); and Cmax , 218.7 (68.6), 435.7 (202.1), and 553.4 (152.8) ng/mL. The plasma glucuronidated genistein concentrations were directly proportional to the administered dose over the range of 50 to 100 mg and increased nonproportionately with the 300-mg dose. No statistically significant differences in pharmacokinetic parameters were found in the fasting group compared with the postprandial group. In the multiple-dose group, the mean (SD) steady-state pharmacokinetic parameters on day 9 were similar to those following a single dose of genistein on day 1 (Tmax, 6.0 [1.0] vs 5.9 [1.5] hours, respectively; tl/2, 9.5 [1.5] vs 9.1 [1.5] hours; AUC0-t, 2830 [1541] vs 2078 [1308] ng/mL · h(-1); Cmax, 203.1 [130.9] vs 168.4 [105.7] ng/mL). All AEs were assessed as mild or moderate and resolved without treatment, with the exception of elevated alanine aminotransferase and aspartate aminotransferase activities in one subject that resolved with treatment. CONCLUSIONS: The pharmacokinetics of glucuronidated genistein appeared to fit the linear-dose range of genistein 50 to 100 mg, but not the 300-mg dose in these healthy Chinese volunteers. Food consumption did not significantly affect the pharmacokinetic properties. No significant differences were observed in the pharmacokinetic parameters after multiple doses of genistein compared with a single dose, suggesting that the drug did not accumulate after multiple doses.

In vivo pharmacokinetic and metabolism studies of ginsenoside Rd.

A high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS(n)) method has been developed to determine ginsenoside Rd in human plasma and to identify its metabolites in rat urine. The plasma and urine samples were pretreated by solid phase extraction (SPE) prior to analyses. In this work, gentiopicroside was used as the internal standard. The lower limit of quantification (LLOQ) for Rd in human plasma was 3 ng/ml. The average half-life time in plasma was detected as 19.29 h, when 10 mg of ginsenoside Rd was administrated intravenously to the volunteers. Seven metabolites including three oxygenated, two combined and two hydrolyzed components were identified in rat urine samples by using LC-MS and MS-MS, when ginsenoside Rd administered either orally or intravenously.

[Determination of ginsenoside rd in human plasma by LC/MS/MS].

OBJECTIVE: To develop a HPLC/MS/MS method for the determination of ginsenoside Rd in human plasma. METHODS: Plasma samples were pretreated by solid phase extraction (SPE). Ginsenoside Rd (m/z 964.6-->m/z 767.5) and gentiopicrin (m/z 374.1-->195.1) was detected by the positive electrospray ionization (ES+I)-MS method under multiple reaction monitoring (MRM) mode. RESULTS: The calibration curve of ginsenoside Rd in plasma was linear over the range of 3.00-5000.00 ng/ml. The limit of quantitation was 3.00 ng/ml. The relative recovery was 81.01-83.39%. The within-day and between-day RSDs were less than 15%. CONCLUSION This method is accurate, sensitive, and specific. It is suitable for the measurement of plasma ginsenoside Rd concentration.

[Pharmacokinetics of ginsenosides Rg1 and Re in Shenmai injection].

AIM: To study the pharmacokinetics of ginsenosides Rg1 and Re after iv infusion of Shenmai injection in human. METHODS: Ginsenosides Rg1 and Re in plasma were determined by LC/MS/MS and the pharmacokinetic parameters were calculated. RESULTS: The linear regressive curves were obtained in the range of 1.023-1023 microg x L(-1) for Rg1 and 1.05-1050 microg x L(-1) for Re. Recoveries using the method of Rg1 and Re were 99%-105% and 99%-104%, respectively. The within-day and between-day RSDs were less than 15%. After iv infusion of Shenmai injection to volunteers, the concentration-time curves of Rg1 and Re fitted to the two-compartment model, T1/2alpha were 0.28 h and 0.10 h, T1/2beta were 2.1 h and 1.2 h, respectively. CONCLUSION: The method is specific, simple, sensitive and suitable for the measurement of plasma Rg1 and Re concentrations. The distribution and elimination of Rg1 and Re were rapid after iv infusion of Shenmai injection in volunteers, the pharmacokinetic characteristics were fitted with the two-compartment model.

[Study on anti-HPV activity of Asarum heterotropoides].

OBJECTIVE: To screen anti-HPV effective fraction from Asarum heterotropoides. METHODS: Asarum heterotropoides was extracted with systematic solvents (petroleum benzine, ether, ethyl acetate, n-butanol, ethanol and distilled water) accordingly. The Polymerase Chain Reaction (FQ-PCR) was applied in the vitro pharmacodynamics for the extracts from Asarum heterotropoides. The amplification of HPV-DNA in the lesions of the isolated Condyloma acuminatum. RESULTS: After the water extracts of Asarum heterotropoides was used for HPV-DNA, the PCR amplification showed negative, with the minimum effective concentration 0.4 g/ml. But the other solvents extracts from Asarum heterotropoides appeared positive. CONCLUSION: The water extracts from Asarum heterotropoides are effective on anti-Human papillomavirus.