Neuroprotective effect of paeoniflorin on okadaic acid-induced tau hyperphosphorylation via calpain/Akt/GSK-3ß pathway in SH-SY5Y cells.

Abnormal phosphorylation of tau, one of the most common symptoms of dementia, has become increasingly important in the study of the etiology and development of Alzheimer's disease. Paeoniflorin, the main bioactive component of herbaceous peony, is a monoterpene glycoside, which has been reported to exert beneficial effects on neurodegenerative disease. However, the effect of paeoniflorin on tauopathies remains ambiguous. SH-SY5Y cells were treated with okadaic acid (OA) for 8 h to induce tau phosphorylation and no cell death was observed. Optical microscopy results showed that paeoniflorin ameliorated okadaic acid induced morphological changes, including cell swelling and synapsis shortening. Western blotting data illustrated that paeoniflorin reversed okadaic acid induced tau hyperphosphorylation, which was enhanced by inhibiting the activities of calpain, Akt and GSK-3ß. Transmission electron microscopy results showed that paeoniflorin alone can reduce the number of autophagosomes and stabilize the microtubule structure. In addition, calpastain and paeoniflorin enhance the effect of paeoniflorin on stabilizing microtubules. In addition, calpastain markedly enhanced the effect of paeoniflorin on reversing okadaic acid-lowered fluorescence intensity of both MAP-2 and ß III-tubulin, two microtubule-associated proteins. This study shows that paeoniflorin protected SH-SY5Y cells against okadaic acid assault by interfering with the calpain/Akt/GSK-3ß-related pathways, in which autophagy might be involved. Besides, paeoniflorin is found to relieve the stress response of the microtubule structure system caused by okadaic acid treatment. The results presented in this study suggest that paeoniflorin potentially plays an important role in tauopathies.

An exploration of the role of a fish-oriented diet in cognitive decline: a systematic review of the literature.

Epidemiological studies have presented inconsistent evidence of the correlation between a fish-oriented dietary intake (FDI) and the risk of cognitive decline. To address these controversies, we performed this systematic review of prospective studies published in December 2016 and earlier using PubMed, Embase, and Web of Science. Two independent researchers conducted the eligibility assessment and data extraction; all discrepancies were solved by discussion with a third researcher. The pooled relative risks (RRs) focused on the incidence of events were estimated with 95% confidence intervals (CIs). Overall, nine studies containing 28,754 subjects were analyzed. When the highest and lowest categories of fish consumption were compared, the summary RR for dementia of Alzheimer type (DAT) was 0.80 (95%CI = 0.65-0.97); i.e., people with a higher intake of fish had a 20% (95%CI = 3-35%) decreased risk of DAT. Additionally, the dose-response synthesized data indicated that a 100-g/week increase in fish intake reduced the risk of DAT by an additional 12% (RR = 0.88, 95%CI = 0.79-0.99). Non-significant results were observed for the risk of dementia of all causes (DAC) and mild cognitive impairment (MCI). Limited evidence involving heterogeneity was found within subgroups or across studies. In conclusion, this review confirmed that a higher intake of fish could be correlated with a reduced risk of DAT. Further research, especially prospective studies that specifically quantify FDI, will help find a more accurate assessment of the different levels of dietary intake.

Determination and pharmacokinetics of amygdalin in rats by LC-MS-MS.

A sensitive and specific liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed for the determination and pharmacokinetics of amygdalin in rats. Rat plasma pretreated by solid-phase extraction was analyzed by LC-MS-MS with negative electrospray ionization in the multiple reaction monitoring mode. Amygdalin and geniposide [the internal standard (IS)] were separated on a C18 column eluted with a mobile phase of methanol and water (85:15; v/v) at a flow rate of 0.25 mL/min in a run time of 3.0 min. The precursor to product ion transitions were monitored at m/z 457.2 → 279.1 for amygdalin and m/z 387.1 → 224.9 for the IS. The calibration curve of amygdalin showed good linearity over a concentration range of 10-2,000 ng/mL. The limit of quantification was 10 ng/mL. Intra-day and inter-day precisions and accuracy (percent relative standard deviation) were both within 10%. The method was fully validated for its selectivity, sensitivity, matrix effect, recovery and stability. This accurate and specific assay produced a useful LC-MS-MS method, which was successfully applied to pharmacokinetic studies after the oral administration of amygdalin to rats.

Effect of borneol on cytochrome P450 3A enzyme and midazolam pharmacokinetics in rats.

Borneol is a commonly used herbal medication in China and Japan. Previous studies have indicated that borneol could reduce the plasma concentrations of oneself and concomitant drugs, and its first-pass metabolism could be catalyzed by the cytochrome P450 3A (CYP3A) enzyme as well. The impact of borneol on CYP3A activity and efficacy in influencing the pharmacokinetics of co-administrated drugs is currently unknown. Therefore, the purpose of the current study is to investigate the effect of borneol on CYP3A enzyme in vivo. After treatment with borneol twice daily for 3 days, rat liver microsomes were exposed to probe substrates to determine CYP3A enzyme activity, protein, and RNA harvested using microsomal testosterone 6ß-hydroxylation as a marker of enzyme activity. To verify the result, the effect of borneol on the pharmacokinetics of the CYP3A model substrate midazolam was further examined. The results showed that borneol treatment had increased CYP3A expression at the mRNA, protein, and activity (testosterone 6ß hydroxylase activity) level in rat liver microsomes. In addition, borneol accelerated the metabolism of midazolam, which was consistent with the enhancement in CYP3A metabolic capacity. The hepatic clearance (Cl) of midazolam injected via the caudal vein in rats following borneol co-administration was higher; however, the area under the curve (AUC0-∞) was lower than the solvent. Hence, it was proposed that borneol could increase the metabolic activity of the CYP3A enzyme, which might cause drug-drug interactions in humans when using Chinese herbal or Western medicine with borneol.

[The anti-portal hypertension effect of oleanolic acid in CCl4-induced cirrhosis rats].

OBJECTIVE: To study the anti-portal hypertension effect of oleanolic acid (OA) in CCl4-induced cirrhosis rats and its mechanism. METHODS: Rats were induced to portal hypertension by CCl4. After treatment with low dose of OA (30 mg/kg) and high dose of OA (60 mg/kg) by intragastrically for a month, the parameters in serum or liver tissue including ALT, AST, MDA, GSH-Px, NOx, eNOS, cGMP and type I collagen were measured. The MAP, PP and HR were determined by hameodynamic method and the eNOS expression in liver was measured by western blot. The pathological changes of liver tissue were also tested by Masson dye. The normal group and model group were given 0.25% of CMC-Na solution. RESULTS: Compared with the model group, treatment with 30 mg/kg and 60 mg/kg OA significantly decreased the levels of ALT, AST, ALP, gamma-GT and MDA and enhanced the level of GSH-Px in liver (P<0.05). Moreover, the collagen content also notably lowered in CCl4-induced cirrhosis rats, thus decreasing the portal pressure (PP). However, the MAP and HR were not affected by OA treatment. In addition, the expression of eNOS in liver markedly increased after one mouth treatment of OA, hereof enhancing the level of cGMP and NOx in the CCl4-induced portal hypertensive rats (P<0.05). CONCLUSION: OA could inhibit the progress of fibrosis and lower the PP in CCl4-induced portal hypertensive rats and the anti-portal hypertension effect might be related to increasing the expression of eNOS and enhance the NOx level in liver.

Simultaneous determination of esculin and its metabolite esculetin in rat plasma by LC-ESI-MS/MS and its application in pharmacokinetic study.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of esculin and its metabolite esculetin in rat plasma. After addition of internal standards scopoletin, the plasma sample was pretreated by solid-phase extraction (SPE), and separated on a reversed phase C(18) column with a mobile phase of 0.01% formic acid in water (solvent A) and methanol (solvent B) using isocratic elution (A:B=20:80, v/v). The detection of target compounds was done in multiple reaction monitoring (MRM) mode. The MRM detection was operated in the negative ESI mode using the transitions of m/z 339.1 ([M-H](-))→176.7 for esculetin, m/z 176.9 ([M-H](-))→133.0 and m/z 191.0 ([M-H](-))→175.9 for scopoletin. The standard curves, which ranged from 25 to 3200 ng/mL for esculin with the lowest limit of quantification (LLOQ) of 0.25 ng/mL and from 1.25 to 160 ng/mL for esculetin with the LLOQ of 1.25 ng/mL, were fitted to a 1/x weighted quadratic regression model. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD<8.73%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to study the pharmacokinetics of esculin and its metabolite esculetin in rat plasma after oral administration of esculin at a dose of 100mg/kg.

Determination of geniposidic acid in rat plasma by LC-MS/MS and its application to in vivo pharmacokinetic studies.

A simple, rapid and sensitive method for the determination of geniposidic acid (GSA) in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Geniposide (GS) was used as the internal standard. Rat plasma pretreated by solid-phase extraction (SPE) was analyzed by LC-MS/MS with negative ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The analytical column was C8 column and the mobile phase was methanol (A) and water (B). The flow rate was set at 0.8 mL/min with split ratio of 1:3, the total run time was 15 min. The MS/MS ion transitions monitored were m/z 373.3-211.1 for GSA and m/z 387.3-225.3 for GS. The quantification limit was 5 ng/mL within a linear range of 10-4000 ng/mL. The inter-day and intra-day accuracy and precision were within ±10%. The method was fully validated for its sensitivity, selectivity, matrix effect, stability study and recovery. The data indicate that our LC-MS/MS assay is an effective method for the pharmacokinetics study of GSA in rat plasma.

Pharmacokinetics of natural borneol after oral administration in mice brain and its effect on excitation ratio.

Previous studies have indicated that borneol has double side effects on the central nervous system (CNS), but the mechanism is unknown. The aim of this study was to clarify the relationship between excitation ratio [contents of excitatory amino acids (AAs) versus that of inhibitory] and the content of natural borneol after a single oral dose. Mice were administered a 1.2 g/kg dose of natural borneol (containing 98% D: -borneol) by oral ingestion. Brain samples were collected before administration and at 0.083, 0.167, 0.25, 0.333, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4 and 5 h after administration. The brain concentration of natural borneol and contents of AA neurotransmitters in mice brain were determined by GC-MS and HPLC-FLU, respectively. After per oral application, natural borneol was absorbed rapidly into the brain and could be determined 5 min after dosing. The maximal brain concentration (86.52 µg/g) was reached after 1 h post-dosing. Natural borneol could affect the contents of AA neurotransmitters in mice brain: L: -aspartic acid increased significantly from 0.083 to 1 h after administration, L: -glutamic acid increased significantly at 0.333 h and decreased from 1.5 to 5 h, gamma-amino-N-butyric acid increased significantly from 0.167 to 5 h, whereas glycine was not affected. The excitation ratio is the contents of excitatory AAs versus that of inhibitory AAs, which reflects the excitatory or inhibitory state of the body. The excitation ratio elevated transitorily and then declined 0.5 h post-dosing; there were significant differences between 1.5-5 h post-dose compared with pre-dose. The present study indicated that natural borneol could affect the contents of AA neurotransmitters, and the change in excitatory ratio led to borneol's double side effects on the CNS.

[Anticoagulative effect and antiplatelet aggregation effect of combination of Hirudo and Tabanus on rat model of blood stasis syndrome].

OBJECTIVE: To observe anticoagulative effect and antiplatelet aggregation effect of the combination of Hirudo and Tabanus with different dose-ratio on rat model of blood stasis syndrome. METHODS: The rat model of blood stasis syndrome was established by subcutaneous injection of adrenaline combined with stimulation of icy water. Then prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) contents and inhibition rate of blood platelet aggregation were determined. RESULTS: Platelet aggregation increases, APTT and PT reduced, and FIB contents increased in model control group significantly (P<0.001). Hirudo, Tabanus and the combination of Hirudo and Tabanus had antiplatelet aggregation effect in varying degrees. APTT and PT were prolonged significantly (P<0.05 and P<0.01, respectively) in Hirudo group, Tabanus group and combination groups, especially in the group with dose-ratio of Hirudo to Tabanus being 4:3. FIB contents decreased significantly in combination group with dose-ratio being 3:1 (P<0.05). CONCLUSIONS: The combination groups of Hirudo and Tabanus have better effect of anticoagulation and antiplatelet aggregation than Hirudo group and Tabanus group. While in the four combination groups, the group recommended by classical TCM monograph with dose-ratio of Hirudo to Tabanus being 4:3, has the best anticoagulation effect.

[Effects of Rhizoma zingiberis and Pericarpium citri reticulatae extracts on myocardial ischemia in rats].

OBJECTIVE: To observe the effects of Rhizoma Zingiberis and Pericarpium Citri Reticulatae extracts on acute myocardial ischemia rats and explore the mechanism. METHODS: The model of myocardial ischemia in rats was established by ligating the front descending anterior branch of the coronary artery. With Fufang Danshen Pill as positive control drug,the effects of Rhizoma Zingiberis and Pericarpium Citri Reticulatae extracts on the electrocardiogram (ECG), the extension of myocardial infarction, the hemorheology indexes, lactic dehydrogenase (LDH), creatine kinase (CK), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in rats were evaluated. RESULTS: Rhizoma Zingiberis and Pericarpium Citri Reticulatae extracts decreased the ST-segment of ECG (P < 0.01), reduced the extension of myocardial infarction (P < 0.05), decreased the contents of CK and LDH in serum (P < 0.01 or P < 0.05), improved hemorheology (P < 0.05), increased SOD and GSH-Px activity and decreased MDA content (P < 0.05). CONCLUSION: Rhizoma Zingiberis and Pericarpium Citri Reticulatae extracts have protective effect on myocardial ischemia in rats, and its mechanism may be related to inhibiting lipid peroxidation.

Plasma pharmacokinetics and tissue distribution of [6]-gingerol in rats.

[6]-Gingerol is one of the pungent components in ginger which has been found to possess various pharmacological effects. However, there is insufficient information on the properties of [6]-gingerol based on controlled pharmacokinetic studies. The aim of this study was to clarify distribution profiles of [6]-gingerol in blood and biological tissues of experimental rats. Rats were administered a 240 mg/kg dose of Gs (a ginger extract, containing 53% [6]-gingerol) by oral ingestion. Plasma samples were collected at 2.5, 5, 7.5, 10, 15, 20, 30, 45 min, and 1, 1.5, 2, 3, 4 h after dosing (eight samples per time point), and brain, heart, lung, spleen, liver, kidney, stomach and small intestine tissues were collected at 5, 15, 30 min and 1, 2, 4 h after dosing (five animals per time point). Samples were prepared by a liquid-liquid extraction procedure and the extracts were assayed by HPLC-UV. After per oral application, [6]-gingerol was absorbed rapidly into the plasma, and the maximal concentration (4.23 microg/ml) was reached after 10 min post dosing. [6]-Gingerol plasma concentrations declined with time in a biexponential pattern. The elimination half-time at the terminal phase was 1.77 h and the apparent total body clearance was 40.8 l/h. When administered orally, [6]-gingerol was well distributed to the tissues examined, with the highest concentrations found in the gastrointestinal tract. Maximal concentrations of [6]-gingerol were reached in most tissues at 0.5 h post-dosing. The concentrations of [6]-gingerol in tissues all were higher than in plasma with corresponding tissue to plasma ratios greater than 1 after 0.25 h post-dose, showing high tissue partitioning and extensive distribution.

In vitro characterization of borneol metabolites by GC-MS upon incubation with rat liver microsomes.

The metabolism of borneol is studied by the analysis of incubations of in vitro-prepared rat liver microsomes. A sensitive gas chromatography (GC)-mass spectrometry (MS) method is developed for the identification of borneol and its metabolites. Four novel metabolites, which have not previously been reported, are isolated and confirmed by comparison of the GC-MS method. The biotransformation pathway of borneol in rat liver microsomes is proposed based on the in vitro results.

Pharmacokinetics of dihydroartemisinin in Artekin tablets for single and repeated dosing in Chinese healthy volunteers.

Aim. To study the pharmacokinetics of dihydroartemisinin (DHA) in Artekin (compound dihydroartemisinin) tablets in Chinese healthy volunteers. Methods. Eighteen healthy volunteers (9 males, 9 females) received Artekin tablets for oral administration. The plasma samples of DHA were analysed by liquid-liquid extraction and determined by HPLC/ESI/MS. Results. The plasma DHA concentration-time curves of single dose and repeated doses of DHA were fitted to a two-compartment open model. The mean pharmacokinetic parameters of DHA in a single dose were: t(1/2(beta))=1.245 +/- 0.495 h, C(max)=243.6 +/- 56.15 microg/l, AUC(0 --> infinity)=450 +/- 69 h x microg/l, V(d)=5.75 +/- 2.2 l/kg and Cl=3.245 +/- 0.38 l/h/kg, while in repeated doses they were: t(1/2(beta))=1.085 +/- 0.298 h, AUC(0 --> infinity)=444.35 +/- 80.43 h x ng/ml, V(d)=4.62 +/- 1.128 ml/kg, Cl=3.0125 +/- 0.875 ml/h/kg, respectively. Conclusion. The study showed that DHA in Artekin was rapidly absorbed, distributed and eliminated in the healthy subjects. The pharmacokinetic properties of DHA in Artekin were not affected by gender in a single dose. While in repeated doses accumulation of DHA did not appear after repeated doses.

Determination of CQP propionic acid in rat plasma and study of pharmacokinetics of CQP propionic acid in rats by liquid chromatography.

A sensitive method for the determination of CQP propionic acid in rat plasma was developed and validated after solid-phase extraction. Chromatographic separation was achieved on a reversed-phase Alltima C18 column with the mobile phase of methanol-0.15% (v/v) phosphoric acid solution (pH 2.5) and step gradient elution resulted in a total run time of about 20min. The analytes were detected by using UV detector at 345nm. A good linear relationship was obtained in the concentration range of 50-12,800ng/mL (r=0.9998). The intra-day RSDs and the inter-day RSDs at the concentration of 200, 800, 6400 and 12,800ng/mL were less than 7.0% and 11.0%, respectively. The intra-day accuracy ranged from 96.3 to 106.5% and the inter-day accuracy ranged from 98.6 to 113.4%, respectively. Average extraction recoveries ranged from 83.6 to 94.3% in plasma at the concentrations of 200, 800, 6400 and 12,800ng/mL. This method was successfully applied to the pharmacokinetic studies on rats.

[Effect of Wuling Powder on rats with renal hypertension].

OBJECTIVE: To observe the therapeutic effect of Wuling Powder extract on rats with renal hypertension and to evaluate the influence of it on the volume of urine and the concentrations of Na(+), K(+), Cl(-). METHODS: Reformed Gold-blatt hypertension rat model (G-2K1C) was established. The rats were divided into 6 groups as follows: sham-operation group; model group, Wuling Powder high dosage group (80 g/kg), Wuling Powder middle dosage group (40 g/kg), Wuling Powder low dosage group (20 g/kg), and hydrochlorothiazide (HCT) group (25 mg/kg). Urine volume of the rats was measured during the experiment. Tail arterial pressure and [Na(+)], [K(+)], [Cl(-)] in serum of the rats were detected after 30 days of treatment. RESULTS: The blood pressure of the G-2K1C rats was decreased in the three Wuling Powder groups (P<0.05 or P<0.01), but higher than that of the false-operation group (P<0.01), and there was no difference between each of the Wuling Powder groups and the HCT group (P>0.05). Diuretic effect of the three dosages of Wuling Powder was weaker than that of the HCT (P<0.05 or P<0.01). The effects of the three dosages of Wuling Powder and HCT on [Na(+)] and [Cl(-)] in the serum were not obviously different (P>0.05), but [K(+)] of the HCT group was significantly decreased compared with that of the false-operation group and the three Wuling Powder groups (P<0.01). CONCLUSION: Wuling Powder extract had satisfying therapeutic effects in increasing the discharge of urine, decreasing the blood pressure and keeping the balance of the serum electrolyte contents in rats with renal hypertension.