Revealing the anti-inflammatory ingredients in wine-processed Radix et Rhizoma Rhei using immobilized cysteinyl leukotriene receptor type 1 as the stationary phase.

Despite the tremendous progress of wine-processed Radix et Rhizoma Rhei (Jiudahuang, JDH) in removing toxic heat from the blood in the upper portion of the body for hundreds of years, the deep understanding of its functional material basis of the anti-inflammatory ingredients remains unclear due to the lack of high specific and efficient methods. Herein, taking Cysteinyl leukotriene receptor type 1(CysLT1R) as the target protein, we established a chromatographic method based on the immobilized CysLT1R using haloalkane dehalogenases (Halo) at the C-terminus of the receptor in one step. After careful characterization by X-ray photoelectronic spectroscopy, immune-fluorometric analysis, and chromatographic investigations, the immobilized receptor was used to screen the anti-inflammatory ingredients in JDH. Aloe-emodin, rhein, emodin, chrysophanol, and physcion were identified as the main anthraquinone exerting anti-inflammatory effects in the drug. The association constants for the five compounds to bind with the receptor were calculated as (0.30 ± 0.06)× 105, (0.35 ± 0.03)× 105, (0.46 ± 0.05)× 105, (1.05 ± 0.14)× 105, and (1.66 ± 0.17)× 105 M-1 by injection amount-dependent method. Meanwhile, hydrogen bonds were identified as the main driving force for the five compounds to bind with CysLT1R by molecular docking. Based on these results, we believe that the immobilized receptor chromatography preserves historic significance in revealing the functional material basis of the complex matrices.

Rapid separation and binding configuration prediction of the components in Danshen decoction to endothelin A receptor using affinity chromatography and molecular dynamics simulation.

As a famous traditional Chinese formula, Danshen Decoction has the potential to relieve the pain of pulmonary arterial hypertension patients, however, the functional components remain unknown. Herein, we reported a method to screen the functional components in Danshen Decoction targeting endothelin receptor A, an accepted target for the treatment of the disease. The receptor was functionalized on the macroporous silica gel through an epidermal growth factor receptor fusion tag and its covalent inhibitor. Using the affinity gel as the stationary phase, the bioactive compound was identified as salvianolic acid B by mass spectrometry. The binding kinetic parameter (dissociation rate constants kd ) of salvianolic acid B with the receptor was determined via peak profiling. Using the specific ligands of the receptor as probes, the binding configuration prediction of salvianolic acid B with the receptor was performed by molecular dynamics simulation. Our results indicated that salvianolic acid B is a potential bioactive compound in Danshen Decoction targeting the receptor. This work showed that receptor chromatography in combination with molecular dynamics simulation is applicable to predicting the binding kinetics and configuration of a ligand to a receptor, providing crucial insight for the rational design of drugs that recognize functional proteins.

Neuroprotective effects of the aerial parts of Polygala tenuifolia Willd extract on scopolamine-induced learning and memory impairments in mice.

Alzheimer's disease is a common neurodegenerative disease characterized by progressive cognitive dysfunction and behavioral impairment. Aerial parts of Polygala tenuifolia Willd (APT) is a traditional Chinese medicine used for the treatment of amnesia. The present study aimed to investigate the protective effects of APT on scopolamine-induced learning and memory impairments in mice. Scopolamine-induced mice were used to determine the effects of APT on learning and memory impairment. Mice were orally administered with APT (25, 50 and 100 mg/kg) and piracetam (750 mg/kg) for 14 days, and intraperitoneally injected with scopolamine (2 mg/kg) from days 8 to 14. Morris water maze and step-down tests were performed to evaluate learning and memory. Levels of acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), interleukin (IL)-1ß, IL-10 and brain-derived neurotrophic factor (BDNF) in the hippocampus and frontal cortex were measured by ELISA. Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) were measured via biochemical detection. The results demonstrated that APT ameliorated learning and memory impairment in scopolamine-induced mice. Correspondingly, APT significantly increased ACh and ChAT levels in the hippocampus and prefrontal cortex of scopolamine-induced mice. Additionally, treatment with APT significantly increased BDNF and IL-10 levels, and decreased IL-1ß and AChE levels in the same mice. Furthermore, APT significantly increased SOD activity and GSH content, and decreased MDA levels in brain tissue. These results indicated that APT may ameliorate learning and memory impairment by regulating cholinergic activity, promoting BDNF and inhibiting neuroinflammation and oxidative stress.

Extract of the Aerial Part of Polygala tenuifolia Attenuates d-Galactose/NaNO2-induced Learning and Memory Impairment in Mice.

Alzheimer's disease, one of the most common types of age-related dementia, is characterized by memory deterioration and behavior disorder. The aboveground part of Polygala tenuifolia is a traditional Chinese medicine used for the treatment of amnesia. This study was conducted to investigate the ameliorating effect of the aerial part of P. tenuifolia on d-galactose/NaNO2-induced learning and memory impairment in mice. d-galactose (120 mg/kg) and NaNO2 (90 mg/kg) were injected intraperitoneally for 60 days to induce learning and memory impairment in mice. The aerial part of P. tenuifolia (25, 50, and 100 mg/kg) and piracetam (200 mg/kg) were simultaneously administered orally on days 15 - 60. Results of this study showed that aerial part of P. tenuifolia significantly decreased the latency time and increased the number of platform crossings in the Morris water maze compared with the Model group. Moreover, the aerial part of P. tenuifolia significantly increased the latency time and decreased the error frequency in the step-down and step-through tests compared with the Model group. Meanwhile, the aerial part of P. tenuifolia was able to regulate the cholinergic system by increasing the levels of ACh and ChAT and decreasing the level of AChe. The aerial part of P. tenuifolia also significantly attenuated the levels of interleukin-1 beta and malonaldehyde and enhanced the interleukin-10 and glutathione levels and superoxide dismutase activity. Furthermore, treatment with aerial part of P. tenuifolia increased the protein and mRNA expression of brain-derived neurotrophic factor and tropomyosin receptor kinase B in the hippocampus. These results suggest that the aerial part of P. tenuifolia can ameliorate learning and memory impairments by modulating cholinergic activity, inhibiting neuroinflammation and oxidative stress, and regulating the brain-derived neurotrophic factor and tropomyosin receptor kinase B signaling pathway.

Simultaneous Quantification of Seven Constituents from Zaoren Anshen Prescription and Four Endogenic Components in Rat Plasma by UHPLC-TSQ-MS/MS and the Application of the Correlation Study.

A highly rapid and sensitive ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-TSQ-MS/MS) method was developed for the simultaneous determination of 7 constituents from Zaoren Anshen prescription (ZAP) and 4 endogenic components in rat plasma. The proteins in the plasma samples were removed using acetonitrile. The separation of the 11 components was performed on an Alltima C18 column (150 × 4.6 mm, 5 µm) with acetonitrile and 0.01% formic acid water as the mobile phase. Quantification of the 11 components was performed by multiple reaction monitoring (MRM), and the electrospray ion source polarity was switched between positive and negative modes. The method exhibited good linearity for the 11 components (R2 > 0.9942). The lower quantitative limit for the 11 components was in the range from 0.90-9.95 ng/mL. The precision was evaluated by intraday and interday assays, with all relative standard deviation (RSD)% values within 14.92%. The relative error of the accuracy ranged from -9.90 to 14.93%. The recovery ranged from 73.94 to 101.06%, and the matrix effects of the 7 components ranged from 80.06 to 105.70%. The developed method was successfully applied for correlation analysis for the simultaneous quantification of the 7 constituents from ZAP and 4 endogenic components in rat plasma after ZAP treatment.

Development and validation of an LC-MS/MS method for the simultaneous quantification of seven constituents in rat plasma and application in a pharmacokinetic study of the Zaoren Anshen prescription.

A sensitive, specific and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of seven constituents of the Zaoren Anshen prescription (ZAP) in rat plasma after oral administration of the ZAP: spinosin, salvianic acid A, 6'''-feruloylspinosin, protocatechualdehyde, salvianolic acid B, schisandrin and deoxyschisandrin. The plasma samples and the internal standard (IS) sulfamethoxazole were extracted using acetonitrile. Chromatographic separation was performed with an Agilent HC-C18 column using a gradient elution profile and a mobile phase consisting of 0.01% formic acid in water (A) and acetonitrile (B). The analytes were quantified simultaneously in a single run using an ion trap mass spectrometer operated in the multiple reaction monitoring mode and electrospray ion-source polarity in the positive and negative modes. The calibration curves for spinosin, salvianic acid A, 6'''-feruloylspinosin, protocatechualdehyde, salvianolic acid B, schisandrin and deoxyschisandrin were linear over the concentration ranges of 2.90-1160, 2.50-1000, 1.80-720, 0.65-260, 2.50-1000, 8.00-1600 and 1.30-520 ng/mL, respectively. The intra- and inter-day precisions in terms of relative standard deviation were <18.9%, and the accuracies in terms of relative error were within ±14.2%. Consequently, the proposed method was successfully applied to the pharmacokinetic analysis of these seven major active compounds in rats administered ZAP. These results will facilitate research aiming to predict the effectiveness of the optimal dose of ZAP and might be beneficial for the therapeutic use of ZAP in the clinical setting.

Purification and activity characterization of polysaccharides in the medicinal lichen Umbilicaria tornata from Taibai Mountain, China.

Water-soluble polysaccharides from Umbilicaria tornata (UTP) were purified and preliminarily characterized. The antioxidant and antitumor activities of crude UTP and two purified fractions (UTP-1 and UTP-2) were evaluated using in vitro experiments. The results showed that the molecular weights of UTP-1 and UTP-2 were 84.86 and 28.66 kDa, respectively. Both UTP-1 and UTP-2 were composed of glucose and xylose, with their molar ratios being 1.3:0.9 and 0.9:4.6, respectively. In addition, crude UTP, UTP-1 and UTP-2 showed dose-dependent DPPH and hydroxyl radical scavenging and reducing activities. However, crude UTP exhibited stronger antioxidant activity than UTP-1 and UTP-2, particularly in terms of DPPH radicals. Crude UTP and the two purified fractions inhibited the growth of HeLa, HepG2, A375, MCF-7, SGC7901 and Caco2 cancer cells in vitro. Compared with UTP-1 and UTP-2, crude UTP presented significantly higher antitumor activity in vitro against HeLa and HepG2 cells (p < 0.05). These findings provide a scientific basis for the deeper exploration and resource development of U. tornata.

Protective effect of gentiopicroside from Gentiana macrophylla Pall. in ethanol-induced gastric mucosal injury in mice.

Gentiopicroside isolated from gentiana macrophylla Pall. belongs to iridoid glycosides. This study aimed to evaluate the protective effect of gentiopicroside against ethanol-induced gastric mucosal injury in mice. Mice were proactively administrated with gentiopicroside by intragastric administration once a day for 3 consecutive days. On the 3rd day, gastric ulcer in mice was induced with 70% ethanol after the last intragastric administration. The stomach tissues were submitted for evaluation of the severity of gastric mucosal alterations. Gentiopicroside administrated orally ameliorated the severity of gastric mucosal alterations. Oral administration of gentiopicroside significantly increased heat shock protein-70 and glutathione levels and superoxide dismutase activity, normalized epidermal growth factor and vascular endothelial growth factor levels, and decreased the levels of tumour necrosis factor-α, interleukin-6 and malondialdehyde, and myeloperoxidase activity in gastric tissue. These findings demonstrated that gentiopicroside has protective effect against ethanol-induced gastric mucosal injury in mice through the improvements of antioxidative and anti-inflammatory effects, as well as up-regulation of heat shock protein-70 level and normalization of epidermal growth factor and vascular endothelial growth factor levels. The results presented in this study provide some evidence for the development of a novel antigastric ulcer agent.

[Pharmacokinetic effect of combined administration on spinosin and ferulic acid in monarch drug Ziziphi Spinosae Semen kernel].

To study the pharmacokinetic effect of different combined administration with monarch drug Ziziphi Spinosae Semen on its main components in rats. Sprague-Dawley (SD) rats were randomly divided into Ziziphi Spinosae Semen group, Ziziphi Spinosae Semen-Fructus Schisandrae Chinensis group, Ziziphi spinosae Semen-Salviae Miltiorrhize Radix et Rhizoma group and Zaoren Ansheng prescription group. After oral administration, HPLC was eluted with the mobile phase of acetonitrle-0.03% phosphate acid water in a gradient mode. The detection wavelength was 280 nm. The pharmacokinetic parameters of spinosin and ferulic acid were calculated by DAS 2. 0 software. Compared with Ziziphi Spinosae Semen group, Ziziphi Spinosae Semen-Fructus Schisandrae Chinensis group, Ziziphi Spinosae Semen-Salviae miltiorrhizae Radix et Rhizoma group showed a lower maximum plasma concentration (C(max)) and area under curve (AUC(0-t)) for spinosin and ferulic acid but higher clearance speed (CL/F); whereas the Zaoren Ansheng prescription group showed higher maximum plasma concentration (C(max)) and area under curve (AUC(0-t)) for spinosin and ferulic acid but lower clearance speed (CL/F). Compared with Ziziphi Spinosae Semen group, prescription group showed slower metabolism of spinosin and ferulic

Simultaneous Quantitative and Qualitative Analysis of Flavonoids from Ultraviolet-B Radiation in Leaves and Roots of Scutellaria baicalensis Georgi Using LC-UV-ESI-Q/TOF/MS.

Scutellaria baicalensis Georgi is one of the most widely used traditional Chinese herbal medicines. It has been used for anti-inflammatory, anticancer, antibacterial activities, and so forth. Long-term enhanced ultraviolet-B (UV-B) radiation caused more effect on leaves than on roots of the plant. Liquid chromatography-ultraviolet detection coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-UV-ESI-Q/TOF/MS) method was applied for simultaneous quantitative and qualitative analysis of flavonoids in leaves and roots of S. baicalensis by enhanced UV-B radiation. Both low-intensity radiation and high-intensity radiation were not significantly increaseing the contents of baicalin, wogonoside, and wogonin in roots. However different intensity of radiation has different effects on several flavonoids in leaves. Both low-intensity radiation and high-intensity radiation had no significant effect on contents of baicalin and tectoridin in leaves; the content of scutellarin was significantly decreased by low-intensity radiation; chrysin was detected in low-intensity radiation and high-intensity radiation, and chrysin content is the highest in low-intensity radiation, but chrysin was not detected in control group. Different changes of different flavonoids under enhanced UV-B radiation indicate that induction on flavonoids is selective by enhanced UV-B radiation.

Potential antiarrhythmic effect of methyl 3,4,5-trimethoxycinnamate, a bioactive substance from roots of polygalae radix: suppression of triggered activities in rabbit myocytes.

3,4,5-Trimethoxycinnamic acid (TMCA), methyl 3,4,5-trimethoxycinnamate (M-TMCA) and p-methoxycinnamic acid (PMCA) have been identified as the major bioactive components in the serum collected from rats treated with oral administration of Polygalae Radix ("YuanZhi," the roots of Polygala tenuifolia WILLD.), a traditional Chinese medicine used to relieve insomnia, anxiety and heart palpitation. The present study was designed to investigate its direct electrophysiological effects on isolated ventricular myocytes from rabbits. Whole-cell configuration of the patch-clamp technique was used to measure action potential (AP) and membrane currents in single ventricular myocytes enzymatically isolated from adult rabbit hearts. Ca(2+) transients were recorded in myocytes loaded with the Ca(2+) indicator Fluo-4AM. Among three bioactive substances of Polygala metabolites, only M-TMCA (15-30 µM) significantly shortened action potential duration at 50% and 90% repolarization (APD(50) and APD(90)) in cardiomyocytes in a concentration-dependent and a reversible manner. M-TMCA also inhibited L-type calcium current (I(Ca,L)), but showed effect on neither transient outward potassium current (I(to)) nor steady-state potassium current (I(K,SS)). Furthermore, M-TMCA abolished isoprenaline plus BayK8644-induced early afterdepolarizations (EADs) and suppressed delayed afterdepolarizations (DADs) and triggered activities (TAs). This potential anti-arrhythmic effects were likely attributed by the inhibition of I(Ca,L) and the suppression of intracellular Ca(2+) transients, which consequently suppress the generation of transient inward current (I(ti)). These findings suggest that M-TMCA may protect the heart from arrhythmias via its inhibitory effect on calcium channel.

[Advances in influence of UV-B radiation on medicinal plant secondary metabolism].

Stratospheric ozone depletion results in an increased level of solar UV-B radiation (UV-B, 280-320 nm) reaching the earth surface. By the effect of UV-B radiation, various medicinal active ingredients changed because of the change of gene expression, enzyme activity and secondary metabolism, clinical effect is also changed. The research status of UV-B radiation and the accumulation of plant secondary metabolites in the past 10 years were summarized in this paper to supply reference for cultivation and exploitation of the medicinal plants.

[Correlation between environmental factors and liposoluble and hydrophilic constituents of Polygalae Radix].

OBJECTIVE: To analyze the correlation between environmental factors and the lipophilic and hydrophilic constituents of Polygalae Radix. METHOD: The contents of lipophilic constituent were determined by GC-MS and hydrophilic constituents by HPLC. Geographical factors were collected by on-site inspection and climate factors by the local meteorological data. The relationship between the content of the lipophilic and hydrophilic constituents and the factors were analyzed by SPSS 18.0. RESULT: There was linear relationship between the content of lipophilic constituent and climate factors such as average temperature of July, average temperature of January. There was also linear relationship between hydrophilic constituents and climate factors such as annual average temperature, latitude, annual average rainfall. CONCLUSION: The main climate factors that affect liposoluble constituent content were average temperature of July, average temperature of January, and the main climate factors that affect hydrophilic constituent content were annual average temperature, latitude, annual average rainfall. The study would contribute to the quality evaluation and suitability of origin of Polygalae Radix.

[Effect of processing on metabolism of amygdalin from bitter almond in rat].

OBJECTIVE: To study the influence of processing on metabolism of the main component of bitter almond-amygdalin in rat. METHOD: The blood was collected at different times after amygdalin given by injection and oral, bitter almond and its processed production given by oral respectively, and then detected by both HPLC and HPLC-MS(n) methods after extraction pretreatment. RESULT: After injection, amygdalin was absorbed in prototype to blood rapidly, while the other three kinds of medicine given by oral were all not detected the prototype of amygdalin, but two metabolites were detected which were isomers of prunasin confirmed by mass spectrometry. The metabolic pathway of prunasin in processed bitter almond group was markedly different from the bitter almond group. CONCLUSION: Processing has a significant effect on bitter almond metabolic processes in rats.

[Effect of Santalum album on tissue distribution of danshensu in rabbits by HPLC].

OBJECTIVE: To investigate the influence of Shi herb (Santalum album, SA) to the tissue distribution of danshensu (DSS) which is the main hydrosoluble component of Jun herb (Salvia miltiorrhiza, SM) in rabbits by HPLC. METHOD: Rabbits were oral administrated decoction of SM and SM-SA, respectively. Perchloric acid (10%) was used to precipitate the tissue samples of rabbits heart, brain, liver, kidney, acetic ether was used to extracte supernatant, and the internal standard was p-hydroxybenzoic acid. The content of DSS of SM in tissues was detected. RESULT: The content of DSS reached the highest point close to 50 min in the mentioned tissues. Before and after co-administration, the sequences of average concentration of DSS in tissues were C(kidney) > C(heart) > C(brain) > C(liver) and C(kidney) > C(liver) > C(brain) > C(heart) respectively. Compared with SM administrated singly, the content of DSS in every tissues of co-administration was higher. CONCLUSION: In Danshenyin Formulae, SA can increase concentration of DSS in target tissues significantly, and therefore therapeutic effect of SM for cardiovascular disease is raised.

[Germplasm resources of Polygala tenuifolia and determination of presenegenin in processing products].

OBJECTIVE: To research the germplasm resources and the contents of senegenin in processing products of Polygala tenuifolia. METHOD: The contents of senegenin in wild Polygala tenuifolia and cultivated samples of Polygala tenuifolia were determined by RP-HPLC, and compared. RESULT: The contents of senegenin in wild reduce gradually along Shaanxi, Shanxi, Hebei to Dongbei. The contents of senegenin in cultivated three-year samples of three year Polygala tenuifolia from five main place was similar, 0.44%-0.49%. The content of senegenin were 0.44%-0.64% in the wand and 0.03%-0.09% in the residual part of stem, and the content of senegenin in Polygala tenuifolia was more than that in processing products. CONCLUSION: There is a correlation between the content of senegenin in Polygala tenuifolia and ecology environment that show a is inverse proportion with the quality grade, and the contents in the processing products were decreased. Senegenin can be used as a characteristic marker in range. This research provides a reference for search a index for quality control of Radix polygala and its processing products.

Co-administration of Radix curcumae alters the tissue distribution of danshensu in Radix salviae miltiorrhiae in rabbits.

The tissue distribution profile of danshensu, one of the bioactive components in Radix salviae miltiorrhiae, was investigated after oral administration of Radix salviae miltiorrhiae coupled with Radix curcumae in rabbits. Eighteen rabbits were evenly divided into 3 groups. Rabbits of the groups were orally administrated with carboxymethyl cellulose, Radix salviae miltiorrhiae (10.0 g/kg) and Radix salviae miltiorrhiae (10.0 g/kg) coupled with Radix curcumae (5.0 g/kg), respectively. High performance liquid chromatography-electrospray ionic trap mass spectrometry (HPLC-ESI-MS(n)) was used for the determination of danshensu in the tissues including heart, liver, kidney and brain. The statistical results demonstrated that the characteristic profiles of danshensu were c(kidney) > c(heart) > c(liver) > c(brain) and c(kidney) > c(brain) > c(liver) > c(heart) after administration of Radix salviae miltiorrhiae and Radix salviae miltiorrhiae coupled with Radix curcumae, respectively, indicating that Radix curcumae could obviously increase the concentration of danshensu in brain. This result would provide substantial foundation for the harmonizing effect of shi herb in a Chinese compound prescription.