Radix Ilicis Pubescentis total flavonoids combined with mobilization of bone marrow stem cells to protect against cerebral ischemia/reperfusion injury

Previous studies have shown that Radix Ilicis Pubescentis total flavonoids have a neuroprotective effect, but it remains unclear whether Radix Ilicis Pubescentis total flavonoids have a synergistic effect with the recombinant human granulocyte colony stimulating factor-mobilized bone marrow stem cell transplantation on cerebral ischemia/reperfusion injury. Rat ischemia models were administered 0.3, 0.15 and 0.075 g/kg Radix Ilicis Pubescentis total flavonoids from 3 days before modeling to 2 days after injury. Results showed that Radix Ilicis Pubescentis total flavonoids could reduce pathological injury in rats with cerebral ischemia/reperfusion injury. The number of Nissl bodies increased, Bax protein expression decreased, Bcl-2 protein expression increased and the number of CD34-positive cells increased. Therefore, Radix Ilicis Pubescentis total flavonoids can improve the bone marrow stem cell mobilization effect, enhance the anti-apoptotic ability of nerve cells, and have a neuroprotective effect on cerebral ischemia/reperfusion injury in rats.

Extraction optimization of coumarins from radix angelicae pubescentis by HPLC-DAD coupled with uniform design

Uniform design was used to optimize extraction condition of direct refluence extraction of coumarins from the Chinese traditional medicine of radix angelicae pubescentis(Duhuo); the sum peak area of coumarins separated with high performance liquid chromatography(HPLC) at detection wavelength of 320nm was considered as detection index, two factors of solvent concentration and extraction time were mainly studied at extraction temperature of 85℃ and a volume ratio of solvent to sample of 10∶1. Optimal subclass, quadric polynomial step by step aggression and neural network method were applied to process the experimental results. The results show that the first and second methods give the same factors combination (concentration of ethanol: 95%, extraction time: 3.6 h) and the second method is much better than the first one. The extraction model is consequently developed.

Radix Ilicis Pubescentis total flavonoids ameliorates neuronal damage and reduces lesion extent in a mouse model of transient ischemic attack

Flavonoids are a major component in the traditional Chinese medicine Radix Ilicis Pubescentis.Previous studies have shown that the administration of Radix Ilicis Pubescentis total flavonoids is protective in cerebral ischemia.However,to our knowledge,no studies have examined whether the total flavonoids extracted from Radix Ilicis Pubescentis prevent or ameliorate neuronal damage following transient ischemic attacks.Therefore,Radix Ilicis Pubescentis total flavonoids question and the potential underlying mechanisms.Thus,beginning 3 days before the induction of a mouse model of transient ischemic attack using tert-butyl hydroperoxide injections,mice were intragastrically administered 0.3,0.15,or 0.075 g/kg of Radix Ilicis Pubescentis total flavonoids daily for 10 days.The results of spectrophotometric analyses demonstrated that Radix Ilicis Pubescentis total flavonoids enhanced oxygen free radical scavenging and reduced pathological alterations in the brain.Hematoxylin-eosin staining results showed that Radix Ilicis Pubescentis total flavonoids reduced hippocampal neuronal damage and cerebral vascular injury in this mouse model of transient ischemic attack.These results suggest that the antioxidant effects of Radix Ilicis Pubescentis total flavonoids alleviate the damage to brain tissue caused by transient ischemic attack.

Effect of Radix Ilicis Pubescentis on the Rats with Hyperlipidemia

[Objectives]To study the hypolipidemic effect of Radix Ilicis Pubescentis and to provide experimental scientific basis for the study of hypolipidemic mechanism of Radix Ilicis Pubescentis.[Methods]60 healthy(SD)male rats were selected,10 rats were given basic feed and pure water as blank control group,and the other 50 rats were fed with high-fat diet for 4 weeks.After establishing the hyperlipidemic rat model,the rats were randomly divided into model group,high,middle and low dose groups(1000,800,500 mg/kg),and positive control group(simvastatin 0.4 mg/mL).The corresponding drugs were intragastrically administered to the rats in each group(blank control group and model group intragastrically administered with normal saline).Four weeks later,blood samples from abdominal aorta were taken to detect serum total cholesterol(TC),serum triglyceride(TG),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C).[Results]Compared with the blank control group,TC,TG and LDL-C increased while HDL-C decreased in the model group(P<0.05);compared with the model group,TC,TG and LDL-C in Radix Ilicis Pubescentis group decreased to varying degrees,and the content of HDL-C in serum of rats had no statistical significance.Compared with the blank control group,the serum TC and TG contents of the model group,positive control group,high,middle and low dose groups of Radix Ilicis Pubescentis extract significantly increased(P<0.01 or 0.05),and the serum LDL-C content of rats decreased(P<0.05).[Conclusions]The extract of Radix Ilicis Pubescentis had a certain lipid-lowering effect on hyperlipidemia in rats,and had obvious preventive and therapeutic effect on hyperlipidemia.

Effect of Ermiao Recipe (二妙方)with Medicinal Guide Angelicae Pubescentis Radix on Promoting the Homing of Bone Marrow Stem Cells to Treat Cartilage Damage in Osteoarthritis Rats

Objective： To investigate the effect of Ermiao Recipe （二妙方, EMR） with medicinal guide Angelicae Pubescentis Radix （APR） on the homing of bone marrow stem cells （BMSCs） to focal zone in osteoarthritis （OA） rats. Methods： Forty-eight Sprague-Dawley rats were randomly assigned to the sham-operated, model, EMR, and EMR plus APR groups （12 rats in each group）. The OA rat model was induced by anterior cruciate ligament transection and medial meniscus resection. All rats were injected with recombinant human granulocyta colony- stimulating factor [rhG-CSF, 30 μg/（kg.d） for continuous 7 days], and rats in the EMR and EMR plus APR groups were treated with EMR or EMR plus APR at 1.6 or 1.9 g/（kg.d） for 3 or 6 weeks, respectively. Cartilage histopathologic changes were observed by hematoxylin and eosin staining. Chondrocytes apoptosis and cartilage matrix components were tested by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay and special staining. Interleukin-1β （IL-1 β）, tumor necrosis factor α （TNF-α）, bone morphogenetic protein 2 （BMP-2）, and transforming growth factor beta-1 （TGF-β1） in serum were detected by enzyme-linked immunosorbent assay or radioimmunoassay assay. Matrix metalloproteinase （MMP）-13, tissue inhibitors of metalloproteinase （TIMP）-1, bromodeoxyuridine （BrdU）, cluster of differentiation 34 （CD34）, and stromal cell derived factor 1 （SDF-1） were measured by immunohistochemistry assay. Results： EMR and EMR plus APR significantly inhibited articular cartilage damage and synovium inflammation in OA rats at 3 or 6 weeks of treatment, the most obvious changes in these parameters were found in the EMR plus APR group. At 6 weeks, compared with EMR treatment, EMR plus APR remarkably inhibited chondrocytes apoptosis and the release of IL-1β and TNF- a, obviously decreased MMP-13 expression, and significantly increased expressions of proteoglycan, collagen, type 11 collagen and TIMP-1, serum levels of BMP-2 and TGF- 131 as well as expressions of BrdU, CD34 and SDF-1 in cartilage articular （P〈0.01 or P〈0.05 ）. Conclusion： The medicinal guide APR improved the therapeutic effects of EMR on OA rats by promoting directional homing of BMSCs to focal zone.

EGFR/cell membrane chromatography-online-high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Radix Angelicae Pubescentis

The intracellular kinase domains of the epidermal growth factor receptor(EGFR) in some tumor cells are significant targets for drug discovery.We have developed a new EGFR cell membrane chromatography(EGFR/CMC)-online-high performance liquid chromatography/mass spectrometry(HPLC/MS) method for screening anti-EGFR antagonists from medicinal herbs such as Radix Angelicae Pubescentis.In this study,the HEK293 EGFR cells with high expression of EGFR were used to prepare cell membrane stationary phase(CMSP) in the EGFR/CMC model.The retention fractions on the EGFR/CMC model were directly analyzed by combining a 10 port columns switcher with a HPLC/MS system online.As a result,osthole from Radix Angelicae Pubescentis was found to be the active component acting on EGFR like dasatinib as the control drug.There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro.This new EGFR/CMC-online-HPLC/MS method can be applied for screening anti-EGFR antagonists from TCMs,for instance,Radix Angelicae Pubescentis.It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.

Analysis of the chemical composition of Angelicae Pubescentis Radix by ultra-performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry

Angelicae Pubescentis Radix (APR, Duhuo) is a commonly used traditional Chinese medicine and usually used for the treatments of rheumatic diseases. To assess the chemical composition of APR extract, a sensitive and reliable UPLC-Q?TOF-MS method was used for qualitative analysis. The separation was achieved on an Agilent SB-Ci8 column (1.8 pm, 2.1 mm><50 mm) with a gradient elution system consisting of acetonitrile and water containing 0.1% formic acid. An electrospray ionization (ESI) was used for mass spectrometer, and the data were collected in the positive ion mode, which was operated in a full-scan mode at m/z 100-800. A total of 49 compounds including 46 coumarins were identified according to the MS and MS/MS data. Among them, two were new compounds, and isoangenomalin, scoparone, 4-methyl-umbelliferyl acetate, suberenol,/raw5-dehydroosthol, and oroselone were first reported in APR.

Simultaneous quantifi cation and evaluate the differences of two skeleton components in raw,salt and wine Angelicae Pubescentis Radix by UPLC-MS/MS in negative/positive modes coupled with chemometrics

The aim of this study was to establish an ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method with positive and negative ion exchange scanning mode for simultaneous quantification of two different skeleton components to reveal the differences among raw and processed Angelicae Pubescentis Radix. The results of methodology showed that each compound had good linearity and recovery rates. And the chemometrics results indicate that there were differences not only in Angelicae Pubescentis Radix samples from different sources but also in raw and processed Angelicae Pubescentis Radix. The results of this study confirm that the method has been successfully applied in simultaneous determination and discovering the difference of two different skeleton components between raw and processed Angelicae Pubescentis Radix for the first time. In short, the method could be an effective tool for detecting the quality of traditional Chinese medicine, and can better reveal the difference between raw and processed Chinese medicine from different sources.

正交试验优化超声波提取独活有效成分的工艺研究

[目的]优选超声波提取独活有效成分的工艺条件。[方法]采用紫外分光光度法分析总香豆素含量,以独活中总香豆素为评价指标,通过正交试验优选影响提取的3个主要因素(溶剂倍量、提取时间以及乙醇浓度)的最佳条件。[结果]乙醇浓度对总香豆素提取率的影响最为明显,其次是溶剂倍量,提取时间对总香豆素提取率无明显影响。正交试验优选的超声波提取独活总香豆素工艺条件为:料液比1∶10(W/V,g/ml,下同)、提取时间20 min、乙醇浓度60%;在此条件下,独活中总香豆素的提取率为14.095 2 mg/g。[结论]超声波提取独活总香豆素时间短,溶剂用量少,提取效率高。

Quantitative determination of ilexgenin A in rat plasma by liquid chromatography coupled with mass spectrometry and its pharmacokinetics

A sensitive and selective high performance liquid chromatography coupled with electrospray ionization mass spectrometry （LC-MS） was developed for the quantitative determination of ilexgenin A （IA）,a major component in Radix Ilicis Pubescentis,in rat plasma.Chromatographic separation was performed on a C 18 column,with methanol-5 mM ammonium acetate （80：20,v/v） as the mobile phase.Mass spectrometer was set in negative mode with target ions at m/z 501.1→501.1 for IA and m/z 779.4→779.4 for digoxin （internal standard,IS）.Rat plasma was extracted with ethyl acetate after addition of phosphoric solution and the organic layer was evaporated and reconstituted with mobile phase for LC-MS analysis.The proposed method was validated with a linear range of 1.05-525.5 ng/mL for IA with limit of quantitation （LOQ） at 1.05 ng/mL.Intra-and inter-day precision expressed as relative standard deviation （RSD） were less than 10% at LOQ level and overall recovery was over 80%.This validated method was used successfully for the pharmacokinetic study of IA in rats after oral dosing of IA （100 mg/kg） and some main pharmacokinetic parameters of IA in rats were obtained.