Effects and its possible mechanism of Radix Saposhnikoviae on rat colonic smooth muscle in vitro

Objective： To determine the effect of different concentrations of Radix Saposhnikoviae （RS） on the contraction of smooth muscle strips and the Ca2＋ mobilization of cultured smooth muscle ceils of rat colon and its possible mechanism of action. Methods： Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell- experiments, intracellular Ca2＋ was assessed using fluorescent intensity （FI） of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results： In the in vitro experiment, RS （0.02, 0.2, 2, 20 g/L） inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations （P 〈 0.01） for both Peak （the maximal contraction amplitude） and Area （the area under curves）. Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant （P 〈 0.01）, as was the inhibition of the Area values in all RS groups （P 〈 0.05）. Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect （P 〈 0.01）. In experiments using primary smooth muscle cell cultures in Ca2＋ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values （P 〈 0.05）, and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant （P 〈 0.01）. However, in Ca2＋-free buffer, FS had no significant effect on cell fluorescence. Conclusion： RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α -adrenoceptors, but not β -adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca2＋ from extracellular sources, but may had no effect on the release of Ca2＋ from sarcoplasmic reticulum and endoplasmic reticulum.

Network pharmacology and verification experiment-based prediction of active components and potential targets of Alpiniae Oxyphyllae Fructus-Saposhnikoviae Radix(Yizhiren-Fangfeng)for treatment of diabetic kidney disease

Background:In this study,we analyzed the potential active components,related crucial targets and possible signaling pathway mechanisms of Alpiniae Oxyphyllae Fructus and Saposhnikoviae Radix(AOF-SR)herb pairs in the treatment of diabetic kidney disease(DKD)using network pharmacology and verification experiments.Methods:The active compounds and potential targets of AOF-SR were derived from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,The Encyclopedia of Traditional Chinese Medicine,and PubChem databases,and the potential therapeutic targets of DKD were derived from the OMIM,Drugbank,and DisGeNET databases.The“compounds-diseases-targets”network was constructed using Cytoscape 3.6.0.ClusterMaker functionality in Cytoscape is being used to screen important targets for AOF-SR treatment of DKD.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of important targets were performed using DAVID database.In addition,according to the predicted results of network pharmacology,HK-2 cells were used to construct DKD model for verification experiment.HK-2 cells were divided into control group,high glucose(HG)group and AOF-SR(HG+AOF-SR)group to detect survival rate and expression of key proteins in NF-κB and PI3K/Akt signaling pathways.Results:A total of 38 compounds were selected from AOF-SR,of which 23 were Alpiniae Oxyphyllae Fructus and 15 were Saposhnikoviae Radix.Through enrichment analysis of 82 important targets,88 signaling pathways were identified;some of these pathways,such as the NF-κB,PI3K-Akt,IL-17,and JAK/STAT signaling pathways,regulate the pathological process of DKD.In verification experiment,the HK-2 cells survival rate was higher in the HG+AOF-SR group than in the HG group(P<0.05).Moreover,western blotting results showed that the expression levels of NF-κB,p-PI3K,and p-Akt in HG+AOF-SR group were significantly lower than those in HG group(P<0.05).Conclusion:Overall,this study revealed the active compounds,important targets and possible mechanisms of AOF-SR treatment for DKD,and conducted preliminary verification experiments on its correctness,provided novel insights into the treatment of DKD by AOF-SR.

Exploring the mechanism of Saposhnikoviae Radix and Chuanxiong Rhizoma treating Parkinson's disease based on network pharmacology and molecular docking

Backgroud:Parkinson’s disease(PD)is a neurodegenerative disorder with an increasing global prevalence.However,the development of drugs for PD treatment has not kept pace with the continuously growing number of patients.Currently,the search for new effective substances from natural drugs is a major research direction.Two Chinese medicinal materials,Saposhnikoviae Radix(Fangfeng)and Chuanxiong Rhizoma(Chuanxiong),are commonly used in the treatment of PD in China.However,the mechanism of their combination is not clear,and further research is needed.Methods:Data were collected from publicly available databases:TCMSP,UnitProt,GeneCards OMIM,PharmGKB,Therapeutic Target Database and DrugBank.Network pharmacology and molecular docking methods was used to analyze the data to discover the possible pharmacological effects of the two drugs in the treatment of PD.Results:Beta-sitosterol,Mandenol and Wallichilide were the active components of Saposhnikoviae Radix and Chuanxiong Rhizoma(FC),and they stably bonded with PD targets,including PTGS2,CASP3,AKT1 and JUN.The target genes of FC were significantly enriched in PD-associated pathways,including calcium signaling and apoptosis pathways.Moreover,the study revealed that the active components of FC may affect cellular structures,such as membrane rafts,membrane microdomains,membrane regions,and postsynaptic membranes,which,in turn,affect a variety of molecular functions and biological processes.Conclusion:The results of this study indicate the direction for clarifying the pharmacodynamic substances of FC,the extraction method of pharmacodynamic substances,as well as the mechanism and efficacy of pharmacodynamic substances.Importantly,this study provides a strategy for developing new therapeutic drugs for PD.

Quality evaluation of heat stress treated Radix saposhnikoviae using pharmacokinetic and pharmacologic methods

The quality of Radix saposhnikoviae（RS） had deteriorated due to the adoption of cultivated plants rather than wild plants. High temperature treatment increases the content of several chromones, however using one or more individual constituents would be difficult to determine the medical quality of RS. In this paper, we used pharmacokinetic and pharmacologic approaches to evaluate the quality of RS. The active constituents were analysed using pharmacokinetic parameters of the chromone derivatives, and the antipyretic, analgesic, and anti-inflammatory effects were evaluated by pyretic animal model, hot plate test, and ear edema model, respectively. Only cimifugin was found in plasma after RS and heat-stress-RS were administered to rats, with a 50.6% increase in AUC0–24 h of cimifugin in the latter. Likewise, more potent anti-pyretic, analgesic, and anti-inflammatory activities were also found in the latter. Exposure of S. divaricata fresh roots to high temperatures enhanced the antipyretic, analgesic, and anti-inflammatory effects of RS by promoting the absorption of cimifugin.

Exploration of the effective components and mechanism of Alpinia oxyphylla-Saposhnikovia Radix in the treatment of diabetic kidney disease based on network pharmacology

Objective:Network pharmacology was used to predict the main targets and related mechanism pathways of the key compounds and active ingredients of the Alpinia oxyphylla-Saposhnikovia Radix based on the therapeutic strategy of tonifying kidney and dispelling pathogenic wind for the treatment of diabetic kidney disease(DKD).Methods:The effective components of Alpinia oxyphylla-Saposhnikovia Radix were screened through the data platform of Traditional Chinese Medicine(TCM).After screening effective targets,the disease target gene database was used to obtain targets for DKD,and then gene enrichment analysis to predict relevant signalling pathways after obtaining the key target genes through Venn analysis.Results:There were 324 active components of Alpinia oxyphylla-Saposhnikovia Radix through the data platform of TCM.In addition,Venn analysis showed that there were 48 key target genes for the treatment of DKD.The biological pathways for the action of Alpinia oxyphylla-Saposhnikovia Radix in DKD were closely related to many signaling pathways including NF-kappaB,classical MAP kinase,JNK and P38 MAP kinase,and ERK5.Conclusions:Treatment of DKD with Alpinia oxyphylla-Saposhnikovia Radix based on the therapeutic strategy of tonifying kidney and dispelling pathogenic wind has the characteristics of multi-components,multi-targets and multi-pathways,which could provide new ideas and methods for further researches on the treatment of DKD with Alpinia oxyphylla-Saposhnikovia Radix.

Optimization of Supercritical Fluid Extraction and Rapid Resolution LC-MS/ESI Identification of Chromones from Saposhnikoviae Radix through Orthogonal Array Design

Objective To establish a rapid and effective supercritical fluid extraction（SFE） and rapid resolution liquid chromatography method coupled with diode-array detector（RRLC-DAD） to quantify the chromones in a species. Methods The effects of four parameters including ethanol concentration（50%-90%）, pressure（25-45 MPa）, temperature（40-60 ℃）, and time（30-90 min） on the chromones yields, namely prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside, and sec-O-glucosylhamaudol, were investigated using SFE system with orthogonal array design（OAD）. Furthermore, the extracts were analyzed using rapid resolution liquid chromatography coupled with diode-array detector（RRLC-DAD） system to confirm the results. Results Under the optimized conditions, i.e., 35 MPa of pressure, 60 ℃ of temperature, 70% ethanol, and 60 min of time, the yields of prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside, sec-O-glucosylhamaudol, and total chromones were 3.514, 0.132, 6.242, 0.342, and 10.231 mg/g, respectively. In comparison with ultrasonic assisted extraction（UAE）, SFE was able to yield a 20.7% increase in the total chromones from Saposhnikoviae Radix. Conclusion SFE is an alternative and promising method to extract chromones from this species, and the established RRLC-DAD method could serve as a rapid and effective method for the identification of chromones from Saposhnikoviae Radix.

Specification and grade of Saposhnikoviae Radix(Saposhnikovia divaricata)

Objective:Saposhnikoviae Radix(Fangfeng in Chinese),the roots of Saposhnikovia divaricata,lacks commodity specification and grade standardization in the current market.This study investigated the existing specifications and grades of Saposhnikoviae Radix to provide a standardized scientific reference for its market use.Methods:Based on a textual research of Chinese herbal medicine from the Han Dynasty to the present,medicinal materials of different specifications and grades obtained from Saposhnikoviae Radix in the main producing areas of China were collected and the markets for these materials were investigated.Field investigations were performed in the major producing areas such as Northeast China,Hebei Province,and Inner Mongolia.Four major Chinese herbal medicine markets in China were investigated.Sensory indices were used to categorize the two specifications(wild and cultivated)according to the shape,color,texture,and cross-section.High-performance liquid chromatography was performed to determine the active components.Vernier calipers and measuring tape were used to measure the diameter and length,respectively,of 41 samples.Using Excel and the R Language software,cluster analysis and descriptive statistical analysis were performed to assist in the application of new specifications and grades based on physical characteristics,pharmacological activity,and chemical composition.Results:The two specifications(wild and cultivated)of Saposhnikoviae Radix were divided into three grades each based on the length and diameter.Prim-O-glucosylcimifugin,5-O-methylvisamminoside,and the length of Saposhnikoviae Radix can be used as a basis for classifying the commodity specifications and grades.The specifications and grade standards of Saposhnikoviae Radix were established based on the following eight aspects:shape,surface characteristics,texture,cross section,taste,prim-Oglucosylcimifugin content,5-O-methylvisamminoside content and length.Conclusion:The formulation of this standard stipulates the commodity specification level of Saposhnikoviae Radix.It is also suitable for the evaluation of commodity specifications in the process of production,circulation and use of Saposhnikoviae Radix.

Explore the direct and/or the synergistic antihypertensive effects of wind-dispelling herbs involving Fangfeng and Baizhi on hypertensive rats with liver-yang hyperactivity based on vasoactive substances

Objective： To explore the direct and/or the enhancing antihypertensive effects of wind-dispelling herbs involvingFangfeng （Saposhnikoviae Radix） and Baizhi （Angelicae dahuricae Radix） using liver-yang hyperactivity renalhypertension rat. Methods： Model rats with hyperactivity of liver-yang hyperactivity were prepared using male SD rats.Once successful, the rats were randomly divided into groups and given medicine by gavage for 4 weeks. Blood wascollected from the abdominal aorta to prepare serum. Serum nitric oxide （NO） concentration was determined bychemical colorimetry. The contents of ET, TXB2 and 6-K-PGF1a were determined by enzyme linked immunosorbentassay （ELISA）. Results： Compared with the control model group, the blood pressure, serum ET and TXB2 of rats in thewind dispelling medicine group was not significantly reduced, while those of rats in captopril group, Tianma GoutengDecoction I group （TGD group） decreased significantly （P 〈0.05 for both）. Interestingly, the blood pressure, serum ETand TXB2 of rats in the combination group （wind dispelling medicine ＆ TGD） decreased significantly compared withboth wind dispelling medicine group and TGD group. Meanwhile, the levels of serum NO and 6- K-PGF1a in thecombination group was much higher than those in the wind dispelling medicine group or TGD group （P 〈0.05 for both）.However, the levels of serum NO and 6- K-PGF1a in the wind dispelling medicine group was not significantly increasedcompared with the model group. Conclusion： There is no direct pressure lowering effects of wind dispelling medicinealone, while wind dispelling medicine could potentiates the antihypertensive effects of Tianma Gouteng Decoction I.Besides, the synergistic effects may be related to decreased ET and TXB2 levels and the increased NO and 6- K-PGF1alevels.