Methanolic extract of Ephedra alata inhibits breast cancer cells in vitro and in vivo

Objective:To determine the anticancer potential of the methanolic extract from Ephedra alata against breast cancer both in vitro and in vivo.Methods:The effects of the methanolic extract of Ephedra alata on the viability,migration as well as apoptosis of breast cancer 4T1 cells were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,Transwell assay,and annexin V-FITC staining assay,respectively.Histological examination was also carried out.Moreover,a murine breast cancer model was established to evaluate the inhibitory effect of the extract.Biochemical parameters including hepatic and non-hepatic enzymes,malondialdehyde,and glutathione were investigated.Results:The methanolic extract of Ephedra alata showed a strong anti-proliferative and anti-migratory activity against 4T1 cells in a dose-dependent manner.It also induced apoptosis in 4T1 cells.In an in vivo mouse model,the extract markedly inhibited tumor growth,reduced malondialdehyde,and hepatic and non-hepatic enzymes as well as increased glutathione level.Conclusions:The methanolic extract of Ephedra alata inhibits breast cancer in vitro and in vivo,which may be a promising anticancer agent.

Preparation of Ephedra houttuynia Granule and Its Therapeutic Mechanism of Anti-Mycoplasma gallisepticum Infection Based on Network Pharmacology

The use of Chinese herbal medicines can replace antibiotics that cause drug-resistance problems,which are currently necessary for disease control.In this paper,a traditional Chinese medicine compound named Ephedra houttuynia granule for the treatment of Mycoplasma galliscepticum(MG)infection was prepared.Furthermore,its action mechanism was explored through network pharmacology.The optimal extraction and granulation processes of the compound were determined by high performance liquid chromatography(HPLC)method and L9 orthogonal test,and in the treatment experiment,Ephedra houttuynia granule showed a significant therapeutic effect on MG infection.In the study of network pharmacology,the results showed that the core targets of Ephedra houttuynia granule against MG infection were vascular endothelial growth factor(VEGFA),fos proto-oncogene(FOS),prepro-coagulation factor II(F2),etc.,the gene ontology/kyoto encyclopedia of genes and genomes(GO/KEGG)analysis results indicated that the signaling pathways of neuroactive ligand receptor interaction,cAMP,IL-17,T cell receptor,and tumor necrosis factor(TNF)might involve in anti-MG infection.In conclusion,this study would provide a new idea for elucidating the action mechanism of other diseases in veterinary clinic,which had a certain guiding significance.

Ontogeny and Metamorphic Patterns of Female Reproductive Organs of Ephedra sinica Stapf (Ephedraceae)

Ontogeny and metamorphic. development of female reproductive organs in Ephedra sinica Stapf were surveyed. At the end of April, female cones began to initialize from the vegetative buds. Pollination occurred in mid-May and seeds matured at the beginning of July. The ontogenetic pattern of female reproductive organs of E. sin ca is basically similar to that of E. distachya L. The foliar nature of the outer envelope of the ovule in Ephedra is corroborated. Reduction of ovule number as a tendency of speciation in the genus is substantiated by the occurrence of tri-ovulate cones coupled with comprehensive characters of the genus. The metamorphic patterns as well as the leaf nature of the outer envelope indicate that female cones of Ephedra are compound while the female reproductive units of the ovulate cone have been reduced from secondary reproductive shoots similar to those of cordaites by phylogenetic transformation, fusion and reduction. Each fertile bract together with its axillary female reproductive unit composed the Bract Scale and Seed Scale Complex of Ephedra.

A fossil-calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America

Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions （amphitropical range）. Great heterogeneity in the substitution rates of Gnetales （Ephedra, Gnetum, and Welwitschia） has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species （and 7736 aligned nucleotides） to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil-based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long-distance dispersal, but finer-scale studies are needed to test this hypothesis.

蒙古族利用草麻黄（Ephedra sinica Stapf）资源的民族植物学研究

应用民族植物学的研究方法，记载和描述了蒙古族对草麻黄资源在经济利用、药物利用和文化利用方面的知识和经验，对民间利用的合理性和意义及草原地区草麻黄资源的开发和持续利用作了分析和探讨．

Identification of Two Cold Water-Soluble Polysaccharides from the Stems of Ephedra sinica Stapf

Two polysaccharides (ESP-A1 and ESP-A2) were isolated from the cold water extract of Ephedra sinica Stapf and purified through ethanol precipitation, deproteinization and by ion exchange and gel-filtration chromatography. Their molecular weight was determined using high performance size exclusion chromatog-raphy and evaporative light scattering detector (HPSEC-ELSD) and their monosaccharide composition was analyzed by high performance capillary electrophoresis (HPCE) based on pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). It was shown that ESP-A1 consisted of xylose, arabinose, glucose, mannose and galactose and ESP-A2 consisted of xylose, arabinose, rhamnose and galactose, in a molar ratio (%) of 3.2: 61.1: 11.1: 12.9: 11.6 and 20.6: 67.7: 5.0: 6.7, respectively. The molecular weights (Mw) of ESP-A1 and ESP-A2 were 5.83 × 104 Da and more than 200 × 104 Da, respectively. To the best of our knowledge, two neutral polysaccharides are now being reported for the first time in this study.

Effects of ephedra asarum aconite decoction on Th1/Th2 balance in two animal models of allergic rhinitis

Objective:Ephedra asarum aconite decoction is used in the clinical treatment of allergic rhinitis.We investigated the effects of ephedra asarum aconite decoction on Th1/Th2 balance in two animal models of allergic rhinitis and in activated T cells in vitro.Methods:Animal models of simple and compound kidney yang deficiency types of allergic rhinitis were treated with ephedra asarum aconite decoction to observe the efficacy of this traditional Chinese medicine formula on allergic symptoms,serum cytokines,and tissue RNA levels.We also cultured and activated T cells from the mouse.After treatment with ephedra asarum aconite decoction,we tested the proliferation and differentiation of T cells.Results:Ephedra asarum aconite decoction relieved allergy symptoms in both types of animal models,and was more effective in the compound kidney yang deficiency model In addition to its anti-allergic efficacy,ephedra asarum aconite decoction significantly improved the health status of animals in the compound kidney yang deficiency groups,significantly made their bodyweight rising up,and significantly increased the T-bet mRNA/GATA-3 mRNA ratio.Moreover,ephedra asarum aconite decoction promoted the proliferation of activated T cells and inhibited the differentiation to Th2 cells in vitro.Conclusions:Ephedra asarum aconite decoction regulates the balance of Th1/Th2 to ameliorate allergic conditions.

Effects of the ephedra alkaloid methylephedrine on the basal evoked potential transportation and the long-term potentiation (LTP) in the rat hippocampal dentate granule cells in vivo

Objective The effect of the ephedra/ephedrine alkaloid methylephedrine(dl-methylephedrine hydrochloride for testing in this paper)on cognitive related synaptic plasticity was investigated by recording extracellular field evoked potentials and its LTP in hippocampal dentate granule cells in urethane-anaesthetized rats in vivo.Methods Single pathway recording of evoked field potentials was made from the dentate granule cells of hippocampal hemisphere in response to stimulation of the ipsilateral medial perforant path(MPP).Two parameters,the amplitude of population spike(PS amplitude)and the latency of the PS,were employed to evaluate the effects of drug on the overall changes in cellular responses.Results The present study show that methylephedrine 90 mg·kg-1 intraperitoneally,about 1/3 LD50,could increase the latency of the PS in hippocampal dentate granule cells by constant single stimulation of the MPP as the basal ransportation.However,the 30 mg·kg-1 and 10 mg·kg-1 dosage had no effect on the latency,and there are no influences of PS amplitude for all examinational groups.The methylephedrine 90 mg·kg-1 group significant enhanced the development of amplitude LTP in hippocampal dentate granule cells that induced by 60 Hz,60 pulses conditional tetanus in medial perforant path area.Also,the 30 mg·kg-1 group can promoted the maintenance of LTP induced by this tetanus,but no promotion on PS amplitude LTP appeared in this dosage and no any changes been found in 10 mg·kg-1 dosage group.Conclusions The ephedra/ephedrine alkaloid methylephedrine can modulate the synaptic plasticity in the lateral perforant path.A possible mechanism of methylephedrine on hippocampal LTP is been discussed.

Herbivory Effects on <i>Ephedra</i>spp. in the Chihuahuan Desert

Two species of Ephedra: E. trifurca and E. torreyana inhabit shrub and grassland habitats in the northern Chihuahuan Desert. E. torreyana is limited to black grama grasslands where grasses are taller than the shrub. E. torreyana is heavily browsed by vertebrates and E. trifurca is browsed during some years. We established an experiment with cylindrical exclosures that excluded rabbits and rodents, rabbits but accessible to rodents, for comparison with E. torreyana plants available to all herbivores. Plants accessible to all vertebrate herbivores were significantly smaller with shorter stem lengths than plants in exclosures. We concluded that E. torreyana in black grama grassland are largely hidden from vertebrate herbivores and that intense herbivory reflects the degraded state of the study site which makes the E. torreyana evergreen shrubs apparent to vertebrates.

Maintenance of Ephedra alata Seeds Viability via Storage Containers

Sequential incubation of seed samples yielded 20 fungal species belonging to 13 genera. The prevalent genera were Aspergillus (A. flavus and A. parasiticus), Fusarium (F. moniliforme and F. oxysporum) and Penicillium (Penicillium sp.). Such seedborne fungi differ in their colonization in different parts of seeds with most of them are colonized in seed coat, endosperm, and embryo of the seed. The usage of different storage containers for storing seeds indicated that the cotton cloth bags were the most favorable ones as they maintain seed moisture content (SMC) below the critical level resulting in minimum seed deterioration compared with other seed storage containers.

Action mechanism of Ephedra in treating allergic rhinitis based on network pharmacology

Objective:To study the mechanism of ephedra in treating allergic rhinitis by network pharmacology.Methods:the chemical components and corresponding targets of ephedra were searched and screened by TCMSP database.With"allergic rhinitis"as the key word,disease-related genes were searched in GeneCards and OMIM database.Using Venny2.1.0 mapping,Venn diagram and drug-disease common targets,network visualization software Cytoscape 3.6.1 track production elements-targets-disease network diagram,String database build their proteins interact with each other(PPI),and using R language to do gene ontology(GO)functional enrichment and pathway analysis and kyoto encyclopedia of genes and genome(KEGG)pathway enrichment analysis.Results:ephedrine was screened for 136 efficacy components and 88 common targets,and was predicted to intervene and regulate allergic rhinitis through AGE-RAGE signaling pathway in diabetic complications,Fluid shear stress and atherosclerosis,IL-17 signaling pathway,TNF signaling pathway and other signaling pathways in diabetic complications.Conclusion:preliminary prediction of the target and pathway of ephedra in the treatment of allergic rhinitis through network pharmacology can provide direction and evidence for the next experiment and clinic.

Study on the mechanism of action of Ephedra Herba Decoction against influenza A virus based on network pharmacology

Objective To predict the main bioactive components and potential mechanisms of Ephedra Herba Decoction(Chinese Pinyin abbreviated as MHT)in treating influenza A virus(IAV)based on network pharmacology.Methods Multiple online databases were used to search and screen out the active components from MHT,the related targets of active components of MHT and the genes related to IAV.Search the corresponding genes name of target through UniProt database.Cytoscape 3.7.2 was used to construct the drug-component-target network diagram.Venn diagram was used to screen the intersection genes of the active components corresponding to the target and disease-related genes,and the intersection genes were imported into the STRING Database Online platform to obtain the protein-protein interaction(PPI)network.Then,the PPI network was imported into Cytoscape 3.7.2 to obtain the core targets.Finally,Wei Sheng Xing(http://www.bioinformatics.com.cn/)was used to do GO function enrichment analysis and KEGG signaling pathway enrichment analysis for the intersection genes,and the results of GO and KEGG were visualized.Results A total of 116 active components and 253 potential targets were screened from MHT.Quercetin,Kaempferol,and Luteolin are the main active components,and AKT1,TNF,TP53,IL6,and JUN are the core targets of 253 potential targets.There are 2906 targets of influenza A,including 121 intersection genes.Enrichment analysis showed that there were 131 entries of molecular function,89 entries of cell component,1645 entries of biological process and 195 entries of signaling pathway.PI3K-Akt,MAPK,and JAK-STAT were the main signaling pathways.Conclusion MHT plays an important role in the prevention and treatment of influenza A by acting on multiple targets and multiple signaling pathways.

Discovery and characterization of naturally occurring covalent inhibitors of SARS-CoV-2 M~(pro)from the antiviral herb Ephedra

The Chinese herb Ephedra(also known as Mahuang)has been extensively utilized for the prevention and treatment of coronavirus-induced diseases,including coronavirus disease 2019(COVID-19).However,the specific anti-SARS-CoV-2 compounds and mechanisms have not been fully elucidated.The main protease(M~(pro))of SARS-CoV-2 is a highly conserved enzyme responsible for proteolytic processing during the viral life cycle,making it a critical target for the development of antiviral therapies.This study aimed to identify naturally occurring covalent inhibitors of SARS-CoV-2 M~(pro)from Ephedra and to investigate their covalent binding sites.The results demonstrated that the non-alkaloid fraction of Ephedra(ENA)exhibited a potent inhibitory effect against the SARS-CoV-2 M~(pro)effect,whereas the alkaloid fraction did not.Subsequently,the chemical constituents in ENA were identified,and the major constituents'anti-SARS-CoV-2 M~(pro)effects were evaluated.Among the tested constituents,herbacetin(HE)and gallic acid(GA)were found to inhibit SARS-CoV-2 M~(pro)in a time-and dose-dependent manner.Their combination displayed a significant synergistic effect on this key enzyme.Additionally,various techniques,including inhibition kinetic assays,chemoproteomic methods,and molecular dynamics simulations,were employed to further elucidate the synergistic anti-M~(pro)mechanisms of the combination of HE and GA.Overall,this study deciphers the naturally occurring covalent inhibitors of SARS-CoV-2 M~(pro)from Ephedra and characterizes their synergistic anti-M~(pro)synergistic effect,providing robust evidence to support the anti-coronavirus efficacy of Ephedra.

Ephedra Herb extract ameliorates adriamycin-induced nephrotic syndrome in rats via the CAMKK2/AMPK/mTOR signaling pathway

This study aimed to investigate the effect and mechanisms of Ephedra Herb(EH)extract on adriamycin-induced nephrotic syndrome(NS),providing an experimental basis for the clinical treatment of NS.Hematoxylin and eosin staining,creatinine,urea nitrogen,and kidn injury molecule-1 were used to evaluate the activities of EH extract on renal function.The levels of inflammatory factors and oxidative stress were detected by kits.The levels of reactive oxygen species,immune cells,and apoptosis were measured by flow cytometry.A network pharmacological approach was used to predict the potential targets and mechanisms of EH extract in the treatment of NS.The protein levels of apoptosis-related proteins and CAMKK2,p-CAMKK2,AMPK,p-AMPK,mTOR and p-mTOR in the kidneys were detected by Western blot.The effective material basis of EH extract was screened by MTT assay.The AMPK pathway inhibitor(compound C,CC)was added to investigate the effect of the potent material basis on adriamycin-induced cell injury.EH extract significantly improved renal injury and relieve inflammation,oxidative stress,and apoptosis in rats.Network pharmacology and Western blot results showed that the effect of EH extract on NS may be associated with the CAMKK2/AMPK/mTOR signaling pathway.Moreover,methylephedrine significantly ameliorated adriamycin-induced NRK-52e cell injury.Methylephedrine also significantly improved the phosphorylation of AMPK and mTOR,which were blocked by CC.In sum,EH extract may ameliorate renal injury via the CAMKK2/AMPK/mTOR signaling pathway.Moreover,methylephedrine may be one of the material bases of EH extract.

Effects of harvesting time and processing on the contents of five alkaloids in the herbaceous stems of Ephedra sinica

In the present study,we studied the changes of the contents of alkaloids in the herbaceous stems of Ephedra sinica during different harvest periods as well as before and after processing.The alkaloid contents of 39 batches of ephedra herb,prepared slices ephedra and honey-fried ephedra,24 batches of ephedra herb with different harvesting periods,which were all collected from cultivation base in Inner Mongolia,and 38 batches of prepared slices ephedra purchased from the market were detected by taking norephedrine(NE),norpseudoephedrine(NPE),ephedrine(E),pseudoephedrine(PE)and methylephedrine(ME)as indicators by using HPLC method.The content of total alkaloid in prepared slices ephedra(1.71%–3.14%)was higher than that in ephedra herb(1.20%–2.53%)and honey-fried ephedra(1.52%–2.99%).Contents of different alkaloids in these three types of samples were significantly different.Prepared slices ephedra and honey-fried ephedra showed significant differences in the contents of NE,NPE and ME(P<0.05),and the contents of E were significantly different between ephedra herb and honey-fried ephedra(P<0.05).The total alkaloid content of ephedra herb was the highest in September(3.10%).Alkaloid contents of prepared slices ephedra collected in the market were uneven and 13%–91%lower than those collected from cultivation base.The results provided a basis for the quality evaluation of ephedra herb and its processed products,and had certain guiding significance for the selection of processed ephedra according to different drug purposes in clinical application.It also provided data support for the harvesting time of ephedra herb.

Diploid species phylogeny and evolutionary reticulation indicate early radiation of Ephedra in the Tethys coast

Reconstructing a robust species phylogeny and disentangling the evolutionary and biogeographic history of the gymnosperm genus Ephedra,which has a large genome and rich polyploids,remain a big challenge.Here we reconstructed a transcriptome-based phylogeny of 19 diploid Ephedra species,and explored evolutionary reticulations in this genus represented by 50 diploid and polyploid species,using four low-copy nuclear and nine plastid genes.The diploid species phylogeny indicates that the Mediterranean species diverged first,and the remaining species split into three clades,including the American species(Clade A),E.rhytidosperma,and all other Asian species(Clade B).The single-gene trees placed E.rhytidosperma sister to Clade A,Clade B,or Clades A+B in similar proportions,suggesting that radiation and gene flow likely occurred in the early evolution of Ephedra.In addition,reticulate evolution occurred not only among the deep nodes,but also in the recently evolved South American species,which further caused difficulty in phylogenetic reconstruction.Moreover,we found that allopolyploid speciation was pervasive in Ephedra.Our study also suggests that Ephedra very likely originated in the Tethys coast during the late Cretaceous,and the South American Ephedra species have a single origin by dispersal from Mexico or North America.

膜果麻黄(Ephedra przewalskii)和白刺(Nitraria tangutorum)幼枝水势及影响因素

为探究干旱环境下植物的水势日变化特征及其与内外因素的相关性,测定了民勤绿洲耐旱植物膜果麻黄(Ephedra przewalskii)和白刺(Nitraria tangutorum)幼枝的水势、含水量及K+、Ca2+、Na+、Mg2+含量。结果表明:膜果麻黄和白刺的水势日变化趋势大体呈U型,日最低水势分别为-4.91 MPa和-4.35 MPa。膜果麻黄体内的K+和Na+含量日变化不显著,而Ca2+和Mg2+含量变化趋势呈U型;白刺体内K+、Na+、Mg2+含量日变化明显,基本呈U型,Ca2+含量日变化不明显。膜果麻黄水势与光照强度及其体内的Ca2+、Mg2+含量密切相关;白刺水势与气温、体内Mg2+含量及含水量密切相关。影响这2种植物水势的因子并不完全一样,但Mg2+离子对二者的水势均具有显著影响。

Comparative analysis of essential oil components in Ephedra and its processed products by GC-MS

The similarities and differences of essential oil components in the raw ingredients of Ephedra （RIE） and processed products of Ephedra obtained by stir-baking with honey or stir-baking without any supplements were analyzed by GC-MS.Wet distillation （WD） and supercritical fluid extraction （SFE） were used to extract essential oil components from the samples.Total 48,57 and 48 compounds were found in the extracted essential oils using WD from RIE,the products obtained by stir-baking with honey from Ephedra （SBHE） and the products obtained by stir-baking without any supplements from Ephedra （SBE）,respectively whereas total 22,36 and 28 compounds were identified in the extracted essential oils using SPE from these three samples,respectively.In addition,14 and 9 new compounds were found in the essential oils extracted using WD from SBHE and SBE,whereas 15 and 23 new compounds were found in the essential oils extracted by SFE from SBHE and SBE,respectively.The composition and concentration of the essential oil components in the processed products were significantly different from RIE.Such changes in essential oil components might affect drug actions,which is dependent on the manner in which the sample is processed.The findings in this study may shed some lights on the understanding and further exploration of Ephedra processing.

以麻黄为主药系列经方在儿童过敏性疾病辨治中的运用

以麻黄为主药系列经方在儿童过敏性疾病辨治中的运用。儿童过敏性疾病多年来是临床治疗的难点,以麻黄为主药系列方治疗该病临床疗效确切肯定。儿童过敏性疾病多因正气不足,肺、脾、肾功能低下,痰饮等伏邪宿根停留于体内,外邪引动而发病。麻黄为治疗咳喘之圣药,通过合理的配伍,麻黄与杏仁宣降相成,麻黄与石膏寒温并举,麻黄与附子温通表里。为了确保疗效,要重视遵循经方的原配伍比例、剂量、组方、煎服法和调护法。此外,还要明确麻黄类方及方证应用,即对方证,抓主证。