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The paper aims to compare the protective effect of Salvia miltiorrhiza and Anemarrhena asphodeloides on AD cell model and investigate its protective mechanism by cell metabolomics platform. AD cell model was established by the abnormal phosphorylation of Tau protein in SH-SY5Y cells induced by okadaic acid. The protective effect of the extract of Salvia miltiorrhiza and Anemarrhena asphodeloides on the model was evaluated by cell proliferation-toxicity experiment. The metabolomics platform was used to study the efficacy of Salvia miltiorrhiza and Anemarrhena asphodeloides comprehensively, explore the potential biomarkers related to AD and the effect of drugs on the potential biomarkers. Salvia miltiorrhiza extract had a certain protective effect on the AD model (P < 0.05), while the Anemarrhena asphodeloides extract had no significant protective effect (P > 0.05). 45 significant differential metabolites and the related 12 metabolic pathways were identified using UHPLC-QTOF/MS platform, which were related to the AD cell model. After administration of Salvia miltiorrhiza extract, 30 different metabolites appeared callback, while after intervention of Anemarrhena asphodeloides extract, 7 metabolites appeared callback. The results showed that the extracts of Salvia miltiorrhiza and Anemarrhena asphodeloides had certain protective effects on the AD cell model with Tau protein abnormal phosphorylation, but Salvia miltiorrhiza had more extensive targets and could significantly improve the cell viability. The mechanism may be related to the regulation of the metabolic pathways of AD cell model induced by okadaic acid.
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Objective:To establish ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） for the analysis of 18 components of five categories, namely spirosteroid saponins （timosaponin AⅠ，timosaponin AⅡ，timosaponin AⅢ，anemarrhenasaponin Ⅲ，sarsasapogenin），furostane saponins（anemarrhenasaponin Ⅰ，anemarrhenasaponin Ⅰa，anemarsaponin E，officinalisinin I，timosaponin B-Ⅱ，timosaponin B-Ⅲ，anemarsaponin C），flavonoids（icarisin I，baohuoside I），bisphenone（meomangiferin，mangiferin，isomangiferin），and hydroquinone glycoside （β-arbutin）,in order to analyze the differences between the main root and fibrous root of Anemarrhena asphodeloides from different sources and provide reference for the sustainable development of A. asphodeloides resources. Method:0.25 g sample was refluxed and extracted with 25 mL dilute ethanol for 30 min. The chromatographic separation was carried out on Waters Acquity Uplc BEHHILI C18 column（2.1 mm×100 mm，1.7 μm），with 0.1% formic acid water-acetonitrile as the mobile phase for gradient elution，and the volume flow rate was 0.2 mL·min-1. Electrospray ion source（ESI+，ESI-），and multi-reaction ion monitoring（MRM） were used for detection，external standard method was used to calculate the content of the tested components in medicinal samples，and SMICA14.1 software was used to analyze the differences between the main root and fibrous root samples of A. asphodeloides. Result:The tested components showed a good linear relationship in their respective linear ranges，with a good precision，repeatability and stability. The recovery rate of samples was between 95.22%-101.42%，and RSD was less than 4%. The experimental results showed great differences between the main root and fibrous root of A. asphodeloides, when the multivariate statistical analysis was carried out with 18 main components. Conclusion:This study provides experimental basis for the reuse of fibrous root of A. asphodeloides resources and the quality control of A. asphodeloides.
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OBJECTIVE：To establish the fingerprint of ethanol extract and acetone extract from Anemarrhena asphodeloides and its different processed products ，and to investigate the spectrum-effect relationship between the fingerprint and the antioxidant activity. METHODS ：HPLC method and HPLC-ELSD method were adopted. The determination was performed on Thermo BDS Hypersil C 18 column with mobile phase consisted of acetonitrile- 0.2％ acetic acid at the flow rate of 1.0 mL/min. The column temperature was 30 ℃，and the detection wavelength was set at 258 nm. The sample size was 10 μL. The determination was performed on XDB-C 18 columnwith mobile phase consisted of acetonitrile-0.1％ acetic acid （gradient elution ）at the flow rate of 0.9 mL/min. The column temperature was 30 ℃ . The temperature of atomizer was 40 ℃ and the flow rare of N 2 was 1.6 mL/min. The sample size was 10 μL. Using mangiferin and timosaponin B Ⅱ as reference ，Fingerprint Similarity Eva- com luation System of TCM Chromatogram （2004A edition ）was adopted to draw the fingerprint of ethanol extract and acetoneextract from 20 batches of A. asphodeloides and its different processed products to confirm common peaks. Using scave nging rate of 1，1-diphenyl-2-trinitrophenylhydrazine（DPPH）radical as index，antioxidant activities of ethanol extract and acetone extract from 20 batches of A. asphodeloides and its processed products were investigated. Using scavenging rate of DPPH radical as dependent variable ，common peak area as independent variable ，PLSR was used to analyze the spectrum-effect relationship of ethanol extract and acetone extract from A. asphodeloides with antioxidantion activity. RESULTS ：Eight peaks （M1-M8）were identified in the fingerprints of ethanol extracts from 20 batches of processed A. asphodeloides . Mangiferin （chromatogram peak M 7）was identified with similarity of 0.389-1.000；seven comon peaks （S1-S7）and timosaponin B Ⅱ（peak S 5）were identified in the fingerprint of acetone extract ，and the similarity was 0.044-0.999. DPPH radical scavenging rate of ethanol extract from 20 batches of A. asphodeloides and its processed products was 21.23％- 81.39％，and A. asphodeloides was significantly lower than salt-processed A. asphodeloides with salt wine-processed A. asphodeloides （P＜0.001）；and that of acetone extract was 49.73％-83.78％，and A. asphodeloides was significantly higher than stir-baked A. asphodeloides with salt ，wine or fire （P＜0.001）. The standardized regression coefficients of peaks M 2-M7 in the spectrum of ethanol extract from A. asphodeloides were all greater than 0，which was positively correlated with antioxidant activity. Only the variable importance projection （VIP）value of peak M 7 was greater than 1，which had an important contribution. The standardized regression coefficients of peaks S 4-S7 in the acetone extract spectrum of A. asphodeloides were greater than 0，and were positively correlated with antioxidant activity. The order of VIP values was peak S 5＞S6＞S4，and the VIP values were all greater than 1. CONCLUSIONS：The fingerprint of the different processed products A. asphodeloides and its antioxidant activity spectral effect relationship were successfully established ；mangiferin（peak M 7）may be the main antioxidant substance of ethanol extract from A. asphodeloides . Timosaponin B Ⅱ（peak S 5），peak S 6 and peak S 4 may be the main antioxidant substance in acetone extract from A. asphodeloides .
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Objective: To screen potential α-glucosidase inhibitors from Anemarrhena asphodeloides. Methods: Response surface methodology employing Box-Behnken design was used to optimize conditions for the extraction of α-glucosidase inhibitory active compounds from A. asphodeloides. The powders (20.0 g) of A. asphodeloides were extracted under the optimized conditions. The extract was applied to a D-101 macroporous resin column. It was eluted with ethanol and water to give six fractions. Compounds from the active fraction were identified by UPLC-Q-TOF-MS. The structure-activity relationship was discussed based on grey relational analysis. Results: The optimum extraction conditions were as follows: ethanol concentration, 100%; extraction temperature, 51 °C; and solvent to solid ratio, 23 mL/g. It indicated that the active compounds were concentrated into 80% ethanol fraction. Twenty five steroid saponins from 80% ethanol fraction were identified by UPLC-Q-TOF-MS. Peaks 19 and 23 were tentatively identified as new structures. The predicted α-glucosidase inhibitory activities of the compounds were 7 > 2 > 1 > 22 > 23 > 3 > 9 > 21 > 24 > 4 > 13 > 8 > 14 > 16 > 17 > 25 > 6 > 19. Conclusion: The fraction eluted by 80% ethanol showed the best inhibitory activity. After analyzing the data of UPLC-Q-TOF-MS, 25 steroid saponins were tentatively identified in this fraction.
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Five compounds were isolated from the fibrous roots of Anemarrhena asphodeloides by silica gel, Sephadex LH-20 and semi-HPLC column chromatography. On the basis of physic-chemical properties and spectroscopic data analysis, these compounds were identified as methyl 2-[2,4-dihydroxy-3-(4-hydroxybenzoyl)-6-methoxyphenyl]acetate(1), 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoate(2), perlolyrine(3),syringaresinol-4'-O-β-D-glucoside(4) and 4',6-dihydroxy-4-methoxybenzophenone-2-O-(2″),3-C-(1″)-1″-desoxy-α-L-fructofuranoside(5). Among them, 1 was a new benzophenone. Compounds 2-5 were isolated from this plant for the first time. Compound 1 was tested for neuroprotective effects against H_2O_2-induced damage in SH-SY5 Y cells.
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Humans , Anemarrhena , Chemistry , Benzophenones , Pharmacology , Cell Line , Chromatography, High Pressure Liquid , Neuroprotective Agents , Pharmacology , Phytochemicals , Pharmacology , Plant Roots , ChemistryABSTRACT
Objective:To explore the macroporous resin adsorption and the membrane separation technologies for the purification of saponins water extract of Anemarrhena asphodeloides. Methods:Ten-fold amount of water was used to extract twice for 120 min each time to extract saponins from Anemarrhena asphodeloides. The macroporous resin adsorption(HP-20,HPD-600,D101,AB-8) and the membrane separation technologies (ceramic microfiltration membranes 0.8 μm and 0.05 μm, and hollow fiber ultrafiltration mem-branes 50,10 and 6 kDa) were adopted to purify the saponins water extract liquid. The physicochemical parameters including electri-cal conductivity,viscosity and turbidity were measured,as well as the contents of total saponins,proteins and polysaccharides were de-termined. Results:The viscosity and turbidity decreased,the value of pH increased and the electrical conductivity of the saponins puri-fication liquid changed irregularly after the membrane filtration. The microfiltration membrane was more advantageous than the ultrafil-tration membrane in removing macromolecular substances. The smaller the pore diameter of microfiltration membrane, the smaller the intercepted molecular weigh,the higher the removal ratio of proteins and the higher the penetration rate of the total saponins,while the polysaccharides content was stable, which was consistent with the results of physicochemical parameters. The ceramic microfiltration membrane could obtain clearer extract,while the ultrafiltration membrane was more suitable for the enrichment of saponins when the in-tercepted molecular weight was 6 kDa. The macroporous resin HPD-600 was the best for the purification of timosaponin water extraction liquid.Conclusion:The selection of membrane for the separation and purification of different substances is particularly important. The change of physicochemical parameters and the content decrease of macromolecular substances have obvious corresponding relationship. Ultrafiltration membrane is better than microfiltration membrane for the purification of timosaponin water extract liquid.
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Investigations into the development of new therapeutic agents for lung inflammatory disorders have led to the discovery of plant-based alternatives. The rhizomes of Anemarrhena asphodeloides have a long history of use against lung inflammatory disorders in traditional herbal medicine. However, the therapeutic potential of this plant material in animal models of lung inflammation has yet to be evaluated. In the present study, we prepared the alcoholic extract and derived the saponin-enriched fraction from the rhizomes of A. asphodeloides and isolated timosaponin A-III, a major constituent. Lung inflammation was induced by intranasal administration of lipopolysaccharide (LPS) to mice, representing an animal model of acute lung injury (ALI). The alcoholic extract (50–200 mg/kg) inhibited the development of ALI. Especially, the oral administration of the saponin-enriched fraction (10–50 mg/kg) potently inhibited the lung inflammatory index. It reduced the total number of inflammatory cells in the bronchoalveolar lavage fluid (BALF). Histological changes in alveolar wall thickness and the number of infiltrated cells of the lung tissue also indicated that the saponin-enriched fraction strongly inhibited lung inflammation. Most importantly, the oral administration of timosaponin A-III at 25–50 mg/kg significantly inhibited the inflammatory markers observed in LPS-induced ALI mice. All these findings, for the first time, provide evidence supporting the effectiveness of A. asphodeloides and its major constituent, timosaponin A-III, in alleviating lung inflammation.
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Animals , Humans , Mice , Acute Lung Injury , Administration, Intranasal , Administration, Oral , Alcoholics , Anemarrhena , Bronchoalveolar Lavage Fluid , Herbal Medicine , Lung , Models, Animal , Plants , Pneumonia , RhizomeABSTRACT
Objective: To optimize the extraction of total saponins from fibrous root of Anemarrhena asphodeloides (TSFAA) and explore its protective effect on PC12 cells induced by oxygen-glucose deprivation (OGD). Methods: Single factor experiment and Box-Benhken response surface method were used to select the best extraction technology. The model of PC12 cells induced by OGD was established and treated with the total concentration of 20, 40, and 80 mg/L TSFAA. Inverted microscope was used to observe the morphology of PC12 cells, and cell viability was measured by MTT assay. Fluorescence probe was used to detect the intracellular oxygen free radical (ROS), and Annexin V/PI double staining method was performed to measure the apoptotic rate. The apoptotic protein Bcl-2 and Bax expression were detected by Western blotting. Results: The optimum conditions were as follows: The concentration of solvent was 72.22%; The ratio of material to liquid was 1:11; And the extraction time was 73.33 min. Under this condition, the theoretical calculated extraction rate was 8.38% and the measured value was 8.33%. PC12 cells viability was significantly decreased after OGD injury for 4 h. However, TGA showed a dose-dependent protective effect on OGD-induced cell damage; Flow cytometry analysis showed that TSFAA significantly reduced the content of ROS and apoptotic rate of PC12 cells. Also, Western blotting showed that TSFAA up-regulated the expression of Bcl-2 and down-regulated Bax expression. Conclusion: The extraction process for TSFAA optimized by response surface method has high yield and good extraction effect. TSFAA has protective effect on PC12 cells injured by OGD. The mechanism may be related to the decrease in the content of ROS in PC12 cells induced by OGD and the inhibition of mitochondrial apoptosis pathway.
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Objective: To establish a simultaneous quantitative analysis method of 58 pesticide residues in Anemarrhena asphodeloides based on QuEChERS-GC-QQQ-MS/MS technique. Methods: This study was performed on a Shimadzu GC-QQQ-MS/MS spectrometer, equipped with a SHIMADZU SH-Rxi-5Sil MS column (30 m × 0.25 mm, 0.25 μm). The temperature of injection port was 250 ℃, the injection volume was 1.0 μL, the injection mode was splitless, the injection pressure was 250 kPa; The carrier gas was high purity helium and the carrier gas control mode was constant linear velocity mode; The column flow rate was 1.69 mL/min, the line speed was 47.2 cm/s, the purge flow rate was 5 mL/min. The temperature rise method was the gradient program: The initial temperature was 50 ℃, hold the state for 1 min, first raise the temperature to 125 ℃ with 25 ℃/min, then raise the temperature to 300 ℃ with 10 ℃/min, keep the state for 15 min; The balance time was 0.5 min. The QuEChERS method was used to purify the test sample solutions, and the calibration was carried out using the standard curve of blank matrix matching, using a stable isotope internal standard for quantitative. Results: Quantitative determination of 58 pesticide residues in 30 batches of A. asphodeloides samples was carried out. The results showed that there were five batches of samples detecting a small amount of p,p’-DDE, three batches of samples were detected trifluralin, two batches of samples were detected chlorpyrifos, and one batch of samples were detected butralin. Conclusion: The development and validation quantitative analysis method of 58 pesticide residues based on QuEChERS-GC-QQQ-MS/MS has good applicability, which is of reference value for the establishment of other pesticide residues in herbs.
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AIM To explore the therapeutic effects of polysaccharide from Anemarrhena asphodeloides Bunge (PAA) on diabetic rats and potential mechanism of action.METHODS The rat model for diabetes was established by intraperitoneal injection of STZ (60 mg/kg),and sixty rats were assigned to control-,model-,glibenclamide-(25 mg/kg) groups and three groups of PAA-(50,100,200 mg/kg).Drugs were intragastrically administrated to rats once a day for 28 days.The rat body weight,glucose tolerance,fasting blood glucose (FBG),fasting insulin (FINS),IL-6,TNF-α,CAT,SOD,MDA,insulin receptor substrate (IRS1),Phospho-IRS1 and glucose transporter protein 4 (Glut4) were tested.RESULTS PAA could increase rat body weight and FINS level,reduce FBG level,and improve the glucose tolerance.The levels of IL-6 and TNF-α in serum,and MDA content in hepatic tissue were decreased,and the activities of CAT and SOD in hepatic tissue were increased.Meanwhile,PAA could also reduce the Phospho-IRS1 expression,and increase the Glut4 expression in hepatic tissue.CONCLUSION PAA has an anti-diabetic effect,whose mechanism is involved in anti-inflammatory,antioxidation,decreasing Phospho-IRS1 expression,and increasing Glut4 expression.
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AIM To explore the therapeutic effects of polysaccharide from Anemarrhena asphodeloides Bunge (PAA) on diabetic rats and potential mechanism of action.METHODS The rat model for diabetes was established by intraperitoneal injection of STZ (60 mg/kg),and sixty rats were assigned to control-,model-,glibenclamide-(25 mg/kg) groups and three groups of PAA-(50,100,200 mg/kg).Drugs were intragastrically administrated to rats once a day for 28 days.The rat body weight,glucose tolerance,fasting blood glucose (FBG),fasting insulin (FINS),IL-6,TNF-α,CAT,SOD,MDA,insulin receptor substrate (IRS1),Phospho-IRS1 and glucose transporter protein 4 (Glut4) were tested.RESULTS PAA could increase rat body weight and FINS level,reduce FBG level,and improve the glucose tolerance.The levels of IL-6 and TNF-α in serum,and MDA content in hepatic tissue were decreased,and the activities of CAT and SOD in hepatic tissue were increased.Meanwhile,PAA could also reduce the Phospho-IRS1 expression,and increase the Glut4 expression in hepatic tissue.CONCLUSION PAA has an anti-diabetic effect,whose mechanism is involved in anti-inflammatory,antioxidation,decreasing Phospho-IRS1 expression,and increasing Glut4 expression.
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Two new steroidal saponins, named timosaponin P (1) and timosaponin Q (2), were isolated from the rhizome parts of Anemarrhena asphodeloides Bunge using various chromatographic methods. Their structures and absolute configurations were elucidated by a combination of spectroscopic and spectrometric data, including 1D, 2D NMR, HR-ESI-MS and ECD calculations, and this is the first time the absolute configuration of C-23 of steroidal saponin was confirmed by ECD calculations.
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Anemarrhena , Chemistry , Drugs, Chinese Herbal , Chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Saponins , Chemistry , Steroids , ChemistryABSTRACT
Objective To develop a method for the determination of five furostanol saponins(timosaponin N,timosaponin L, timosaponin BⅡ,25R-timosaponin BⅡ,and 25S-officinalisnin-Ⅰ)in rhizome and fibrous root of Anemarrhena asphodeloides Bge. by HPLC with the charged aerosol detector(CAD). Methods The analysis was performed on TechMate C18-ST-II(250 mm×4.6 mm,5μm)with acetonitrile:water(22:78,V/V),the flow rate of 1.0 ml/min and column temperature at 30℃. The Corona parameters were as follows:sampling rate 10 Hz,filter 5 s,and the nebulizer temperature 55℃. Results The approach showed good linearity for five saponins. The correlation coefficients(r2)for calibration curves varied from 0.9992 to 0.9998. The limits of detection(LOD)were 0.28,0.92,0.92,0.92 and 0.92 ng for five steroidal saponins,respectively. The limits of quantitation(LOQ)were found to be 0.92, 2.77,2.77,2.77 and 2.76 ng,respectively. RSD calculated from peak area of precision,repeatability and stability in 48 h were all less than 3.0%. The average recoveries of timosaponin N,timosaponin L,timosaponin BⅡ,25R-timosaponinBⅡ,and 25S-officinalis-nin-Ⅰwere 98.17%,101.37%,98.53%,97.63%,and 98.17%,respectively. Conclusion The developed method is accurate,reli-able,which could be applied to the quality control of multiple components in A. asphodeloides Bge.
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<p><b>OBJECTIVE</b>To assess the biological effects of the six-herb mixture Anti-Insomia Formula (AIF) extract using caffeine-induced insomnia Drosophila model and short-sleep mutants.</p><p><b>METHODS</b>Caffeineinduced insomnia wild-type Drosophila and short-sleep mutant flies minisleep (mns) and Hyperkinetic(Y) (Hk(Y)) were used to assess the hypnotic effects of the AIF in vivo. The night time activity, the amount of night time sleep and the number of sleep bouts were determined using Drosophila activity monitoring system. Sleep was defined as any period of uninterrupted behavioral immobility (0 count per minute) lasting > 5 min. Night time sleep was calculated by summing up the sleep time in the dark period. Number of sleep bouts was calculated by counting the number of sleep episodes in the dark period.</p><p><b>RESULTS</b>AIF at the dosage of 50 mg/mL, effectively attenuated caffeine-induced wakefulness (P<0.01) in wild-type Canton-S flies as indicated by the reduction of the sleep bouts, night time activities and increase of the amount of night time sleep. AIF also significantly reduced sleeping time of short-sleep Hk(Y) mutant flies (P<0.01). However, AIF did not produce similar effect in mns mutants.</p><p><b>CONCLUSION</b>AIF might be able to rescue the abnormal condition caused by mutated modulatory subunit of the tetrameric potassium channel, but not rescuing the abnormal nerve firing caused by Shaker gene mutation. This study provides the scientific evidence to support the use of AIF in Chinese medicine for promoting sleep quality in insomnia.</p>
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Animals , Caffeine , Chromatography, High Pressure Liquid , Disease Models, Animal , Drosophila melanogaster , Physiology , Hypnotics and Sedatives , Pharmacology , Therapeutic Uses , Mutation , Genetics , Potassium Channels , Genetics , Sleep , Sleep Initiation and Maintenance Disorders , Drug Therapy , WakefulnessABSTRACT
AIM@#To develop and validate a high performance liquid chromatography (HPLC) coupled with diode array and evaporative light scattering detectors (DAD-ELSD) method for the quantitative determination and fingerprint analysis of ten active constituents in three chemical classes (namely, xanthone glycosides, steroidal saponins, and alkaloids) in Zhimu-Huangbai herb pair (ZB).@*METHOD@#Chromatographic separation was performed on a Diamonsil C18 column (4.6 mm × 250 mm, 5 μm, Dikma) by gradient elution using acetic acid in acetonitrile solution at a flow rate of 1.0 mL·min(-1) at 260 nm. The drift tube temperature of ELSD was set to 60 °C and nebulizer gas pressure was 4.0 Bar. Method validation was performed to assure its linearity, limits of detection and quantification, precision, repeatability, stability, and accuracy.@*RESULTS@#The HPLC-DAD-ELSD method allowed the quantification of ten compounds (phellodendrine, jatrorrhizine, palmatine, berberine, neomangiferin, mangiferin, timosaponin E-I, timosaponin B-II, timosaponin B, and timosaponin A-III), and was successfully applied to fingerprint analysis for ten batches of ZB samples.@*CONCLUSION@#This was the first time to apply the combination of DAD and ELSD for the simultaneous determination of ten active ingredients in ZB. The results showed that the combination of quantitative analysis for marker ingredients and chemical fingerprint for the TCM herb pair provides a potentially powerful, widely introduced, and internationally accepted strategy for assessment of complex TCM formulas.
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Alkaloids , Chromatography, High Pressure Liquid , Methods , Drugs, Chinese Herbal , Glycosides , XanthonesABSTRACT
During a screening program to search the anticolitic herbal medicines, 80% ethanol extract of the rhizome of Anemarrhena asphodeloides (AA) was found to potently inhibit the expression of proinflammatory cytokines TNF-alpha and IL-1beta, as well as the activation of NF-kappaB in LPS-stimulated colonic macrophages, followed by that of the rhizome of C. chinensis (CC). AA also potently inhibited TNBS-induced colitic markers, shortening of the colon and increase of macroscopic score, myeloperoxidase activity, TNF-alpha, IL-1beta, and IL-6, in mice. The synergistic effect of CC against the anticolitic effect of AA was investigated. CC synergistically inhibited the anticolitic effect of AA. AC-mix (AA+CC, 1:1) potently inhibited them. AC-mix also inhibited the activation of NF-kappaB, as well as the expression of TNF-alpha, IL-1beta, IL-6, iNOS and COX-2. The effects of AC-mix against oxazolone-induced colitis were investigated in mice. AC-mix also potently inhibited oxazolone-induced inflammatory markers, colon shortening, macroscopic score, myeloperoxidase activity, NF-kappaB activation and proinflammatory cytokines. Overall, the anti-colitic effect of AC-mix was superior to that of mesalazine. Based on these findings, AC-mix may improve colitis by inhibiting NF-kappaB activation.
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Animals , Mice , Anemarrhena , Colitis , Colon , Cytokines , Ethanol , Interleukin-6 , Macrophages , Mass Screening , Mesalamine , NF-kappa B , Peroxidase , Rhizome , Tumor Necrosis Factor-alphaABSTRACT
Objective: The tissue culture of Anemarrhena asphodeloides was preliminarily studied to establish A. asphodeloides regeneration system. Methods: The establishment of A. asphodeloieds sterile system, tiller bud proliferation, tiller callus induction and its re-differentiation as well as transplanting of regenerated plantlets were studied by plant tissue culture and single factor test method. Results: The best disinfection way of A. asphodeloides seeds was firstly dealt with 75% ethanol for 30 s and then dealt with 0. 1% HgCl2 for 15 min; The best medium of bud proliferation for A. asphodeloides tillers was MS+KT 1 mg/L+NAA 0.5 mg/L; The best medium of A. asphodeloides tiller callus induction was MS+KT 2 mg/L+NAA 0.5 mg/L; The best medium of A. asphodeloides tillers callus redifferentiation was MS+ KT 2 mg/L+NAA 0.1 mg/L; The best rooting medium of A. asphodeloides callus regeneration buds was 1/2 MS+NAA 0.5 mg/L; The best transplanting substrate of A. asphodeloides plantlets was humus soil. Conclusion: The regeneration system of A. asphodeloides is established, which provides a technological basis for factory production of A. asphodeloides plantlets.
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AIM To investigate the neuroprotective effects and possible mechanisms of saponins from Anemarrhena asphodeloides Bge. (SAaB) on neuronal damage induced by amyloid β-protein fragments 25-35 (Aβ25-35). METHODS Cultured mouse peritoneal macrophages were stimulated with Aβ25-35 (20 μmol·L-1) for 0.5, 1, 2 and 6 h or preincubated with SAaB (10, 30 and 100 μmol·L-1)for 10 min or mitogen-activated protein kinase (MAPK) specific inhibitors (p38 MAPK inhibitor SB 203580 and MEK specific inhibitor PD98059) for 30 min prior to the addition of Aβ25-35(20 μmol·L-1). After stimulation with Aβ25-35 for the indicated times, total cellular extracts were prepared for Western blotting of extracellular signal-regulated kinase (ERK) and p38 MAPK. After stimulation with Aβ25-35 for 48 h, the supernatants of cultured macrophages were collected for quantification of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) and protein expression of inducible nitric oxide synthase (iNOS) in macrophages was determined by immunocytochemical staining. To determine whether SAaB has protective effect against neuronal apoptosis mediated by Aβ25-35-induced macrophages activation, macrophages were stimulated with Aβ25-35 in the presence or absence of SAaB (10, 30 and 100 μmol·L-1) for 48 h and then the cell-free supernatant of Aβ25-35-stimulated macrophages was transferred to the culture of cerebellar granule neurons for 72 h. Neuronal apoptosis was quantitated by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining. RESULTS Aβ25-35(20 μmol·L-1) significantly induced increase in phospho-ERK1/2 and phospho-p38 MAPK protein expression without affecting total protein levels and in the production of TNF-α and NO in cultured macrophages. Aβ25-35-induced increase of TNF-α production in macrophages involved activation of ERK1/2 signal pathway. Importantly, TNF-α and NO generated by cultured macrophages after Aβ25-35 stimulation may be responsible for the majority of the neuronal apoptosis. SAaB (30 and 100 μmol·L-1) significantly suppressed Aβ25-35-induced increase in phospho-ERK1/2 and phospho-p38 MAPK protein. In addition, SAaB (10, 30 and 100 μmol·L-1) also decreased the level of TNF-α and NO in supernatants of cultured macrophage and inhibited Aβ25-35-induced increase in iNOS protein expression of macrophages. Neuronal apoptosis mediated by Aβ25-35-induced macrophage activation was also significantly attenuated by treatment with SAaB (10, 30 and 100 μmol·L-1). CONCLUSION SAaB protects neurons against the neuronal cell death induced by Aβ25-35. The beneficial effects of SAaB may be related to the reduction of TNF-α and NO from activated macrophage induced by Aβ25-35.
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Saponin and sapogenin are main components in chinese medicine Anemarrhena asphodeloides Bge.Modern research has shown that Anemarrhena asphodeloides Bge and its effective components could markedly enhance the ability of learning and memory in dementia animals,as well as improve the descent of brain function.The effect was related with many factors such as inhancing the cholinergic receptor(M,N),improving the activity of ChAT and inhibiting the activity of AChE,regulating the balance of ? receptor-cAMP and M receptor-cGMP,improving the metabolite of free radicals in model animal brains and so on.