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Saponins and sterones are two main characteristic components in Achyranthis Bidentatae Radix. In order to control the quality of Achyranthis Bidentatae Radix more effectively, a high-performance liquid chromatography (HPLC) method was established by using double external standards calibration method (DESCM) for simultaneous determination of the contents of achyranthoside C, achyranthoside D, β-ecdysterone, 25R-inokosterone and 25S-inokosterone in Achyranthis Bidentatae Radix. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 (150 mm × 4.6 mm, 2.7 µm) using 0.1% phosphoric acid in water and 0.1% phosphoric acid in acetonitrile as mobile phase. The flow rate was 0.8 mL·min-1 and the column temperature was set as 35 ℃, the injection volume was 5 μL and the total analytical time was 30 min. β-Ecdysterone was used as the reference to calculate the relative correction factors (RCF) and relative retention time (RRT) of 25R-inokosterone and 25S-inokosterone, achyranthoside D was used for achyranthoside C. The RCFs of 25R-inokosterone, 25S-inokosterone, and achyranthoside C were 1.116, 1.056, and 0.888 1, respectively. The double external standards calibration method (DESCM) and external standard method (ESM) were used to calculate the contents of five ingredients in Achyranthis Bidentatae Radix samples from different sources and the variation between the results was within acceptable limits (RE ≤ 5%). The results showed that the contents of two saponins and three sterones of Achyranthis Bidentatae Radix were 0.597%-1.916% and 0.044%-0.150% respectively. The total content of saponins was about 10 times that of sterones. In conclusion, the established DESCM allowed simultaneous determination of five ingredients (achyranthoside C, achyranthoside D, β-ecdysterone, 25R-inokosterone, and 25S-inokosterone) in Achyranthis Bidentatae Radix, providing a scientific and feasible overall quality evaluation method for Achyranthis Bidentatae Radix.
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BACKGROUND: β-ecdysterone as a “phytoestrogen” has the ability to stimulate protein synthesis, promote carbohydrate and lipid metabolism, relieve hyperglycemia and hyperlipidemia, protect endothelial cells from apoptosis and induce their proliferation. Some scholars have reported that it also plays an important role in the treatment of osteoporosis, fractures and other bone inflammatory diseases. OBJECTIVE: To observe the effect of β-ecdystrone on the proliferation of mouse pre-osteoblasts(MC3T3-E1 cells) in vitro, and to explore whether β-ecdysterone can induce osteogenic differentiation of MC3T3-E1 at a safe dose. METHODS: The fourth generation MC3T3-E1 cells were cultured in the osteogenic induction medium for 7, 10, 14, 21, and 28 days. The osteogenic differentiation proteins(alkaline phosphatase, type I collagen, osteopontin, and calcified nodules) were detected at different time points, to identify whether the cells have the ability of osteogenic differentiation. MC3T3-E1 cells were then seeded into the induction medium containing different final concentrations of β-ecdysterone(0.01, 0.1, 1, 10, 100 µmol/L). The proliferation activity of the cells was detected by cell counting kit-8 method at days 1, 2, 3, 4, 5, 6, and 7 after induction. The control group(general induction medium group) and the experimental group(general induction medium + β-ecdysterone) were cultured under the same conditions, and the expression levels of osteogenic marker proteins in each group of cells at different time periods were determined. RESULTS AND CONCLUSION: In the MC3T3-E1 cells stimulated by the osteogenic induction medium, alkaline phosphatase staining and type I collagen florescence staining showed higher expression at day 10 of induction, and this was also confirmed by detection of alkaline phosphatase activity(P 0.05). The expression of alkaline phosphatase and type I collagen was higher in the experimental group than in the control group at day 10 of induction. The expression of osteopontin and osteocalcin in the cells was higher at day 14 of induction, and there was no significant difference in the calcified nodule staining between the two groups at day 28 of induction. These findings indicate that β-ecdysterone can promote the proliferation of MC3T3-E1 cells in vitro and induce MC3T3-E1 cells to differentiate into osteoblasts at a safe dose.
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Objective: To study the chemical constituents from the roots of Pfaffia paniculata. Methods: The compounds were isolated and purified by means of chromatographic separation techniques and their structures were identified based on spectral features. Results: Fourteen known compounds, including 11 30-noroleanane triterpenoids and three ecdysterones, named pfaffianol A (1), 3-oxo-akebonoic acid (2), 16β-hydroxyl-3-oxo-akebonoic acid (3), pfaffiaglycoside A (4), pfameric acid (5), 2α,3β,20α-trihydroxy-30-norolean-12-en-28-oic acid (6), 2α,3β-dihydroxy-23-oxo-30-norolean-12,20(29)-dien-28-oic acid (7), 2α,3β-dihydroxy-30-norolean-12,20(29)-dien-28-oic acid (8), 2α,3α-dihydroxy-30-norolean-12,20(29)-dien-28-oic acid (9), quinatic acid (10), 3β-hydroxy-30-norhederagenin (11), diaulusterol B (12), 20,22-didehydrotaxisterone (13), and ecdysterone (14) were isolated from the roots of P. paniculata. Conclusion: Compounds 10-12 are obtained from P. paniculata for the first time. Compounds 6-9, and 13 are isolated from the genus of Pfaffia for the first time.
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Objective:To study the role of endoplasmic reticulum stress(ERS)on ecdysterone(EDS)influenced protective effect in H2O2induced myocardial injury.Methods: The H9c2 cells were randomly divided into control group(normal cells),H2O2 groups(the cells treated with H2O2at concentrations of 1×10-3,1×10-4,1×10-5mol/L,respectively);EDS group(the cells were exposed to H2O2and treated with EDS at concentrations of 25,50,100 μmol/L respectively).The cell activity and apoptosis were measured by MTT assay and flow cytometry,respectively.The protein levels of Bcl-2,Bax,cleaved-caspase-3,caspase-4,caspase-7, caspase-12,GRP78 and CHOP were deter mined by Western blot.The MDA content and SOD activity were detected by the MDA and SOD detection kits.Results:The cell activity was decreased,cell apoptosis was increased,the content of MDA was elevated,the activity of SOD was inhibited and the protein expression of GRP78 and CHOP were increased in H2O2group as compared with control group, which were all significantly reversed by EDS treatment(P<0.05).Conclusion: Ecdysterone exhibits a protective effect on the cardiomyocytes with H2O2induced injury by inhibiting endoplasmic reticulum stress.
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AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress.METHODS:H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H2O2 group.H9c2 cardio-myocytes in H 2 O2 group and high , middle and low doses of EDS groups were exposed to H 2 O2 for 6 h to establish the model of oxidative stress.The viability of the H9c2 cells was detected by CCK-8 assay.The apoptosis of H9c2 cells was analyzed by flow cytometry.The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorime-try.The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cy-tometry and confocal laser scanning microscopy .The protein levels of Bax , Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot .RESULTS:Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells.Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H 2 O2 , but were decreased by EDS treatment in a dose-dependent man-ner.The levels of SOD and mitochondrial membrane potential of the H 9c2 cells in H2 O2 group were reduced significantly compared with control group , but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mito-chondrial membrane potential in H 2 O2-treated H9c2 cells.The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H2 O2 group showed significant elevation in comparison with control group , and the protein expression of Bcl-2 de-clined in H2 O2 group compared with control group , but high, middle and low doses of ecdysterone treatments down-regula-ted the protein levels of Bax , cleaved caspase-3 and up-regulated the expression of Bcl-2 in H2 O2-treated H9c2 cells. CONCLUSION:Ecdysterone attenuates the effect of H 2O2-induced oxidative stress on H9c2 cardiomyocytes.The mecha-nism may be involved in scavenging oxidative stress products , increasing antioxidant enzyme activity and improving mito-chondrial function .
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Objective: To investigate the chemical constituents from the flowers of Rhaponticum uniflorum. Methods: The chemical constituents were separated and purified by macroporous resin, silica gel, Sephadex LH-20, and MCI column chromatographies. Their structures were determined by physicochemical properties and spectral data. Results: Seventeen compounds were isolated from the ethanol extract in the flowers of R. uniflorum. Among them, eleven flavones were identified as 5,7,4'-trihydroxy-3'-methoxyflavone (1), quercetin-3'-O-methyl ether (2), apigenin (3), kaempferol (4), luteolin (5), quercetin (6), apigenin-7-O-β-D-glycuronate ethyl ester (7), kaempferol-3-O-α-L-rhamnoside (8), quercetin-3-O-α-L-rhamnoside (9), apigenin-7-O-β-D-glucopyranoside (10), and apigenin-6,8-di-C-β-D-glucoside (11); Two liganans were hemislin B glucoside (12) and hemislin B (13); Two phytoecdysones were ecdysterone (14) and turkesterone (15); Others were ursolic acid (16) and 3,5-O-dicaffeoyl quinic acid (17). Conclusion: Compounds 1, 2, 7-10, 12 and 13 are isolated from the plants of Rhaponticum Cass. for the first time, and the compounds 1, 2, and 7-17 are found from the flowers of R. uniflorum for the first time.
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OBJECTIVE:To establish a method for the content determination of ecdysterone in different parts of Radix serratu-lae. METHODS:HPLC was performed on the column of ZORBAX Eclipse XDB-C18 with mobile phase of methanol-water at a flow rate of 1.0 ml/min(46∶54,V/V),detection wavelength was 248 nm,column temperature was 30℃,and the injection volume was 10 μl. Compare the contents of ecdysterone in different parts of R. serratulae. RESULTS:The linear range of ecdysterone was 0.12-1.21 μg;RSDs of precision,stability and reproducibility tests were lower than 1.5%;recovery was 98.4%-101.9%(RSD=1.64,n=6). The content of ecdysterone in roots was found at the highest level,followed by tubers,and lower in stems and leaves, and it was not detected in seeds. CONCLUSIONS:The method is simple and rapid with good accuracy and reproducibility,and suitable for the content determination of ecdysterone in different parts of R. serratulae. It is feasible to develop the medicinal parts of R. serratulae from roots to roots,flowers,tubers and buds on tubers.
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Objective To study the effect of ecdysterone (EDS) on rabbits chondrocytes that is injuried by lipopolysac?charide (LPS). Methods Aricular chondrocytes were isolated from rabbits and randomly divided into three groups:control group;chondrocytes with LPS induced injury (LPS group);injury chondrocytes treated with EDS (LPS+EDS group). The cell proliferation and cell apoptosis of chondrocytes were determined by MTT method and flow cytometry assay respectively. The mRNA and protein expression levels of inducible nitric oxide synthase (iNOS) were detected by RT-PCR and western blot. In addition, the content of NO and IL-1βwere measured by nitric acid reductase assay and enzyme-linked immunosorbent assay (ELISA) respectively. Results Attenuated proliferation, increased cell apoptosis, iNOS, NO and IL-1βwere seen in LPS group , but all these changes were significantly reversed by EDS treatment (P<0.05). Conclusion Ecdysterone exhib?ited a protective effect on LPS induced rabbits chondrocytes injury through inhibiting the expression of iNOS.
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Aim To screen the express-altered proteins before or after effect of Ecdysterone on HepG_2 cell model of insulin resistance by the strategy of comparative proteomics, which may approach new proves exploring the target of sensitizer.Methods HepG_2 cells were incubated with 5×10~(-7) mol·L~(-1) insulin for 16 h, and glucose consumption was determined. After treatment, the insulin resistant cells were incubated with 10~(-5) mol·L~(-1) Ecdysterone for 24 h.Then glucose consumption contents were determined. The proteins of two groups before and after treatment with Ecdysterone were extracted by lysis buffers. The express-altered proteins were screened by 2-DE technique.Some of them were analyzed by MALDI-TOF-MS mass spectrometry and MS-Fit database.Results 53 express-altered protein spots of insulin resistant HepG_2 cells before and after treated by Ecdysterone were screened by 2-DE technique,in which 35 ones were up-regulated and the others down-regulated, 6 spots of which were analyzed by MALDI-TOF-MS mass spectrometry and MS-Fit database.Conclusion The target of Ecdysterone as a sensitizer involves many proteins and kinases which correlate insulin resistant. These results lay a foundation for further studies on the function of these target proteins.
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Neste trabalho foi avaliado, em roedores, o efeito depressor das frações clorofórmio (CHCl3), acetato de etila (EtOAc) e n-butanol, obtidas das partes subterrâneas de Pfaffia glomerata, empregando-se o teste de tempo de sono barbitúrico como referência. Somente a fração lipofílica (CHCl3:EtOAc, 1:1, m/m) (i.p. 500 mg/kg; v.o. 1000 mg/kg) potenciou o tempo de sono induzido por pentobarbital. A ecdisterona foi isolada e identificada como constituinte majoritário (1,4 por cento m/m) desta fração, através de cromatografia líquida de alta eficiência e métodos espectroscópicos, respectivamente. Este composto potenciou o tempo de sono barbitúrico (100 mg/kg, i.p.; 400 mg/kg, v.o), sem causar hipotermia. Nestas mesmas doses, a ecdisterona não alterou a performance dos animais no rota-rod, esquiva inibitória e labirinto em cruz-elevado, além de não alterar o padrão de convulsões induzidas por pentilenotetrazol. Este perfil indica que esta substância, nestas doses, não apresenta perfil ansiolítico ou neurotóxico. Estes resultados indicam que a ecdisterona é o componente responsável pela ação hipnótica apresentada pela fração lipofílica obtida das partes subterrâneas de P. glomerata.
In this study the depressant effect of fractions from P. glomerata was initially evaluated using the mice barbiturate sleeping time test as reference. The fractions tested were the CHCl3, the EtOAc, the n-BuOH and the aqueous fraction obtained from P. glomerata subterraneous parts. Only the pretreatment with the lipophilic fraction (CHCl3: EtOAc, 1:1, w/w) increased the barbiturate sleeping time (i.p 500 mg/kg; v.o. 1000 mg/kg). Ecdysterone, the main substance isolated from this lipophilic fraction, was identified by spectroscopic methods and its content in the ethanol extract was determined as 1.4 percent (w/w) by HPLC. In order to investigate the hypothesis of ecdysterone displaying a depressant effect on nervous central system, an evaluation toward the hypnotic-sedative and anxiolytic effects of this drug was carried out. Ecdysterone 100 mg/kg, i.p, increased the barbiturate sleeping time without provoking hypothermia; when administered by oral route its minimal effective dose was 400 mg/kg. On the other hand, ecdysterone (100 mg/kg, i.p; 400 mg/kg, p.o) did not impair motor coordination and was ineffective on pentylenetetrazole-induced convulsion, elevated plus-maze and step-down inhibitory avoidance tests, indicating that at these doses the drug does not present an anxiolytic profile and does not cause manifest neurotoxic effects as well. In conclusion, the lipophilic fraction from P. glomerata presents a hypnotic effect being ecdysterone one of the compounds responsible for this CNS activity.
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Objective To observe the effect of ecdysterone(EDS)on the level of VEGF protein in the brain,angiogenesis and neurologic function after focal cerebral ischemia in rats.Methods Rat with focal cerebral ischemia were established by occluding their middle cerebral artery.The established rats(n=36)were randomly and equally divided into EDS treatment group and ischemia group.EDS(20 mg?kg-1?d-1 for 7 d)was intraperitoneally injected into the rats of EDS treatment group 2 h after operation,and the animal of ischemia group received an intraperitoneal injection of the same solvent as in EDS group.Another 6 rats served as normal control.Rats were sacrificed in 7,14 and 21 d after operation,and the VEGF protein level and microvessel density(MVD)was detected with immunohistochemical methods and analyzed quantitatively with image system.Effect of EDS on neurologic recovery following brain ischemia were assessed using the neurologic severity scores(NSS).Results VEGF expression was not seen in normal control,and was higher in ischemia group than in the EDS treatment group at day 7 and 14,but the significant difference was only observed at day 7(P
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Aim To investigate whether ecdysterone is able to exert glucose-lowering effect on hepatocytes or stimulate the secretion of insulin.Methods For glucose consumption studies,the amounts of glucose disappeared from the culture medium of HepG2 cells within 24 h were determined.?TC3 cells were also used to monitor insulin secretion.Results The glucose concentrations decreased significantly by ecdysterone 1?10~(-6)～10~(-4) mol?L~(-1),while glucose consumption increased by 44%～77% with ecdysterone.Glucose consumption declined as its concentrations increased.Insulin had no effect on glucose-lowering of ecdysterone.?TC3 cells were not stimulated by ecdysterone.Conclusion Ecdysterone is able to exert glucose-lowering effect on hepatocytes which is insulin independent,but has no effect on insulin secretion.
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Objective To apply accelerated solvent extraction (ASE) technique to extract Achyranthes bidentata and to explore the application of this technique in quality control of Chinese materia medica. Methods Investigation of single factor was used to optimize the conditions that affected the efficiency for ASE from A. bidentata by RP-HPLC using ecdysterone as a quantitative marker. Results The optimized conditions for ASE of A. bidentata were obtained as follows: methanol as solvent, particle size between 0.3 and 0.45 mm, temperature at 100 ℃, pressure under 10.34 MPa, 6 min duration and once extraction. Conclusion ASE technique can be used to extract A. bidentata quickly and effectively.
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Objective To determine ecdysterone content in Radix Achyranthis Bidentalae by near-infrared diffuse reflectance spectroscopy (NIRDRS) combined with partial least squares (PLS). Methods NIRDRS and PLS. Results The correlation coefficient of the quantitative mathematics model between the prediction and the true values was 0.948 9. It is feasible to appling the above established method to determination of chemical constituents in Chinese materia medica. Conclusion Compared with other methods, the method is simple, rapid, high efficient, low expenditure, no pollution and with preparation undone and is suit for large quantity of unknown samples.
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Objective To determine the relationship and significance between the acute ischemia, hypoxia and the production of VEGF in rat myocardium and the influence of EDS on expression of VEGF protein in myocardium Methods To establish the model of AMI in rats, Wistar rats were randomly divided into control group, AMI group (1,3,7 day) and EDS group (0, 40 mg/day) Myocardial enzymes, VEGF protein, microvascular density and infarct size were detected Rat cardiac myocytes cultured primarily received EDS 100 ?g/ml in normal and hypoxia condition After 24 hours, VEGF was detected with immunohistochemical technique Results The production of VEGF was higher with ischemia and hypoxia time, the positive relationship was found between the time of AMI and the production of VEGF EDS reduced the concentration of serum myocardial enzymes (CK MB), enhanced the formation of collateral circulation,microvascular density and cardiac function; decreased infarct size In addition, EDS could enhance expression of VEGF protein in cardiac myocytes of rat in normal and hypoxia condition Conclusion Acute ischemia strongly stimulated the production of VEGF in myocardium, which played an important role for autoprotection of ischemic myocardium EDS could promote the establishment of cardiac collateral circulation and enhance microvascular density in infarct zone by upregulation of VEGF protein expression
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As raízes secas da espécie brasileira Pfaffia tuberosa (Spreng.) Hicken foram submetidas a extração com metanol. Deste extrato metanólico foram isolados a ecdisterona e o ácido oleanólico, através de processos de separação por HPLC e cromatografia em coluna. Tanto a ecdisterona como o ácido oleanólico foram identificados comparativamenve com os respectivos padrões. Análise quantitativa por HPLC mostrou 0,23 por cento de ecdisterona. Este teor foi considerado baixo em relação a outra espécie, também brasileira, a Pfaffia iresinoides.
In this paper, we wish to report the isolation and identification of ecdysterone from Pfaffia tuberosa (Spreng.) Hicken. The roots of Pfaffia tuberosa (Spreng.) Hicken were colected in the Mato Grosso State - Brazil. The dried and sliced roots (197g) were extracted with hot MeOH. A suspension of the resulting MeOH extract in the water was treated with n-BuOH. The n-BuOH layer was concentrated in vacuo to give a brown viscous oil (5g), wich was chromatographed on silicagel (65g) using CHCl3 - MeOH - H2O (80:20:10) lower phase, and 15ml fractions were collected. Fractions 11-13 were concentrated and the residual solid was recrystallized from EtOH to afford oleanolic acid (65mg). The crystalline residue obtained from fractions 44-56 was recrystallized from EtOAC-MeOH to give ecdysterone (87mg). Each compound was identified by direct comparison with an authentic sample. Further HPLC quantitative analysis showed lower content of ecdysterone (0,23 percent) compared with that of Pfaffia iresinoides Spreng.
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AIM To explore the effect of hypoxia on apoptosis in human umbilical vein endothelial cells(HUVECs) and the role of ecdysterone(EDS)in inhibition of apoptosis. METHODS ① Culture and identification of HUVECs;② Hypoxic model(0,12,24,48 h) in HUVECs was established. While HUVECs was incubated 24 h with EDS(100 mg?L -1 ) under hypoxic condition. Apoptosis in HUVECs was detected by TUNEL;③ HUVECs received EDS 100 mg?L -1 in normal and hypoxic condition. After 24 h, vascular endothelial growth factor (VEGF) was detected with immunohistochemical technique. RESULTS The apoptosis percentages are different under different hypoxic condition. The longer hypotic the time was, the higher the apoptosis percentage was. EDS reduced apoptosis of HUVECs. EDS could enhance expression of VEGF protein in cardiac myocytes of rat in normal and hypoxic condition. CONCLUSION The role of hypoxia in HUVECs apoptosis is more significant along with prolonging of hypoxic time. VEGF play an important role in protective effect of EDS on endothelial cell apoptosis.
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Aim This study examined the uptake and transport of ecdysterone(EDS) using Caco-2 cell monolayers as a model of human intestinal mucosa.Methods Two kinds of Caco-2 cell monolayer model(Caco-2 cell monolayers;CYP3A4 expressing Caco-2 cell monolayers) were set up to study the uptake and transport of EDS.Results Compared bi-directional charaterizaton of Caco-2 cell,the apparent permeability coefficients(Papp) values of EDS were between 0.1?10-6 cm?s-1 and 1?10-6 cm?s-1.EDS absorption was 1%~10% for two kinds of Caco-2 models.The RPapp values were all less than 1.5 for 4 h.Conclusions The uptake and transport of EDS was a passive transcellular diffusion mechanism.Ecdysterone was not influenced by CYP3A4 mediate mechanism.
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Aim To investigate the effects of ecdysterone on insulin sensitivity and glucose metabolism in the insulin resistant HepG2 cell model induced by high concentration of insulin. Methods The insulin-stimulated glucose incorporation rate was determined with ~3H-D-glucose uptake test which was used to estimate insulin sensitivity of HepG2 cell, and glucose consumption, the amounts of glucose disappeared from the culture medium of HepG2 cells within 24 h were determined. Results The incubation of insulin resistance cells with ecdysterone 1?10~ -6 ～10~ -4 mol?L~ -1 could significantly increase the glucose uptake and glucose consumption of the cells compared with that of control cells(P0.05). Conclusion Ecdysterone could improve the insulin sensitivity in the cell model and can attenuate the aggression of insulin resistance. The effect to improve the insulin sensitivity with ecdysterone is similar to that with pioglitazone.