Study on isoflavones isolated from stems of Wisteria sinensis / 中草药

Objective: To investigate the chemical constituents from the stems of Wisteria sinensis. Methods: The 14 chemical constituents were isolated and purified by silica gel, polyamide, Sephadex LH-20 column chromatography, and semi-preparative HPLC, and their chemical structures were identified by physico-chemical constants and spectral data. Results: Fourteen isoflavones were isolated from the methanol extract of the stems of W. sinensis and identified as tectorigenin-7-O-β-D-(6″-O-acetyl)-glucoside (1), 7,3',4'-trihydroxyisoflavanone (2), formononetin (3), afromosin (4), prunetin (5), biochanin A (6), gliricidin (7), 8-O-methylreyusin (8), tectoridin (9), genistin (10), sissotrin (11), ononin (12), kakkanin (13), and kushenol O (14). Conclusion: Compound 1 is a new compound named as tectoridin A, and compounds 2, 5, 7, 9-11, 13, and 14 are obtained from the genus Wisteria for the first time.

Study on Synthesis and Anti-tumor Activity of 6 Kinds of Tectorigenin Mannich Base Derivatives / 中国药房

OBJECTIVE： To conduct structural modification of tectorigenin to search for new compounds with anti-tumor activity. METHODS： Tectorigenin was used as a lead compound， and then added into amine reagents as ethanolamine， methylamine， ethylamine， dimethylamine， diethylamine， n-propylamine and formaldehyde solution. Tectorigenin Mannich base derivatives were synthesized by mannich reaction with as the lead compound. The structures of the derivatives were identified according to IR， UV， MS and NMR data. Solubility of tectorigenin and its derivatives were investigated by solubility test method. MTT assay was used to investigate the inhibitory effects of tectorigenin and its derivatives on the proliferation of human colon cancer cell line HCT116， human lung cancer cell line A549 and human hepatoma cell line HepG2， and half inhibitory concentration （IC50） was calculated. The inhibition rate of tectorigenin and its derivatives （100 mg/kg） on H22 hepatoma-bearing mice in vivo was studied. RESULTS： Totally of 6 kinds of tectorigenin mannich base derivatives were synthesized， such as 8-（N-hydroxyethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-methyl）-methyleneamino-5，7，4′-trihydroxy-6- methoxyisoflavone， 8-（N， N-diethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N， N-dimethyl）-methyleneamino- 5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-ethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-propyl）- methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone （compounds 1-6 in turn）. Compared with tectorigenin， the water solubility of six derivatives was significantly improved， and the solubility was 5-20 times higher than that of tectorigenin. IC50 of compounds 1， 3 and 5 to HCT116 cells were （34.82±3.27）， （16.21±4.13）， （33.12±3.25） μmol/L， which were stronger than that of tectorigenin [（45.23±5.74） μmol/L]； IC50 of compounds 1， 3 and 5 to A549 cells were （37.05±5.74）， （26.88±4.52）， （30.13±6.23） μmol/L， which were stronger than that of tectorigenin [（53.24±6.34） μmol/L]； IC50 of compounds 1， 3 and 5 to HepG2 cells were （23.74±1.45）， （18.96±2.34）， （30.95±2.87） μmol/L， which were stronger than that of tectorigenin [（48.98±2.58） μmol/L]. Compounds 1， 3 and 5 showed higher inhibition rates （55.51％， 57.20％ and 49.15％） than tectorigenin （33.05％） on H22 hepatoma-bearing mice， respectively. The other three compounds had no obvious advantage over tectorigenin in anti-tumor activity. CONCLUSIONS： In this study， compounds 1， 3 and 5 of six tectorigenin mannich base derivatives synthesized in this study have stronger antitumor activity than tectorigenin.

Pharmacokinetics of tectorigenin in rabbit / 中成药

AIM To develop a HPLC method for determining tectorigenin content in rabbit plasma for pharmacokinetics.METHODS As self crossover control,the rabbits,six Japanese giant ear rabbits were subject to a singledose intragastric administration of 60 mg/kg (tectorigenin 40 mg/kg) pueraria isoflavone.The free and enzymatic tectorigenin contents in plasma were measured by HPLC,and then pharmacokinetic parameters were calculated by PKsolver 2.0 pharmacokinetic program.RESULTS The AUC0-t,Tmax,Cmax after intragastric administration of pueraria isoflavone were (46.78 ±5.12) μg · min/mL,(5.39 ±0.54) min,(0.84 ±0.21) μg/mL for free tectorigenin,respectively;(485.48 ±23.53) μg · min/mL,(20.12 ±2.84) min,(2.95 ±0.67) μg/mL for total tectorigenin,respectively.CONCLUSION HPLC method is suitable for tectorigenin pharmacokinetic study.Tectorigenin is present mainly as glucuronide conjugates in plasma after intragastric administration pueraria isoflavone.

Decoction of Isoflavone Content and Its Acid Hydrolysis Conversion Rate in the Flower of Pueraria lobata / 中国药房

OBJECTIVE:To establish a method for the decoction of isoflavone content and its acid hydrolysis conversion rate in the flower of Pueraria lobata. METHODS:Using the flowers of Pueraria lobata as raw material,the isoflavone with main com-ponent of tectoridin in the flower of P. lobata was prepared with ethanol,ethyl acetate extracted,ethanol recrystallized and puri-fied,and it was converted to tectorigenin with hydrolysis in hydrochloric acid. By screening the solvent and wavelength,UV spec-trophotometry was established to determine tectovidin and tectorigenin,and calculate the isoflavone content and acid hydrolysis con-version rate of tectoridin(expressed by the relative percentage of tectorigenin). It was compared with HPLC detection results,the accuracy of UV method was evaluated. RESULTS:The solvent was 70％ethanol solution containing 1％triethylamine,and the iso-flavone content was detected at wavelength of 339,274 nm. The linear range of tectoridin was 8.80-29.33 nmol/mL(r=0.9999). RSDs of precision(n=6),stability(n=5)and reproducibility(n=6)tests were lower than 1.94％;average recovery was 99.7％(RSD=1.77％,n=9). There were no statistical significances in the contents of total flavonoids (UV:17.64-25.55 nmol/mL vs. HPLC:17.39-24.40 nmol/mL) and the relative percentage of tectorigenin (UV:57.65％-87.59％ vs. HPLC:55.62％-91.14％). CONCLUSIONS:The established method is accurate,reliable,and can be used for the rapid determination of acid hydrolysis con-version rate of tectoridin.

Eeffect of tectorigenin on myocardial fibrosis in rats and its mechanism / 吉林大学学报(医学版)

Objective:To explore the effect of tectorigenin on myocardial fibrosis(MF) in the rats and clarify the related mechanism,and to provide reference for its clinical application. Methods:Sixty Wistar rats were randomly divided into normal control group,model group,positive drug (capropril) control group, and low,middle,high doses of tectorigenin groups(n=10).Except normal control group, the rats in other groups were used to construct MF models by subcutaneous injection of 5 mg·kg -1·d -1 isoproterenol (Iso) for 7 d.The rats in tectorigenin groups and captopril group were intragastricly administrated with different doses of tectorigenin (25,50,100 mg·kg-1·d-1)and captopril(10 mg·kg -1·d -1) from the second day after modeling for consecutive 28 d.Bl-420E+ biological function experiment system was used to detect the heart function;Heart mass index (HMI) and left ventricular mass index (LVMI) were measured after experiment.UV detection was used to measure the levels of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) in myocardial tissue.Microplate reader was used to measure the activities of lactic dehydrogenase(LDH) and creatine kinase(CK) and the levels of nitric oxide (NO)in serum.ELISA were used to detect the levels of collagen typeⅠ(ColⅠ) and collagen type Ⅲ (Col Ⅲ) in myocardium tissue of the rats.The pathological changes of myocardium tissue of the rats in various groups were observed by HE staining.Results:Compared with normal control group,the HR of rats in model groups was increased,and the left ventricular systolic pressure (LVSP) was decreased(P<0.01);the HMI and LVMI were increased(P<0.05),the levels of MDA in left ventricular myocardial tissue was increased(P<0.01),and the activity of SOD was decreased,the levels of serum ColⅠ,Col Ⅲ and the activities of LDH , CK were also increased(P<0.01);the level of NO in serum was decreased(P<0.01).Compared with model groups, the HR were decreased,LVSP were increased, and HMI and LVMI of the rats in different doses of tectorigenin groups were decreased in a dose-dependent manner;the levels of MDA were reduced;the activities of SOD were increased in myocardium tissue,and the CK activities and the ColⅠ and ColⅢ levels were decreased(P<0.05 or P<0.01);the LDH activities in middle and high doses of tectorgenin groups were decreased(P<0.01);and the levels of NO in serum in different doses of tectorigenin groups were significantly increased(P<0.05 or P<0.01) .Conclusion:Tectorigenin could inhibit the MF induced by Iso in the rats, and its mechanism may be related to antioxidation,scavenging free radical and inhibition of collagen synthesis.

Study on UPLC Fingerprint of Gehua Formula Granules / 中国药房

OBJECTIVE:To establish UPLC fingerprint of Gehua formula granules. METHODS:UPLC method were adopted. The determination was performed on Zorbax Eclipse XDB-C18 column with mobile phase consisted of acetonitrile-water at the flow rate of 0.5 mL/min. The detection wavelength was set at 264 nm,column temperature was 25 ℃,and the sample size was 1 μL. Using tectorigenin as reference substance,UPLC chromatograms of 10 batches of Gehua formula granules were determined. The common peak identification and similarity evaluation were conducted by TCM Chromatogram Fingerprint Similarity Evaluation Sys-tem(2004 A edition). RESULTS:14 common peaks were identified in UPLC chromatograms of 10 batches of Gehua formula gran-ules and similarities were all higher than 0.90. UPLC chromatograms of 10 batches of samples were in good agreement with control fingerprint. CONCLUSIONS:Established UPLC fingerprint can provide reference for identification and quality evaluation of Gehua formula granules.

Correlation of blood concentration and drug dose and reducing lipid efficacy of tectorigenin in rabbits / 中国药学杂志

OBJECTIVE: To investigate the correlation of blood concentration and drug dose and reducing lipid efficacy of tectorigenin in atherosclerosis rabbits. METHODS: Forty-eight Japanese white rabbits were randomly divided into six groups, the normal control group, model group, high-, middle-and low-dose (50, 25, 12.5 mg·kg-1·d-1) tectorigenin groups and atorvastatin (5 mg·kg-1· d-1) group. The normal control group was fed with routine forage, and the other groups were fed with high fat forage for the establishment of atherosclerosis model. After 8 weeks, tectorigenin blood concentration was detected by HPLC. After 12 weeks, blood lipids were detected with automatic biochemical analyzer. We sheared the thoracic and abdominal aorta to observe the formation of atherosclerotic plaque and make pathologic examination. RESULTS: Steady-state blood concentration increased with the increase of drug dose, there was a positive relationship (r = 0.969 1) between them, but not the same proportion. After treatment, serum total cholesterol (TC), triglyceride(TG), low density lipoprotein cholesterol (LDL-C) levels were decreased and high density lipoprotein cholesterol (HDL-C) were increased in the tectorigenin each dose group, but the effect was weaker than atorvastatin group. CONCLUSION: Tectorigenin has obvious effect on reducing blood lipid and anti-atherosclerosis in rabbits, and it is related to the blood concentration and the drug dose.

In vivo pharmacokinetics of tectorigenin floating sustained-release tablets in rabbits and evaluation of in vitro-in vivo correlation / 中草药

Objective: To evaluate therelease characteristicsin vitro, pharmacokinetics in rabbits and in vivo-in vitro correlation of tectorigenin floating sustained-release tablets (TFSRT). Methods: The release characteristics of TFSRTin vitro was detected with HPLC in the artificial gastric fluid. Six Japanese Giant Ear Rabbits as self crossover control, which were given TFSRT and suspension liquid (200mg). The concentration of tectorigenin in plasma was determined with HPLC and the data were processed with PKsolver 2.0 software. Results:The cumulative releaserate of TFSRTin vitro was over 70% in 10 h.The pharmacokineticsin rabbits showed that TFSRT and tectorigenin suspension liquid conformed to the single compartment model and the pharmacokinetic parameters were obtained: tmax: (2.809±0.371) and (0.442±0.138)h, Cmax: (6.317±1.337) and (9.662±2.759) μg/mL, AUC0-t: (74.156±10.420) and (57.059±13.309) μg∙h/mL. The relative bioavailability of TFSRT was (134.63±27.94)%, so there was significant difference between them. Conclusion: TFSRT can release slowly, so it increase the relative bioavailability significantly. The correlation between the absorption in vivo and release in vitro is fine (r=0.9879), so the release rate in vitro can control the quality of TFSRT.

Preparation and release mechanism of tectorigenin intragastric floating sustained-release tablets / 中国中药杂志

To investigate the preparation technology and release mechanism of tectorigenin intragastric floating sustained-release tablets. The tablet was produced by wet granulation compression method, with hydroxypropyl methyl cellulose (HPMCK15M), cross-linked polyvinyl pyrrolidone (PVPP), octadecanol and sodium bicarbonate as excipient. The prescriptions were screened and optimized by orthogonal experimental design with in vitro floating capacity and drug release characteristics as the evaluation indexes. The optimization results were as follows: tectorigenin 33.3%, HPMCK15M 16.7%, PVPP 20.0%, octadecanol 13.3%, sodium bicarbonate 5%, and starch gel 10.7%. The prepared tablet can be floated within 10 s in the artificial gastric juice, lasting for 12 h in vitro, with a cumulative release rate of 70% in 10 h. The analysis of Rritger-Peppas equation showed that the sustained-release tablet had two advantages of both drug diffusion and skeleton dissolution. The tablet had good appearance and compressibility, as well as favorable floating capacity and drug release characteristics.

Pharmacokinetics of tectorigenin in rabbit / 中成药

AIM To develop a HPLC method for determining tectorigenin content in rabbit plasma for pharmacokinetics.METHODS As self crossover control,the rabbits,six Japanese giant ear rabbits were subject to a singledose intragastric administration of 60 mg/kg (tectorigenin 40 mg/kg) pueraria isoflavone.The free and enzymatic tectorigenin contents in plasma were measured by HPLC,and then pharmacokinetic parameters were calculated by PKsolver 2.0 pharmacokinetic program.RESULTS The AUC0-t,Tmax,Cmax after intragastric administration of pueraria isoflavone were (46.78 ±5.12) μg · min/mL,(5.39 ±0.54) min,(0.84 ±0.21) μg/mL for free tectorigenin,respectively;(485.48 ±23.53) μg · min/mL,(20.12 ±2.84) min,(2.95 ±0.67) μg/mL for total tectorigenin,respectively.CONCLUSION HPLC method is suitable for tectorigenin pharmacokinetic study.Tectorigenin is present mainly as glucuronide conjugates in plasma after intragastric administration pueraria isoflavone.

Preparetion of tectorigenin self micro-emulsifying drug delivery system and evaluation on its in vitro dissolution / 中草药

Objective: To prepare self micro-emulsifying drug delivery system (SMEDDS) of tectorigenin (TG), and investigate its dissolution. Methods: The formulation was optimized using Design Expert based on D-optimal design. The microemulsion's physicochemical and in vitro dissolution were evaluated after self-microemulsification. Results: The particle size and Zeta potential of the final formulation were (14.95 ± 0.31) nm and (-12.53 ± 0.80) mV after it was diluted by 10 times with pure water. The microemulsion appeared to be spheres with homogeneous size, which can be observed through a transmission electron microscope. The drug loading capacity was 20 mg/g, and the average content was (99.03 ± 0.70)%. The results of in vitro dissolution study showed that the accumulative dissolution could be close to 100% after 10 min in both hydrochloric acid solution (pH 1.2) and PBS (pH 6.8). Conclusion: D-optimal design could be used to optimize the formulations of TG-SMEDDS successfully. The TG-SMEDDS exhibits a larger accumulation dissolution than TG. This formulation would be easier absorbed through gastrointestinal tract compared to TG. The results of this study are expected to offer data support and reference for the TG's formulation design and clinical application.

Protective Effect of an Isoflavone, Tectorigenin, Against Oxidative Stress-induced Cell Death via Catalase Activation

BACKGROUND: Isoflavones are biologically active compounds that occur naturally in a variety of plants, with relatively high levels in soybean. Tectorigenin, an isoflavone, protects against hydrogen peroxide (H2O2)-induced cell damage. However, the underlying mechanism is unknown. METHODS: The MTT assay was performed to determine cell viability. Catalase activity was assessed by determining the amount of enzyme required to degrade 1 μM H2O2. Protein expression of catalase, phospho-extracellular signal-regulated kinase (ERK), IκB-α, and NF-κB were evaluated by Western blot analysis. A mobility shift assay was performed to assess the DNA-binding ability of NF-κB. Transient transfection and a NF-κB luciferase assay were performed to assess transcriptional activity. RESULTS: Tectorigenin reduced H2O2-induced death of Chinese hamster lung fibroblasts (V79-4). In addition, tectorigenin increased the activity and protein expression of catalase. Blockade of catalase activity attenuated the protective effect of tectorigenin against oxidative stress. Furthermore, tectorigenin enhanced phosphorylation of ERK and nuclear expression of NF-κB, while inhibition of ERK and NF-κB attenuated the protective effect of tectorigenin against oxidative stress. CONCLUSIONS: Tectorigenin protects cells against oxidative damage by activating catalase and modulating the ERK and NF-κB signaling pathway.

Optimized extraction technology of flos Puerariae lobata isoflavone using orthog-onal test / 药学实践杂志

Objective To optimize the extraction technology of flos Puerariae lobata isoflavone .Methods The flos Pu-erariae lobata isoflavone was distilled by ethanol circumfluence .Total flavonoids ,tectoridin and tectorigenin extracted from Puerariae using the UV and HPLC spectromertry methods were taken as evaluation indexes .Extraction technology was opti-mized with L9 (34 ) orthogonal test on the base of single observation of ethanol concentration ,solvent dosage and distilling time . Results The best extraction technology of flos Puerariae lobata isoflavone was :to add 12 times the amount of 70% ethanol for 90 minutes for the first time ,and 10 times the amount of 70% ethanol for 60 minutes for the second time .Conclusion The op-timized extraction process of flos Puerariae lobata isoflavone is reasonable and feasible ,and it can offer reference to actual pro-duction .

Anti-Helicobacter pylori Compounds from Maackia amurensis

Eight isoflavonoid compounds were isolated from the EtOAc fraction of Maackia amurensis which had shown the highest anti-Helicobacter pylori activity among the fractions, using medium pressure liquid chromatography and recrystallization. Based on the spectroscopic data including 1H-NMR, 13C-NMR, HMBC and MS data, the chemical structures of the isolates were determined to be (-)-medicarpin (1), afromosin (2), formononetin (3), tectorigenin (4), prunetin (5), wistin (6), tectoridin (7) and ononin (8). Anti-H. pylori activity of each compound was evaluated with broth dilution assay. As a result, (-)-medicarpin (1), tectorigenin (4) and wistin (6) showed anti-H. pylori activity. (-)-Medicarpin (1) exhibited the most potent growth inhibitory activity against H. pylori with the minimal inhibitory concentration (MIC)90 of 25 microM, and tectorigenin (4) with MIC90 of 100 microM ranked the second. This is the first study to show the anti-H. pylori activity of M. amurensis, and it is suggested that the stem bark of M. amurensis or the EtOAc fraction or the isolated compounds can be a new natural source for the treatment of H. pylori infection.

Tectorigenin inhibits the inflammation of LPS-induced acute lung injury in mice / 中国天然药物

AIM@#In a previous study, the anti-inflammatory effects of tectorigenin were disclosed. In this study, the anti-inflammatory effects of tectorigenin on acute lung injury using a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model were investigated@*METHOD@#The cell-count in the bronchoalveolar lavage fluid (BALF) was measured. The animal lung edema degree was evaluated by the wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) activity and myeloperoxidase (MPO) activity was assayed using SOD and MPO kits, respectively. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 were assayed using an enzyme-linked immunosorbent assay method. Pathological changes of lung tissues were observed through HE staining. The inflammatory signal pathway related protein nuclear factor NF-κB p65 mRNA expression was measured by real-time PCR, and the protein level of NF-κB p65 was measured using Western blotting analysis.@*RESULTS@#The data showed that treatment with the tectorigenin markedly attenuated the inflammatory cell numbers in the BALF, decreased nuclear factor NF-κB p65 mRNA level and protein level in the lungs, and improved SOD activity and inhibited MPO activity. Histological studies showed that tectorigenin substantially inhibited LPS-induced neutrophils in lung tissue compared with the model group.@*CONCLUSION@#The results indicated that tectorigenin had a protective effect on LPS-induced ALI in mice.

Quick Detection of Four Isoflavone Compounds from Extracts of Pueraria Lobata Flowers by HPLC / 中国药师

Objective:To establish an HPLC method for the determination of four kinds of isoflavone compounds including daid-zin, tectoridin, daidzein and tectorigenin in the extracts of Pueraria lobata flowers. Methods:The separation was performed on an Agi-lent C18 column(250 mm × 4. 6 mm, 5 μm) using a mobile phase of methanol-acetonitrile-water(2∶1∶2). The flow rate was 1. 0 ml· min-1 ,and the detection wavelength was 264nm. The column temperature was 25℃ and the sample size was 20 μl. Results:The de-tection could be accomplished within 10 minutes with good separation and specificity of four isoflavone compounds with the retention timeof3.4,3.8,5.7and7.2min,respectively. Thelinearrangewas0.784-78.440 μg·ml-1(daidzin),2.000-200.000 μg· ml-1(tectoridin) and 0.800-80.020 μg·ml-1 (daidzein and tectorigenin),and the relative coefficient was 0.999 9, 0.999 8, 0. 999 7 and 0. 999 9, respectively. The average recovery was 100. 54%(RSD=1. 66%,n=6),100. 03%(RSD=1. 00%, n=6), 99. 48%(RSD=1. 76%, n=6) and 100. 92%(RSD=2. 26%, n=6), respectively. Conclusion: The method is simple, rapid and accurate with good repeatability, which can be used in the rapid determination of isoflavone compounds in the flowers of Pueraria loba-ta.