Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast u...Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.展开更多
In this study, a novel and simple high performance liquid chromatography with diode array detection （HPLC-DAD） method for the simultaneous qualitative and quantitative determination of 12 bioactive components in Geg...In this study, a novel and simple high performance liquid chromatography with diode array detection （HPLC-DAD） method for the simultaneous qualitative and quantitative determination of 12 bioactive components in Gegen Qinlian pill was developed. The separation was performed on a Kromasil C18 column （250 mm×4.6 ram, 5.0 μm） by gradient elution with acetonitrile and 0.02 mol/L ammonium acetate （containing 0.3% triethylamine, and adjusted pH to 4.3 using 1% glacial acetic acid） as the mobile phase at a flow rate of 0.7 mL/min. Three different detection wavelengths （250, 280, 346 nm） were set at the maximum UV absorption wavelengths of these components. Twelve components （puerarin, daidzin, baicalin, wogonoside, liquiritin, berberine, palmatine, jatrorrhizine, glycyrrhizin, baicalein, wogonin and daidzein） were identified and determined using the developed method. All calibration curves showed good linear regression （r〉0.9995） within tested ranges. The injection precision, intra-day precisions and analysis repeatability were evaluated with the RSD values, which were no more than 0.97%, 1.69% and 1.71%, respectively. The recoveries were ranged from 97.4% to 100.2% with RSD values less than 1.87%. This readily available, low-cost and reliable HPLC-DAD method would improve the quality control of Gegen Qinlian pill.展开更多
OBJECTIVE:To investigate serum bone biomarkers in rats with intrauterine growth restriction(IUGR)in order to determine the effects of puerarin on bone metabolism.METHODS:A rat model of IUGR was induced using a low pro...OBJECTIVE:To investigate serum bone biomarkers in rats with intrauterine growth restriction(IUGR)in order to determine the effects of puerarin on bone metabolism.METHODS:A rat model of IUGR was induced using a low protein diet during pregnancy.The offspring were given puerarin or an identical volume of saline via subcutaneous abdominal injection.All rats were studied at 1,3,and 8 weeks of age.Serum biomarkers of bone formation,including insulin-like growth factor-1(IGF-1),bone-specific alkaline phosphatase(BALP),osteocalcin(OC),osteoprotegerin(OPG),receptor-activator of nuclear factor-κB ligand(RANKL),as well as blood levels of calcium and phosphorus were measured.RESULTS:Serum BALP,OPG,IGF-1,and OC levels,as well as the OPG/RANKL ratio,were lower in the IUGR group compared with the control group at 1week of age(P=0.024,0.011,0.014,0.004,and0.002,respectively).At 3 weeks of age,the serum BALP and OC levels were higher in the protein-re-stricted group compared with the control group(P=0.003 and 0.001,respectively).A comparison between the IUGR plus puerarin intervention group and the IUGR group revealed differences in the levels of BALP and IGF-1 at 3 weeks of age(P=0.008 and 0.003,respectively).In addition,serum OPG and OC levels and the OPG/RANKL ratio were higher at 8 weeks of age(P=0.044,0.007,and0.016,respectively).No differences in serum calcium and phosphorus levels were observed among the three groups.CONCLUSION:Our study demonstrates that the bone microenvironment of the fetus can be altered by a low protein maternal diet and that puerarin can reverse these effects.These results indicate that the nutritional environment plays an important role in early skeletal development and that the bone turnover of IUGR rats can be altered by puerarin treatment.展开更多
We developed an HPLC method to study the pharmacokinetics ofpuerarin in Beagle dogs after oral administration of puerarin or puerarin mixed micelles （PMMS）. Beagle dogs were divided into two groups randomly, and the...We developed an HPLC method to study the pharmacokinetics ofpuerarin in Beagle dogs after oral administration of puerarin or puerarin mixed micelles （PMMS）. Beagle dogs were divided into two groups randomly, and the blood samples were collected at fixed time intervals after single oral administration ofpuerarin or PMMS at a dosage of 120 mg/kg. Following sample preparation, analytes were separated on a C18 column （DIKMA, 150 mm×4.6 mm, 5 μm） with a guard column （DIKMA, 8 mm×4 mm） and eluted with methanol-water （25：75, v/v）. Theophylline was used as the internal standard. WinNonlin 6.1 （Pharsight, USA） was used to calculate the pharrnacokinetic parameters. The pharmacokinetic parameters for puerarin and PMMS in Beagle dogs were as follows： AUC, 515.96 and 2796.43 min μg/mL; Tmax, 61.48 and 202.91 min; CL/F, 232.58 and 42.91 mL/min/kg, respectively. The relative bioavailability of PMMS to puerarin was 542.0%. Our results showed that the mixed micelle preparation significantly improved the bioavailability of puerarin by delaying absorption and reducing clearance.展开更多
文摘Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.
基金National Natural Science Foundation of China (Grant No.30572376)
文摘In this study, a novel and simple high performance liquid chromatography with diode array detection （HPLC-DAD） method for the simultaneous qualitative and quantitative determination of 12 bioactive components in Gegen Qinlian pill was developed. The separation was performed on a Kromasil C18 column （250 mm×4.6 ram, 5.0 μm） by gradient elution with acetonitrile and 0.02 mol/L ammonium acetate （containing 0.3% triethylamine, and adjusted pH to 4.3 using 1% glacial acetic acid） as the mobile phase at a flow rate of 0.7 mL/min. Three different detection wavelengths （250, 280, 346 nm） were set at the maximum UV absorption wavelengths of these components. Twelve components （puerarin, daidzin, baicalin, wogonoside, liquiritin, berberine, palmatine, jatrorrhizine, glycyrrhizin, baicalein, wogonin and daidzein） were identified and determined using the developed method. All calibration curves showed good linear regression （r〉0.9995） within tested ranges. The injection precision, intra-day precisions and analysis repeatability were evaluated with the RSD values, which were no more than 0.97%, 1.69% and 1.71%, respectively. The recoveries were ranged from 97.4% to 100.2% with RSD values less than 1.87%. This readily available, low-cost and reliable HPLC-DAD method would improve the quality control of Gegen Qinlian pill.
基金Supported by the Administration of Traditional Chinese Medicine of Hunan Province(Effect of Puerarin on Low Birth Weight Associated with Adulated Osteoporosis,No.201110)
文摘OBJECTIVE:To investigate serum bone biomarkers in rats with intrauterine growth restriction(IUGR)in order to determine the effects of puerarin on bone metabolism.METHODS:A rat model of IUGR was induced using a low protein diet during pregnancy.The offspring were given puerarin or an identical volume of saline via subcutaneous abdominal injection.All rats were studied at 1,3,and 8 weeks of age.Serum biomarkers of bone formation,including insulin-like growth factor-1(IGF-1),bone-specific alkaline phosphatase(BALP),osteocalcin(OC),osteoprotegerin(OPG),receptor-activator of nuclear factor-κB ligand(RANKL),as well as blood levels of calcium and phosphorus were measured.RESULTS:Serum BALP,OPG,IGF-1,and OC levels,as well as the OPG/RANKL ratio,were lower in the IUGR group compared with the control group at 1week of age(P=0.024,0.011,0.014,0.004,and0.002,respectively).At 3 weeks of age,the serum BALP and OC levels were higher in the protein-re-stricted group compared with the control group(P=0.003 and 0.001,respectively).A comparison between the IUGR plus puerarin intervention group and the IUGR group revealed differences in the levels of BALP and IGF-1 at 3 weeks of age(P=0.008 and 0.003,respectively).In addition,serum OPG and OC levels and the OPG/RANKL ratio were higher at 8 weeks of age(P=0.044,0.007,and0.016,respectively).No differences in serum calcium and phosphorus levels were observed among the three groups.CONCLUSION:Our study demonstrates that the bone microenvironment of the fetus can be altered by a low protein maternal diet and that puerarin can reverse these effects.These results indicate that the nutritional environment plays an important role in early skeletal development and that the bone turnover of IUGR rats can be altered by puerarin treatment.
基金Foundation of Independent Subject of Beijing University of Chinese Medicine (Grant No. JYB22-JS025)Compound Chinese Medicine Preparation Research Innovation Team (Team No. 2011-CXTD-13)
文摘We developed an HPLC method to study the pharmacokinetics ofpuerarin in Beagle dogs after oral administration of puerarin or puerarin mixed micelles （PMMS）. Beagle dogs were divided into two groups randomly, and the blood samples were collected at fixed time intervals after single oral administration ofpuerarin or PMMS at a dosage of 120 mg/kg. Following sample preparation, analytes were separated on a C18 column （DIKMA, 150 mm×4.6 mm, 5 μm） with a guard column （DIKMA, 8 mm×4 mm） and eluted with methanol-water （25：75, v/v）. Theophylline was used as the internal standard. WinNonlin 6.1 （Pharsight, USA） was used to calculate the pharrnacokinetic parameters. The pharmacokinetic parameters for puerarin and PMMS in Beagle dogs were as follows： AUC, 515.96 and 2796.43 min μg/mL; Tmax, 61.48 and 202.91 min; CL/F, 232.58 and 42.91 mL/min/kg, respectively. The relative bioavailability of PMMS to puerarin was 542.0%. Our results showed that the mixed micelle preparation significantly improved the bioavailability of puerarin by delaying absorption and reducing clearance.
