2009 Vol. 40 No. 3
2009, 40(3): 193-199.
Abstract：
Single nucleotide polymorphism(SNP) is a major source of genetic differences，and the number of SNP is extremely large.Consequently，a high-throughput，rapid and cost-effective technique is essential for SNP typing.Recently，a set of solutions aimed directly at the bottlenecks in SNP detection were put forward，which including direct PCR from human whole blood，adapter-ligation mediated allele-specific amplification，gene polymorphism detection in one tube，microplate array parallel gel electrophoresis and microchip electrophoresis.These methods have been successfully applied in pharmacogenomics，disease-related research，herbal medicine identification and quality control.The results showed that the advantages of the genotyping platforms are highly specific，sensitive，easy to operate and inexpensive with a good prospect of application.The principles and applications of the genotyping platforms in detail were described in this paper.
Single nucleotide polymorphism(SNP) is a major source of genetic differences，and the number of SNP is extremely large.Consequently，a high-throughput，rapid and cost-effective technique is essential for SNP typing.Recently，a set of solutions aimed directly at the bottlenecks in SNP detection were put forward，which including direct PCR from human whole blood，adapter-ligation mediated allele-specific amplification，gene polymorphism detection in one tube，microplate array parallel gel electrophoresis and microchip electrophoresis.These methods have been successfully applied in pharmacogenomics，disease-related research，herbal medicine identification and quality control.The results showed that the advantages of the genotyping platforms are highly specific，sensitive，easy to operate and inexpensive with a good prospect of application.The principles and applications of the genotyping platforms in detail were described in this paper.
2009, 40(3): 200-204.
Abstract：
Aim ：To search for novel α1-adrenoceptor(α1-AR) antagonists. Methods ：On the basis of hybridization principle with silodosin as the lead compound，twelve 5-［2-［4-［(substituted phenoxy)alkyl］piperazin-1-yl］propyl］indoline compounds were designed and synthesized by maintaining indoline while incorporating the 1-［(substituted phenoxy)alkyl］piperazine group. Results ：The structures of synthesized target compounds were confirmed by the elemental analysis，IR，ESI-MS and 1H NMR.Preliminary pharmacological test showed that pA2 values of six target compounds were greater than 7.50，which suggested that the compounds possessed considerable α1-AR antagonic activity. Conclusion ：5-［2-［4-［(substituted phenoxy)alkyl］piperazin-1-yl］propyl］indoline compounds is potentially a new candidate for α1-AR antagonist.
Aim ：To search for novel α1-adrenoceptor(α1-AR) antagonists. Methods ：On the basis of hybridization principle with silodosin as the lead compound，twelve 5-［2-［4-［(substituted phenoxy)alkyl］piperazin-1-yl］propyl］indoline compounds were designed and synthesized by maintaining indoline while incorporating the 1-［(substituted phenoxy)alkyl］piperazine group. Results ：The structures of synthesized target compounds were confirmed by the elemental analysis，IR，ESI-MS and 1H NMR.Preliminary pharmacological test showed that pA2 values of six target compounds were greater than 7.50，which suggested that the compounds possessed considerable α1-AR antagonic activity. Conclusion ：5-［2-［4-［(substituted phenoxy)alkyl］piperazin-1-yl］propyl］indoline compounds is potentially a new candidate for α1-AR antagonist.
2009, 40(3): 205-208.
Abstract：
Aim ：To improve the synthesis for 2，5-anhydro-3，4，6-tri-O-benzyl-D-glucitol. Methods ：Dehydration of D-mannitol，followed by a reaction sequence of selective protection，benzylation，deprotection，tritylation，benzylation and removal of the trityl group，afforded the title compound. Results ：Based on the improvement of the reported synthetic route，2，5-anhydro-3，4，6-tri-O-benzyl-D-glucitol was synthesized in seven steps from D-mannitol with a total yield of 8.3%. Conclusion ：This new method features mild reaction conditions and facile work-up.
Aim ：To improve the synthesis for 2，5-anhydro-3，4，6-tri-O-benzyl-D-glucitol. Methods ：Dehydration of D-mannitol，followed by a reaction sequence of selective protection，benzylation，deprotection，tritylation，benzylation and removal of the trityl group，afforded the title compound. Results ：Based on the improvement of the reported synthetic route，2，5-anhydro-3，4，6-tri-O-benzyl-D-glucitol was synthesized in seven steps from D-mannitol with a total yield of 8.3%. Conclusion ：This new method features mild reaction conditions and facile work-up.
2009, 40(3): 209-212.
Abstract：
To study the antitumor activities of the constituents of Cephalotaxus fortunei distributed in Guizhou province. Methods ：The constituents were isolated by column chromatography and identified by physical and spectral analysis.Meanwhile，the anti-tumor activities of some compounds were evaluated by sulforhodamine B(SRB) and MTT assay. Results ：Eleven compounds were isolated and identified as apigenin (I)，β-sitosterol (II)，acetylcephalotaxine (III)，chrysoeriol (IV)，drupacine (V)，1-hentriacontanol (VI)，7，3′，4′-trihydroxyflavone (VII)，sugiol (VIII)，cephalotaxine (IX)，wllsonine (X)，and hainanolide (XI)，respectively.Biological screening Results demonstrated that some of the tested compounds exhibited the antitumor activities in vitro. Conclusion ：Compounds II，VI-VIII were isolated from this plant for the first time.Compound XI has a better inhibitory activity on cell line A549 and K562 .
To study the antitumor activities of the constituents of Cephalotaxus fortunei distributed in Guizhou province. Methods ：The constituents were isolated by column chromatography and identified by physical and spectral analysis.Meanwhile，the anti-tumor activities of some compounds were evaluated by sulforhodamine B(SRB) and MTT assay. Results ：Eleven compounds were isolated and identified as apigenin (I)，β-sitosterol (II)，acetylcephalotaxine (III)，chrysoeriol (IV)，drupacine (V)，1-hentriacontanol (VI)，7，3′，4′-trihydroxyflavone (VII)，sugiol (VIII)，cephalotaxine (IX)，wllsonine (X)，and hainanolide (XI)，respectively.Biological screening Results demonstrated that some of the tested compounds exhibited the antitumor activities in vitro. Conclusion ：Compounds II，VI-VIII were isolated from this plant for the first time.Compound XI has a better inhibitory activity on cell line A549 and K562 .
2009, 40(3): 213-217.
Abstract：
Aim ：To prepare ropivacaine hydrochloride multivesicular liposomes，and to study the physicochemical properties and drug release behavior in vitro. Methods ：Ropivacaine hydrochloride multivesicular liposomes were prepared by the multiple emulsion method.Single factor experiments were utilized to study the factors which affect the encapsulation efficiency of multivesicular liposomes.The formulation and pharmaceutical process were optimized by Box-Behnken experimental design，with the factors of encapsulation efficiency as the criteria.Three batches of the optimized multivesicular liposomes were prepared， and the encapsulation efficiency and in vitro release behavior were studied. Results ：The particle size of the optimized multivesicular liposomes was uniform and 85% of them were well distributed in the range of 7-30 μm.The encapsulation efficiency was up to 90% when the ratios of lipid to drug，phospholipids to cholesterol and the amount of triolein was 1.328∶1(w/w)，1.5∶1(w/w) and 6 mmol/L，respectively.The release profile in vitro fitted to a first-order kinetics with the period of release up to 48 h in PBS buffer under 37 °C. Conclusion ：Ropivacaine hydrochloride multivesicular liposomes showed high encapsulation efficiency and significant sustained-release feature.
Aim ：To prepare ropivacaine hydrochloride multivesicular liposomes，and to study the physicochemical properties and drug release behavior in vitro. Methods ：Ropivacaine hydrochloride multivesicular liposomes were prepared by the multiple emulsion method.Single factor experiments were utilized to study the factors which affect the encapsulation efficiency of multivesicular liposomes.The formulation and pharmaceutical process were optimized by Box-Behnken experimental design，with the factors of encapsulation efficiency as the criteria.Three batches of the optimized multivesicular liposomes were prepared， and the encapsulation efficiency and in vitro release behavior were studied. Results ：The particle size of the optimized multivesicular liposomes was uniform and 85% of them were well distributed in the range of 7-30 μm.The encapsulation efficiency was up to 90% when the ratios of lipid to drug，phospholipids to cholesterol and the amount of triolein was 1.328∶1(w/w)，1.5∶1(w/w) and 6 mmol/L，respectively.The release profile in vitro fitted to a first-order kinetics with the period of release up to 48 h in PBS buffer under 37 °C. Conclusion ：Ropivacaine hydrochloride multivesicular liposomes showed high encapsulation efficiency and significant sustained-release feature.
2009, 40(3): 218-221.
Abstract：
Aim ：To prepare the influenza vaccine lyophilized liposomes and to characterize its particle distribution，encapsulation efficiency and immunogenicity. Methods ：Flu vaccine liposome based on the method of thin-film evaporation was prepared using phospholipids ，cholesterol and the purified influenza virus split vaccine，and was further subjected to frozen-drying.The polymorph was observed by microscope；the particle distribution and the average size were analysed by transmission electron microscope；its encapsulation efficiency was determined by Lowry method and the antibody titers were assessed by hemagglutination-inhibition after pulmonary delivery to mice. Results ：The reconstitated influenza vaccine liposome under electronic microscope were round or elliptic particles evenly distributed at a mean size of 2.14 μm，with the encapsulation efficiency of more than 80%.The antibody titer through pulmonary delivery was higher than that through intraperitoneal injection. Conclusion ：The prepared influenza vaccine lyophilized liposomes possess high encapsulation efficiency，better particle distribution and marked immunogenicity through pulmonary delivery to mice.Pulmonary delivery of influenza vaccine liposomes is a potential immunization approach worthy of further exploitation.
Aim ：To prepare the influenza vaccine lyophilized liposomes and to characterize its particle distribution，encapsulation efficiency and immunogenicity. Methods ：Flu vaccine liposome based on the method of thin-film evaporation was prepared using phospholipids ，cholesterol and the purified influenza virus split vaccine，and was further subjected to frozen-drying.The polymorph was observed by microscope；the particle distribution and the average size were analysed by transmission electron microscope；its encapsulation efficiency was determined by Lowry method and the antibody titers were assessed by hemagglutination-inhibition after pulmonary delivery to mice. Results ：The reconstitated influenza vaccine liposome under electronic microscope were round or elliptic particles evenly distributed at a mean size of 2.14 μm，with the encapsulation efficiency of more than 80%.The antibody titer through pulmonary delivery was higher than that through intraperitoneal injection. Conclusion ：The prepared influenza vaccine lyophilized liposomes possess high encapsulation efficiency，better particle distribution and marked immunogenicity through pulmonary delivery to mice.Pulmonary delivery of influenza vaccine liposomes is a potential immunization approach worthy of further exploitation.
2009, 40(3): 222-226.
Abstract：
Aim ：To establish a linear additive model for the predication of in vitro sinomenine hydrochloride release from the combination of immediate release，enteric-coated and sustained-release pellets based on the release profiles of each pellet type. Methods ：Immediate release pellets were manufactured by extrusion/spheronization technology.The operation of bottom-spraying in the fluid-bed equipment was conducted to enteric-coating using Eudragit?L-30D-55 and sustained-release coating using Surelease.In vitro sinomenine hydrochloride release profiles of both uncoated and coated pellets were fitted to the chosen mathematical equations offered by the curve fitting toolbox of Matlab? before a linear additive model was created based upon the best-to-fitting equations.The proportion of each pellet type in the combined format to generate the desired 24 h sinomenine hydrochloride release profile was solved by Matlab?.The predicted and assayed sinomenine hydrochloride release from the polled pellets was compared. Results ：It was shown that the actual sinomenine hydrochloride release profiles of each pellet type were approximate to those of predicted ones.A linear additive model of the appropriate mathematical equations of each pellet was proven to be capable of controlling in vitro release of sinomenine hydrochloride multiple-unit pellets. Conclusion ：A multiple-unit combined system of the selected pellets，as a novel sustained-release system，was successfully prepared.In vitro release performance of the calculated combination of each pellet type could be guaranteed by this approach in designing sustained-release drug delivery system.
Aim ：To establish a linear additive model for the predication of in vitro sinomenine hydrochloride release from the combination of immediate release，enteric-coated and sustained-release pellets based on the release profiles of each pellet type. Methods ：Immediate release pellets were manufactured by extrusion/spheronization technology.The operation of bottom-spraying in the fluid-bed equipment was conducted to enteric-coating using Eudragit?L-30D-55 and sustained-release coating using Surelease.In vitro sinomenine hydrochloride release profiles of both uncoated and coated pellets were fitted to the chosen mathematical equations offered by the curve fitting toolbox of Matlab? before a linear additive model was created based upon the best-to-fitting equations.The proportion of each pellet type in the combined format to generate the desired 24 h sinomenine hydrochloride release profile was solved by Matlab?.The predicted and assayed sinomenine hydrochloride release from the polled pellets was compared. Results ：It was shown that the actual sinomenine hydrochloride release profiles of each pellet type were approximate to those of predicted ones.A linear additive model of the appropriate mathematical equations of each pellet was proven to be capable of controlling in vitro release of sinomenine hydrochloride multiple-unit pellets. Conclusion ：A multiple-unit combined system of the selected pellets，as a novel sustained-release system，was successfully prepared.In vitro release performance of the calculated combination of each pellet type could be guaranteed by this approach in designing sustained-release drug delivery system.
2009, 40(3): 227-231.
Abstract：
t Aim ：To establish the HPLC and LC-MC Methods for the quality analysis of the different species of Radix Paeoniae Alba cultivated in Bozhou. Methods ：The LC-MS method was applied to analyze the chemical composition of the cultivated species，the HPLC method was applied to determine the contents of paeoniforin，benzoic acid and paeonivayin in crude drugs and processing products. Results ：10 characteristic peaks were identified by LC-MS with reference standards.The significant difference was observed in the chemical compositions of 3 cultivated species.The significant difference was observed in the chemical compositions of 3 cultivated species.The content of paeoniflorin was increased significantly after traditional processing.The Results indicated the 4-year-old “pubang” and 5-year-old “xiantiao” are the best cultivated species. Conclusion ：The Methods are simple，rapid and precise to assess and control the quality of different species of Radix Paeoniae Alba cultivated in Bozhou.
t Aim ：To establish the HPLC and LC-MC Methods for the quality analysis of the different species of Radix Paeoniae Alba cultivated in Bozhou. Methods ：The LC-MS method was applied to analyze the chemical composition of the cultivated species，the HPLC method was applied to determine the contents of paeoniforin，benzoic acid and paeonivayin in crude drugs and processing products. Results ：10 characteristic peaks were identified by LC-MS with reference standards.The significant difference was observed in the chemical compositions of 3 cultivated species.The significant difference was observed in the chemical compositions of 3 cultivated species.The content of paeoniflorin was increased significantly after traditional processing.The Results indicated the 4-year-old “pubang” and 5-year-old “xiantiao” are the best cultivated species. Conclusion ：The Methods are simple，rapid and precise to assess and control the quality of different species of Radix Paeoniae Alba cultivated in Bozhou.
2009, 40(3): 232-237.
Abstract：
Aim ：To investigate the chemical components in Zhizidahuang decoction(ZZDHD) to reveal the possible material therapeutic basis. Methods ：High performance liquid chromatography coupled with photodiode array detector and tandem mass spectrometry(HPLC-PDA-MS/MS) was applied to simultaneously characterize the chemical components and their structures in ZZDHD.The analysis was preformed on a Lichrospher C18 column using a binary eluent of 0.1% acetic acid(A) and methanol(B) mixed under the gradient mode；UV spectra were scaned from 210 nm to 480 nm；negative ESI experiments in data-dependent scan mode were performed. Results ：HPLC-PDA-MS/MS chromatogram of ZZDHD was achieved.Based on UV spectral data，information of molecular weight and mass fragmentation behaviors connected with extracted ion current(EIC) chromatogram，twenty kinds of components were detected and identified，including flavonoids，anthraquinones，iridoids and other constituents. Conclusion ：The method presented in this study，which combined HPLC with UV and MS，allowes the characterization of compounds in the complex herbal system even without the reference standards.
Aim ：To investigate the chemical components in Zhizidahuang decoction(ZZDHD) to reveal the possible material therapeutic basis. Methods ：High performance liquid chromatography coupled with photodiode array detector and tandem mass spectrometry(HPLC-PDA-MS/MS) was applied to simultaneously characterize the chemical components and their structures in ZZDHD.The analysis was preformed on a Lichrospher C18 column using a binary eluent of 0.1% acetic acid(A) and methanol(B) mixed under the gradient mode；UV spectra were scaned from 210 nm to 480 nm；negative ESI experiments in data-dependent scan mode were performed. Results ：HPLC-PDA-MS/MS chromatogram of ZZDHD was achieved.Based on UV spectral data，information of molecular weight and mass fragmentation behaviors connected with extracted ion current(EIC) chromatogram，twenty kinds of components were detected and identified，including flavonoids，anthraquinones，iridoids and other constituents. Conclusion ：The method presented in this study，which combined HPLC with UV and MS，allowes the characterization of compounds in the complex herbal system even without the reference standards.
2009, 40(3): 238-243.
Abstract：
Aim ：To evaluate the effects of bioactive extracts from Fructus Jujubae in attenuating the inflammation induced by Radix Kansui. Methods ：Exoteric splenic lymphocyte(SPL) of mouse and peritoneal macrophage(PMφ) of rat were used as cell model to evaluate the anti-inflammatory effects of fractions from Fructus Jujubae.MTT method was used to assay SPL proliferation，and Griess method was used in NO release of PMφ.Firstly， Radix Kansui fraction was applied to induce inflammatory effects of the cells，and then，the anti-inflammatory effects of five extracts of Fructus Jujubae were investigated to determine the active fractions in Fructus Jujubae，which probably contribute to the attenuation of Radix Kansui.The effects of Fructus Jujubae extract in interfering the gastrointestinal acute damages caused by the extract of Radix Kansui were observed according to the pathological sections of mice. Results ：The extract RK-3(diterpenes) exhibited the most significant pro-inflammatory effect.The bioactive extracts B(micromolecule saccharides and amino acids)，D(flavonoid glycosides)，and E(triterpenes) of Fructus Jujubae could attenuate the action of RK-3.The effective extract of Fructus Jujubae protected stomach and duodenum of mouse from acute damages caused by the extract of Radix Kansui. Conclusion ：Fraction RK-3(diterpenes) of Radix Kansui had pro-inflammatory effects，and some extracts of Fructus Jujubae could attenuate this effect of Radix Kansui.The results helped to clarify that Fructus Jujubae attenuates the toxic effect of Radix Kansui in certain cases，and the bioactive extracts of Fructus Jujubae discovered in the experiments offer the basis for further research to the active components of Fructus Jujubae.
Aim ：To evaluate the effects of bioactive extracts from Fructus Jujubae in attenuating the inflammation induced by Radix Kansui. Methods ：Exoteric splenic lymphocyte(SPL) of mouse and peritoneal macrophage(PMφ) of rat were used as cell model to evaluate the anti-inflammatory effects of fractions from Fructus Jujubae.MTT method was used to assay SPL proliferation，and Griess method was used in NO release of PMφ.Firstly， Radix Kansui fraction was applied to induce inflammatory effects of the cells，and then，the anti-inflammatory effects of five extracts of Fructus Jujubae were investigated to determine the active fractions in Fructus Jujubae，which probably contribute to the attenuation of Radix Kansui.The effects of Fructus Jujubae extract in interfering the gastrointestinal acute damages caused by the extract of Radix Kansui were observed according to the pathological sections of mice. Results ：The extract RK-3(diterpenes) exhibited the most significant pro-inflammatory effect.The bioactive extracts B(micromolecule saccharides and amino acids)，D(flavonoid glycosides)，and E(triterpenes) of Fructus Jujubae could attenuate the action of RK-3.The effective extract of Fructus Jujubae protected stomach and duodenum of mouse from acute damages caused by the extract of Radix Kansui. Conclusion ：Fraction RK-3(diterpenes) of Radix Kansui had pro-inflammatory effects，and some extracts of Fructus Jujubae could attenuate this effect of Radix Kansui.The results helped to clarify that Fructus Jujubae attenuates the toxic effect of Radix Kansui in certain cases，and the bioactive extracts of Fructus Jujubae discovered in the experiments offer the basis for further research to the active components of Fructus Jujubae.
2009, 40(3): 244-249.
Abstract：
Aim ：To study the effects of berberine(Ber) on the rapidly activating component(I Kr )，the slowly activating component(I Ks) of the delayed rectifier potassium current and the inward rectifier potassium current(I K1) in cardiomyopathic guinea pig ventricular myocytes. Methods ：After guinea pigs were ip L-thyroxine 0.5 mg/kg for 10 d，their hearts were cardiomyopathic.Then whole cell patch-clamp recording technique was used to observe the effect of 30 μmol/L Ber on the I Kr ，I Ks and I K1in cardiomyopathic guinea pig ventricular myocytes. Results ：In cardiomyopathic guinea pig ventricular myocytes，Ber 30 μmol/L markedly inhibited I Kr and I Ks by 22.8% and 29.5% at +10 mV and +80 mV，respectively.The effect of Ber on I Ks was greater than that on I Kr .Ber 30 μmol/L also inhibited the inward component of I K1 by 29.1% at+120 mV，but the reverse potential of I K1was unaffected.Ber(1- 300 μmol/L) was shown to inhibit I Kr and I Ks in a concentration-dependent manner.Their IC50 were 76.74 μmol/L and 55.37 μmol/L，respectively. Conclusion ：Ber inhibited I Kr ，I Ks and I K1in cardiomyopathic guinea pig ventricular myocytes，which may be important in understanding the antiarrhythmic effects of this drug.
Aim ：To study the effects of berberine(Ber) on the rapidly activating component(I Kr )，the slowly activating component(I Ks) of the delayed rectifier potassium current and the inward rectifier potassium current(I K1) in cardiomyopathic guinea pig ventricular myocytes. Methods ：After guinea pigs were ip L-thyroxine 0.5 mg/kg for 10 d，their hearts were cardiomyopathic.Then whole cell patch-clamp recording technique was used to observe the effect of 30 μmol/L Ber on the I Kr ，I Ks and I K1in cardiomyopathic guinea pig ventricular myocytes. Results ：In cardiomyopathic guinea pig ventricular myocytes，Ber 30 μmol/L markedly inhibited I Kr and I Ks by 22.8% and 29.5% at +10 mV and +80 mV，respectively.The effect of Ber on I Ks was greater than that on I Kr .Ber 30 μmol/L also inhibited the inward component of I K1 by 29.1% at+120 mV，but the reverse potential of I K1was unaffected.Ber(1- 300 μmol/L) was shown to inhibit I Kr and I Ks in a concentration-dependent manner.Their IC50 were 76.74 μmol/L and 55.37 μmol/L，respectively. Conclusion ：Ber inhibited I Kr ，I Ks and I K1in cardiomyopathic guinea pig ventricular myocytes，which may be important in understanding the antiarrhythmic effects of this drug.
2009, 40(3): 250-253.
Abstract：
Aim ：To study the effects of Clostridium difficile toxin A on the cytotoxity and apoptosis of human hepatoma cell line SMMC-7721. Methods ：Clone inhibiting experiment and MTT calorimetric assay were used to assay SMMC-7721 proliferation. Morphological changes relevant to the apoptosis and the DNA damage were analyzed by transmission electron microscope and single cell gel assay. The number of apoptosis cells and the expression of Bcl-2 and p53 protein were detected by the flow cytometry. Results ：0.018- 4.690 mg/L toxin A significantly decreased the colony information of SMMC-7721 cells，and greatly inhibited SMMC-7721 proliferation in a time- and concentration-dependent manner. Morphological changes related to apoptosis were evident under transmission electron microscope and DNA damage was detected by single cell gel assay when SMMC-7721 cultured with 4.690 mg/L toxin A for 48 h. Toxin A 0.073-4.690 mg/L induced apoptosis in SMMC-7721 cells from 6.8% to 41.8%. In addition，Toxin A 0.293-4.690 mg/L significant decreased Bcl-2 protein expression and increased p53 protein expression in SMMC-7721. Conclusion ：These results showed that Clostridium difficile toxin A had a significantly cytotoxicity in human SMMC-7721 which was attributed to toxin A induced apoptosis.
Aim ：To study the effects of Clostridium difficile toxin A on the cytotoxity and apoptosis of human hepatoma cell line SMMC-7721. Methods ：Clone inhibiting experiment and MTT calorimetric assay were used to assay SMMC-7721 proliferation. Morphological changes relevant to the apoptosis and the DNA damage were analyzed by transmission electron microscope and single cell gel assay. The number of apoptosis cells and the expression of Bcl-2 and p53 protein were detected by the flow cytometry. Results ：0.018- 4.690 mg/L toxin A significantly decreased the colony information of SMMC-7721 cells，and greatly inhibited SMMC-7721 proliferation in a time- and concentration-dependent manner. Morphological changes related to apoptosis were evident under transmission electron microscope and DNA damage was detected by single cell gel assay when SMMC-7721 cultured with 4.690 mg/L toxin A for 48 h. Toxin A 0.073-4.690 mg/L induced apoptosis in SMMC-7721 cells from 6.8% to 41.8%. In addition，Toxin A 0.293-4.690 mg/L significant decreased Bcl-2 protein expression and increased p53 protein expression in SMMC-7721. Conclusion ：These results showed that Clostridium difficile toxin A had a significantly cytotoxicity in human SMMC-7721 which was attributed to toxin A induced apoptosis.
2009, 40(3): 254-257.
Abstract：
Aim ：To study the therapeutic effects of ZK14，a novel nitric oxide donor，on hepatic fibrosis in rats. Methods ：Rats were injected intraperitoneally with CCl4 and induced to hepatic fibrosis，followed by intragasrtic administration with high dose of 20 mg/kg and low dose of 10 mg/kg of ZK14for 4 weeks.The effects were evaluated in the content of nitric oxide of serum and nitric oxide synthases of liver，in serum levels of the hepatic functions indices and hepatic fibrotic index and in liver histopathology. Results ：The biochemical indices and pathology grade were effectively improved in the group of ZK14 20 mg/kg，and liver function was also effectively protected in this group. Conclusion ：ZK14，a novel nitric oxide donating biphenyldicarboxylate derivative，showed preferable therapeutic effects on hepatic fibrosis of rats induced with CCl4，and it may be of potential value in the treatment of hepatic fibrosis.
Aim ：To study the therapeutic effects of ZK14，a novel nitric oxide donor，on hepatic fibrosis in rats. Methods ：Rats were injected intraperitoneally with CCl4 and induced to hepatic fibrosis，followed by intragasrtic administration with high dose of 20 mg/kg and low dose of 10 mg/kg of ZK14for 4 weeks.The effects were evaluated in the content of nitric oxide of serum and nitric oxide synthases of liver，in serum levels of the hepatic functions indices and hepatic fibrotic index and in liver histopathology. Results ：The biochemical indices and pathology grade were effectively improved in the group of ZK14 20 mg/kg，and liver function was also effectively protected in this group. Conclusion ：ZK14，a novel nitric oxide donating biphenyldicarboxylate derivative，showed preferable therapeutic effects on hepatic fibrosis of rats induced with CCl4，and it may be of potential value in the treatment of hepatic fibrosis.
2009, 40(3): 258-262.
Abstract：
Aim ：To study the pharmacokinetic effects of salvianolic acid B(Sal B) on danshensu(DSS) in Danshen injection in rats.Method：Following the intravenous administration of Danshen injection and Danshen injection added with Sal B to rats， the plasma kinetic study，tissue distribution and urine excretion were studied.The plasma kinetic study was also investigated by giving DSS and DSS in combination with Sal B to rats.Plasma，tissue and urine drug levels of danshensu were analyzed by LC-MS. Results ：Compared with danshensu given alone，no significant difference of the pharmacokinetic behavior of danshensu was found when danshensu was given in combination with salvianolic acid B.However，compared with Danshen injection given alone，the pharmacokinetic behavior of danshensu changed remarkably when Danshen injection was given in combination with salvianolic acid B which might be caused by the decrease of DSS in kidney distribution and urine excretion. Conclusion ：The pharmacokinetic effects of salvianolic acid B on danshensu depend on the existence of multiple components in Danshen injection.Results suggest that the pharmacokinetic interactions of the multiple components are closely related to the integrity of herbal medicines.
Aim ：To study the pharmacokinetic effects of salvianolic acid B(Sal B) on danshensu(DSS) in Danshen injection in rats.Method：Following the intravenous administration of Danshen injection and Danshen injection added with Sal B to rats， the plasma kinetic study，tissue distribution and urine excretion were studied.The plasma kinetic study was also investigated by giving DSS and DSS in combination with Sal B to rats.Plasma，tissue and urine drug levels of danshensu were analyzed by LC-MS. Results ：Compared with danshensu given alone，no significant difference of the pharmacokinetic behavior of danshensu was found when danshensu was given in combination with salvianolic acid B.However，compared with Danshen injection given alone，the pharmacokinetic behavior of danshensu changed remarkably when Danshen injection was given in combination with salvianolic acid B which might be caused by the decrease of DSS in kidney distribution and urine excretion. Conclusion ：The pharmacokinetic effects of salvianolic acid B on danshensu depend on the existence of multiple components in Danshen injection.Results suggest that the pharmacokinetic interactions of the multiple components are closely related to the integrity of herbal medicines.
2009, 40(3): 263-268.
Abstract：
Aim ：To determine the uptake characteristics of salvianolic acid B(Sal B) in myocardial cells and blood vessel endothelial cells. Method ：The effects of various factors，such as time，temperature，drug concentration，pH of the medium，on the uptake of Sal B in myocardial cells and aorta endothelial cells were investigated.LC/MS was employed to determine the intracellular concentration of Sal B. Results ：Uptake kinetics of Sal B in the myocardial cells and aorta endothelial cells fitted well to the logarithmic model at 37 ℃ and 4 ℃.The amount of uptake was in direct proportion to the extracellular concentration of Sal B in the experimental concentration range.Uptake of Sal B both in the myocardial cells and blood vessel endothelial cells would significantly increase while the medium pH decreased，and some water-soluble components extracted from danshen would also facilitate the uptake of Sal B both in the myocardial cells and blood vessel endothelial cells obviously.The energy metabolism inhibitors would significantly inhibit the uptake of Sal B in the myocardial cells and blood vessel endothelial cells.When lactic acid and fatty acid were added to the incubation solution，the uptake of Sal B both in the myocardial cells and aorta endothelial cells increased more than 20%. Conclusion ：pH is the most important factor influencing the cellular uptake of Sal B，and the amount of uptake tends to increase in acidic medium.Results suggest that the uptake of Sal B would increase in the acidified internal environment induced by myocardial ischemia，thus exerting better cardiovascular activities.
Aim ：To determine the uptake characteristics of salvianolic acid B(Sal B) in myocardial cells and blood vessel endothelial cells. Method ：The effects of various factors，such as time，temperature，drug concentration，pH of the medium，on the uptake of Sal B in myocardial cells and aorta endothelial cells were investigated.LC/MS was employed to determine the intracellular concentration of Sal B. Results ：Uptake kinetics of Sal B in the myocardial cells and aorta endothelial cells fitted well to the logarithmic model at 37 ℃ and 4 ℃.The amount of uptake was in direct proportion to the extracellular concentration of Sal B in the experimental concentration range.Uptake of Sal B both in the myocardial cells and blood vessel endothelial cells would significantly increase while the medium pH decreased，and some water-soluble components extracted from danshen would also facilitate the uptake of Sal B both in the myocardial cells and blood vessel endothelial cells obviously.The energy metabolism inhibitors would significantly inhibit the uptake of Sal B in the myocardial cells and blood vessel endothelial cells.When lactic acid and fatty acid were added to the incubation solution，the uptake of Sal B both in the myocardial cells and aorta endothelial cells increased more than 20%. Conclusion ：pH is the most important factor influencing the cellular uptake of Sal B，and the amount of uptake tends to increase in acidic medium.Results suggest that the uptake of Sal B would increase in the acidified internal environment induced by myocardial ischemia，thus exerting better cardiovascular activities.
Abstract：
Aim ：To prepare a fully human anti-VEGF165(vascular endothelial growth factor 165) monoclonal antibody with antitumor activity from five-feature mice which express human immunoglobin loci. Methods ：A routine method for the generation of monoclonal antibodies(mAbs) against the human VEGF165 was developed.The immunizing effect between five-feature mice and BALB/c was observed and the mAb was purified through MBP IgM affinity chromatography.The effect of mAbs on antitumor was tested in vitro by T24 cell line. Results ：Four hybridomata secreting mAbs steadily were isolated successfully，and the serum titer of mAb in BALB/c mice was almost 10 times higher than that in five-feature mice.The indirect ELISA method for mAb titer determination was also established.The anti-VEGF165mAb was purified to homogeneity by precipitation with ammonium sulfate followed by the affinity chromatography on MBP IgM purification column.Moreover，both purified human IgM V2，V75 and mouse ascites were characterized by SDS-PAGE and Western blotting.Proliferation of T24 cell line was considerably inhibited by V2 and V75. Conclusion ：Five-feature mice could be used to produce fully human monoclonal antibody.The fully human anti-VEGF mAb is potential in the cancer treatment.
Aim ：To prepare a fully human anti-VEGF165(vascular endothelial growth factor 165) monoclonal antibody with antitumor activity from five-feature mice which express human immunoglobin loci. Methods ：A routine method for the generation of monoclonal antibodies(mAbs) against the human VEGF165 was developed.The immunizing effect between five-feature mice and BALB/c was observed and the mAb was purified through MBP IgM affinity chromatography.The effect of mAbs on antitumor was tested in vitro by T24 cell line. Results ：Four hybridomata secreting mAbs steadily were isolated successfully，and the serum titer of mAb in BALB/c mice was almost 10 times higher than that in five-feature mice.The indirect ELISA method for mAb titer determination was also established.The anti-VEGF165mAb was purified to homogeneity by precipitation with ammonium sulfate followed by the affinity chromatography on MBP IgM purification column.Moreover，both purified human IgM V2，V75 and mouse ascites were characterized by SDS-PAGE and Western blotting.Proliferation of T24 cell line was considerably inhibited by V2 and V75. Conclusion ：Five-feature mice could be used to produce fully human monoclonal antibody.The fully human anti-VEGF mAb is potential in the cancer treatment.
2009, 40(3): 273-278.
Abstract：
Aim ：To study the effect of Eudragit S-100，a pH-responsive polymer，on protein refolding level， using recombinant human keratinocyte growth factor-2(rhKGF-2) as a model protein. Methods ：The refolding of rhKGF-2 was performed by directly diluting denatured rhKGF-2 into a refolding buffer containing different concentrations of Eudragit.The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTT assay，reverse phase HPLC，fluorescence emission spectroscopy and circular dichroism spectroscopy. Results ：The addition of Eudragit S-100 in the refolding buffer significantly increased the rhKGF-2 refolding yield to 71%，when dilution refolding was conducted at 0.5 mg/mL rhKGF-2.The outcome from the refolding study showed possibility of a special interaction between rhKGF-2 and Eudragit，suggesting that the refolding-enhancing ability of Eudragit S-100 was due to this interaction between Eudragit S-100 and rhKGF-2.Mean while，the result showed that the concentration of urea was also an important factor for the optimization of the refolding in the presence of Eudragit. Conclusion ：Eudragit S-100 can significantly increase the refolding level of rhKGF-2.
Aim ：To study the effect of Eudragit S-100，a pH-responsive polymer，on protein refolding level， using recombinant human keratinocyte growth factor-2(rhKGF-2) as a model protein. Methods ：The refolding of rhKGF-2 was performed by directly diluting denatured rhKGF-2 into a refolding buffer containing different concentrations of Eudragit.The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTT assay，reverse phase HPLC，fluorescence emission spectroscopy and circular dichroism spectroscopy. Results ：The addition of Eudragit S-100 in the refolding buffer significantly increased the rhKGF-2 refolding yield to 71%，when dilution refolding was conducted at 0.5 mg/mL rhKGF-2.The outcome from the refolding study showed possibility of a special interaction between rhKGF-2 and Eudragit，suggesting that the refolding-enhancing ability of Eudragit S-100 was due to this interaction between Eudragit S-100 and rhKGF-2.Mean while，the result showed that the concentration of urea was also an important factor for the optimization of the refolding in the presence of Eudragit. Conclusion ：Eudragit S-100 can significantly increase the refolding level of rhKGF-2.
2009, 40(3): 279-283.
Abstract：
HIV-1 protease inhibitors play a very important role in AIDS chemotherapy，but with the rapid emergence of drug resistance as a result of the residue mutation of HIV-1 protease，developing effective protease inhibitors with superior activity against drug-resistant variants is becoming the research hotspot in AIDS drug design. Meanwhile，some molecular designing strategies of anti-drug-resistant HIV-1 protease inhibitors were put forward and applied to develop anti-AIDS drugs.The purpose of the research is introducing these molecular designing strategies to develop potent HIV-1 protease inhibitors to combat drug resistance，including substrate envelope hypothesis，strengthening the binding of inhibitors to HIV-1 protease and searching inhibitors acting in novel sites of HIV-1 protease.
HIV-1 protease inhibitors play a very important role in AIDS chemotherapy，but with the rapid emergence of drug resistance as a result of the residue mutation of HIV-1 protease，developing effective protease inhibitors with superior activity against drug-resistant variants is becoming the research hotspot in AIDS drug design. Meanwhile，some molecular designing strategies of anti-drug-resistant HIV-1 protease inhibitors were put forward and applied to develop anti-AIDS drugs.The purpose of the research is introducing these molecular designing strategies to develop potent HIV-1 protease inhibitors to combat drug resistance，including substrate envelope hypothesis，strengthening the binding of inhibitors to HIV-1 protease and searching inhibitors acting in novel sites of HIV-1 protease.
2009, 40(3): 284-288.
Abstract：
The extracts of marine mollusks（Abalone，Aplysia，Sepia，Arcagranosa linnaeus，Cyclina sinensis，Mytilus coruscus，Chlamys farrei，Meretrix meretrix，Ostrea，Sinonovacula constricta and Solen gradis）classified as polysaccharides，proteins and peptides，possess the antitumor，anti-aging，and anti-bacterial activities.Cancer patients undergoing long-term chemotherapy usually suffer from the damage to vital organs，and resistances affecting the outcomes of chemotherapy.Therefore，it is imperative to search for anti-cancer drugs with low toxicity and high efficiency.At present，several marine mollusks are being studied.Summary on the antitumor activities of the marine mollusks extrats is presented，which might provide a basis for the development of marine anti-cancer drugs.
The extracts of marine mollusks（Abalone，Aplysia，Sepia，Arcagranosa linnaeus，Cyclina sinensis，Mytilus coruscus，Chlamys farrei，Meretrix meretrix，Ostrea，Sinonovacula constricta and Solen gradis）classified as polysaccharides，proteins and peptides，possess the antitumor，anti-aging，and anti-bacterial activities.Cancer patients undergoing long-term chemotherapy usually suffer from the damage to vital organs，and resistances affecting the outcomes of chemotherapy.Therefore，it is imperative to search for anti-cancer drugs with low toxicity and high efficiency.At present，several marine mollusks are being studied.Summary on the antitumor activities of the marine mollusks extrats is presented，which might provide a basis for the development of marine anti-cancer drugs.
