Comparison of Differential Saponin Contents and Pro-angiogenic Activities on Zebrafish Between Freeze-dried and Sun-dried Panacis Quinquefolii Radix Decoction Pieces / 中国实验方剂学杂志

ObjectiveTo analyze and determine the differential components of freeze-dried and sun-dried Panacis Quinquefolii Radix（PQR）， and to compare the differences in their pro-angiogenic activities. MethodFingerprints of freeze-dried and sun-dried PQR were established based on ultra performance liquid chromatography（UPLC）， and chemometrics methods such as principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were combined to determine the differential saponin composition of the two decoction pieces， and six representative saponins were selected and their contents in freeze-dried and sun-dried PQR were determined by UPLC. Transgenic zebrafish line Tg（fli1a∶EGFP） embryos fertilized for 24 h were selected， and different doses of 70% methanol extracts of freeze-dried and sun-dried PQR（10， 30 mg·L-1） were used to intervene in normal zebrafish and in a zebrafish model of intersegmental vascular（ISV） injury induced by vascular endothelial growth factor（VEGF） receptor tyrosine kinase inhibitor Ⅱ（PTK787）， then the development of subintestinal vein（SIV） and ISV of zebrafish was observed， SIV diameter， mean number of crossings and mean number of germinations were determined， and the ISV vascular index was calculated， in order to compare the pro-angiogenic activities of the two decoction pieces. ResultThe similarity of the fingerprints of freeze-dried and sun-dried PQR decoction pieces was>0.950， and 17 common peaks were identified， of which 6 common peaks were designated as peak 6（ginsenoside Rg1）， peak 7（ginsenoside Re）， peak 8（ginsenoside Rb1）， peak 11（ginsenoside Rc）， peak 13（ginsenoside Rb2）， and peak 16（ginsenoside Rd）， respectively. A total of 11 differential saponin components were screened by PCA and OPLS-DA， indicating that there were some differences in the contents of the components in the two decoction pieces. The results of determination showed that the contents of ginsenosides Rg1， Re， Rb1 and Rb2 in freeze-dried PQR were higher than those in sun-dried PQR， while the contents of ginsenosides Rc and Rd were lower than those in sun-dried PQR（P<0.05， P<0.01）. In the study of the pro-angiogenic effect on normal zebrafish embryos， compared with the blank group， and the SIV vessel diameter， mean germination rate and mean crossover rate were significantly higher in the high-dose groups of freeze-dried and sun-dried PQR（P<0.01）， and the vessel diameter， mean numbers of crossings and germinations in the freeze-dried PQR group were higher than those of the sun-dried PQR group（P<0.05）. In the study of the pro-angiogenic effect on zebrafish embryos with ISV injury， the development of ISV in the model group was significantly inhibited when compared with the blank group， compared with the model group， different dose groups of freeze-dried and sun-dried PQR could promote the growth and sprouting of ISV， and the number of normal blood vessels in the freeze-dried PQR group was significantly higher than that in the sun-dried PQR group at the same dosage（P<0.05）. ConclusionFreeze-drying can effectively avoid the loss and secondary transformation of ginsenosides in PQR， and its angiogenic activity is better than that of sun-dried PQR， which can provide a reference for the production and development of high-quality PQR decoction pieces.

Analysis of Chemical Constitutions of Volatile Oil in Opisthopappus taihangensis by GC-MS Combined with Retention Index / 中国实验方剂学杂志

ObjectiveTo study the composition of volatile oil in Opisthopappus taihangensis （Taihangju）， and provide a reference for comprehensive development of this medicine. MethodTaking Chrysanthemum morifolium （Xiaobaiju）， C. morifolium （Xiaohuangju） and C. indicum （Yejuhua） as control， the qualitative and quantitative analysis of volatile oil in Taihangju and three control varieties were completed by gas chromatography-mass spectrometry （GC-MS） combined with retention index method. The GC conditions were as following：programmed temperature （initial temperature at 60 ℃， kept for 2 min； up to 120 ℃ with the heating rate of 5 ℃·min-1， still kept for 2 min； up to 180 ℃ with the heating rate of 2 ℃·min-1， kept for 3 min； and then up to 240 ℃ with the heating rate of 8 ℃·min-1， kept for 5 min； finally up to 280 ℃ with the heating rate of 10 ℃·min-1， kept it for 5 min and finished）， high-purity helium as the carrier gas， the split ratio of 50∶1. MS conditions were as follows：electron impact ion source （EI）， ion source temperature of 230 ℃， electron collision energy of 70 eV and scanning range of m/z 30-445. Principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were used to obtain the characteristic components between Taihangju and the three control varieties. ResultA total of 86， 96， 112 and 109 compounds including 73 common components were identified in Taihangju， Xiaobaiju， Xiaohuangju and Yejuhua， respectively. The contents of volatile components in Taihangju were significantly different from that of the control varieties. In which， the relative contents of α-thujone， eucalyptol and terpinen-4-ol were high in Taihangju， and eucalyptol， camphor and α-terpinyl acetate were the main compositions in the control varieties. In addition， 11 compounds were screened as characteristic components to distinguish Taihangju and the three control varieties by PCA and OPLS-DA， including main differential components of chamazulene and δ-cadinene. ConclusionThe main components of volatile oil in Taihangju includes alcohols， terpenes， ketones and esters with high medicinal value. The accuracy of qualitative analysis of volatile oil is improved by GC-MS combined with retention index method， which provides scientific reference for development and utilization of Taihangju.

Determination of UV-Vis Spectrophotometric Method of Metal Complexes Stoichiometry between Cu(II), Ca(II), Cd(II), Mg(II) and Zn(II) with Bupropion Hydrochloride

In this study, simple, rapid and accurate UV-Vis spectrophotometric method was developed for the determination ofcomplexes stoichiometry of Cu(II)-bupropion hydrochloride, Ca(II)-bupropion hydrochloride, Cd(II)-bupropionhydrochloride, Mg(II)-bupropion hydrochloride and Zn(II)-bupropion hydrochloride. The spectrums of the complexesformed between bupropion hydrochloride with Cu (II), Ca (II), Cd (II), Mg (II) and Zn (II) metal cations were taken in therange of 500 to 190 nm and the stoichiometry of the complexes formed between bupropion hydrochloride with Cu (II), Ca(II), Cd (II), Mg (II) and Zn (II) metal cations were determined by using the mole ratio method at maximum wavelength(λmax=252 nm). The stoichiometry of Cu(II)-bupropion hydrochloride, Ca(II)-bupropion hydrochloride, Cd(II)-bupropionhydrochloride and Zn(II)-bupropion hydrochloride were calculated as 1:1, but Mg(II)-bupropion hydrochloride complexwas calculated as 1:2. The recovery was found 95.0% for bupropion hydrochloride. Lambert-Beer’s obeyed theconcentration range of 1.40-6.91 µg/mL for bupropion hydrochloride. The LOD and LOQ were found to be 13.81 and 1.38µg/mL for bupropion hydrochloride, respectively. The interaction between bupropion hydrochloride with Cu (II), Ca (II), Cd(II), Mg (II) and Zn (II) were investigated by applying UV-visible spectrophotometric method, and satisfactory results wereobtained

Discussion on medicinal value of Saposhnikoviae Radix based on difference of ketone content in cortex and wood of Saposhnikoviae Radix / 中国中药杂志

In order to confirm the tradition that bolting Saposhnikoviae Radix could not be used as medicine,the content of four chromone components in the cortex and wood of Saposhnikoviae Radix was analyzed by high performance liquid chromatography( HPLC),and the chemical fingerprints were established,12 common peaks were calibrated. The similarity analysis found that the similarity between batches was 0. 115-0. 995,it indicates that the cortex and wood of Saposhnikoviae Radix have certain differences. On this basis,systematic clustering analysis,principal component analysis and orthogonal partial least squares discriminant analysis were carried out with the content of four chromone components and whether they met the pharmacopoeia criteria as the original variables. The results showed that the content of the four components in the cortex of Saposhnikoviae Radix was much higher than that in the wood,and the four components detected were able to distinguish the cortex and the wood of Saposhnikoviae Radix. The results of the study reveal the tradition that bolting Saposhnikoviae Radix should not be used as medicine dut to decreased quality.

A screening method and practice for alternative resources of Paridis Rhizoma based on overall similarity of components and activities / 中草药

Objective To study the overall similarity of composition-activity of different kinds of Paridis Rhizoma, and establish a new method for selecting alternative resources. Methods The HPLC method was used to detect the content of polyphyllin I, II, VI, and VII, and pharmacological model of analgesic and hemostatic were used to detect the efficacy. The data were standardized using standard deviation method. Using polyphyllin I, II, VI, and VII at minimum 0.6%, aspirin and Yunnan Baiyao Group analgesic and hemostatic efficacy data as standard controls, we also established component and activity indexes. SPSS 20.0 Software was used to analyze the principal component analysis (PCA) and hierarchical cluster analysis (HCA) of these seven medicinal plants. Results The total content of the main saponins in seven medicinal plants of Paris genus ranged from 0.125% to 1.649%, and the maximum content (1.649%) was found in the P. forrestii while the minimum content (0.125%) was found in P. daliensis. The study also disclosed that P. polyphylla var. yunnanensis has the most active analgesic activity, and the time of bleeding (BT) in mice and the time of coagulation (CT) in mice were the shortest in P. polyphylla. The shortest mice activated partial thrombin time (APTT) was P. forrestii and the longest was P. thibetica. The shortest prothrombin time (PT) in mice was P. vietnamensis. The longest BT in mice was P. daliensis. The longest CT and PT in mice was P. polyphylla. PCA and HCA results showed that P. polyphylla var. yunnanensis, P. vietnamensis and P. thibetica were in the same class, while P. pubescens, P. daliensis, P. polyphylla, and P. forrestii were in another class. It is indicated that the overall similarity of component-activity of P. vietnamensis, P. thibetica, and P. polyphylla var. yunnanensis was higher, thus they can be considered as alternative sources. Conclusion The quality relations of different kinds of Paris genus were evaluated by the overall similarity of the component and activity index, which provides ideas and methods for the search of the replacement resources of the endangered plants.

Pharmacological activities of α3β2 and α3β4 nicotinic acetycholine receptors with different α and β subunit stoichiometries / 中国病理生理杂志

AIM:To compared the differential sensitivity of nicotinic acetycholine receptors (nAChRs) consisting of α and β subunits with different ratios.METHODS:The cRNA of α and β subunits was obtained by in vitro transcription.The α3β2 and α3β4 nAChR subtypes were expressed in Xenopus laevis oocytes by microinjection of cRNA coding α and β subunits at α∶β ratios of 1∶10, 1∶1 and 10∶1.The pharmacological activities of nAChRs to agonist acetycholine (ACh) and antagonist α-conotoxin (CTx) RegⅡA were investigated by two-electrode voltage-clamp techniques.RESULTS:For α3β2 nAChR expressed at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 91.2 μmol/L, 104.4 μmol/L and 130.6 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 40.2 nmol/L, 36.4 nmol/L and 42.3 nmol/L, respectively.For α3β4 nAChR at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 44.0 μmol/L, 110.0 μmol/L and 230.0 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 226.8 nmol/L, 71.5 nmol/L and 49.4 nmol/L, respectively.CONCLUSION:The results imply that the α3 and β4 subunit stoichiometry can change the structure and pharmacological activity of α3β4 nAChR, but the stoichiometry of α3 and β2 subunits has no effect on α3β2 nAChR.

Sensitivity of α-conotoxin TxID on stoichiometry of α3β4 nicotinic acetylcholine receptors / 中国药学杂志

OBJECTIVE: To interrogate differential sensitivity of α-conotoxin TxID on stoichiometry of α3β4 nicotinic acetylcholine receptors(nAChRs). METHODS: Oocytes of Xenopus laevis were used to express rat α3β4 nAChRs with different stoichiometries by altering α3:β4 RNA injection ratios of 1:1, 1:10 or 10:1. Sensitivity of α-conotoxin TxID on these different stoichiometry receptors were evaluated and compared. RESULTS: The three stoichiometry receptors of α3β4 nAChRs were expressed in oocytes successfully. α-Conotoxin TxID showed differential sensitivity on α3β4 nAChR stoichiometries. Inhibition of 1:10 injection ratio by TxID was similar with regular 1:1 α3β4 nAChRs within 2-fold difference. While potency of 10:1 injection ratio by TxID decreased 5-fold significantly comparing with 1:1 α3β4 nAChRs. CONCLUSION: α-Conotoxin TxID exhibits distinct sensitivity on different stoichiometry of α3β4 nAChRs, which could reflecting different stoichiometries of α and β subunits. The RESULTS would be helpful for elucidation of structure and physiology function of α3β4 nAChRs.

A influência do tempo de detenção celular e decaimento endógeno na estequiometria de reações em processos biológicos de tratamento de águas residuárias: I - Crescimento quimioheterotrófico / The influence of cell detention time and endogenous decay on the stoichiometry reactions of biological processes in wastewater treatment: I - Chemoheterotrophic growth

No metabolismo quimioheterotrófico, um mesmo substrato orgânico é utilizado como fonte de energia e síntese celular. Este artigo apresenta uma metodologia que pode ser usada para calcular a estequiometria do metabolismo quimioheterotrófico considerando o tempo de detenção celular e o decaimento endógeno. O método se baseia em princípios da bioenergética e é útil para o desenvolvimento de balanços de massa em processos biológicos de tratamento de águas residuárias. Também serve para se estimar as necessidades de nutrientes, alcalinidade e aceptores de elétrons para as reações desejadas, assim como a biomassa formada e gases produzidos.

In chemohetrotrophic metabolism, an organic substrate is used as energy and cell synthesis sources. This article presents a methodology that can be used to calculate the stoichiometry of chemoheterotrophic metabolism, considering solids detention time and endogenous decay. The method is based on bioenergetic principles, and is useful for mass balance calculations for biological processes used in wastewater treatment. It is also applied to estimate nutrients, alkalinity and electron acceptor requirements for the intended reactions, as well as biomass and gas production.

A possible molecular metric for biological evolvability.

Proteins manifest themselves as phenotypic traits, retained or lost in living systems via evolutionary pressures. Simply put, survival is essentially the ability of a living system to synthesize a functional protein that allows for a response to environmental perturbations (adaptation). Loss of functional proteins leads to extinction. Currently there are no universally applicable quantitative metrics at the molecular level for either measuring ‘evolvability’ of life or for assessing the conditions under which a living system would go extinct and why. In this work, we show emergence of the first such metric by utilizing the recently discovered stoichiometric margin of life for all known naturally occurring (and functional) proteins. The constraint of having well-defined stoichiometries of the 20 amino acids in naturally occurring protein sequences requires utilization of the full scope of degeneracy in the genetic code, i.e. usage of all codons coding for an amino acid, by only 11 of the 20 amino acids. This shows that the non-availability of individual codons for these 11 amino acids would disturb the fine stoichiometric balance resulting in non-functional proteins and hence extinction. Remarkably, these amino acids are found in close proximity of any given amino acid in the backbones of thousands of known crystal structures of folded proteins. On the other hand, stoichiometry of the remaining 9 amino acids, found to be farther/distal from any given amino acid in backbones of folded proteins, is maintained independent of the number of codons available to synthesize them, thereby providing some robustness and hence survivability.

Aplicação de princípios de bioenergética no cálculo da estequiometria de reações biológicas em processos de tratamento de águas residuárias / Application of bioenergetic principles to the stoichiometry of biological reactions in wastewater treatment processes

O tratamento biológico de águas residuárias é realizado por microrganismos que utilizam determinado substrato para obtenção de energia e crescimento celular através de reações de oxidação-redução e fermentação. Esses mecanismos podem ser expressos por meio de reações estequiométricas desenvolvidas a partir de princípios da termodinâmica. As reações são dependentes dos compostos doadores e aceptores de elétrons. As equações balanceadas resultantes permitem estimar as necessidades de nutrientes, a variação de pH/alcalinidade, a produção de biomassa e gases e o coeficiente de produção celular associados aos sistemas biológicos de tratamento. Esta nota técnica descreveu a metodologia para cálculo estequiométrico de reações bioquímicas usando princípios de bioenergética, apresentando exemplos para diferentes substratos e aceptores de elétrons.

Biological wastewater treatment is performed by microorganisms by means of oxidation-reduction and fermentation reactions that release energy for growth and maintenance. These processes can be represented by stoichiometric reactions developed based on thermodynamic principles. Reactions are dependent on the compounds used as electron donor and acceptor. The overall stoichiometric reactions allow estimations of nutrient requirements, alkalinity/pH changes, biomass and gas production, and yield coefficient. This article described a methodology for stoichiometry calculations using bioenergetic principles, presenting examples with different substrates and electron acceptors.

Bacterial citrate lyase.

Bacterial citrate lyase, the key enzyme in fermentation of citrate, has interesting structural features. The enzyme is a complex assembled from three non-identical subunits, two having distinct enzymatic activities and one functioning as an acyl-carrier protein. Bacterial citrate lyase, si-citrate synthase and ATP-citrate lyase have similar stereospecificities and show cofactor cross-reactions. On account of these common features, the citrate enzymes are promising markers in the study of evolutionary biology. The occurrence, function, regulation and structure of bacterial citrate lyase are reviewed in this article.