A microcalorimetric method to determine antimicrobial effects of two bile acid derivatives on Staphylococcus aureus

Using microcalorimetry, the characteristic metabolic heat flow power-time curves of S. aureus growth affected by Ursodesoxycholic acid and Hyodeoxycholic acid were measured at 37?°C. The thermal-kinetic parameters such as, growth rate constant k, the maximum power output (P _m), the time corresponding to the maximum power output (t _p), total heat-production Q _t , half inhibitory concentration of the drugs (IC ₅₀) were calculated from the growth curves. For both HDCA and UDCA, with the increasing of concentration, k, P _m, and Q _t decreased, meanwhile, k?Cc fit a linear equation, t _p was prolonged correspondingly. Principle component analysis, the results indicated t _p might be the main parameter in evaluating the antibacterial activity of HDCA and UDCA in microcalorimetric method. Combining with t _p and IC ₅₀, the results revealed that the differences and trends of antibacterial activity of these bile acid derivatives were: HDCA?>?UDCA. Structure-activity relationship (SAR) analysis showed that the ??-OH at C-3 and C-6 position at equal pace on the steroid nucleus enhanced the hydrophilicity of HDCA, which led to a stronger antibacterial effect than UDCA. In this study, a useful tool was provided to accurately evaluate the antibacterial effects of bile acid derivatives. The thermolysis curve recorded by microcalorimetry could provide a lot of kinetic and thermodynamic information for the study of growth process of living microbial, which could be helpful in the screening of high efficacy antibacterial agents.     

Microcalorimetric investigation on antibacterial activity of the peptide from Plutella xylostella

The antibacterial activities of a kind of novel peptide from Plutella xylostella (pxCECA1) on methicillin-resistant staphylococcus aureus (MRSA) and Escherichia coli (E. coli) growth were investigated by microcalorimetry. The heat flow power–time curves of MRSA and E. coli growth in the presence of pxCECA1 were recorded using the 3114/3115 Thermal Activity Monitor Air Isothermal Calorimeter based on ampoule mode at 37 °C. Some parameters including growth rate constant k, maximum power output P _max, total heat output Q_t, generation time t _g, growth inhibitory ratio I, and half-inhibitory concentration of the drugs IC₅₀ were obtained to elucidate the antibacterial activity of pxCECA1. The results showed that k, P _max, and Q _t decreased, but I and t _g increased or delayed with the increase in pxCECA1 concentration. The IC₅₀ of pxCECA1 on E. coli was 6.122 μg mL^?1 and MRSA was 7.809 μg mL^?1. It could be concluded that pxCECA1 had stronger inhibitory effect on E. coli than MRSA. In vivo test was simultaneously performed using an E. coli and MRSA infection model to validate the antibacterial activity of pxCECA1. The results revealed that pxCECA1 with broad spectrum antimicrobial activities hopefully represented a class of promising substitute of antimicrobial agents.     

Microcalorimetry coupled with principal component analysis for comparing the effects of two Panax species on mice splenic lymphocytes

A powerful microcalorimetric method based on the cell heat production was applied to evaluate the effects of two Panax species on mice splenic lymphocytes growth. Some qualitative and quantitative information, such as the metabolic power-time curves, growth rate constant k, maximum heat-output power P _max, appearance time for the highest peak t _max, total heat production Q _t for all the metabolic progress of mice splenic lymphocytes were obtained to present the effects of Panax ginseng and American Ginseng on these cells. Coupled with principal component analysis (PCA) on these quantitative thermokinetic parameters, the effects of the two Panax species on mice splenic lymphocytes could be quickly evaluated by analyzing the change of the main parameter k. From the values of k, it could be concluded quickly and accurately that Panax ginseng and American Ginseng both showed strong inhibitory effects on mice splenic lymphocytes, and the inhibitory effects was strengthened with increasing concentration of the two Panax species in the concentration range of 0–3.2 mg mL^?1. Panax ginseng with IC ₅₀ of 1.38 mg mL^?1 showed stronger inhibitory effect on mice splenic lymphocytes growth than American Ginseng with IC ₅₀ of 2.08 mg mL^?1. This study indicates that microcalorimetry is a powerful tool for evaluating the drugs’ efficiency on living system, providing some useful references for the application of Panax ginseng and American Ginseng in practice.     

微量热法比较黄连中四种生物碱的抗菌作用

采用微量热法研究和比较中药黄连中四种生物碱(BAs)的抗菌(大肠杆菌)作用。这四种生物碱分别为小檗碱、黄连碱、巴马汀和药根碱。用LKB-2277生物活性检测系统,以停留法测定了37 ℃时大肠杆菌在BAs作用下的热流功率-时间曲线,并记录生长速率常数k, 指数生长期和稳定期的最大热功率Pm, log、Pm, stat, 生长抑制率I, 传代时间tg,指数生长期的总产热量Qt, log,半数抑菌浓度IC50等热动力学参数。结果显示这四种生物碱具有相异的抗大肠杆菌生长代谢的作用,k, Pm, log和Qt, log值随BAs浓度的增加而相应的减少。综合分析k, Pm, log, Qt, log, I 和IC50值可以看出,BAs抑制大肠杆菌生长代谢强度按小檗碱、黄连碱、巴马汀、药根碱的顺序依次减弱。构效关系研究表明,母环C-2和C-3上连接的亚甲二氧基比甲氧基更能显著增强相应化合物的抗菌作用,而C-9和C-10上连接的亚甲二氧基或甲氧基对这种抗菌作用影响不大。     

Anti-bacterial effect of four extracts from leaves of Dracontomelon dao on Escherichia coli growth using microcalorimetry coupled with principal component analysis

The anti-Escherichia coli activities of four extracts in leaves of Dracontomelon dao, a traditional folk herb in China were investigated and compared by microcalorimetry. The four extracts are PE fraction, CHCl₃ fraction, EtOAc fraction, and n-BuOH fraction. The heat flow power–time (HFP–time) curves of E. coli growth in the presence of the four extracts were measured using an ampoule method. Then the nine thermal kinetic parameters were obtained from the curves. From the result of principal component analysis, it can be seen that parameters k ₁, k ₂, P ₁, and Q _p2 might be the main parameters in evaluating the anti-E. coli effects. In the presence of CHCl₃ fraction, EtOAc fraction, and n-BuOH fraction, k ₂, Q _p2 of E. coli decreased with increasing concentrations of the extracts. The EtOAc fraction was observed to have the strongest anti-bacterial activity with half-inhibitory concentration IC50 of 98.5 μg mL^?1. So, it can be concluded that EtOAc fraction can be further developed as anti-bacterial bioactive fraction of leaves of Dracontomelon dao.     

Microcalorimetric Studies on Influence of Sm^3＋, Dy^3＋ on Growth and Sporulation of Bacillus thuringiensis

By using an LKB-2277 Bioactivity Monitor and cycle-flow method, the thermogenic curves of aerobic growth for Bacillus thuringiensis cry Ⅱ strain at 28 ℃ have been obtained. The metabolic thermogenic curves of Bt cry Ⅱ contain two distinct parts: the first part reflects the changes of bacterial growth phase and the second part corresponds to sporulation phase. From these thermogenic curves in the absence or presence of Sm^3 , Dy^3 ions, the thermokinetic parameters such as the growth rate constants k. the interval time τ1, the maximum power PMAX1 and heat-output QLOG for log phase, the maximum power PMAX2 and heat-output QSTAT for stationary phase, the heat-output QSPOR for sporulation phase and total heat effects QT were calculated. Sm^3 and Dy^3 ions have promoting action on the growth of Bt cryⅡ in their lower concentration range, on the other hand, they have inhibitory action on the sporulation of Bt in their higher concentration range, It has also been found that the effects of Sm^3 and Dy^3 ions on Bt during the sporulation phase were far greater than those during the bacterial growth phase. It was concluded that the application of Bt for controlling insecticide could not be affected by the presence of the rare-earth elements in the environmental ecosystem.     

Microcalorimetric Study of the Biological Effects of Zn^＋ on Bacillus thuringiensis Growth

A microcalorimetric technique was used to investigate the influence of Zn^2 on the growth metabolism of Bacillus thuringiensis .LKB-2277 Bioactivity Monitor was employed to obtain the power-time curves,from which the maximum peak-heat output power(Pmax) in the log phase,the growth rate constants(k), the inhibitory ratios(I) ,the generational time(tG) and the total heat effect (Qtotal) in 23 h for the growth metabolism of Bacillus thuringiensis at 28℃ can be evaluated,The results indicate that the concentration of Zn^2 affects its growth obviously,Low concentration (0-50μg/mL) of Zn^2 promotes the growth of Bacillus thuringiensis while high concentration (50-500μg/mL) of Zn^2 inhibits its growth .When the concentration reached up to 600μg/mL,it can not grow at all.     

Microcalorimetric investigation of water vapor adsorption on silica gel

In this study, the activities of four ginsenosides Rc, Re, Rd, and Rf on splenic lymphocytes growth were studied by microcalorimetry. Some qualitative and quantitative information, such as the metabolic power–time curves, growth rate constant k, maximum heat-output power of the exponential phase P _max and the corresponding appearance peak time t _max, total heat output Q _t, and promotion rate R _p of splenic lymphocytes growth affected by the four ginsenosides were calculated. In accordance with thermo-kinetic model, the corresponding quantitative relationships of k, P _max, t _max, Q _t, R _p, and c were established, . Also, the median effective concentration (EC₅₀) was obtained by quantitative analysis. Based on both the quantitative quantity–activity relationships (QQAR) and EC₅₀, the sequence of promotion activity was Rc > Re > Rd > Rf. The analysis of structure–activity relationships showed that the number, type, and position of sugar moieties on the gonane steroid nucleus had important influences on the promotion activity of Rc, Re, Rd, and Rf on splenic lymphocytes growth. Microcalorimetry can be used as a useful tool for determining the activity and studying the quantity–activity relationship of drugs on cell.     

Investigation of the Effect of Four Organic Acids in Radix Isatidis on E. coli Growth by Microcalorimetry

The inhibitory effects of four organic acids (OAs) in Radix Isatidis, a traditional Chinese medicinal herb, on Escherichia coli (E. coli) growth were investigated by microcalorimetry. The power‐time curves of E. coli growth with and without OAs were acquired, meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by growth rate constants (k₁, k₂), half inhibitory ratio (IC₅₀), maximum heat output (P_max) and peak time (t_p). The values of k₁ and k₂ of E. coli growth in the presence of the four OAs decreased with the increasing concentrations of OAs. Moreover, P_max was reduced and the value of t_p increased with increasing concentrations of the four drugs. The sequence of anti‐microbial activity of the four OAs was: syringic acid>2‐amino‐benzoic acid>salicylic acid>benzoic acid. IC₅₀ of the four OAs was respectively 56 µg/mL for syringic acid, 75 µg/mL for 2‐amino‐benzoic acid, 86 µg/mL for salicylic acid and 224 µg/mL for benzoic acid. The existence of the functional groups on phenyl ring improves the anti‐microbial activity compared to benzoic acid. The functional groups methoxyl at C(3) and C(5) improve anti‐microbial activity more strongly than the other functional groups, and the functional group amino at C(2) improve anti‐microbial activity more strongly than hydroxyl at C(2) on phenyl ring.     

Studies on the nongrowth metabolism of the different strains of Tetrahymena cells by isothermal microcalorimetry

The nongrowth metabolic processes of Tetrahymena strains with the different cell densities were monitored by isothermal microcalorimetry, including Tetrahymena thermophila BF₁, Tetrahymena thermophila SB210, Tetrahymena pyriformis GL, the mixed cells of T. thermophila BF₁ and T. thermophila SB210, and the mixed cells of T. thermophila BF₁ and T. pyriformis GL. All the typical power–time curves showed a decreasing trend on the whole. It was found that total heat production (Q _t) and maximum heat output (P _m) decreased significantly with the decrease of cell density. Cell density did not influence significantly the maximum heat output per cell (P _cell) of Tetrahymena cells, which was probably due to enough oxygen. The P _cell and metabolic decrease rate constant (K) values for the mixture of T. thermophila BF₁ and T. pyriformis GL, and that of T. thermophila BF₁ and T. thermophila SB210 were similar to the mean values of P _cell and K for the corresponding single Tetrahymena strain, respectively. It was speculated that the cell mixing did not possibly influence significantly their P _cell and K values because of no competition for nutrition, oxygen, and room, and the low conjunction percentages.     

Isothermal microcalorimetric investigation of the toxic action of the effective constituents of Podophyllum emodi Wall on Tetrahymena thermophila BF5

The effects of podophyllotoxin (PPT), etoposide (VP16), and teniposide (VM26) on the growth of Tetrahymena thermophila BF₅ (T.t.BF₅) was investigated by the TAM AIR isothermal microcalorimetric system. The extent and duration of toxic effects on T.t.BF₅ metabolism were evaluated by studying the growth rate constant (k), inhibitory ratio (I), maximum heat-output power (P _max), peak time of maximum heat-output power (t _p), and total heat production (Q _t). Experimental results showed that the value of t _p increased and the value of k and P _max decreased with the increasing compound concentrations. Furthermore, the growth rate constant k was linear with compound concentration. The corresponding I was obtained from different k values. According to the IC₁₀ (the concentration of inhibitor when the inhibitory ratio is 10 %), the relative toxicity of the three compounds was PPT (IC₁₀ = 49.6 μg mL^?1) > VP16 (IC₁₀ = 117.5 μg mL^?1) > VM26 (IC₁₀ = 359.1 μg mL^?1). The preliminary investigation of structure–activity relationships showed that the thienyl group was likely responsible for reducing the toxicity of the compounds.     

Microcalorimetric study of the opposing effects of ginsenosides Rg<Subscript>1</Subscript> and Rb<Subscript>1</Subscript> on the growth of mice splenic lymphocytes

Splenic lymphocytes play an important role in host acute or chronic diseases. The abnormality of these cells in the spleens of humans might lead to some riskful diseases for human. Hence, in this study, the effects of two ginsenosides Rg₁ and Rb₁ on splenic lymphocytes growth were studied by microcalorimetry. Some qualitative and quantitative information, such as the metabolic power-time curves, growth rate constant k, maximum heat-output power of the exponential phase P _max, total heat output Q _t of splenic lymphocytes were obtained to present the effects of Rg₁ and Rb₁ on these cells. The values of k, P _max, and Q _t from the thermogenic growth curves of splenic lymphocytes were found to increase in the presence of Rg₁, while the change was adverse for Rb₁, illustrating that Rg₁ had promotion effect and Rb₁ had inhibitory effect on splenic lymphocytes growth and these promotion or inhibitory effects were enhanced with increasing the concentration of the two compounds, respectively. The microcalorimetric results were confirmed by MTT assay for determining the MTT optical density (OD) value and [³H] Thymidine incorporation assay ([³H]-TdR) for determining the count per minute (cpm) value: Rg₁ could increase the MTT OD value and the cpm value of [³H]-TdR incorporation into splenic lymphocytes, and these values were increased with increasing the concentration of this compound, while Rb₁ had the adverse results. The structure–activity relationships showed that the glucopyranoside and hydroxyl groups at the dammarane-type mother nucleus skeleton might play a crucial role for the opposing effects of the two ginsenosides on splenic lymphocytes. Compared with the other two assay methods, the microcalorimetric method provided more useful and reliable information for quickly and objectively evaluating the effects of drugs or compounds on the living cells, which would be a highly promising analytical tool for the characterization of the biological process and the estimation of the drugs’ efficiency.     

Microcalorimetric Investigation of Toxic Effects of Three Injectable Solubilizing Excipients on Tetrahymena thermophila BF5 Growth

Using microcalorimetry, thermal metabolic curves of Tetrahymena thermophila BF₅ (T. thermophila BF₅) growth at 28°C affected by three injectable solubilizing excipients (ISE) including tween 80, hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) and poloxamer 188 were measured. Meanwhile, the toxicities of three ISE were evaluated by dynamic parameters of thermal metabolic curves. In addition, the irritative effects of the ISE on myoblast L₆ cells were investigated to show their cytotoxicities by biochemical method. The results indicated that the effects of the ISE on T. thermophila BF₅ varied for different ISE. 5% inhibition concentration values (IC₅) of the ISE were 1.33, 1.83 and 1.64 mg/mL for tween 80, HP‐β‐CD and poloxamer 188, respectively. By the principal component analysis (PCA), the total quantity of heat (Q), growth rate constant (k) and second maximum power (P₂) were selected as the main characteristic parameters to present their toxicities, there were good linear relationships between Q, k, P₂ and concentrations c, suggesting that the toxicities of the ISE on T. thermophila BF₅ were closely linked to their concentrations. The results of creatine kinase (CK) bioassay of myoblast L₆ cells indicated that the sequence of irritative effects of the ISE was HP‐β‐CD相似文献   

Microcalorimetric investigation of six alkaloids from Radix Aconite Lateralis Preparata (Fuzi) on the metabolic activity of mitochondria isolated from rat liver

Using the TAM air isothermal microcalorimeter, the thermogenic power–time (HP–time) curves of isolated mitochondrial energy metabolism were measured, and the effects of six alkaloids from Radix Aconite Lateralis Preparata (Fuzi) including mesaconitine, aconitine, hypaconitine, benzoylmesaconine, benzoylaconine, and benzoylhypaconine on mitochondrial energy metabolism were investigated. Some quantitative information, such as k, P _max, T _max, Q, P _av, and T _lag, was obtained from the HP–time curves. The results revealed that mesaconitine, aconitine, and hypaconitine had inhibitory effect on rat-isolated mitochondrial energy metabolism, and their IC₅₀ value was 1.82, 2.22, and 2.48 μg mL⁻¹, respectively, while benzoylmesaconine, benzoylaconine, and benzoylhypaconine promoted mitochondrial energy metabolism, and their EC₅₀ value was 30.95, 14.36, and more than 30 μg mL⁻¹, respectively. Further structure–activity relationship analysis showed that the ester bond in C(8) position, hydroxyl in C(3) position, and ethyl in N atom could significantly influence the energy metabolism. These results provide scientific basis for the treatment of mitochondrial abnormalities and energy dysfunction diseases using Fuzi, and it will be of great interest to doctors and pharmacists.

     

微量热法研究亚砷酸钠对鲫鱼肝脏线粒体的影响

Metabolic thermogenic curves of mitochondria isolated from liver tissue of Carassius auratus and the effect of different concentration of NaAsO2 on it were investigated by TAM air isothermal microcalorimeter, ampoule method, at 28.00 ℃. From the thermogenic curves, activity recovery rate constants k, the maximum heat production rate （Pmax） and the total heat produced （Q） were obtained. The values of k and Pmax decline gradually with the increase of the concentration of NaAsO2, and both of the values of Pmax and k are highly correlated to the concentration of NaAsO2. When concentration of NaAsO2 reached 16.0 μg/mL, the maximum heat production rate dropped to 70.8% of the control group, and the corresponding percentage of k was 71.4% of the control group. This experimental result indicates that the addition of NaAsO2 has restrained the metabolic activities of mitochondria in vitro.     

Microcalorimetric investigation of the antibacterial activity of curcumin on Staphylococcus aureus coupled with multivariate analysis

The antibacterial effect of Curcumin on Staphylococcus aureus growth was evaluated by microcalorimetry. The heat flow power?Ctime curves and nine quantitative parameters of the S. aureus growth were applied to investigate the inhibitory effect with Curcumin. By analyzing these curves and some quantitative parameters using multivariate analytical methods, similarity analysis and principal component analysis, the antibacterial activity of Curcumin on S. aureus could be accurately evaluated from the change of the two main parameters, the second exponential growth rate constant k ₂ and the maximum heat flow power P _m ² . The main two thermal parameters played more important role in the evaluation: at low concentration (0?C10.5???g?mL^?1), Curcumin hardly influence the growth of S. aureus, while at high concentration (10.5?C43.4???g?mL^?1) it could notably inhibit the growth. All these illustrated that the antibacterial activity of Curcumin on S. aureus was enhanced with the increase of the concentration of this compound. This study might provide an useful method and idea accurately evaluate the antibacterial effects of Curcumin, which provides some useful methods for evaluate the nature antibacterial agents.     

Microcalorimetric studies on the antimicrobial actions of different cephalosporins

Microcalorimetry was applied to study the effect of cephalosporins (cefazolin sodium and cefonicid sodium) on the E. coli growth. The microbial activity was recorded as power-time curves through an ampoule method with a TAM Air Isothermal Microcalorimeter at 37°C. The parameters such as the growth rate constant (k), inhibitory ratio (I), the maximum power output (P _m) and the time corresponding to the maximum power output (t _m) were calculated. The change tendencies of k, with the increasing of concentration (C) of the two cephalosporins, are similar which show that cefazolin sodium and cefonicid sodium have the same inhibitory mechanism. The experimental results reveal that cefonicid sodium has a stronger antibacterial activity towards E. coli than that of cefazolin sodium and this was coincide with the clinical manifestations.     

A microcalorimetric study of the biologic effect of Mn(II) on Bacillus thuringiensis growth

A microcalorimetric technique based on the bacterial heat-output was explored to evaluate the effect of Mn(II) on Bacillus thuringiensis. The power-time curves of the growth metabolism of B. thuringiensis and the effect of Mn(II) on it were studied using an LKB-2277 BioActivity Monitor, ampoules method, at 28°C. For evaluation of the results, the maximum peak-heat output power (P _max) in the growth phase, the growth rate constants (k), the log phase heat effects (Q _log ), and the total heat effect in 23 h (Q _T) for B. thuringiensis were determined. Manganese has been regarded as the essential biological trace element. Mn(II) of different concentration have different effects on B. thuringiensis growth metabolism. High concentration (800-1600 μg mL^-1) of Mn(II) can promote the growth of B. thuringiensis; low concentration (500-800 μg mL^-1) can inhabit its growth. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microcalorimetric performance of the growth in culture of Escherichia coli,Proteus mirabilis and their mixtures in different proportions

Microcalorimetry is a technique that determines the heat flow produced as a result of microbial activities. The heat variations resulting from chemical reactions, which take place during metabolism, can be used to monitor bacterial growth in a culture medium. However, there are very few studies using calorimetry to investigate the relationships between two bacteria. In this work, we studied the interaction between E. coli and P. mirabilis, two bacteria belonging to the family Enterobacteriaceae. We have prepared three samples, mixing both enterobacteria at a concentration of 10³ CFU mL^?1 but in different proportions. Experimental equipment used was a Calvet microcalorimeter, where a constant temperature of 309.65 K was maintained. Then, we compared the shape of the heat flow–time curves of single microorganisms and their mixtures. Also, we calculated the thermokinetic parameters such as growth constant (k), generation time (G), detection time (t _d) and the amount of heat released (Q). The results obtained showed that when E. coli and P. mirabilis were put together in the culture medium, the growth profile of P. mirabilis seemed to dominate, even at low proportions in the sample.