Abstract
In this paper, crude monkshood polysaccharide was isolated from Radix Aconiti Lateralis Preparata. The effects of crude monkshood polysaccharide on Escherichia coli and Staphylococcus aureus were investigated by microcalorimetry. The power-time curves of the bacterial growth at various concentrations (c) of crude monkshood polysaccharide were plotted with a TAM air isothermal microcalorimeter at 37 °C. The growth rate constant (μ), inhibitory ratio (I), peak-height (P m), and peak-time (t m) were calculated. From the data, the relationship between μ and c also was established. The growth rate constant μ decreased with the increasing concentrations of crude monkshood polysaccharide. Moreover, P m reduced and t m increased with increasing concentrations. The experimental results revealed that crude monkshood polysaccharide had inhibitory activity towards S. aureus and E. coli. Results obtained from our study strongly suggest that microcalorimetry is a fast, simple, and more sensitive technology that can be easily performed to study the effect of drugs on bacteria.
Keywords: Crude monkshood polysaccharide, Microcalorimetry, Escherichia coli, Staphylococcus aureus, Inhibitory
1. Introduction
Radix Aconiti Lateralis Preparata (also known as monkshood or aconite), is the daughter root of Aconitum carmichaeli Debx. It is a traditional Chinese medicinal (TCM) herb that belongs to the family of Ranunculaceae. It has been reported that Radix Aconiti Lateralis Preparata presents some pharmacological activities, including reviving yang for resuscitation, alleviating pain, anti-inflammatory activity, hypoglycemic effects, and so on (China Pharmacopoeia Committee, 2005). In recent research, more attention has been paid to the polysaccharides. Some activities of crude monkshood polysaccharide have been reported, including immunological activity (Li et al., 2008), anti-cancer activity (Ren et al., 2008), and hypoglycemic effect (Konno et al., 1985; Yu and Wu, 2009). Few studies concerning the effect of crude monkshood polysaccharide on bacteria have been reported. Thus, our aim is to investigate this further.
Microcalorimetry, as a quantitative, inexpensive, and versatile method for measuring heat production, has been successfully applied in biochemistry, biophysics, and environmental sciences in recent years (Wadsö, 1995; 1997; 2001). It allows for interaction in a heterogeneous medium, monitoring the process without disturbing the system, measuring the thermal effect of the system, and giving abundant thermodynamic and kinetic information (Critter et al., 2001; Yan et al., 2007). It is a good method to study the effect of drugs on bacteria. The heat output can be recorded in real time. Then the power-time curves of bacteria can be plotted. Using a mathematical growth model, a series of kinetic parameters can be calculated, such as the growth rate constant (μ), generation time (t), inhibitory ratio (I), minimal inhibitory concentration (MIC), peak-height (P m), peak-time (t m), and total heat (Q tot). Microcalorimetry appears as a suitable technique to study the microbial activity (Li et al., 2001; 2002; Wadsö, 2002; Wu et al., 2005; Kong et al., 2008). The purpose of this study is to investigate the effect of crude monkshood polysaccharide on Escherichia coli and Staphylococcus aureus by microcalorimetry.
2. Materials and methods
2.1. Instrument
A 3114/3236 TAM air microcalorimeter (Thermometric AB, Sweden) was used to determine the metabolism of E. coli and S. aureus. The TAM air microcalorimeter is an eight-channel heat conduction calorimeter for heat flow measurements under isothermal conditions. Reactions could be carried out in the temperature range 5 to 90 °C. The temperature error was ±0.02 °C. The detection limit was 2 µW and baseline stability was 2×10−6 µW over a period of 24 h (Kong et al., 2008; Yang et al., 2008).
2.2. Materials
Crude monkshood polysaccharide was isolated from Radix Aconiti Lateralis Preparata through water extraction and alcohol precipitate, and sequentially purified through Sevage method and dialysis (Yang et al., 2007). It was a white powder, easily soluble in water.
Bacteria E. coli (CMCC (B) 44102) and S. aureus (CMCC (B) 26003) were used as the tested bacteria, provided by the Shandong Institute for Drug Control. They were routinely cultured in a Luria-Bertani (LB) culture medium, which contained 5 g/L NaCl, 5 g/L yeast extract, and 10 g/L peptone. Medium pH was adjusted to 7.0–7.2. The LB culture medium was sterilized by autoclaving at 121 °C for 20 min before the experiment.
2.3. Methods
The microcalorimetric measurement was made with the ampoule method. The LB culture medium, containing bacteria, was placed in 20 ml glass ampoules. Then different concentrations of crude monkshood polysaccharide were inoculated into each ampoule with a final volume of 10 ml. One ampoule without crude monkshood polysaccharide was used as the blank control. The ampoules were sealed and put into an eight-channel calorimeter block. The temperature was controlled at 37 °C. The power-time signals were recorded at an interval of 1 min until the recorder returned to the baseline (Burt, 2004; Yang et al., 2008).
3. Results and discussion
3.1. Thermokinetics
In the growth phase, theoretical model is in accordance with the following law (Burt, 2004):
| dNt/dt=μNt−βNt2, | (1) |
where Nt is the bacterial number at time t, μ is the growth rate constant, and β is the fungi static rate constant. The integral of Eq. (1) is given by
| Nt=K/(1+αe−μt), | (2) |
where K is the maximum density, and α is integral constant. If the power produced by every bacterium is P, then we can obtain PNt=KP/(1+αe−μt). Making Pt=PNt and P m=PK, where Pt is the power output at time t, and P m the maximum power output, then Eq. (2) can changed to
| Pt=Pm/(1+αe−μt). | (3) |
Eq. (3) is the logistic equation. According to the data Pt and t obtained from the bacterial growth curves, the rate constant μ can be calculated.
The inhibitory ratio I is an excellent index to indicate the inhibition of crude monkshood polysaccharide on E. coli and S. aureus, and it can be defined as:
| I=[(μ0−μc)/μ0]×100%, | (4) |
where μ 0 and μ c are the growth rate constants of bacteria without and with crude monkshood polysaccharide, respectively.
The corresponding values of μ, t m, P m, and I are shown in Table 1.
Table 1.
Parameters of bacteria growth at different concentrations of crude monkshood polysaccharide
| Bacteria | c (mg/ml) | μ (min−1) | r | tm (min) | Pm (mW) | I (%) |
| E. coli | 0 | 0.3584 | 0.9998 | 80 | 1.2313 | |
| 0.1 | 0.3387 | 0.9998 | 81 | 1.2226 | 5.50 | |
| 0.4 | 0.3000 | 0.9997 | 89 | 1.1938 | 16.29 | |
| 0.8 | 0.2770 | 0.9997 | 98 | 1.1678 | 22.71 | |
| 1.6 | 0.2516 | 0.9997 | 100 | 1.1410 | 29.80 | |
| 2.0 | 0.2341 | 0.9998 | 111 | 0.9403 | 34.68 | |
| S. aureus | 0 | 0.2380 | 0.9998 | 120 | 0.4860 | |
| 0.2 | 0.2068 | 0.9997 | 122 | 0.4757 | 13.11 | |
| 0.4 | 0.1734 | 0.9997 | 133 | 0.4438 | 27.14 | |
| 0.8 | 0.1380 | 0.9996 | 140 | 0.4226 | 42.02 | |
| 1.6 | 0.1256 | 0.9998 | 143 | 0.4006 | 47.22 | |
| 2.0 | 0.1150 | 0.9999 | 154 | 0.3900 | 51.68 |
c: concentration of monkshood polysaccharide; μ: growth rate constant; r: correlation coefficient; t m: peak-time value; P m: maximum heat output; I: inhibitory ratio
3.2. Power-time curves
The power-time curves of E. coli and S. aureus growths of black control were plotted in Fig. 1. We could see that the shapes of the curves were different. The focal points were power-time curves for the exponential growth, which were plotted in Fig. 2. It could be seen that the increase of the heat output became slower with increasing concentration of crude monkshood polysaccharide. High concentration of crude monkshood polysaccharide needed more time to reach the same heat output.
Fig. 1.
Power-time curves of E. coli (a) and S. aureus (b)
Fig. 2.
Power-time curves for the exponential growths of E. coli (a) and S. aureus (b) affected by various concentrations of crude monkshood polysaccharide
3.3. Relationship between μ and c
The values of the growth rate constant μ in Table 1 showed that crude monkshood polysaccharide had potent antibacterial activity against E. coli and S. aureus. The relationships between μ and c were demonstrated in Fig. 3. The μ of the bacterial growth declined with increasing of the c. That was mainly because some bacteria were killed, and some metabolized continuously at a lower heat production rate. This rate directly depended on the concentration of crude monkshood polysaccharide. The correlations between μ and c could be formulated according to following equations: for E. coli: μ=−0.0324c 3+0.1252c 2−0.1822c+0.3569 (r=0.9993); for S. aureus: μ=−0.0325c 3+0.1433c 2−0.2189c+0.2406 (r=0.9984).
Fig. 3.
Relationship between rate constant μ and concentration c for E. coli (a) and S. aureus (b)
3.4. Relationship between I and c
According to Eq. (4) and the relation between μ and c, the inhibitory ratio I was calculated. As shown in Table 1, the inhibition ratio increased with increasing concentration of crude monkshood polysaccharide. It demonstrated that crude monkshood polysaccharide had inhibitive effect on E. coli and S. aureus. At the same concentration, the inhibitory ratio I was larger on S. aureus than on E. coli. The phenomenon illustrated that crude monkshood polysaccharide had a greater inhibitive effect on S. aureus than on E. coli.
3.5. Relationship between P m and c
From Table 1, it could be seen that the max heat output P m with crude monkshood polysaccharide was less than that of blank control, and a decrease in the value of P m was observed when c was increasing. The main reason could be that the number of the survivors decreased with increasing concentration of crude monkshood polysaccharide. There was a similar effect on E. coli and S. aureus. The results indicated that crude monkshood polysaccharide had an inhibitory effect on the growths of E. coli and S. aureus.
3.6. Relationship between t m and c
Experimental results for the t m as a function of c are presented in Table 1. The peak-time t m of crude monkshood polysaccharide was longer than that of blank control. With increasing concentration of crude monkshood polysaccharide, the t m prolonged. This was mainly because, after the treatment with crude monkshood polysaccharide, there was a partial inhibition of the bacteria and the remaining survivors maintained growth and metabolized at a lower rate. There was a similar effect on E. coli and S. aureus. The results also indicated that crude monkshood polysaccharide had an inhibitory effect on the growths of E. coli and S. aureus.
4. Conclusions
As shown in this work, the inhibitory effect of crude monkshood polysaccharide on the growths of E. coli and S. aureus was found by microcalorimetry. The power-time curves of E. coli and S. aureus at different concentrations of crude monkshood polysaccharide were plotted. The parameters such as the growth rate constant (μ), inhibitory ratio (I), peak-height (P m), and peak-time (t m) were calculated. The relationships between μ and c were also established. The μ decreased with increasing concentration of crude monkshood polysaccharide. Crude monkshood polysaccharide showed stronger inhibitory effect on S. aureus than on E. coli. Microcalorimetry is a useful technique that can be applied to study microbial growth and estimate the efficiency of drugs.
Footnotes
Project supported by the Natural Science Foundation of Shandong Province (No. Y2007C141), the Independent Innovation Foundation of Shandong University (No. 2010TS041), and the Award Program for Outstanding Young Scientist of Shandong Province (No. 2007BS02002), China
References
- 1.Burt S. Essential oils: their antibacterial properties and potential applications in foods—a review. Int J Food Microbiol. 2004;94(3):223–253. doi: 10.1016/j.ijfoodmicro.2004.03.022. [DOI] [PubMed] [Google Scholar]
- 2.China Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China. Beijing, China: China Chemical Industry Press; 2005. pp. 132–133. (in Chinese) 1st Div. [Google Scholar]
- 3.Critter SAM, Freitas SS, Airoldi C. Calorimetry versus respirometry for the monitoring of microbial activity in a tropical soil. Appl Soil Ecol. 2001;18(3):217–227. doi: 10.1016/S0929-1393(01)00166-4. [DOI] [Google Scholar]
- 4.Kong WJ, Zhao YL, Shan LM, Xiao XH, Guo WJ. Microcalorimetric studies of the action on four organic acids in Radix isatidis on the growth of microorganisms. Chin J Biotechnol. 2008;24(4):646–650. doi: 10.1016/S1872-2075(08)60033-3. [DOI] [PubMed] [Google Scholar]
- 5.Konno C, Murayama M, Sugiyama K, Arai M, Murakami M, Takahashi M, Hikino H. Isolation and hypoglycemic activity of aconitans A, B, C and D, glycans of Aconitum carmichaeli roots. Planta Med. 1985;51(2):160–161. doi: 10.1055/s-2007-969436. [DOI] [PubMed] [Google Scholar]
- 6.Li FS, Xu HG, Li MY, Liu H. Study on extraction of polysaccharide from Rhizoma Typhonium Gigantei and its immunological properties. Modern Prev Med. 2008;35(12):2290–2295. (in Chinese) [Google Scholar]
- 7.Li X, Liu Y, Wu J, Qu SS. The effect of the selenomorpholine derivatives on the growth of Staphyylococcus aureus studied by microcalorimetry. Thermochim Acta. 2001;375(1-2):109–113. doi: 10.1016/S0040-6031(01)00515-9. [DOI] [Google Scholar]
- 8.Li X, Liu Y, Wu J, Liang HG, Qu SS. Microcalorimetric study of Staphylococcus aureus growth affected by selenium compounds. Thermochim Acta. 2002;387(1):57–61. doi: 10.1016/S0040-6031(01)00825-5. [DOI] [Google Scholar]
- 9.Ren LY, Gao LL, Li Y, Zeng SP. The research advances in the monkshood polysaccharide. J Practical Tradit Chin Med. 2008;24(6):406–407. (in Chinese) [Google Scholar]
- 10.Wadsö I. Microcalorimetric techniques for characterization of living cellular systems. Will there be any important practical applications? Thermochim Acta. 1995;269/270:337–350. [Google Scholar]
- 11.Wadsö I. Isothermal microcalorimetry near ambient temperature: an overview and discussion. Thermochim Acta. 1997;294(1):1–11. doi: 10.1016/S0040-6031(96)03136-X. [DOI] [Google Scholar]
- 12.Wadsö I. Isothermal microcalorimetry: current problems and prospects. J Therm Anal Calorim. 2001;64(1):75–84. doi: 10.1023/A:1011576710913. [DOI] [Google Scholar]
- 13.Wadsö I. Isothermal microcalorimetry in applied biology. Thermochim Acta. 2002;394(1-2):305–311. doi: 10.1016/S0040-6031(02)00263-0. [DOI] [Google Scholar]
- 14.Wu YW, Gao WY, Xiao XH, Liu Y. Calorimetric investigation of the effect of hydroxyanthraquinones in Rheum officinale Baill on Staphylococcus aureus growth. Thermochim Acta. 2005;429(2):167–170. doi: 10.1016/j.tca.2005.03.008. [DOI] [Google Scholar]
- 15.Yan D, Jin C, Xiao XH, Dong XP. Investigation of the effect of berberines alkaloids in Coptis chinensis Franch on Bacillus shigae growth by microcalorimetry. Sci China Ser B: Chem. 2007;50(5):638–642. doi: 10.1007/s11426-007-0094-9. [DOI] [Google Scholar]
- 16.Yang F, Hao AG, Xu HG, Li FS, Li MY. Determination of polysaccharide content of Rhizoma Typhonium Gigantei . J Dalian Med Univ. 2007;29(5):453–454. (in Chinese) [Google Scholar]
- 17.Yang LN, Xu F, Sun LX, Zhao ZB, Song CG. Microcalorimetric studies on the antimicrobial actions of different cephalosporins. J Therm Anal Calorim. 2008;93(2):417–421. doi: 10.1007/s10973-007-8680-9. [DOI] [Google Scholar]
- 18.Yu L, Wu WK. The monkshood polysaccharide’s infection on the insulin resistance of uptake of glucose for fat cell model. Asia-Pacific Tradit Med. 2009;5(7):11–12. (in Chinese) [Google Scholar]