Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Investigation of the anti-fungal activity of coptisine on Candida albicans growth by microcalorimetry combined with principal component analysis
W.-J. Kong1,2, Y.-L. Zhao1, X.-H. Xiao1, Z.-L. Li2, C. Jin1 and H.-B. Li1
1 PLA Institute of Chinese Materia Medica, Hospital of Peoples Liberation Army, Beijing, China 2 Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu Sichuan, China

Keywords anti-fungal activity, Candida albicans, coptisine, microcalorimetry, principal component analysis. Correspondence Xiao-He Xiao, PLA Institute of Chinese Materia Medica, 302 Hospital of Peoples Liberation Army, 100 West 4th Ring Middle Road, Beijing 100039, China. E-mail: kongwj302@126.com

Abstract Aims: This study investigated the anti-fungal activity of coptisine on Candida albicans growth. Methods and Results: The metabolic power-time curves of Candida albicans growth at 37C affected by coptisine were measured by microcalorimetry using an LKB-2277 Bioactivity Monitor with stop-ow mode. Then, the diameter of inhibitory zones in the agar layer was observed using agar cup method, and the minimal inhibitory concentration (MIC) of coptisine on Candida albicans growth was determined by serial dilution method. From the principal component analysis on nine quantitative parameters obtained from the power-time curves, we could easily evaluate the anti-fungal activity of coptisine by analysing the change of values of the main two parameters, growth rate constant k and maximum power output in the log phase Pm, log. The results showed that coptisine had strong anti-fungal activity: at a low concentration (45 lg ml)1) began to inhibit the growth of Candida albicans and at a high concentration (500 lg ml)1) completely inhibited Candida albicans growth. Coptisine gave big inhibitory zones with diameters between 11 and 43 mm within test range, and the MIC of it was 1000 lg ml)1. Conclusions: Coptisine had strong anti-fungal activity on Candida albicans growth. The method of microcalorimetry applied for the assay of anti-fungal activity of coptisine was quantitative, sensitive and simple. Signicance and Impact of the Study: This work will provide useful information for the development of chemical biology policy in the use of anti-microbials in food and drug production. automatically and continuously, the microcalorimetric method could reveal more qualitative and quantitative information about the metabolism of the living system (Critter et al. 2004; Yao et al. 2005) than the existing methods, for example, serial dilution, micro-dilution, antimicrobial circle and agar cup method (Batovska et al. 2007; Leng et al. 2007) do. It has recently been demonstrated that calorimetric methods can be used for fundamental studies of bacterial growth (Liu et al. 1996). Candida albicans (C. albicans) is the major human pathogen giving rise to opportunistic oral and vaginal infections. It is a leading cause of invasive fungal disease in premature infants, diabetics, surgical, AIDS and other

2008 2041: received 27 November 2008, revised 22 January 2009 and accepted 26 January 2009
doi:10.1111/j.1365-2672.2009.04292.x

Introduction In any living systems including fungi, the various metabolic events occur within the cells involving heat-producing reactions. Therefore, by monitoring the heat effects with sufciently sensitive calorimeters, the metabolic processes of living cells can be observed and it can be seen that the thermogenic curve reects time-dependent changes in growth patterns (Yao et al. 2003). Thus, the calorimeter is a powerful tool for observing living cells, with which information on their metabolism can be obtained. As of high sensitivity and accuracy of the monitor and the fact that the whole metabolism of the living system can be examined
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immunocompromised patients (Batovska et al. 2007). In these cases, the intensive prophylactic use of anti-fungal drugs such as azoles (Zhou and Zhou 2006), plagiochin E (Leng et al. 2007) leads to emergence of resistant strains of C. albicans. This causes a great concern for nding suitable new therapeutics and anti-fungal compounds and drugs. Also, the study on the metabolic process of C. albicans with and without drugs using suitable method is important for researching the mode of action of C. albicans, the anti-fungal compounds and drugs and the antifungal activities of these compounds and drugs. Coptisine, an isoquinoline alkaloid originally isolated from rhizome of Coptis japonica, has extensive pharmacological actions including anti-microbial (Cui et al. 2006; Park et al. 2006; Yan et al. 2008), anti-cancer (Tanabe et al. 2006; Ma and Yang 2007) and anti-oxidative (Ro et al. 2001) effect, etc. Moreover, the protection effect of coptisine on gastricmucous membrane has also been reported (Li et al. 2007). Although the anti-bacterial activities of coptisine on Staphylococcus aureus, Shigella dysenteriae growth, et al. by slip diffusion and anti-microbial circle method (Yang et al. 2007) has been reported, the anti-fungal activity of coptisine on C. albicans growth investigated by microcalorimetry has not been reported. In recent years, the application of microcalorimetry in biochemistry, biophysics and environmental science has drawn increasing attention. It allows the study of biology at the molecular and cellular levels with the power-time curves generating many kinetic information. By analysis of the power-time curves, kinetic parameters, such as rate constant for bacterial growth, peak power for microbial activity, can be obtained and the actions of compounds and drugs on the microbes can be evaluated (Zhao et al. 2004; Zhang et al. 2006; Kong et al. 2008a,b; Yao et al. 2008). So, in this study, an LKB-2277 bioactivity monitor has been applied to investigate the anti-fungal activity of coptisine on C. albicans growth. Meanwhile, the agar cup method was used to observe the diameter (d) of inhibitory zone, and serial dilution method was used to determine the minimal inhibitory concentration (MIC) of coptisine on C. albicans growth. Our objective is to evaluate the anti-fungal activities of coptisine and provide a simple, fast, useful and sensitive method and more foundations for the study of anti-fungal activity of other compounds and materials. Materials and methods Instrument A microcalorimeter, LKB-2277 Bioactivity Monitor manufactured by LKB corporation (Bromma, Sweden) was used to obtain the metabolic power-time curves of

C. albicans growth. The microcalorimeter was thermostated at 37C. A differential or twin detector system was used to minimize the systematic error and disturbance effect. This system is very sensitive; the detection limit is 015 lW and the baseline stability (over a period of 24 h) is 02 lW. In the monitoring system, two precision resistors for electrical calibration are built into each measuring cylinder, one for each detector. When a known electronic current is passed through the appropriate resistor, the detector can be calibrated easily. Other methods for calibration include suitable internally calibrated radioactive sources and chemical reactions. Using one of these techniques, a calorimetric constant can be evaluated. The time constant (f) of this instrument is about 120 s. For details of the performance and structure of the instrument, see the instruction and report of Xie et al. (Xie et al. 1988). Materials Strain C. albicans (64550) was provided by China Center of Type Culture Collection (Wuhan University, Wuhan 430072, P. R. China). It was grown in Lactose Broth (LB) culture medium which was a solution per 1000 ml (pH 7274) containing: NaCl 5 g, peptone 10 g, beef extract 6 g and was sterilized in high pressure steam at 121C for 30 min. Coptisine was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100051, P.R. China. The purities of coptisine exceeded 98%, and its structure was given in Fig. 1. Water was puried using a Milli-Q water purication system (Milipore, Bedford, MA, USA). All other chemicals are of analytical purity. Microcalorimetric study The power-time curves of C. albicans growth at 37C with and without coptisine were measured by this microcalorimeter using ow-stop mode. In all of the microcalorimetric experiments, the equipment was thermostated at

O N

O
Figure 1 The structure of coptisine. Coptisine is an isoquinoline alkaloid with quaternary nitrogen atom and two methylenedioxy groups at the aromatic ring.

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37C, and the ow-mixed cell was completely cleaned and sterilized. The clean procedure was as follows: sterilized distilled water, 01 mol l)1 HCl, 01 mol l)1 NaOH and sterilized distilled water were pumped into this microcalorimeter in sequence by an LKB-2132 microperpex peristaltic pump through the cell, each for 15 min at a ow rate of 50 ml h)1. After the system was cleaned and sterilized and the baseline had been stabilized, the bacterial suspension, initially containing 2 106 cells ml)1 and coptisine solution at different concentrations was pumped into this microcalorimeter through the calorimetric cell with an LKB-2132 perpex peristaltic pump at a ow rate of 50 ml h)1. When the ow cell (volume 06 ml) was lled, the pump was stopped and the monitor was used to record the power-time curves of C. albicans growth. In this experiment, the C. albicans were suspended in the LB culture medium. Coptisine solution was added at the beginning of the experiment, i.e., it was introduced as soon as the C. albicans were inoculated in the culture medium. The solutions of coptisine were prepared in sterilized distilled water at different concentrations and were prepared freshly each time. Agar cup method Two hundred microlitre suspension of the fungus (107 cells ml)1) was plated on agar layer in Petri dishes (10 cm in diameter). The sample solution of coptisine was diluted, respectively, with LB culture medium to get the diluted solution of 2000, 1000, 500, 250, 125, 625, 3125 lg ml)1. Five wells per dish were prepared, each 10 mm in diameter. One hundred microlitre of each diluted solution was added to the appropriate well. For prediffusion, the Petri dishes were placed at 4C for 2 h. Positive control experiments were carried out with microbial suspension and no coptisine, while blank group with coptisine and no microbial suspension in the pure culture medium. The anti-fungal activity was estimated by the diameter (d) of inhibitory zones in the agar layer after incubation at 37C for 48 h (Spooner et al. 1972). Measurement of minimal inhibitory concentration (MIC) MIC was determined by serial dilution of each coptisine sample solution (002000 lg ml)1) in test tubes using fresh LB culture medium. Each test tube was inoculated with fungal suspension containing 105 cells ml)1 and incubated at 37C for 24 h. The lowest dilution that visibly showed no growth compared with drug-free culture medium inoculated with microbial suspension was considered the MIC. For more precise detection, tubes that showed no visible growth were streaked on fresh LB agar
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plates, incubated at 37C for 24 h and checked for growth (Hindler and Isenberg 1992; Yang et al. 2007). Principal component analysis (PCA) on quantitative thermogenic parameters From the thermogenic power-time curves of C. albicans growth, many quantitative parameters could be obtained to represent the anti-fungal activity of this compound. To reduce the parameters and nd the change potency of this anti-fungal activity, the main parameters, which best and fastest reected this potency, should be obtained. PCA (Mardia et al. 1979) is a sophisticated technique widely used for reducing the dimensions of multivariate problems. As a nonparametric method of classication, it makes no assumptions about the underlying statistical data distribution (Karisa et al. 2005; Kannel et al. 2007). It reduces the dimensionality of the original dataset by explaining the correlation amongst a large number of variables in terms of a smaller number of underlying factors (principal components or PCs), without losing much information (Jackson 1991). Here, PCA was performed on the many quantitative parameters obtained from the thermogenic power-time curves to nd out the main parameters using software of Ubscrambler 97 from Camo AS (Trondheim, Norway). By analysing the main parameters, the anti-fungal activity and the action potency of this compound could be represented well and swiftly. Results Power-time curves of C. albicans growth The thermogenic power-time curve of C. albicans growth at 37C without any substance was shown in Fig. 2. It was a typical power-time curve of C. albicans and could
4 Log phase 2 0 Decline phase 2 4 0 Lag phase 100 200 t (min) 300 400 Stationary phase

Figure 2 The power-time curves of C. albicans growth at 37C without any substance. It was a typical power-time curve of C. albicans culturing in LB culture medium supplemented without any substance.

2009 The Authors Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 10721080

ln P

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The anti-fungal activity of coptisine on C. albicans growth

40 a 30 P (W) 20 10 0 0 150 b d f g h k 300 450 t (min) 600 c e i j

Table 1 Thermo-kinetic equations for C. albicans growth affected by different concentrations of coptisine at 37C
c (lg ml)1) 0 45 90 135 180 225 270 315 360 405 500 ln P$t ln ln ln ln ln ln ln ln ln ln P P P P P P P P P P = = = = = = = = = = )46935 + 005692 t )46714 + 004921 t )52080 + 004597 t )30892 + 004140 t )36655 + 003852 t )28272 + 003453 t )45354 + 002986 t )6.3480 + 002535 t )98722 + 002152 t )48717 + 001678 t k(min)1) 005692 004921 004597 004140 003852 003453 002986 002535 002152 001678 R* 09978 09998 09990 09990 09984 09974 09986 09976 09978 09964

Figure 3 The power-time curves of C. albicans growth affected by different concentrations of coptisine. The concentrations of coptisine were 0 lg ml)1 (a), 45 lg ml)1 (b), 90 lg ml)1 (c), 135 lg ml)1 (d), 180 lg ml)1 (e), 225 lg ml)1 (f), 270 lg ml)1 (g), 315 lg ml)1 (h), 360 lg ml)1 (i), 405 lg ml)1 (j) and 500 lg ml)1 (k),respectively.

*Correlation coefcient.

be divided into a lag phase, a log (exponential) phase, a stationary phase and a decline phase. Next, the corresponding power-time curves of C. albicans growth affected by different concentrations of coptisine solutions were shown in Fig. 3. As it can be seen from the height change of the highest peak of these curves, the growth of C. albicans was signicantly inhibited by this compound solution. Thermo-kinetic data for C. albicans growth In the log phase, the power-time curves obeyed the following equation: Pt P0 expkt or ln P0 kt 1

t for C. albicans growth at different concentrations of coptisine were shown in Table 1. Good reproducibility was obtained under identical experimental conditions. The generation times (tG), which are (ln2) k, were also obtained and shown in Table 2. Coptisine solution inhibited growth of C. albicans with the growth rate constant decreasing, and the inhibitory ratio (I) could be dened as: I k0 kc =k0 100% 2

where P0 represents the heat-output power at time t = 0 and Pt represents the power at time t. Using this equation, the growth rate constants k of all the experiments could be calculated by analysing the log phase. The values of k and the thermo-kinetic equations between ln Pt and

Where k0 is the growth rate constant of C. albicans without coptisine, kc is the growth rate constant in the log phase of C. albicans inhibited at inhibitor concentration c. When the inhibitory ratio I is 50%, the corresponding concentration of inhibitor is called half inhibitory concentration, IC50. The values of I and IC50, the maximum power output in the log phase Pm, log and stationary phase Pm, sta, the appearance time of the maximum power output in the log phase tm, log and stationary phase tm, sta, the heat output in the log phase Qlog and total heat output Qt obtained from the power-time curve of C. albicans growth affected by coptisine were also shown in Table 2.

Table 2 Thermo-kinetic data for C. albicans growth at 37C affected by different concentrations of coptisine
c (lg ml)1) 0 45 90 135 180 225 270 315 360 405 500 k (min)1) 005692 004921 004597 004140 003852 003453 002986 002535 002152 001678 0 I (%) 0 1355 1924 2727 3233 3934 4754 5546 6219 7052 100 tG (min) 122 141 151 167 180 201 232 273 322 413 Pm ,
log

(lW)

Pm

sta (lW)

tm,

log

(min)

tm,

sta

(min)

Qlog (J) 0083 0066 0062 0057 0053 0044 0036 0027 0021 0013

Qt (J) 0140 0129 0112 0105 0102 0092 0085 0072 0066 0048

3440 3080 2439 2329 2195 2114 1744 1540 1316 1075

4016 3647 3467 3316 3257 3235 3175 3030 2818 2433

146 170 154 168 144 204 196 142 278 300

304 322 292 315 305 359 356 309 470 538

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006

100 75 P (W) 50
l (%)

40

k (min1)

004

30

002 25 00 0 IC50 150 300 C (g ml1) 450 0

20

10 0 100 200 300 C ( g ml1) 400

Figure 4 Relationships between the growth rate constant ( k ), inhibition ratio ( I h ) and c. The IC50 obtained from kc and Ic relationship, respectively was almost the same, showing the good reproducibility and reliability of this microcalorimetric method.

Figure 5 Relationships between maximum power output in the log phase ( Pm, log n ), stationary phase ( Pm, sta ) and c The values of Pm, log and Pm, sta decreased with the increasing concentration of coptisine, and the relationships between Pm, log, Pm, sta and c were both nearly linear with R of )09808 and )09771, respectively.

Relationships between k, I and concentration c The values of k in Table 1 illustrated that the inhibition of coptisine on C. albicans growth was clearly dosedependent. The rate constants decreased gradually with increasing concentration of coptisine. Figure 4 showed the relationship between k and c which could be expressed as the following equation: k 00563 1027 104 c 0 500 lg ml1 ; R 09910 When k was equal to k0 2, we could obtain IC50. According to the above k c equation, IC50 of coptisine was 2710 lg ml)1. From the data in Table 2, it could be seen that the inhibition ratio I increased with the increase in concentration of coptisine, illustrating that the anti-C. albicans activities strengthened. When the concentration of coptisine reached 500 lg ml)1, the growth of C. albicans was completely inhibited during the measurement time and the inhibitory ratio (I) is 100%. This could also be expressed from the relationship between I and c in Fig. 4 and I c equation. IC50 of 2717 lg ml)1 of coptisine was calculated from this equation and marked in Fig. 4. I 0180c 108770 500 lgml1 ; R 09910 The values of IC50 of coptisine on C. albicans were almost the same from the k c and I c relationship, respectively, showing the good reproducibility and reliability of this microcalorimetric method. Relationships between Pm,
log,

stationary phase Pm, sta of C. albicans growth decreased with the increasing of the coptisines concentration. The relationships between Pm, log, Pm, sta and c were both nearly linear, which were as follows: Pm;log 321713 00538c 0 500 lg ml1 ; R 09808 Pm;sta 393249 00347c 0 500 lg ml1 ; R 09771 Relationships between tm,
log, tm, sta

and c

Usually, the time (tm) corresponding to maximum power output Pm for growth phase was delayed with the increasing concentration of compound or drug. Figure 6 showed that tm increased gently in the concentration range of

500 400
tm (min)

300 200 100 0 100 200 300 C (g ml1) 400

Pm,

sta

and c

By analysing the Table 2 and Fig. 5, one could see that the maximum power output in log phase Pm, log and
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Figure 6 Relationships between appearance time of maximum power output in the log phase ( tm, log n ), stationary phase ( tm, sta ) and c The relationships between tm, log, tm, sta and c were not linear, and the linear equations of tm, log c, tm, sta c were not obtained.

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The anti-fungal activity of coptisine on C. albicans growth

0315 lg ml)1 of coptisine, and when the concentration reached 360 lg ml)1, the tm increased drastically. It demonstrated that the inhibitory effect of coptisine against C. albicans enhanced with increasing concentration of coptisine, especially when the concentration was above 360 lg ml)1. But the relationships between tm, log, tm, sta and c were not linear and the linear equations of tm, log c, tm, sta c were not obtained. Relationships between Qlog , Qt and c To further show the results in a more quantitative way, we calculate the heat output in the log phase Qlog and total heat output Qt in the growth phase of power-time curves of C. albicans at different concentrations of coptisine. These results were shown in Fig. 7 and the Qlog c, Qt c equations were as follows: Q log 007871 1605 104 c 0 500 lg ml1 ; R 0 9922 Q t 013707 2072 104 c 0 500 lg ml1 ; R 09911 From Fig. 7, it was clear that Qlog, Qt decreased with increasing concentrations of coptisine, which were the same trends as k and Pm. Results of PCA From the thermogenic power-time curves of C. albicans growth affected by coptisine, nine quantitative parameters, such as k, I, tG, Pm, log, Pm, sta, tm, log, tm, sta, Qlog and Qt, were obtained to represent the anti-fungal activity of coptisine. By analysing the relationships between the nine parameters and c, it could be seen that the anti-fun015

gal activity of coptisine was enhanced with the increase in concentration of this compound. But, the change tendency of these relationships was not signicant and same, and the absolute values of correlation coefcient R of these relationships were all <09922, for example the relationship between tm, log, tm, sta and c was not linear, which was an exception. So, it was necessary to nd out the main parameter(s), which played the most important pole in representing the anti-fungal activity of coptisine. By analysing the change tendency of the main parameter(s), the anti-fungal activity could be evaluated better and faster. PCA had a good ability to summarize multivariate variation. It allowed visualizing the information of the dataset in a few principal components retaining the maximum possible variability within that set. Therefore, the PCA is recommended to reduce the computation burden. In this study, the nine quantitative parameters were analysed by PCA. The results were shown in Fig. 8. For the log centred dataset with 7954% of explained variance by the rst two principal components, the scatter plot of the scores showed a good distribution of these parameters. This plot indicated that parameters 1 (k) and 4 (Pm, log) may be the main two parameters and played more important pole in evaluating the antifungal activity of coptisine. From the values of k and Pm, log in Table 2 and the relationship of k, Pm, log and c in Figs 4 and 5, we could fast and clearly nd that the anti-fungal activity of coptisine on C. albicans growth was strengthened with the increasing concentration of this compound. The inhibitory zone diameter (d) and MIC The results showed that the blank control group had no growth of microbes, but the microbes grew signicantly
06
+2

+3

010 Q (J) P (2)

03 0 03
+8 +1 +4 +5 +6

+9

005

06 0 100 300 200 C ( g ml1) 400 04 02 0 P (1) 02

+7

04

Figure 7 Relationships between heat output in the log phase Qlog, total heat output Qt and c The values of Qlog ( n ) and Qt ( ) decreased with the increasing concentration of coptisine and the relationships between Qlog, Qt and c were both nearly linear with R of )09922 and )09911, respectively.

Figure 8 The scatter plot for principal component analysis (PCA) on nine quantitative parameters. The scatter plot obtained by PCA was obtained using software of UBSCRAMBLER 9.7 from Camo AS (Trondheim, Norway). The main two parameters were marked with a circle.

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40

d (mm)

30 d = 00155 c + 1490 R = 09545 20

10 0 500 1000 1500 C ( g ml1) 2000

Figure 9 Relationships between the diameter (d) of inhibitory zones and c. The diameter (d) of inhibitory zones in the agar layer of coptisine on C. albicans growth was measured with a sliding calliper.

in the diluted solution and positive control group. The diameter (d) of inhibitory zones in the agar layer of coptisine on C. albicans growth was measured with a sliding calliper, and the relationship between d and c was shown in Fig. 9. From the d-c equation, it could be seen that coptisine gave big inhibitory zones with diameters between 11 and 43 mm within the test range. And this compound was highly active when tested in liquid media (LB culture medium) yielding MIC around 1000 lg ml)1. Discussion Some methods, such as serial dilution, micro-dilution, anti-microbial circle and agar cup method have been applied for the study of anti-microbial activity of compounds and other materials. But these methods had more or less some disadvantages in evaluating qualitatively and quantitatively the anti-microbial activity. They could not provide more information or references for this study. For example, the agar cup method used in this paper for determining the diameter (d) of inhibitory zones in the agar layer could only show the anti-fungal activity of coptisine qualitatively. And only the MIC of coptisine was obtained quantitatively using the serial dilution method. It was not enough to represent the anti-fungal activity of coptisine using these two methods. As a useful tool, the microcalorimetric method with its high sensitivity and accuracy could examine automatically and continuously the whole metabolism of the living system, such as microbes and provide more qualitative and quantitative information about the metabolism of this living system affected by compound and other materials than the above two methods. From the power-time curves of C. albicans growth under the action of coptisine, nine quantitative parameters were obtained to evaluate the anti-fungal activity
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of coptisine. By analysing the power-time curves and quantitative parameters, it could be seen that with increasing concentrations of coptisine, the lag phase became longer, tm,log increased and the generation time tG delayed, which indicated that the fungal culture took longer time to produce a sufcient number of cells for a detectable signal and that excess berberine inhibited the growth of C. albicans or killed the fungus. The results of PCA showed that parameters k and Pm, log may be the main two parameters and played more important pole in evaluating the inhibitory effect of coptisine on C. albicans growth. The values of k and Pm, log in Table 2 and the relationship of k, Pm, log and c in Figs 4 and 5, suggested that coptisine had strong capacity to inhibit the growth metabolism of C. albicans to different extents, and the inhibitory effect was concentration-depended: the antiC. albicans activities were strengthened with the increasing concentration of coptisine. When the concentration of coptisine reached 500 lg ml)1, C. albicans would not grow: the growth is inhibited completely. This phenomenon probably resulted from the fact that the quaternary nitrogen atom and the two methylenedioxy groups at the aromatic ring played a signicant role in the anti-fungal activity of coptisine. The action of the drugs on the microbe depended on the structure of the compounds and drugs. Factors that determine the characteristics of a dose-response curve were the mode of action of the compound at cells, the number of its target sites and its afnity to those target sites etc. The quaternary nitrogen atom and the methylenedioxy groups at the aromatic ring of coptisine had a higher afnity to the fungal cell, which made coptisine easily enter into the fungal cell. In all, at the cellular level, the probable action mechanism was that coptisine integrated with the cell wall of C. albicans and the cell membrane penetrability was changed and the transport of nutrients and wastes crossing the membrane was disrupted, then coptisine diffused into the cell and further combined with the cell membrane and phospholipids of the cell nucleus resulting in the disappearance of the cell organ and the damage of the structure and functions of cells (Iwasa et al. 1996; Donlan and Costerton 2002; Yang et al. 2007). So, the growth of these cells was inhibited or stopped. In conclusion, our experiments showed that microcalorimetry is a powerful tool for monitoring and controlling the growth process of microbes. It provides kinetic and thermodynamic information that cannot be obtained by conventional bacteriological techniques, and all this information is signicant for investigating the anti-microbial activity of compound and other materials. The results are also important for the studies of toxicology and microbiology research.

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Acknowledgements The project was supported by National Basic Research Program of China (973 project) (2007CB512607 and 2006CB504703), Fond of State Youth Science (30625042) and National Natural Science Fond (no. 30772740). References
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