Preliminary screening for extraction techniques and bioactive fraction of Coptidis Rhizoma based on microcalorimetry

Abstract

Context: Extraction techniques may alter the antibacterial activity of Coptidis Rhizoma (C. Rhizoma), which is regarded as characteristic of this herb.

Objective: To explore the best extraction techniques of C. Rhizoma and the fraction(s) with the strongest antibacterial activity.

Methods: Using microcalorimetry, the influence of different extraction fractions of C. Rhizoma obtained by decoction, reflux, and ultrasound techniques on Escherichia coli growth was investigated by analyzing the power–time curves and some thermokinetic parameters. Then the antibacterial activities of each fraction of C. Rhizoma were compared by analysis of variance (ANOVA). Meanwhile, the minimum inhibitory concentration (MIC) of the strongest antibacterial fraction was determined by 2-fold dilution method.

Results and conclusion: The petroleum ether (PE), chloroform (CHCl₃), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH) fraction, water extract, and the residue after extraction all inhibited the growth of E. coli. The potency of the inhibitory effects was as follows: n-BuOH fraction > EtOAc fraction > CHCl₃ fraction > PE fraction > residue after extraction > water extract. The decoction technique was regarded as the optimum extraction technique. The n-BuOH fraction from the decoction technique was observed to have the strongest antibacterial fraction with half-inhibitory concentration IC₅₀ of 1.68 mg/mL and MIC of 200 μg/mL.

Keywords::

• Coptidis Rhizoma
• extraction technique
• bioactive fraction
• microcalorimetry
• Escherichia coli

Introduction

Coptidis Rhizoma (C. Rhizoma, Huanglian in Chinese), the dried rhizome of Ranunculaceae plants such as Coptis chinensis Franch. and Chaenomeles japonica Makino, is officially listed in the Chinese Pharmacopoeia (China Pharmacopoeia Committee, 2005). It possesses anti-inflammatory and antimicrobial activity as well as desiccatory effects (Mou & Paul, 1987; Lee et al., 2003; Yu et al., 2005). Studies have shown that many pharmacologically active compounds have been isolated from C. Rhizoma (Wei et al., 2007; Xu & Liu, 2007; Ying et al., 2007; Yu et al., 2007). The water extracts have been reported to possess strong antimicrobial activities (Xu et al., 2007; Kong et al., 2009a). The organic solvent extracts with antibacterial activity have been used in treating dysentery, cholera, diabetes, and lung cancer diseases (Ding, 1999). However, there is no report of searching for the best extraction techniques and organic solvent fraction with the strongest antibacterial activity of C. Rhizoma.

In any living system, various metabolic events following heat-producing reactions occur. Therefore, the thermogenic curve reflecting the metabolic processes of living cells can be measured by sensitive calorimeters. Microcalorimetry is a nondestructive and noninvasive technique with high sensitivity. Using this microcalorimetric method, the whole metabolic processes of the living system affected by other substances, such as drugs, heavy metal, and so on (Li et al., 2002; Kong et al., 2008a–c; Yao et al., 2008), can be recorded automatically and continuously. More useful information about the metabolism can be revealed by this method than the existing methods (McGulnness & Barisas, 1991). It has been widely used in biological experiments and different fields of life sciences (Ingemar, 2002; Yao et al., 2003; Yang et al., 2004 Zheng et al., 2006; Kong et al., 2008a,b), and can determine the energy transfer and caloric change during the metabolic processes of organisms such as Escherichia coli B growth affected by other materials (Li et al., 2003; Yu et al., 2004).

So in this study microcalorimetry was applied to investigate the effects of different extraction fractions of C. Rhizoma on E. coli growth to screen for the best extraction techniques of this herb and fraction(s) with the highest bioactivity, moreover to find the minimum inhibitory concentration (MIC) of the strongest bioactive fraction. The work provides a useful tool to study the interaction of drugs and microbe and advances idea of searching for the best extraction technique and bioactive fraction of traditional Chinese medicines (TCM).

Materials and methods

Apparatus

A 3114/3236 Thermal Activity Monitor (TAM) air isothermal calorimeter (Thermometric AB, Sweden) was used to determine the metabolism of cells. As an eight-channel twin instrument, it can continuously monitor a wide variety of processes and complex systems in a temperature range of 5°C–60°C with the detection limitation of 2 µW, and baseline stability (over a period of 24 h) is 6 µW. Each measuring cylinder contains a sample and a reference in separate measuring cups (twin system). The heat output from the sample flows via the thermoelectric detector to a large heat sink (in close contact with the water bath). Picolog software (Pico Technology, USA) is used to process the data. The detailed description about this apparatus has been reported in Wadsö (2002) and Kong et al. (2008b).

Plant material and bacterial strain

C. Rhizoma (No. 070605), the rhizome of C. chinensis Franch. (Ranunculaceae), was collected in June 2007 from Anguo city, Hebei province, China, and was identified by Xiao-he Xiao, plant taxonomist, Institute of Chinese Materia Medica, Beijing. Other chemicals used in the experiments were of analytical grade, and double-distilled water was used to prepare all solutions.

The E. coli strain (E. coli CMCC B44103) was provided by the Chinese Center for Type Culture Collections, NICPBP, Beijing. They were inoculated in Luria-Bertani (LB) culture medium and preserved at 4°C. LB culture medium, a solution (pH 7.0–7.2) containing 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl, was sterilized in high-pressure steam at 121°C for 30 min before use.

Extraction of samples

Extraction by decoction technique

C. Rhizoma were crushed into powder and filtered through an 80-gauge screen mesh. The powder (300 g) was extracted with 3000 mL double-distilled water and decocted in a saucepan. The water decoction was further extracted by 500 mL of each solvent in sequence to get petroleum ether (PE), chloroform (CHCl₃), ethyl acetate (EtOAc), n-butyl alcohol (n-BuOH), and residual fractions.

Extraction by refluxing technique

C. Rhizoma were crushed into powder and filtered through an 80-gauge screen mesh. The powder (300 g) was extracted with 3000 mL double-distilled water using a round-bottom flask. The extraction was further extracted by the organic solvents in sequence to get different extraction fractions.

Extraction by ultrasound

C. Rhizoma were crushed into powder and filtered through an 80-gauge screen mesh. The powder (300 g) was extracted with 3000 mL double-distilled water using an ultrasonic washer. The extraction was further extracted by the organic solvents in sequence to get different extraction fractions.

Determination of the thermogenic curves of E. coli growth

The metabolic thermogenic curves of E. coli growth without and with different fractions were determined by microcalorimetry using the ampoule method. Initially, E. coli were cultivated in the incubator at 37°C. They were inoculated in the prepared 5 mL culture medium using a 20-mL glass ampoule, and the initial population density was 1 × 10⁶ colony-forming units (CFU)/mL. The freshly prepared solution of each extraction fraction was added into the ampoule. The glass ampoule was then sealed with a cap and put into the microcalorimeter. The growth process of E. coli affected by different fractions was monitored continuously by this microcalorimeter and the thermogenic power–time curve was determined. All the experiments were carried out under aseptic condition (Zhu et al., 2006; Kong et al., 2009b).

Statistical analysis

The analysis of variance (ANOVA) followed by Dunnett’s many-to-one multiple comparison tests were applied appropriately to observe whether there were significant differences in the antibacterial activities of these fractions obtained by different extraction techniques. The Wilks’s lambda statistic was preferred to obtain P-values in this analysis and P < 0.05 was considered statistically significant between groups (Duncan, 1957). For this ANOVA, SAS versions 6.12 and 8.0 software for Windows (SAS, Cary, NC) was employed.

Determination of MIC

MIC of n-BuOH fraction on E. coli growth was determined by 2-fold dilution of solution of n-BuOH fraction (0–500.0 μg/mL) in test tubes using fresh LB culture medium. Each test tube was inoculated with bacterial suspension containing 1 × 10⁵ CFU/mL cells and incubated at 37°C for 24 h. The lowest concentration that visibly showed no growth compared with drug-free culture medium inoculated with microbial suspension was considered the MIC (Hindler & Isenberg, 1992).

Results

Growth rate constant (k) of E. coli

The metabolic curve of E. coli growth in culture medium at 37°C without drug was recorded and shown in Figure 1. This power–time curve was a typical growth characteristic of E. coli and could be divided into five phases: a lag phase, the first exponential phase, the second exponential phase, a stationary phase, and a decline phase. The peaks and trough are characteristic of organism growth on LB culture medium and with restricted (only the head space within the ampoule) oxygen availability. It is plausible that the first exponential phase seen represents aerobic metabolism, which is then followed by a change to anaerobic metabolism, that is, the second exponential phase (Michael et al., 2003). In the exponential phase, the cell growth is an exponential mode. If the cell number is n₀ at time 0, and n_t at time t, then

1

Figure 1.  The power–time curve of E. coli growth at 37°C without any substance. E. coli was cultured in LB culture medium and monitored using a TAM air isothermal calorimeter, ampoule method at 37°C.

If the heat power output of each cell is w, then

2

P₀ = n₀w and P_t = n_tw, giving P_t = P₀ exp(kt), or

3

where P₀ represents the heat-output power at time t = 0 and P_t at time t. The exponential phase of E. coli growth corresponded to Equation (3). Using this equation, the growth rate constant k (Table 1) was calculated by fitting ln P_t and t to a linear equation.

Table 1.  Growth rate constants k₁ and k₂ of E. coli growth at 37°C.

Channel	1	2	3	4	5	6	7	8
k1 (/mill)	0.11206	0.11213	0.11201	0.11195	0.11201	0.11193	0.11192	0.11201
Ra	0.9987	0.9989	0.9971	0.9974	0.9990	0.9981	0.9972	0.9988
k2 (/mill)	0.00891	0.00874	0.00869	0.00885	0.00879	0.00901	0.00905	0.00895
R^a	0.9992	0.9982	0.9988	0.9990	0.9992	0.9994	0.9991	0.9989

^aCorrelation coefficient.

Table 1 shows k₁ = (0.112 ± 0.00021)/min and R all exceeded 0.997, k₂ = (0.00887 ± 0.00036)/min and R all exceeded 0.998, indicating good reproducibility and stability of this microcalorimetric method. Subsequent k₁ and k₂ were the growth rate constants of the first and second exponential phases, respectively. The second exponential phase could be regarded as stable growth stage of the bacteria because k₂ was much less than k₁ (Liu et al., 2003).

The power–time curves and thermokinetic parameters of E. coli growth affected by each fraction

The power–time curves of E. coli growth affected by each fraction of C. Rhizoma were shown in Figure 2, and the corresponding thermokinetic parameters were listed in Table 2. The final concentration of each fraction in the LB culture medium was 2 mg/mL.

Table 2.  The thermokinetic parameters of E. coli growth at 37°C affected by C. Rhizoma extractions at 2 mg/mL.

Extraction technique	Extraction fraction	k1 (/mill)	k2 (/miu)	tm (miu)	Pm (MW)
	Control	0.11204	0.00932	812.7	1.740
Decoction technique	Water	0.01069	0.00435	818.8	1.354
	Residue	0.01006	0.00473	880.7	1.348
	PE	0.00823	0.00371	861.7	1.218
	CHC13	0.00710	0.00344	928.0	1.183
	EtOAc	0.00664	0.00277	1084.3	1.129
	n-BuOH	0.00552	0.00242	1219.0	1.008
Refluxing technique	Water	0.01081	0.00576	760.7	1.693
	Residue	0.01031	0.00597	758.7	1.652
	PE	0.00872	0.00551	771.0	1.647
	CHCl3	0.00765	0.00543	788.3	1.638
	EtOAc	0.00707	0.00478	820.0	1.548
	n-BuOH	0.00564	0.00461	850.0	1.495
Ultrasound technique	Water	0.01092	0.00861	892.3	1.429
	Residue	0.01056	0.00905	923.6	1.247
	PE	0.00921	0.00826	941.0	1.240
	CHC13	0.00812	0.00702	997.6	1.221
	EtOAc	0.00709	0.00615	1113.0	1.188
	n-BuOH	0.00585	0.00524	908.3	1.163

Figure 2.  The power–time curves of E. coli growth at 37°C affected by C. Rhizoma fractions obtained from (A) decoction, (B) refluxing, and (C) ultrasound techniques. The various factions were (a) control, (b) water extraction, (c) residue, (d) PE fraction, (e) CHCl₃ fraction, (f) EtOAc fraction, and (g) n-BuOH fraction with the final concentration of 2.00 mg/mL in the LB culture medium.

Figure 2 shows that the profiles of power–time curves of E. coli growth changed with the addition of each C. Rhizoma fraction, illustrating that the fractions all had influences on E. coli growth. Table 2 showed that in comparison with the control (without drug), the lag and exponential phases became longer, k₁ and k₂ and the maximum heat-output power (the height of the highest peak) P_m decreased, while the appearance time of P_m increased, which indicated that the bacterial culture took longer to produce a sufficient number of cells for a detectable signal, and excess drugs inhibited the growth of E. coli or killed the bacterial cells. These data illustrated that each fraction of C. Rhizoma had anti-E. coli activity.

The data in Table 2 were input into the SAS software for ANOVA and the results are shown in Table 3. From this table, it is apparent that the F-values for optional two groups among decoction, reflux, and ultrasound extractions were all greater than F_0.05,4,7 = 4.12 with corresponding P-value smaller than 0.05, showing that there were significant differences for the antibacterial activities of the fractions of these three extraction techniques.

Table 3.  ANOVA results for three extraction groups.

Group	Wilks’ lambda value	F-value	Num DF	Den DF	Pr > F
Decoction and refluxing technique	0.24369860	5.43	4	7	0.0261
Decoction and ultrasound technique	0.84743364	6.32	4	7	0.0362
Refluxing and ultrasound technique	0.76176905	5.55	4	7	0.0075

It has been reported that the smaller the values of k₁, k₂, and P_m and the bigger the values of t_m were, the stronger the antibacterial activity the drug possesses (Li et al., 2003). General comparison of the parameters in Table 2 for each extraction group, it could be found that the values of k₁, k₂, and P_m of the decoction extraction group were the smallest, and t_m were the biggest among the three extraction groups, showing that the antibacterial activities of these fractions obtained from decoction techniques were stronger than those obtained by the other two extraction techniques. These also illustrated that the active components in these fractions obtained by decoction techniques were much more sufficient than the other two extraction techniques. Further, it could be concluded that the fractions obtained from decoction could well reflect the antibacterial characteristics of C. Rhizoma. So decoction was selected as the optimum extraction method of C. Rhizoma.

Returning to the values in Table 2, we found that the potency of antibacterial activity of the fractions was as follows: n-BuOH fraction > EtOAc fraction > CHCl₃ fraction > PE fraction > residue after extraction > water extract. The n-BuOH fraction with the strongest antibacterial activities was selected as the strongest antibacterial fraction of C. Rhizoma. The antibacterial activities of n-BuOH fraction are evaluated specifically in the next section.

The antibacterial activity of n-BuOH fraction

The experimental procedure was the same as above. The final concentration of n-BuOH fraction in each ampoule was 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 mg/mL. The power–time curves of E. coli growth at 37°C affected by n-BuOH fraction got from decoction technique were recorded and shown in Figure 3 and the corresponding thermokinetic parameters were listed in Table 4.

Table 4.  The thermokinetic parameters of E. coli growth at 37°C affected by n-BuOH fraction of C. Rhizoma.

Extraction technique	c (mg/mL)	k1 (/min)	k2 (/min)	tm (min)	Pm (mW)	I (%)	IC50 (mg/mL)
	0	0.10735	0.00769	836.3	1.733	—
Decoction technique	0.50	0.10669	0.00472	778.0	1.602	38.6	1.68
	1.00	0.10631	0.00453	819.7	1.538	41.1
	1.50	0.10628	0.00404	841.0	1.499	47.5
	2.00	0.10615	0.00375	869.7	1.413	51.2
	2.50	0.10599	0.00315	930.7	1.353	59.0
	3.00	0.10584	0.00261	830.3	1.331	66.1
	3.50	0.10491	0.00212	890.7	1.202	72.4

Figure 3.  The power–time curves of E. coli growth at 37°C affected by n-BuOH fractions of C. Rhizoma got obtained by decoction techniques at concentrations of (a) 0, (b) 0.5, (c) 1.0, (d) 1.5, (e) 2.0, (f) 2.5, (g) 3.0, and (h) 3.5 mg/mL.

The power–time curves in Figure 4 showed that the highest peaks were lowered with increasing concentrations of n-BuOH fraction, showing that this fraction had strong inhibitory activities on E. coli, and the inhibitory extent was strengthened with the increased concentration. The values of thermokinetic parameters in Table 4 further illustrate the above results: the values of k₁, k₂, and P_m decreased and t_m increased gradually with increasing the concentration of n-BuOH fraction, showing that the antibacterial activities were enhanced.

Figure 4.  Relationship between inhibition ratio (I) and c. The linear relationship between I and c was good with R of 0.9940. IC₅₀ (1.68 mg/mL) was calculated from this linear relationship.

Growth inhibition ratio I

I was calculated on the basis of growth rate constant and could be defined as:

4

where k₀ and k_c were the growth rate constants of E. coli in the absence of drug and inhibited at inhibitor concentration c, respectively. When I is 50%, the corresponding inhibitory concentration is called half-inhibitory concentration, IC₅₀. IC₅₀ can be regarded as the inhibitory concentration that produced a 50% decrease in growth rate constant. From the relationship between I and c in Figure 4, the value of IC₅₀ (1.68 mg/mL) was calculated and also listed in Table 4.

MIC (200.0 μg/mL) of n-BuOH fraction on E. coli growth also showed the strong antibacterial activity of this fraction.

Discussion and conclusion

In this study, we proposed a useful tool—microcalorimetry—to screen the extraction technique and bioactive fraction of TCM. This tool can determine the metabolic curve of microbes such as E. coli at a constant temperature of 37°C affected by other substances, and can obtain thermokinetic parameters reflecting the influence of these substances on the growth of the microbe. Microcalorimetry not only supplies a new point of view for the evaluation of the bioactivity of drugs but also provides more information about the microbial growth. All this information could describe the microbe–drug reaction when drugs are added into the culture medium, providing some experimental foundations for deeply studying the pharmacodynamic actions of these TCMs.

The antibacterial activities of the extraction fractions of C. Rhizoma obtained from three extraction methods (decoction, reflux, and ultrasound techniques) were investigated by microcalorimetry in this study. The fractions obtained from decoction had much stronger antibacterial activities than those from the other two extraction methods and decoction was screened as the optimal extraction method of this herb. The n-BuOH fraction with the strongest antibacterial activity among the organic solvent fractions was selected as the bioactive fraction of this herb. All these were suitable for the clinical medication principle and provided some references for the extraction of other commonly used TCM herb. The active components were extracted more completely from C. Rhizoma by decoction, fully expressing the pharmacodynamic actions of this herb. However, some problems still existed; for example, the active components and their structures in this herb obtained from decoction might be destroyed or lost through volatilization because of the high temperature of this method; some pharmaceutical actions might be weakened. All these problems must be addressed in a future study.

Declaration of interest

The authors are grateful for the support of the National Basic Research Program of China (973 project) (2007CB512607), the National Youth Science Foundation (30625042), and the National Natural Science Foundation (30772740).