Cold/hot pad differentiating assay of property differences of Mahuang and Maxingshigan decoctions

Abstract

Context: Chinese medicines with different cold/hot properties have various pharmacological actions on multiple organisms.

Objective: The objective of this study was to explore the cold/hot property differences of traditional Chinese medicine formulas of Mahuang and Maxingshigan decoctions.

Materials and methods: A novel cold/hot pad differentiating assay method based on the Intelligent Animal Temperature Tropism Behavior monitoring system at 20 °C (cold pad) and 30 °C (hot pad) was introduced to investigate the variability of temperature tropism among the mice treated by 0.4 mL/20 g (drug volume/body weight) of Mahuang decoction and Maxingshigan decoction, respectively. Meanwhile, the oxygen consumption and activities of adenosine triphosphatase (ATPase) were measured to explore the energy metabolism mechanism.

Results: Results showed that the differences between cold/hot properties of Mahuang decoction and Maxingshigan decoction were significant (p < 0.05). Mahuang decoction produced significant synergic effect (a combination index of 1.60), while Maxingshigan decoction expressed significant antagonistic effect (a combination index of 0.35). The changes of energy metabolism including ATPase activity and oxygen consumption might be the possible factors to result in the differences. Those influences tended to be coherent with the definition of cold/hot properties of Chinese medicines based on traditional Chinese medicinal theory.

Conclusions: The results indicated that the method based on cold/hot pad differentiating array could objectively and quantitatively represent the cold/hot properties of different compatibilities of traditional Chinese medicines in an ethological way according to the changes of animal’s temperature tropism. These findings would provide some experimental basis and data references as well as a novel evaluation method for the study of the regularity of recipe composition.

• Chinese medicine formulas
• cold/hot property
• temperature tropism

Introduction

Chinese medicine has long been the core of traditional Chinese medicine (TCM) theory, but has not yet been proven by some experiences. To some extent, chills or fever potency is the naive materialism abstracted by ancient physicians according to drug action in patients. They illustrated macroscopic reaction of the cold and heat exchange of the body and environment to reflect a physiological body thermal changes in the drug effect or pathological feelings (Xiao & Wang, 2004), so as to perform different behaviors for the heat environment tropism (selectivity).

The interaction of drug combination can be divided into the additivity, synergism, and antagonism effects. To date, scholars have used a variety of evaluation methods to determine the drug compatibility with properties (Vinik et al., 1999). Nevertheless, traditional Chinese medicine, due to the multi-component, multi-link, multi-target properties, often does not have a good dose–effect relationship, or even no dose–effect relationship (Deng et al., 2009). Most of the methods for the dose–effect relationship are tedious. This limits the application of the most mathematical methods in the study of traditional Chinese medicine. Guinness Book of Q-value method (Jin, 1980), also known as the probability sum method, is simple, does not require the characteristics of the dose–effect relationship of the two drugs, and widely used in the evaluation of drug combination (Li et al., 2007; Lu & Zheng, 2006). It is applied to the complex role of traditional Chinese medicine compound compatibility law studies.

In this paper, a novel cold/hot plate differentiating assay method (Zhao et al., 2009; Zhou et al., 2009) was introduced to explore the cold and heat properties of traditional Chinese medicines including Mahuang decoction and Maxingshigan decoction by investigating the variability of temperature tropism among the mice which were treated by the two decoctions. Meanwhile, the oxygen consumption and activities of adenosine triphosphatase (ATPase) were measured to explore the mechanism of energy metabolism. This study will provide basic experimental data and references as well as a novel evaluation method for the study of the regularity of recipe composition.

Materials and methods

Drug preparation

Ephedrae herba (Ephedraceae), Armeniacae semen (Rosaceae), Glycyrrhizae radix (Leguminosae), and Cinnamomi ramulus (Cinnamomum) were purchased from the Taipinghuikang drug store, Beijing, China, which were identified by Prof. Xiaohe Xiao, and the voucher specimen were stored in China Military Institute of Chinese Medicine, 302 Hospital of People’s Liberation Army.

As shown in Table 1, the herbs were weighed and soaked for 30 min in water of 10 times the amount of the herbs, and were decocted twice, and then filtered. The filtrates were mixed together. The resulting fluids were concentrated to 0.2 g/mL of Mahuang decoction (MHD), 0.34 g/mL of Maxingshigan decoction (MXSGD), 0.15 g/mL of consensus compositions group (CCG), 0.05 g/mL of cinnamomi ramulus group (CRG), and were kept in a refrigerator at 4 °C prior to use.

Table 1. Samples used in this study.

Groups	Weight ratio
MHD	Ephedra herba: Armeniacae semen: Glycyrrhizae radix: Cinnamomi ramulus = 3: 2: 1: 2
MXSGD	Ephedra herba: Armeniacae semen: Glycyrrhizae radix: Gypsum = 3: 2: 1: 6
CC	Ephedra herba: Armeniacae semen: Glycyrrhizae radix = 3: 2: 1
G	Gypsum = 6
CR	Cinnamomi ramulus = 2

MHD, Mahuang decoction; MXSGD, Maxingshigan decoction; CC, consensus composition; G, Gypsum; CR, cinnamomi ramulu.

Animals

Ninety healthy KM mice, male, 14–16 g of weight, were purchased from the Laboratory Animal Center, Academy of Military Medical Sciences, with license: SCKX-(army) 2007004. Animal experiments and performance of this study were approved by the Ministry of Science and Technology of China and related ethical regulations on 9 September 2013. All experimental procedures were performed in accordance with the Guidelines of the Experimental Animal Care and Use Committee of Beijing (Beijing, China).

Instruments and reagents

Intelligent Animal Temperature Tropism Behavior (IATTB) Monitoring System (Figure 1) for monitoring animal temperature tropism behavior was designed by our research team and assembled by Beijng Zhongjiao Instrument Company (Chinese patent no. ZL2008200004444.2). Before experiment, several stable temperature zones were generated by a temperature-controlling pad (cold/hot pad), then animals were put on it with free motion across the temperature zones. Parameters for evaluating animal’s temperature tropism, such as the staying time in different temperature zones and distance of movement could be obtained through a real-time computer processing of artificial intelligence to analyze the video determining the coordinates (X, Y) of each animal in each frame of image, and the movement trajectories of animals were obtained by joining the coordinates of each animal in all frames Y_t = f[X_t], followed by computer processing of the duration and frequency of each animal in different zones. The instrument used in this study can monitor six animals at the same time, which increases the repeatability and compatibility of data. Video recognition software was based on Paulo’s algorithms (Xiao & Wang, 2004), and data analysis software was written using Visual Basic 6.0 language.

Figure 1. Intelligent animal temperature tropism behavior monitoring system.

Cary50 Bio UV spectrophotometer (Varian Australia PTY LTD, Mulgrave, Australia), Baiyang B320 low-speed centrifuge (Beijing Ruitaihang Technology and Development Corporation, Beijing, China), ATPase assay kit (20090522), Colorimetric Blue Protein assay kit (20090522), and T-AOC assay kit (20090522) were purchased from Nanjing Jiancheng Biological Engineering Research Institute (Jiangsu, China). All the reagents were of A.R. grade.

Grouping and administration

Before the experiment, the mice were trained to adapt to the temperature of hot/cold pad for 5 d. The drugs were intragastrically given to these mice with 0.4 mL/20 g (drug volume/body weight). During the administration of drugs, mice were given limited drink for 5 mL/20 g (body weight) per day. The experiments were divided into five groups, including (1) the difference between cold/hot properties of MHD and MXSGD, (2) the cause of cold/hot properties of MHD, (3) the cause of cold/hot properties of MXSGD, (4) the influence of the dose of Cinnamomi ramulus on the cold/hot properties of MHD, and (5) the influence of the dose of gypsum on the cold/hot properties of MXSGD, which contains shared experimental groups.

The first experiment: 18 male mice were randomly divided into three groups, namely the control group (C), MHD group, and MXSGD group. And the mice in the C group were administered with physiological saline of the same volume for 6 d.

The second experiment: 48 male mice were randomly divided into eight groups, namely C, MHD, CRG, CC, and G groups. And the mice in the C group were administered with physiological saline of the same volume for 6 d.

Determination of oxygen consumption

Mice were place in an air-tight container with 10 g sodium oxide at the bottom to absorbing CO₂ generated by mice, and a rubber tube with a graduated pipette was connected to the top. The other tip of graduated pipette was inserted into water vertically. All interfaces were sealed with vaseline. The time of breathing for 2.5 mL of air and the air-volume of consuming for 6 min were measured.

Determination of temperature tropism of mice on the cold/hot pad

The experiment was performed on a bi-zone temperature-controlling pad: 20 °C (cold pad) and 30 °C (hot pad), with laboratory temperature of 23 ± 2 °C. Half an hour after drug administration, mice were put into the monitoring channel of the cold/hot pad and their temperature tropism behaviors were remotely monitored and the trajectories of each mouse were recorded. By analyzing the data through the professional software, the remaining time of mice on different-temperature pads was obtained to evaluate the temperature tropism. Remaining rate (RR) (%) = remaining time on cold or hot pad (s)/the total monitoring time (s) × 100%.

Determination of the activity of Na⁺–K⁺ ATPase, Ca²⁺–Mg²⁺ ATPase

The animals were sacrificed by cervical dislocation, and the livers were quickly placed in saline at 4 °C to wash away the blood using the filter paper. Then, the ATPase activity of 2% homogenate of liver tissue phosphorus was determined. The activity of tissue proteins per mg per hour and the ATPase of decomposition ATP was accounted with phosphorus as an ATP enzyme activity unit (μmol mg⁻¹ h⁻¹).

King Q value method and combination interaction

The Guinness Q value method was used to evaluate the drug interactions. Relative drug effect (Re) = [(RR1 − RR2)/RR1] × 100%, where RR1 is the high-temperature dwell rate of mice in the control group, RR2 is the high temperature dwell rate of mice treated with drugs. Guinness Book of Q value method, also known as the probability sum method (Aguiar et al., 2007) on the basis of the formula method of Burgi's correction derived: Q = REA + B/(REA + REB − REA • REB), where REA represents A single use is the relative effect, REB represents the relative effect of B drug used alone, REA + REB − REA • REB represent the expected value of the combined effect of the two drugs, EA + B represent the value of the actual effects of the two drugs.

Statistical analysis

Data were expressed as means ± standard deviation (X ± SD). Statistical analysis of remaining rate in warm pad, ATPase activity, and T-AOC activity was performed by ANOVA and Student's t-test using the software of SPSS 13.0 (SPSS Inc., Chicago, IL). p < 0.05 were considered significant.

Results and discussion

General condition of animals

Compared with the mice in the control group, the body weight of mice in the MHD group increased. The feature of “hot symptoms”, such as bright coat, enhanced moving activity and increased oxygen consumption (p < 0.01), were observed. The body weight of mice in the MXSGD group decreased. The feature of “cold symptoms”, such as less water-intake, withered, and caducous fur, increased thermo-tropism and preference of gathering, fatigue, more oxygen consumption could be observed (p < 0.01).

Changes of temperature tropism of mice

The difference between cold/hot properties of MHD and MXSGD: comparing with the C group, the remaining rate of MHD animals on hot pad was found to be significantly decreased (p = 0), revealing the tropism to low temperature zone, consistent with “hot symptoms”. Compared with MHD, the remaining rate of mice treated with MXSGD on hot pad was found to be significantly increased (p = 0.0439), revealing a thermotropism, also consistent with “cold symptoms”.

The cause of cold/hot properties of MHD: compared with MHD, the remaining rate of mice in CR group on hot pad was found to be significantly increased (p = 0.0007), revealing a thermotropism. But there were no significant differences between mice of MHD and CCG groups. With the calculation, the combined CRG with consensus composition – Mahuang decoction – produced significant synergic effect (combination index Q = 1.60).

The cause of cold/hot properties of MXSGD: comparing with MXSGD, the remaining rate of GG animals on hot pad was found to be significantly increased (p = 0.0411), revealing a thermotropism, and it also increased in CCG. With the calculation, the combined Shigao with consensus compositions produced significant antagonistic effect (combination index Q = 0.35). All these results are listed in Table 2 and Figure 2.

Figure 2. Evaluation of combinative effect of different drugs. (+) Q value of 0.85–1.15 additive effect, and (++) of 1.15–2.0 synergic effect, and (+++) >2.0 significant synergic effect, and (−) of 0.55–0.85 antagonistic effect, and (−−) <0.55 significant antagonistic effect.

Table 2. Remaining time ratio of mice in each group on warm pad.

Groups	Dose (g/kg)	Remaining time ratio on warm pad at 30 °C (%) (X ± SD)
Group 1
C		57.6 + 11.8
MHD	4	11.2 + 10.4**
MXSGD	6.8	18.8 + 10.2**#
Group 2
C		57.6 + 11.8
MHD	4	17.7 + 8.8**
CC	3	37.7 + 18.7**#
CR	1	54.2 + 4.5##
Group 3
C		58.0 + 13.4
MXSGD	6.8	45.8 + 10.9
CC	3	37.5 + 10.3*Δ
G	3.4	54.7 + 13.4ΔΔ

**p < 0.01,*p < 0.05 versus the control group; ##p < 0.01, #p < 0.05 versus the MHD group; ΔΔp < 0.01, Δp < 0.05 versus the MXSGD group.

Activity changes of Na⁺–K⁺–ATPase, Ca²⁺–ATPase, and Mg²⁺–ATPase of liver tissue of mice

From Table 3, it could be found that: (1) after irrigation with MHD, the Na⁺–K⁺–ATPase, Ca²⁺–ATPase, and Mg²⁺–ATPase activities of liver tissue of mice were enhanced; while in MXSGT group, these activities were decreased; (2) compared with MHD group, the Na⁺–K⁺–ATPase, and Mg²⁺–ATPase activities of CCG declined; (3) compared with MXSGD, Na⁺–K⁺–ATPase, Ca²⁺–ATPase, and Mg²⁺–ATPase activities of liver tissue of mice in the CC group increased (p < 0.01). The sequence of ATPase activity is as follows: MHD group > C group > CC group > CR group > MXSGD group > G group.

Table 3. Activity of Na⁺–K⁺–ATPase, Ca²⁺–ATPase, and Mg²⁺–ATPase of liver tissue of mice (X ± SD).

Group	Dose (g/kg)	Na^+–K^+–ATPase (μmol mg^−1 h^−1)	Mg^2+–ATPase (μmol mg^−1 h^−1)	Ca^2+–ATPase (μmol mg^−1 h^−1)
C		1.977 + 0.576	1.779 + 0.0.576	2.358 + 0.424
MHD	4	2.530 + 0.892	1.949 + 0.761	2.640 + 0.627ΔΔ
CR	1	1.548 + 1.135	1.534 + 1.064	2.145 + 0.893
CC	3	1.967 + 0.494	1.106 + 0.572	2.674 + 0.914ΔΔ
MXSGD	6.8	1.522 + 0.356	0.892 + 0.149	1.830 + 0.653##
G	3.4	0.554 + 0.476**##	0.818 + 0.728	1.563 + 0.325##

**p < 0.01, *p < 0.05 versus the control group, ##p < 0.01, #p < 0.05 versus the MHD group, ΔΔp < 0.01, Δp < 0.05 versus the MXSGD group.

Conclusions

The results showed that the mice in the treatment group showed some chemotactic cold to indicate that the drugs have some heat resistance. Mice of all-party group had significantly higher resistance than those in the hot and cold panel in the CR and CC group (p < 0.01 and p < 0.05, respectively). By evaluating the “Q” value according to the Guinness Book, CC group combined with CR group interaction (MHD) showed a synergistic effect, the two combined group is not simply a simple sum of the dose, but actually the impassioned compatibility phase must be used and reported (Deng, 2003); comparison group of all-party group with MXSGD group, a total of smell mice performance on hot and cold plate “increasingly cold” enhanced (p < 0.05) gypsum group performance for “chemotaxis cold” weakened (p < 0.01), “Q” by the Guinness Book value method to evaluate the interaction, the overall trend shows that the CC group combined with gypsum (i.e., the MXSGD soup group) performance antagonism belongs phase impassioned compatibility fear and kill (i.e., to sexual memory), reported in the literature coincide.

At the same time, the present study examined the impact of the drug on mouse ATPase activity to reflect the changes of energy metabolism. The results showed that Na⁺–K⁺–ATPase, Ca²⁺–ATPase, and Mg²⁺–ATPase activities of liver tissue of mice in MHD group expressed objective differences. On this basis, the demolition party study found that the combination of CC group and CR group (MHD group) ATPase showed the highest activity, suggesting CC group and CR group have a synergistic effect when combining; CC group in combination with ATPase activity than the total of the smell lower compared with gypsum enhance suggest that the combination of gypsum and CCG antagonism. The experimental results and the cold/hot plate differentiating method the results are consistent.

In this paper, the cold/hot plate differentiating method for the evaluation of cold and hot properties and determination of the ATPase activity of liver tissue verified that the objective existence of a group of cold and heat potency differences between MHD with MXSGD, respectively, from a macro- and micro-perspective, ephedra the soup group of classes based on the compatibility law of the square chills and fever herbal preliminary results “Treatise on consistent discussed, reflecting the parties make drug full their sexual serve enables drug losing its sex parties” (Xiao & Wang, 2004) were discussed. The results show that ATPase activity of the animal's temperature tending to sexual behavior change was closely linked. This study provided the experimental basis for the study of traditional Chinese medicine compatibility law, the new ideas and methods for the interpretation, and the exploration of traditional Chinese medicine, cold and heat herbal Compatibility Law. According to traditional Chinese medicine, treatment with “flavors” and “gas” has medicinal characteristics.

Declaration of interest

The authors declares that they have no conflicts of interest to disclose.

The authors are grateful for the support from National Science Natural Foundation (81173571) and the Major Projects of the National Science and Technology (2012ZX10005010-002-002).