Research on Fuzi based on animal thermotropism behavior to discover if it has fewer “hot” characteristics without Ganjiang

JTCM |www. journaltcm. com June 15, 2012 |volume 32 | Issue 2 |

Online Submissions:http://www.journaltcm.com J Tradit Chin Med 2012 June 15; 32(2): [email protected] ISSN 0255-2922

© 2012 JTCM. All rights reserved.

Basic Investigation

Research on Fuzi based on animal thermotropism behavior to dis-cover if it has fewer "hot" characteristics without Ganjiang

SUN Zhi-yong 孙志勇, ZHAO Yan-ling 赵艳玲, WANG Jia-bo 王伽伯, ZHANG Lin 张琳, WEI Si-si 魏思思, JIANGFeng-juan江凤娟, JIA Lei贾雷, CHENG Dan-hong程丹红, XIAO Xiao-he肖小河aa

SUN Zhi-yong, ZHANG Lin, WEI Si-si, JIANG Feng-juan,JIA Lei, CHENG Dan-hong, China Military Institute of Chi-nese Materia Medica, 302 Military Hospital of China, Beijing100039, China; Chengdu University of Traditional ChineseMedicine, Chengdu 610075, ChinaZHAO Yan-ling, WANG Jia-bo, XIAO Xiao-he, China Mili-tary Institute of Chinese Materia Medica, 302 Military Hospi-tal of China, Beijing 100039, ChinaSupported by National Natural Sciences Foundation (No.81173571) and National Basic Research Program of China(No. 2007CB512607)Correspondence to: Prof. ZHAO Yan-Ling, China MilitaryInstitute of Chinese Materia Medica, 302 Military Hospital ofChina, Beijing 100039, China. [email protected]: +86-10-66933320Accepted: March 01, 2012

AbstractOBJECTIVE: To investigate whether Fuzi (RadixAconiti Praeparata) has fewer "hot" characteristicswhen administered without Ganjiang (RhizomaZingiberis).

METHODS: Differences in the thermotropism be-haviors of mice treated either with Fuzi (Radix Acon-iti Praeparata), Ganjiang (Rhizoma Zingiberis) orthe combination of the two given intragastricallywere investigated using the Animal ThermotropismBehavior Surveillance System. The water intake vol-ume, oxygen consumption volume, adenosine tri-phosphatase (ATPase) activity, total antioxidant ca-pacity (T-AOC) and total superoxide dismutase(T-SOD) activity were determined during the inves-tigation.

RESULTS: When Fuzi and Ganjiang were adminis-tered together, the rate at which mice remained ona warm plate ("remaining rate") and the times anddistances of their movement were all significantlyreduced (P<0.05). Compared with the Normalgroup, the reduction was 55.1%, 48.3% and 44.8%,while compared with the Fuzi group, the reductionwas 57.6%, 34.3% and 36.0%, indicating that "cold"tropism was significantly increased. Compared withthe Normal and Fuzi groups, the ATPase activityand the respiratory oxygen consumption volumeof the Fuzi + Ganjiang group were significantly in-creased (P<0.05), suggesting an improvement inenergy metabolism and showing a "hot" character-istic when Fuzi and Ganjiang are present together.Additionally, the T-AOC and T-SOD activity were sig-nificantly enhanced (P<0.05).

CONCLUSION: The behavior of mice tending to-ward "cold" tropism can be regarded as a quantita-tive reflection of Fuzi having fewer characteristicsconsistent w ith a "hot" nature when not used withGanjiang, the functional mechanism of which maybe a change in the ATPase activity in liver tissue.

© 2012 JTCM. All rights reserved.

Key words: Animal thermotropic behavior; Objec-tivity; Cold/Hot tropism; Fuzi (Radix Aconiti Praepa-rata); Ganjiang (Rhizoma Zingiberis)

INTRODUCTION"Fuzi (Radix Aconiti Praeparata) showing fewer charac-teristics consistent with a 'hot' nature when not used

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with Ganjiang (Rhizoma Zingiberis)" is a fundamentaltheoretical topic in Chinese Materia Medica. The dis-cussion originated in the medical book Zhengzhi Yao-jue (The Key to Diagnosis and Treatment) written byDAI Yan-li. The question remains as to whether thephenomenon really exists and moreover, how to detectit. Previous studies on Fuzi and Ganjiang concentratedon the material basis and pharmacodynamics[1-6]. Thepresent study investigates the phenomenon of "Fuzi ex-erting fewer hot characteristics without Ganjiang"from the perspective of thermodynamics and energymetabolism and looks at animal thermotropism behav-ior to qualitatively and quantitatively describe the dif-ference between Fuzi and Ganjiang used together andseparately. At the same time, the total antioxidant ca-pacity (T-AOC) and total superoxide dismutase(T-SOD) activity of mice was monitored to explainany observable phenomenon. The Animal Thermotro-pism Behavior Surveillance System was used to showand evaluate the cold/hot natures of Chinese herbalmedicines from the bio-thermodynamics perspective[7].Our scientific research team developed the System,which is patented, proprietary intellectual property. Ithas been highly appraised by the Chinese Materia Med-ica expert group of the State's 973 Plan and recom-mended all across the country to scientific researchgroups interested in this research.

METHODS

Drug preparationThe herbal medicines Fuzi and Ganjiang were pur-chased from Beijing Tongrentang and identified byProf. Xiao Xiao-he. Preparation: Fuzi and Ganjiang,100g each, were soaked for 30 min and then decoctedwith 1000 mL of water for the first time for 1.5 h. Af-ter filtration, the residual was decocted for the secondtime with 800 mL of water for 1 h. After filtration, theresidual was decocted one last time with 800 mL of wa-ter for 0.5 h. The decoctions were mixed, concentrat-ed, dried to powder and kept ready for use.

Experimental animalsHealthy Kunming mice, male, 18-20g in weight, werepurchased from the Laboratory Animal Center, Acade-my of Military Medical Sciences with license:SCKX-(Army) 2007004. They were allowed unlimitedaccess to food and drink.

Instruments and reagentsThe Animal Thermotropism Behavior Surveillance Sys-tem[8] was designed by our research team and assembledby the Beijing Zhongjiao Instrument Company (Pat-ent No: ZL2008200004444.2). The system consists ofthree major parts, the Automatic Temperature Control-ling unit (Figure 1a), the Remote Monitoring unit (Fig-ure 1b), and the Data Processing unit (Figure 1c). Be-

fore the experiment, two stable temperature zones weregenerated by the Automatic Temperature Controllingunit, then the animals were put on the cold/hot platesand allowed to move freely across the two zones. Toevaluate their thermotropic behavior during the experi-ment, the amount of time spent in the different tem-perature zones, the distances the mice moved, etc. wereobserved. The parameters were obtained using remotevideo monitoring software, a type of artificial intelli-gence that captured the movement of the mice in thedifferent zones. The video allowed real-time processingof each frame of the images and the plane projectioncoordinates (X, Y) were plotted from those frames. Thetrajectories of the mice were obtained by joining the co-ordinate data of each animal in all frames Yt = f [Xt]. Inthe computer processing of the duration and frequencyeach animal spent in different zones, the video recogni-tion software was based on Paulo's algorithms[9] andthe data analysis software was written with Visual Basic6.0.A Cary50 Bio UV spectrophotometer (Varian AustraliaPTY LTD), a SIGMA 3-18K centrifuge (Germany),ATPase assay kit (20110422), a Total Superoxide Dis-mutase assay kit (20110414), a Colorimetric Blue Pro-tein assay kit (20110303) and a Total Antioxidant Ca-pability (20110420) were purchased from Nanjing Ji-ancheng Biological Engineering Research Institute. Allthe reagents were of A.R. grade.

Grouping and administrationBefore the experiment, the mice were familiarized withthe temperature of the hot/cold plate and those withabnormal rectal temperatures were removed. The quali-fied mice with normal body temperatures were ran-domly divided into four groups, the Normal group,the Fuzi group, the Ganjiang group and the Fuzi+Gan-jiang group. The dose for medicinal groups was 10graw medicine/kg, which was given intragastrically, 0.2mL/10g body weight. The Normal group was adminis-tered the same volume of physiological saline for sevendays in succession.

Determination of oxygen consumptionMice were put in an airtight container with 10g sodi-um oxide at the bottom for absorbing the CO2 theygenerated and a rubber tube with a graduated pipettewas connected at the top. The other end of the graduat-ed pipette was inserted vertically into water. All inter-faces were sealed with Vaseline. The amount of time ittook to breathe 2.5 mL of air and the volume of airconsumed for six minutes were measured.

Determination of the activities of Na+-K+- ATPaseand Ca2+- Mg2+ATPase of liver tissueMice were killed by cervical dislocation then immedi-ately dissected to retrieve their livers, which wereplaced into physiological saline precooled to 4°C to re-move the blood; water on the liver tissue surface was

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Sun ZY et al. Research on animal thermotropism behavior to discover if Fuzi has fewer hot without Ganjiang

wiped off using filter paper. The liver tissues were thenaccurately weighed and prepared into 2% tissue ho-mogenate to measure the ATPase activity, counted as 1μmol of inorganic phosphorus produced by ATPase in1mg of tissue protein within one hour (μmol·mg-1·h-1).

Determination of the temperature tropism of miceon cold/hot plateThe experiment was performed on a bi-zone tempera-ture-controlled plate, 25° C (cool plate) and 40° C(warm plate), with the laboratory temperature set at23°C±2°C. Ten minutes after medicine administration,mice were put into the monitoring channel of the cold/hot plate, where their temperature tropism behaviorswere remotely monitored and the trajectories of eachmouse recorded. Records were taken once every day,30 min each time, for 7 days in succession. Remainingrate (RR) on the warm plate (%)=remaining time onwarm plate (sec)/the total monitoring time (sec) ×100%.

Determination of the total antioxidant capacity(T-AOC)T-AOC activity of 0.1 mL blood serum was measuredaccording to the instructions of an assay kit and count-ed as the absorbency increased by 0.01 unit of the reac-tion system at 37°C in 1 mL blood serum per minute.

Determination of the activity of total superoxidedismutase (T-SOD)The Xanthine oxidase method was used to measure theT-SOD activity of 10 μL blood serum. One unit of ac-

tivity of SOD was counted as the corresponding quan-tity of SOD when a 50% inhibition ratio of SOD wasobtained in 1mL reaction solution.

Determination of the visceral indices of miceMice were killed by cervical dislocation then immedi-ately dissected to retrieve their livers, hearts, spleens,lungs and kidneys. The viscera were put into physiolog-ical saline at 4°C to remove the blood; water on the tis-sue surface was wiped off using filter paper. They wereweighed and the results were recorded. The livers werekept in a freezer at -80°C. The visceral index (%)= vis-ceral weight/body weight×100%.

Statistical analysisData were expressed as means±standard deviation ( x̄ ±s). Statistical analysis on the remaining rate on thewarm plate, times and distances of movement, ATPaseactivity of liver tissues, respiratory oxygen consump-tion volume, serum T-AOC and T-SOD activity wasperformed using ANOVA and t-tests with SPSS 13.0.Additionally, P values less than 0.05 were consideredsignificant.

RESULTS

General conditions of miceDuring the experiment, skin color, diet, stool, urineand the general behavior of mice were monitored andno abnormal phenomena were found in any of thegroups. Compared with the Normal group, the waterintake of all medicinal groups increased in tendency,

Figure 1 Animal Thermotropism Behavior Surveillance System

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while that of the Fuzi group significantly increased.The body weight of the Fuzi group increased, whilethat of the other medicinal groups did not change sig-nificantly, as compared with the Normal group.

Changes in oxygen consumption of miceThe amount of oxygen consumed in six minutes waschosen as the evaluated index. Compared with the Nor-mal group, the respiratory oxygen consumption of theFuzi group and Fuzi + Ganjiang group was found tohave increased significantly (P<0.01), while that of theGanjiang group was not significantly changed. Com-pared with the Fuzi group, the oxygen consumption ofthe Fuzi + Ganjiang group was found to have signifi-cantly increased (P<0.05) (Figure 2a). The longer ittook the mice to consume the 2.5 mL of oxygen, theless they consumed in six minutes, revealing a similarresult in oxygen consumption, but comparativelyspeaking, the latter were the more sensitive data ele-ment.

Changes in the activities of atpase of liver tissue ofmiceCompared with the Normal group, the Na +-K +-ATPase activity of the Fuzi group was found to have de-creased significantly (P<0.05) and the Ca2 +-Mg2 +-ATPase activity decreased significantly (P<0.01).The Na+-K+- ATPase activity and Ca2+-Mg2+-ATPase ac-tivity of the Ganjiang group increased significantly (P<0.01) and the Na +-K +- ATPase activity andCa2 +-Mg2 +-ATPase activity of the Fuzi + Ganjianggroup increased significantly (P<0.05). Compared withthe Fuzi group, the Na +-K +- ATPase activity andCa2 +-Mg2 +-ATPase activity of the Fuzi + Ganjianggroup increased significantly (P<0.01) (Figure 2b).The combination and compatibility of Fuzi and Ganji-ang could increase the ATPase activity of liver tissue in

mice, suggesting an enhancement of energy metabo-lism.

Changes in the temperature tropism of mice on cold/hot plateCompared with the Normal group, the remaining rateon the warm plate of the Fuzi group and Ganjianggroup was not significantly different (P>0.05), whilethe times and distances of movement were found tohave significantly decreased (P<0.05). The remainingrate on the warm plate and the times and distances ofmovement of the Fuzi+Ganjiang group were decreasedsignificantly (P<0.01) with reductions of 55.1% ,48.3% and 44.8%. Compared with the Fuzi group, theremaining rate on the warm plate and the times anddistances of movement of the Fuzi + Ganjiang groupwere found to have decreased significantly (P>0.01),with reductions of 57.6% , 34.3% and 36.0% (Table1). The increased remaining rate on the cool plate ofthe Fuzi+Ganjiang group manifested as "cold" tropism,suggesting an enhancement of "hot" production whenFuzi and Ganjiang are combined, while the decreasedtimes and distances of movement revealed a reductionin the movement of the mice.

Changes in the T-AOC and T-SOD activity of miceCompared with the Normal group, the T-AOC andT-SOD activity of the Ganjiang group were significant-ly decreased (P<0.05), those of the Fuzi + Ganjianggroup were significantly increased (P<0.01), while thechanges in the Fuzi group were not significant. Com-pared with the Fuzi group, the T-AOC and T-SOD ac-tivity of the Fuzi+Ganjiang group were significantly in-creased (P<0.05) (Table 2). The changes in the T-AOCand T-SOD activity suggest that combining Fuzi andGanjiang could reinforce the antioxidant capacity inmice.

Figure 2 Effect of Fuzi, Ganjiang, and Fuzi+Ganjiang on the respiratory oxygen consumption a) and ATPase activity b) of mice (Ver-sus the normal group, *P<0.05, **P<0.01; vs. the Fuzi group, ∆P<0.05, ∆∆P<0.01).

Table 1 Comparison of reduction in the remaining rate on the warm plate, the times and distances of movement ( x̄ ±s, n=12)

Group

Normal

Remaining rateon warm plate (%)

25.6±6.1

Times ofmovement

9.3±3.6

Distances ofmovement

8.2±2.6

Reduction (%)

- - -

Comparison group

-

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Fuzi

Ganjiang

Fuzi + Ganjiang

27.1±5.0

20.2±3.4*

11.5±4.5**∆∆

7.3±3.2*

6.6±3.3*

4.8±3.1**∆∆

7.1±2.4**

6.3±3.0**

4.5±2.5**∆∆

-6.0

21.0

55.1

57.6

43.2

21.3

28.9

48.3

34.3

27.2

13.7

23.3

44.8

36.0

28.0

Normal

Fuzi

Normal

Fuzi

Ganjiang

Notes: Versus the normal group, *P<0.05, **P<0.01; vs. the Fuzi group, ∆P<0.05, ∆∆P<0.01.Table 2 Effects of Fuzi, Ganjiang and Fuzi + Ganjiang on theT-AOC and T-SOD activity of mice ( x̄ ±s, n=12)

GroupNormalFuziGanjiangFuzi + Ganjiang

T-AOC (U/mL)11.05±0.2111.14±0.559.80±1.02*

12.22±0.70**∆

T-SOD (U/mL)388.72±45.75418.65±54.72338.72±50.76*

543.15±37.05**∆∆

Notes: Versus the normal group, *P<0.05, **P<0.01; vs. the Fuzigroup, ∆P<0.05, ∆∆P<0.01.

DISCUSSIONDrug Nature, the core of Traditional Chinese Medicinetheory, is not only the link between Chinese medicineand drugs, but also the basis of syndrome differentia-tion, treatment and prescription. The four natures,warm, hot, cold and cool, are subtly different, thoughnot qualitatively. Hence, cold and hot natures are con-sidered the primary Drug Natures used in Chinesemedicine[10,11]. Fuzi and Ganjiang are pungent in taste,hot in nature and when used in combination, they mu-tually enhance each other's qualities to warm the mid-dle-jiao, which dispels cold and warm the kidneys,which restores yang. They differ not in cold vs. hot na-ture, but by the extent of their hot nature. For exam-ple, Fuzi is often used in combination with Ganjiangto treat the "exhaustion of yang" syndrome and col-lapse, whose symptoms can include cold limbs, feeblepulse, cold sweats, severe vomiting or severe diarrhea[12].The physiological or pathological reaction of organ-isms to heat changes due to medicinal activity may becaused by the normal transformation of energy con-tained in the medicine itself or in the body's heat-pro-ducing materials. They may also be caused by a seriesof physiological or pathological reactions produced bythe activation of endogenous heat-producing substanc-es contained in the medicine or in related materials inthe body[13]. Energy transfer and thermal changes ac-company all these reactions. Whatever the types of en-ergy transfer or effects of heat, the human body maypresent what are known in TCM as "cold" or "hot"characteristics differently[14-16], demonstrating differenttropism to a hot or cold environment. Using the Ani-mal Thermotropism Behavior Surveillance System toobserve the differences in the "cold" or "hot" character-istics of herbal medicines, we can reveal the trends ofenergy changes in the human body by observing the

compensatory behavior associated with automatic selec-tion.In this investigation, it was found that the remainingrate on the warm plate and the times and distances ofmovement in mice after administering a combinationof Fuzi and Ganjiang were significantly decreased (P<0.05). Compared with the Normal group, the reduc-tion was 55.1% , 48.3% and 44.8% , while comparedwith the Fuzi group, the reduction was 57.6%, 34.3%and 36.0%. This indicates that the "cold" tropism wassignificantly enhanced, causing the mice to present acompensatory "cold" tropism that compensated for thesensation of "hot" and the objective existence of "hot";thus we confirmed that the combination and compati-bility of Fuzi and Ganjiang produces an increase inheat.Physiologically, the human body's chills and fever re-flect the relationship of the balance between generatingand using energy. Related studies have reported that en-ergy transfer might change the course of ATPase activi-ty[17]. "Cool" drugs such as Coptis Chinensis and Pep-permint could decrease the ATPase activity, while"warm" drugs such as Fuzi and Herba Epimedii couldincrease the ATPase activity[18-21]. MA Qing-Cui et al.found that strobal had the function of promoting ener-gy metabolism in experimental animals by measuringenergy intake, digestible energy and metabolizable ener-gy[22]; ZHAO Hai-Ping et al. found that a relationshipcould be drawn between ATPase activity and the"cold" nature of rhubarb and the "hot" nature of strob-al[23]. To some extent, the difference between the "cold"and "hot" natures of drugs can be understood by ob-serving energy metabolism. In this study, there was in-creased oxygen consumption and ATPase activity afterFuzi and Ganjiang were given intragastrically, com-pared with the Normal group and the Fuzi group,which was in keeping with the increase in "hot" charac-teristics.Distinguishing between the "cold" and "hot" naturesof medicines can, to some extent, be made easier by un-derstanding energy metabolism. In this study, wefound that in comparing the Normal group and theFuzi group, the increase of respiratory oxygen con-sumption and the Na +-K +-ATPase and Ca2 +-Mg2 +-ATPase activity of the Fuzi + Ganjiang group were inkeeping with the finding that their heat production in-creased. This indicated that to a certain degree, chang-es in energy metabolism correlate with the "cold" or

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"hot" natures of herbal medicines. The increase ofATPase activity in liver tissues may be one of the mech-anisms by which the combination of Fuzi and Ganji-ang increases heat production.ZHAO Yan-ling et al. found that bodily oxygen con-sumption and SOD activity, a parameter for bodily en-ergy metabolism and feedback regulation, changed aswell[24]. CUI X et al. found that D-galactose exposureinduced an increase in peripheral oxidative stress, in-cluding an increase in MDA and decreases in T-AOC,T-SOD and glutathione peroxidase (GSH-Px)activities[25]. In this study, we investigated the effects ofFuzi, Ganjiang and Fuzi and Ganjiang on T-AOC andT-SOD activity. The Fuzi+Ganjiang group showed in-creased T-AOC and T-SOD activity compared withthe Normal group and the Fuzi group. The Fuzi+Ganji-ang group, enhancing the "hot" nature, activated thetotal antioxidant capability, reflecting an increase in theability to resist an external environment.To sum up, normal animals, when treated with Fuzi,Ganjiang or a Fuzi and Ganjiang combination, showedan increase in cold tropism based on animal thermo-tropic behavior. This phenomenon is associated withan increased level of energy metabolism and antioxi-dant activity. The Animal Thermotropism System ob-jectively and quantitatively characterized the phenome-non of increased heat after treatment with Fuzi andGanjiang, showing a high degree of coincidence be-tween the original state and observed changes. Howev-er, further study is needed to determine if Fuzi exertsfewer "hot" characteristics on its own, without Ganji-ang.

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Research on Fuzi based on animal thermotropism behavior to discover if it has fewer “hot” characteristics without Ganjiang