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      article: The Effects of QIHM and QRHM on Energy Metabolism—An experimental perspective  |  date: 2016-02-22 14:19:41  |  Find articles by this author

     


     

    The Effects of QIHM and QRHM on Energy Metabolism—An experimental perspective

    An Approach to the Nature of Qi in TCM - Qi and Bioenergy (10)

    By Xing-Tai Li [1] and Jia Zhao [2]

    10. The effects of QIHM and QRHM on energy metabolism—An experimental perspective

    ABSTRACT Aims: TCM practitioners usually compose prescriptions made up of Qi-invigorating herbal medicines (QIHM) or Qi-flow regulating herbal medicines (QRHM) for Qi system diseases, and have accumulated abundant clinical experience for a long time. To approach to the nature of Qi in TCM from bioenergetics, the effects of QIHM (ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome) and QRHM (immature bitter orange, magnolia bark, green tangerine and lindera root) on oxidative phosphorylation (OXPHOS), bioenergy level and creatine kinase activities were investigated.

    Methods: QIHM and QRHM were administered by oral gavage daily for 10 days. Mice liver mitochondria were isolated by differential centrifugation. The effects of QIHM and QRHM on energy metabolism were studied from the production, regulation, and storage of bioenergy. Mitochondrial OXPHOS curve was determined by Clark oxygen electrode method. The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in liver cells were determined by reversed-phase high performance liquid chromatography (RP-HPLC), adenylate energy charge (AEC), total adenylate pool (TAP) were calculated. The creatine kinase (CK) activities in mice skeletal muscle were determined by a commercial monitoring kit. The regularity of action of QIHM and QRHM were analyzed and concluded.

    Results: Ginseng and astragalus root can decrease oxygen consuming rate and respiratory control ratio (RCR) of liver mitochondria obviously, we consider this is appearance of lowering standard metabolic rate and is a kind of protective adaptation. QRHM can increase P/O ratio and RCR. Both QIHM and QRHM can stimulate activity of CK significantly in the storage of energy, and QRHM is stronger than QIHM. But it is worth notice that all the four QIHM can increase levels of ATP, AEC and TAP; on the contrary, all the four QRHM can decrease levels of ATP, AEC and TAP in liver cells. In a word, QIHM and QRHM increase and decrease bioenergy level of liver cells respectively in vivo. Therefore, Qi is closely related to bioenergy.

    Conclusion: Qi and bioenergy have common sources and identical functions. QIHM and QRHM are able to improve and decrease the energy state of the body respectively. Qi and bioenergy have general characteristics in many aspects. The experiments provide scientific evidence for Qi in TCM is bioenergy.

    Key words: Qi; bioenergy; Adenosine triphosphate; qi-invigorating herbal medicine; qi-regulating herbal medicine. According to TCM theory, Qi (vital energy) refers to a kind of refined nutritive substance within the body. Qi is one of the most basic, the most important, and the most complicated concept in TCM. We propose a hypothesis that Qi is closely related to bioenergy according to the ancient concept of Qi and modern bioenergetics. TCM practitioners usually compose prescriptions made up of Qi-invigorating herbal medicines (QIHM) or Qi-flow regulating herbal medicines (QRHM) for Qi system diseases, and have accumulated abundant clinical experience for a long time. QIHM is a kind of herbal medicines which can invigorate Qi and treat syndromes of Qi deficiency, they have the effects of invigorating Qi, promoting the production of body fluid and tonifying the spleen and lung etc. QRHM is a kind of herbal medicines which can induce the flow of Qi, regulate the Qi system diseases and treat the syndromes of stagnation of Qi or rebellious Qi etc. They can activate Qi to reduce pain, depress upward-reverse flow of Qi, break the stagnant Qi to remove masses etc. QIHM and QRHM have similar nature and atributive channels, but their flavours are different significantly. QIHM taste sweet while QRHM taste acrid-bitter, and their compositions are also different. Although Qi of TCM is similar to the concept of modern medical bioenergy in some aspects, the energy nature of Qi still lacks convincing evidence. Therefore, we take it as our basic point to approach the characteristics of QIHM and QRHM on energy metabolism. We have approached the rules of QIHM and QRHM from the production (oxidative phosphorylation), storage (creatine kinase activity) and regulation (adenylate energy charge) of bioenergy (ATP). Since there is no direct detection method on Qi, the widely used QIHM (ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome) and QRHM (immature bitter orange, magnolia bark, green tangerine and lindera root) were selected to study the effect on energy metabolism to approach to the nature of Qi in TCM.

    Sasang constitutional medicine (SCM) is a unique traditional Korean therapeutic alternative form of medicine. In both SCM and TCM theories, Qi is the most essential element, the ‘driving force’ that constitutes the body and maintains the activities of life, visceral functions and metabolism. In a generalized scope, the essence of Qi in SCM can be compared with that of energy in modern physiology. The metabolic process in physiology provides energy, kinetic and potential energies, whereas metabolism in SCM produces and regulates Qi. Since catabolism breaks own complex molecules into simple ones and releases kinetic energy, this pathway can be compared with the process of consuming Qi in SCM. Similarly, anabolism, which links together simple molecules to form more complex molecules and stores potential energy, is comparable with the process of producing and storing Qi in SCM. In terms of interior–exterior exchange, the process of taking up raw materials from the external environment to produce Qi in SCM (function of the spleen) corresponds to the process of digestion and absorption of food and water and inhaling air in physiology (Kim and Pham, 2009). To approach to the nature of Qi in TCM from bioenergetics, the effects of QIHM and QRHM on oxidative phosphorylation (OXPHOS), bioenergy level and creatine kinase (CK) activities were investigated.

    Materials and methods

    Animals and materials

    Male Kunming mice (Grade II, Certificate No 2002-5), weighing 22±2.0 g each, were purchased from Experimental Animal Center, Dalian University. All mice were cared for according to the Guiding Principles in the Care and Use of Animals. The experiment was approved by Medical College Council on Animal Care Committee of Dalian University (China) in accordance with NIH guidelines (NIH, 2002). Rodent laboratory chow and tap water were available ad libitum during the period. Spherisorb C18 reversed-phase chromatographic column (4.6 mm×250 mm, 5 µm particle size) was produced by Dalian Institute of Chemistry and Physics, Chinese Academy of Sciences. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), 2-Thiobarbituric acid (TBA), and 1,1,3,3-tetraethoxypropane (TEP) were from Sigma Chemical (St Louis, MO, USA). N-2-Hydroxyethylpiperazine-N’-2-ethane sulfonic acid (HEPES) was from Merck (Darmstadt, Germany). Coomassie Brilliant Blue G-250 (CBBG-250) was purchased from Fluka (Bushs SG, Switzerland). Bovine serum albumin (BSA) was from Boehringer Mannheim Corp. (Indianapolis, IN, USA). Tris(hydroxymethyl)aminomethane (Tris) was from Gibco BRL (Grand Island, NY, USA). A commercial creatine kinase monitoring kit [N-acetyl-Lcysteine(NAC)-activated] was from Beijing Zhongsheng High-Tech Bioengineering Company (Beijing, China). All other chemicals and solvents used in the study were of analytical grade made in China. Ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome, immature bitter orange, magnolia bark, green tangerine and lindera root, are Panax ginseng C.A. Mey (Tongrentang red ginseng), Astragalus membranaceus (Fisch.) Bge.var. mongholicus (Bge.) Hsiao, Codonopsis pilosula (Franch.) Nannf, Atractylodes macrocephala Koidz, Citrus aurantium L, Magnolia officinalis Rehd et Wils, Citrus reticulate Blanco and Lindera aggregate (Sims) Kosterm respectively, were purchased from Beijing Tongrentang Drugstore, and identified by professor Li Jiashi at Beijing University of Traditional Chinese Medicine.

    Preparation of the aqueous extracts of QIHM and QRHM

    Powdered dry ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome, immature bitter orange, magnolia bark, green tangerine and lindera root were immersed in distilled water (the ratio of the drug and distilled water was 1:10) for 0.5 hour and extracted thrice with distilled water for 0.5 hour each in a boiling water bath. The filtrate was collected after filtration with gauze, mixed and condensed to 0.2 g crude drug/ml.

    Animal groups

    Mice in each QIHM and QRHM group (n=10) were administered respective aqueous extracts (4 g crude drug/kg/day) by oral gavage and mice in the control group received an equivalent volume of normal saline for 10 days, there are nine groups all together. All the mice were maintained with free access to food and drinking water.

    Isolation of liver mitochondria

    Mitochondria were isolated by differential centrifugation using a modified version of the protocol of Michele et al. (1992). Mice were dislocated and their livers were removed immediately and placed in an ice-cold isolation medium (containing 0.25 M sucrose, 0.5 mM EDTA and 3 mM HEPES, pH 7.4). Livers were homogenized with a motor-driven Teflon pestle in wet ice at 0°C. Following homogenization, samples were centrifuged at 1,000 g for 10 min. This, and all other centrifugation steps, used a Beckman JA-25.50 rotor and Beckman J2-MC centrifuge at 4°C. Supernatants were removed and centrifuge at 12,000 g for 10 min. The pellets were washed twice in the isolation medium, and respun at 12,000 g. Following the final wash, mitochondria were resuspended in the same medium. Protein determinations were carried out using Bradford (1976) method.

    Measurement of oxidative phosphorylation curve of liver mitochondria

    Respiratory control ratio (RCR) of liver mitochondria was measured using the method described by Estabrook (1967). Oxygen consumption was measured at 30°C in a closed, stirred, and thermostatted glass vessel equipped with a Clark-type oxygen electrode in 2.0 ml respiration buffer. The respiration buffer (pH 7.4) consisted of sucrose 225 mM, EDTA 1 mM, MgCl2 5 mM, KCl 15 mM, KH2PO4 15 mM, Tris 50 mM, L-glutamic acid 5 mM, DL-malate 10 mM, and mitochondrial protein 5 g/L. Respiratory state 3 (S3) was the oxygen (O2) consumption by mitochondria in the presence of substrate after the addition of 0.25 mM adenosine diphosphate (ADP, ADP is a potent stimulator of mitochondrial respiration). Respiratory state 4 (S4) was the oxygen consumption when all the ADP has been phosphorylated. S3 and S4 can be calculated according to the oxidative phosphorylation (OXPHOS) curve. Respiration rates were expressed in nanomoles atom O per minute per milligram of protein. RCR was the ratio of S3 to S4 respiration. P/O ratio is the number of ADP molecules phosphorylated per oxygen atom reduced.

    Determination of creatine kinase activity

    Mice were killed via dislocation, and skeletal muscle from the hind leg was rapidly removed, weighed and made into 1% homogenates with normal saline at 0°C. 2.0 ml homogenate was centrifuged at 2,000 g for 5 min, 100 µl supernatant was added to 900 µl normal saline and mixed, 10 µl of which was used for determination of creatine kinase (CK) activity. CK activity was measured by using a commercial CK monitoring kit [N-acetyl-Lcysteine(NAC)-activated], following the manufacturer’s protocol.

    Measurement of ATP, ADP, and AMP in liver cells by HPLC

    Mice were killed via dislocation, and livers were rapidly removed, weighed and made into 10% homogenates with normal saline at 0°C, 1 ml of ice-cold 0.3 M perchloric acid was added to 1 ml of 10% liver homogenates that were kept on ice for an additional 5 min. Harvested materials were centrifuged at 15,000 g at 4°C for 10 min. The supernatant was neutralized with 80 µl of 3 M KOH, and tubes were kept on ice for an additional 30 min. The resulting precipitate was removed by centrifugation, and the supernatant was stored at -80°C until it was analyzed. 10 µl of neutralized cell extract was used for determination of ATP, ADP, and AMP in liver cells, which was carried out by gradient RP-HPLC (reversedphase high performance liquid chromatography) with ultraviolet detector at room temperature and with mobile phase at a rate of 0.8 ml/min. Mobile phases used for the gradient system were buffer A (0.05 M KH2PO4-K2HPO4, pH 6.0) and buffer B, consisting of buffer A plus 10% methanol (v/v). All buffers and solutions used for HPLC analysis were filtered and degassed through a 0.45 µm filter. Gradient elution procedure: buffer A was used as mobile phase between 0 and 3 min, buffer A was changed from 100% to 0% and buffer B from 0% to 100% between 3 and 6 min, buffer B was mobile phase between 6 and 9 min, buffer A was the mobile phase after 9 min, all the running time was 12 min, the detection wavelength was set at 254 nm. ATP, ADP and AMP quantitation in liver cells was calculated by computing the peak area of them, identification and quantitative measurements of nucleotides were carried out by the injection of standard solutions of nucleotides with known concentrations. Standard curves were plotted for individual compounds and were used to determine the contents of ATP, ADP, and AMP in each sample. Total adenylate pool (TAP) and adenylate energy charge (AEC) were calculated by the following formulas respectively: TAP = [ATP] + [ADP] + [AMP], AEC = ([ATP] + 0.5[ADP])/TAP. AEC represents a linear measure of the metabolic energy stored in the adenine nucleotide system.

    Statistical analysis

    Data were expressed as means±SD and statistical differences between groups were analyzed by Student’s t test which was performed using SPSS 16.0 statistical software (SPSS Inc., Chicago, Illinois, USA). The probability (P) values <0.05 were considered to be statistically significant.

    GroupState 3 (nmol/min/mg)d State 4 (nmol/min/mg)dRCR P/OCK (U/ug)e

    Control

    83±11 19.4±2.6 4.2±0.6 2.61±0.28 2.25±0.28
    Panax ginseng 66±11b 18.3±1.9 3.6±0.4a 2.21±0.30b2.58±0.26a
    Astragalus membranaceus 68±13a 18.0±2.5 3.7±0.4a 2.32±0.24a 2.60±0.29a
    Codonopsis pilosula 80±15 18.3±2.2 4.3±0.6 2.66±0.26 2.59±0.29a
    Atractylodes macrocephala 81±14 19.1±1.8 4.2±0.7 2.60±0.28 2.63±0.32a
    Citrus aurantium 91±10 18.5±2.9 4.9±0.4b 2.89±0.27a 3.32±0.27b
    Magnolia officinalis 99±13b 19.0±2.1 5.1±0.5b 2.92±0.34a 3.20±0.44b
    Citrus reticulate 96±12a 19.3±2.2 4.9±0.6a 2.88±0.22a 3.09±0.49b
    Lindera aggregate 86±16 16.6±2.3a 5.2±0.7b 2.93±0.31a 3.23±0.35b
    d nanomole O2 per minute per milligram protein (nmol O2·min-1·mg protein-1). e Unit of CK activity per microgram protein (U μg protein-1). a P <0.05 vs Control. b P <0.01 vs Control.

    Table 1. Effects of QIHM and QRHM on respiratory function of liver mitochondria and CK activities in vivo (n=10, mean ± standard deviation).

    Group ATP/ (mmol·L-1)ADP/ (mmol·L-1) AMP/ (mmol·L-1) TAP/ (mmol·L-1)AEC
    Control 1.02±0.28 0.78±0.20 0.09±0.07 1.89±0.33 0.745±0.021
    Panax ginseng 1.41±0.36a 0.86±0.24 0.07±0.05 2.34±0.46a 0.786±0.031b
    Astragalus membranaceus 1.36±0.31a 0.84±0.23 0.11±0.05 2.31±0.38a 0.770±0.026a
    Codonopsis pilosula 1.33±0.22a 0.85±0.25 0.12±0.08 2.30±0.32a 0.763±0.015a
    Atractylodes macrocephala 1.25±0.19a 0.81±0.26 0.10±0.06 2.16±0.28 0.764±0.016a
    Citrus aurantium 0.70±0.26a0.53±0.14b 0.25±0.12b 1.48±0.27b 0.651±0.024b
    Magnolia officinalis 0.71±0.23a0.61±0.13a0.19±0.09a1.51±0.23b 0.673±0.033b
    Citrus reticulate 0.75±0.25a0.49±0.14b 0.27±0.12b 1.51±0.26a0.660±0.028b
    Lindera aggregate 0.61±0.22b 0.47±0.16b 0.29±0.13b 1.37±0.21b 0.618±0.027b
    All values are mean±SD (n=10). aP <0.05, bP <0.01 versus Control group. Each value expressed in mmol·L–1 (ATP, ADP, AMP, TAP) or as a ratio (AEC). ATP: adenosine triphosphate; ADP: adenosine diphosphate; AMP: adenosine monophosphate; TAP: total adenylate pool; AEC: adenylate energy charge

    Table 2. The effects of QIHM and QRHM on energy status of mice hepatocytes in vivo (n=10, mean ± standard deviation).

    Effects Items QIHM QRHM
    Same or similarities 1. channel tropism
    2. natures
    3. CK activity
    spleen, lung and stomach
    mild or warm
    increase
    spleen, lung and stomach
    warm
    increase
    Cross effects 1. RCR
    2. oxygen consumption rate
    decrease or no effect decrease or no effect no effect or increase no effect or increase
    Differences 1. tastes
    2. compositions
    3. bioenergy level
    sweet
    more carbohydrate
    increase
    bitter and/or hot
    volatile components
    decrease

    Table 3. Comparisons of the regularity between QIHM and QRHM.

    Results

    The effects of QIHM and QRHM on OXPHOS of liver mitochondria

    The liver is known to be the hub of the metabolism; it plays a major role in controlling glucose storage and flux. It is also known that, during heat stress, both lipids and carbohydrate stores can be mobilized for energy generation to attenuate the stress response (Manoli et al., 2007). In addition, many biochemical studies have been performed using mitochondria from liver cells. The rate of ATP synthesis and oxygen consumption (respiratory state 3) driven by complex I substrates, the respiratory control ratio (RCR) and P/O ratio were reduced in liver mitochondria by ginseng and astragalus root, but there were no significant effect on state 4 (P >0.05) (Table 1). It showed that the efficiency of ATP production via ADP phosphorylation was decreased. In perfectly coupled mitochondria, there would be no proton leak across the inner mitochondrial membrane, and the entire gradient generated by the respiratory chain would be used to generate ATP (Boudina and Dale Abel, 2006). Control of oxidative phosphorylation (OXPHOS) allows a cell to produce only the precise amount of ATP required to sustain its activities. Recall that under normal circumstances, electron transport and ATP synthesis are tightly coupled. The value of P/O ratio (the number of molecules of Pi consumed for each oxygen atom reduced to H2O) reflects the degree of coupling observed between electron transport and ATP synthesis (Mckee and Mckee, 1999). Oxygen consumption increase dramatically when ADP is supplied. The control of aerobic respiration by ADP is referred to as respiratory control. Substrate oxidation accelerates only when an increase in the concentration of ADP signals that the ATP pool needs to be replenished. This regulation matches the rates of phosphorylation of ADP and of cellular oxidations via glycolysis, the citric acid cycle, and the electron-transport chain to the requirement for ATP (Horton, et al., 2002). Ginseng and astragalus root can decrease oxygen consuming rate and RCR of liver mitochondria obviously, I consider this is appearance of lowering standard metabolic rate and is a kind of protective adaptation. Qi deficiency patients need nutritional supplements, adequate rest, and should reduce energy consumption, ginseng and astragalus root can just achieve this goal, while the effect of other QIHM is not obvious. All the four QRHM can increase RCR and P/O ratio (Table 1).

    The effects of QIHM and QRHM on creatine kinase activities

    Although ATP is the instantaneous donor of bio-energy in the body, it can not be stored, but phosphocreatine (PCr) can. Among the energy metabolism enzymes in the muscle cells, creatine kinase (CK, EC 2.7.3.2) plays a significant role in energy homeostasis. CK is distributed in skeletal muscle, heart, brain and other tissues and catalyzes the reversible conversion from ATP and creatine (Cr) to ADP and phosphocreatine (PCr, high energy phosphate able to supply ATP on demand) (Zhao et al., 2007; Brancaccio et al., 2007). CK performs a pivotal physiological role in high energy consuming tissues, by acting as an energy buffering and transport system between the sites of ATP production and consumption by ATPases (Bessman and Geiger, 1981). Creatine kinase rapidly provides ATP to highly energy-demanding processes, the rate of transfer of the phosphoryl group from PCr to ADP by CK is greater than the maximum rate of ATP generation by OXPHOS, and this ensures rapid resynthesis of ATP (Wallimann et al., 1998). High tissue CK activity, whether constitutive, induced, or both, may rather directly enhance contractile responses by enhancing cellular energy and contractile reserve (Brewster et al., 2007). Greater CK activity could bind more ADP and increase the rate of the conversion of ADP to ATP, which could reduce the relative levels of local ADP at the contractile proteins (Clark, 1994). CK enhances ATP buffer capacity. We believe that high CK activity may be quite beneficial for rapid and dynamic energy demand. Thus, increased CK activity in muscle tissue might lead to hyperdynamic activity. Both QIHM and QRHM can stimulate activity of CK significantly in the storage of energy, and QRHM is stronger than QIHM (Table 1).

    The effects of QIHM and QRHM on energy state of mice hepatocyte in vivo

    Adenylate energy charge (AEC) is a sign parameter of cellular energy state (the higher [ATP], the larger the AEC, the higher [AMP], the smaller the AEC), when the tissue's ATP level increased, the pathway for generating ATP would be inhibited; When ATP levels drop due to over consumption of energy by the body, the pathway for generating ATP would be stimulated. AEC represents a linear measure of the metabolic energy stored in the adenine nucleotide system. AEC remained at a fairly narrow range of changes, just like pH value in the cells, energy charge also has a buffering effect, AEC of the most cells fluctuate in the 0.8-0.95 range. Ginseng is commonly known as a high-level herb for tonifying Qi, according to our former study, Panax ginseng polysaccharide could increase levels of ATP, TAP and AEC in liver cells under chronic hypoxia condition, therefore, improving energy status, protect mitochondria by inhibiting mitochondrial swelling (Li et al., 2009). It is worth notice that all the four QIHM can increase levels of ATP, AEC and TAP, the effect of ginseng is the most potent; on the contrary, all the four QRHM can decrease levels of ATP, AEC and TAP in liver cells. All the four QIHM can’t affect the levels of ADP and AMP; while all the four QRHM can decrease levels of ADP, and increase levels of AMP in liver cells. In a word, QIHM and QRHM increase and decrease bioenergy level of liver cells respectively in vivo. Therefore, Qi is closely related to bioenergy. This result shows that the decreased energy state of the body can be improved by taking QIHM and the effect of QRHM is contrary to that of QIHM. Therefore, the effects on energy regulation of two types of Qi system drugs are different (Table 2).

    The similarities and differences in natures, tastes, channel tropism and compositions

    Qi is an important concept in physiology and pathology of TCM, directed towards the two main therapeutic principles of Qi—qi-invigoration and qi-flow regulation are self-evidently extreme important. The two therapeutic principles are closely related, complementary and two-way adjustable, and difficult to substitute by others. If they are used properly, they will play an important clinical role in overcoming various difficult diseases. Since there is no direct detection method on Qi, the widely used QIHM (ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome) and QRHM (immature bitter orange, magnolia bark, green tangerine and lindera root) were selected to study the effect on energy metabolism to approach to the nature of Qi in TCM, Comparison of the regulatory role of QIHM and QRHM are summarized as follows.

    The channel tropism of QIHM and QRHM are all the spleen, lung and stomach channel, this shows that they have common target sites in the body. The natures of QIHM are mild or warm, and QRHM are warm, the properties of the two kinds of medicines are similar. QIHM are sweet taste, medicines with sweet taste have the effects of invigoration, normalizing the function of the stomach and spleen, and buffering emergency, etc. they are usually used for tonifying deficiency, easing the pain, and harmonizing the property of different drugs, they are mostly moist and good at nourishing and moistening dryness evil. QRHM are bitter and/or hot tastes, medicines with bitter taste have the effects of purgation and drying the wetness evil etc. medicines with hot taste have the effects of dispersing, promoting the circulation of qi and blood. Therefore, QIHM and QRHM have obviously different effects due to the different tastes. All the QIHM contain more water-soluble carbohydrate due to the sweet taste, and almost all QRHM don’t contain or contain less water-soluble carbohydrate composition, most of them contain volatile components (Zheng et al., 1998). Therefore, QIHM and QRHM have obviously different components due to the different tastes (Table 3).

    [Click here to continue to section 11, "Conclusion"].


    Endnotes:

    This and the other entries herein appeared in: Xing-Tai Li and Jia Zhao (2012). An Approach to the Nature of Qi in TCM–Qi and Bioenergy, Recent Advances in Theories and Practice of Chinese Medicine, Prof. Haixue Kuang (Ed.), ISBN: 978-953-307-903-5, InTech, DOI: 10.5772/28416. Available from: http://www.intechopen.com

    [1] ^ College of Life Science, Dalian Nationalities University, Dalian, China, is given as the professional location of Xing-Tai Lii.

    [2] ^ Norman Bethune College of Medicine, Jilin University, Changchun China, is given as the professional location of Jia Zhao.

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