According to Chinese medicine theory, Yang/Qi plays a pivotal role in driving physiological functions in the body, these being highly dependent on mitochondrial ATP production. Consistent with this, Yang/Qi-invigorating Chinese tonifying herbs have been found to stimulate mitochondrial ATP generation capacity (ATP-GC) in H9c2 cardiomyocytes. In the present study, we have demonstrated that Yang-invigorating Chinese tonifying herbs (namely, Eucommiae Cortex, Cibotii Rhizoma, Dipsaci Radix, Cynomorii Herba, Cistanches Herba, Cuscutae Semen, EpimediiHerba and Morindae Radix) and Qi-invigorating Chinese tonifying herbs (namely, Ginseng Radix, Pseudostellariae Radix, Quinquefolii Radix, Codonopsis Radix, Astragali Radix, Atractylodis Rhizoma, Juiubae Fructus, Fici Simplicissimae Radix and Dioscoreae Oppositae Radix) act by different mechanisms to stimulate mitochondrial ATP-GC. While Yang-invigorating herbs fluidize mitochondrial membranes and thus stimulate ATP-GC, Qi-invigorating herbs can enhance cellular glutathione status and increase ATP-GC. The different mechanisms by which Yang-invigorating herbs and Qi-invigorating herbs stimulate mitochondrial ATP-GC may serve as the basis for establishing biomarkers for Yang/Qi-invigorating herbs and herbal health products in general.
The Yin-Yang Theory, which is a fundamental conceptual framework of Chinese medicine, views life activities as a result of an interaction between two complementary but opposing forces, namely, Yin and Yang. The dynamic equilibrium between Yin and Yang enables the attainment of a balance of physiological functions in the body and hence a state of optimal health [
As mentioned earlier, Yang and Qi are manifestations of various body functions, all of which are dependent on cellular activities. In this regard, ATP, which is generated by mitochondria (the “power house” of the cell), energizes a wide range of cellular activities. To sustain the capacity to generate ATP, mitochondria are equipped with a glutathione-dependent antioxidant system to protect mitochondrial respiratory complexes against reactive oxygen species (ROS) arising from mitochondrial electron transport processes [
Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). Adenosine diphosphate (ADP), adenosine triphosphate (ATP), sodium pyruvate and sodium malate, the reduced form of glutathione (GSH) and the oxidized form of glutathione (GSSG) were bought from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.
Yang-invigorating herbs and Qi-invigorating herbs (see
Pharmaceutical name | Abbreviation | % yield | |
---|---|---|---|
Yang-invigorating herbs | |||
Eucommiae Cortex | EC | 12.1 | |
Cibotii Rhizoma | CR | 23.0 | |
Dipsaci Radix | DR | 35.0 | |
Cynomorii Herba | CYH | 15.5 | |
Cistanches Herba | CIH | 26.0 | |
Cuscutae Semen | CS | 4.24 | |
Epimedii Herba | EH | 9.00 | |
Morindae Radix | MR | 29.9 | |
Qi-invigorating herbs | |||
Ginseng Radix | GR | 13.0 | |
Pseudostellariae Radix | PR | 47.3 | |
Quinquefolii Radix | QR | 12.0 | |
Codonopsis Radix | CR | 37.2 | |
Astragali Radix | ASR | 27.2 | |
Atractylodis Rhizoma | ATR | 20.5 | |
Juiubae Fructus | JF | 52.7 | |
Fici Simplicissimae Radix | FR | 3.60 | |
Dioscoreae Oppositae Radix | DOR | 53.1 |
H9c2 cells, which area subclone of rat heart myoblast cells, were purchased from the American Tissue Culture Centre (ATCC). Cells were cultured as mono-layers in DMEM, supplemented with 10% (v/v) FBS, 100 IU/mL of penicillin, 100 μg/mL of streptomycin and 1.5 g/L NaHCO3. All cells were grown under an atmosphere of 5% (v/v) CO2 in air at 37˚C.
H9c2 cardiomyocytes were seeded at a density of 1 × 105 cells/well into 12-well microtiter plates or at 5 × 104 cells/well into 24-well microtiter plates. Following attachment, cells were incubated with Yang or Qi-invigorating herbal extracts (at a concentration of 100 µg/mL) for 4 h or 24 h at 37˚C. The herbal extract-containing medium was prepared by adding the sample solution in dimethyl Sulfoxide (DMSO) to the culture medium, with the final concentration of DMSO being less than 0.2% (v/v). The control group received vehicle [DMSO (0.2%, v/v, final concentration)] only.
To determine the role of membrane fluidity in the effect of the Yang-invigorating herbal extract on mitochondrial ATP-GC, cholesterol [3 μM, dissolved in ethanol, the final concentration of solvent in the medium being 0.2% (v/v)], which can decrease mitochondrial inner membrane fluidity [
To determine the role of a glutathione-dependent antioxidant response in the stimulation of mitochondrial ATP-GC by Qi-invigorating herbal extracts, H9c2 cells were co-incubated with L-buthionine-(S, R)-sulfoximine (BSO, which blocks glutathione synthesis by inhibiting γ-glutamylcysteine synthetase, at 12.5 μM) and Qi-invigorating herbal extracts (100 µg/mL) for 24 h, and N,N’-bis (2-chloroethyl)-N-nitrosourea [BCNU, a potent inhibitor of glutathione reductase, at 25 μM in 0.2% ethanol in the culture medium (v/v)] was added (2 µL) in the incubation mixture 1 h prior to the end of the 24-h incubation.
Following the incubation with the herbal extract, the mitochondrial ATP-GC assay was performed as previously described [
After the incubation with herbal extract, the cellular level of GSH was measured using the method of Griffith [
Protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA), using bovine serum albumin as standard.
All data were expressed as mean ± standard error of the mean (SEM), unless otherwise specified. Data were analyzed by Student’s t-test or one-way analysis of variance (one-way Anova), as specified. For one-way Anova, inter-group differences were detected by Tukey, with p < 0.05. The correlation between GSH levels and mitochondrial ATP-GC values after 24 h incubation with Qi-invigorating herbal extracts was analyzed by computing the Pearson’s correlation coefficient using the two-tailed significance test.
A 4-h incubation with all tested Yang-invigorating herbal extracts consistently increased mitochondrial ATP-GC in H9c2 cells (ranging from 13% - 41%,
4-h incubation | 24-h incubation | |
---|---|---|
Yang-invigorating herbal extracts | ||
EC | 120 ± 6.46* | 69.1 ± 5.91* |
CR | 113 ± 4.32* | 104 ± 2.67 |
DR | 124 ± 8.79* | 102 ± 7.17 |
CYH | 131 ± 6.44* | 105 ± 3.08* |
CIH | 141 ± 10.8* | 116 ± 3.06* |
CS | 121 ± 3.98* | 113 ± 1.82* |
EH | 117 ± 9.77* | 93.8 ± 6.29 |
MR | 117 ± 3.89* | 100 ± 1.52 |
Qi-invigorating herbal extracts | ||
GR | 109 ± 4.15* | 121 ± 3.53* |
PR | 122 ± 7.28* | 117 ± 1.65* |
QR | 93.1 ± 1.93* | 140 ± 4.71* |
CR | 94.5 ± 5.93 | 132 ± 3.34* |
ASR | 97.9 ± 5.72 | 122 ± 10.3* |
ATR | 86.3 ± 5.42* | 133 ± 8.48* |
JF | 115 ± 5.42* | 116 ± 8.36* |
FR | 97.1 ± 4.13 | 132 ± 3.51* |
DOR | 98.3 ± 3.96 | 130 ± 4.92 |
Values given are expressed as % Control (mean ± SD). *Significantly different from the respective control by Student’s t test (p < 0.05). The control value of AUC2 of Yang-invigorating herbal extracts = 750 ± 35.1. The control value of AUC2 of Qi-invigorating herbal extracts = 750 ± 45.0.
ATP-GC in H9c2 cells, when compared with cholesterol-pre-incubated controls.
None of the tested Yang-invigorating herbal extracts increased cellular GSH levels in H9c2 cells after a 4-h incubation. Following a 24-h incubation with the herbal extracts (EC, CR, CIH, CS and MR), cellular GSH levels were increased, with the degree of stimulation being 14% - 110%, when compared with the non-herbal-pre-incubated controls (
Incubation with Qi-invigorating herbal extracts for 4 h did not produce identical effects on mitochondrial ATP-GC in H9c2 cells (
Some Qi-invigorating herbal extracts (QR, ASR and FR) were found to enhance cellular GSH levels following a 4-h incubation (12% - 17%,
4-h incubation | 24-h incubation | |
---|---|---|
Yang-invigorating herbal extracts | ||
EC | 105 ± 5.37 | 210 ± 9.85* |
CR | 94.4 ± 5.51 | 118 ± 2.41* |
DR | 100 ± 4.86 | 104 ± 4.51 |
CYH | 101 ± 4.69 | 105 ± 6.04 |
CIH | 95.6 ± 3.06 | 120 ± 6.92* |
CS | 101 ± 3.93 | 126 ± 7.86* |
EH | 99.9 ± 8.48 | 102 ± 4.46 |
MR | 104 ± 1.68 | 114 ± 2.55* |
Qi-invigorating herbal extracts | ||
GR | 93.9 ± 2.92 | 121 ± 0.61* |
PR | 106 ± 4.53 | 118 ± 1.67* |
QR | 112 ± 3.31* | 143 ± 4.04* |
CR | 98.1 ± 4.51 | 129 ± 4.63* |
ASR | 114 ± 5.17* | 114 ± 2.29* |
ATR | 106 ± 3.42 | 179 ± 5.13* |
JF | 103 ± 4.99 | 115 ± 6.81* |
FR | 117 ± 4.86* | 146 ± 12.7* |
DOR | 106 ± 8.28 | 113 ± 5.74* |
Values given are expressed as % Control (mean ± SD). *Significantly different from the respective control by Student’s test (p < 0.05). The control value of Yang-invigorating herbal extracts = 33.9 ± 4.76 (nmol/mg protein). The control value of Qi-invigorating herbal extracts = 30.3 ± 2.47 (nmol/mg protein).
Pearson’s Correlation | R squared | p value | |
---|---|---|---|
Yang-invigorating herbal extracts | ATP-GC (4 h) vs cellular GSH (24 h) | 0.0749 | 0.476 |
Qi-invigorating herbal extracts | ATP-GC (24 h) vs cellular GSH (24 h) | 0.403 | 0.044 |
controls (
According to Chinese medicine theory, Yang/Qi balance plays a pivotal role in modulating physiological functions in the body. The gradual depletion of Yang/Qi is associated with aging and the predisposition to age-related diseases in humans [
On the other hand, while Qi-invigorating herbal extracts failed to stimulate mitochondrial ATP-GC following a 4-h incubation, they invariably stimulated ATP-GC after incubation for 24 h in H9c2 cells. The stimulation of mitochondrial ATP-GC was accompanied by an increase in cellular GSH levels. Correlation analysis showed that the degree of mitochondrial ATP-GC stimulation correlated significantly with the extent of cellular GSH increase in Qi-invigorating herbal extract-pre-incubated H9c2 cells. The finding that BSO/BCNU co-incubation abolishes the stimulatory effect of Qi-invigorating herbal extracts on mitochondrial ATP-GC further confirms the proposal that the increase in mitochondrial ATP-GC is an effect secondary to the enhancement of cellular GSH status. This is consistent with earlier findings indicating that glutathione antioxidant status plays a critical role in preserving mitochondrial structural and functional integrity [
In conclusion, our findings indicate that Yang-invigorating and Qi-invigorating herbs can both stimulate mitochondrial ATP-GC in H9c2 cells but by different mechanisms. While Yang-invigorating herbs act primarily by fluidizing the inner mitochondrial membrane, Qi-invigorating herbs exert their action indirectly by increasing cellular GSH levels. Future studies will focus on the mechanism underlying the capacity of Qi-invigorating herbs to enhance cellular GSH levels. The measurement of mitochondrial ATP-GC and cellular GSH levels in H9c2 cells under the experimental conditions described here could serve as convenient biomarkers for the quality assurance of Yang-invigorating and Qi-invigorating Chinese tonifying herbs and herbal health products in general.
Leong, P.K., Leung, H.Y., Chan, W.M. and Ko, K.M. (2018) Differences in the Mechanisms by Which Yang-Invigorating and Qi-Invigorating Chinese Tonifying Herbs Stimulate Mitochondrial ATP Generation Capacity. Chinese Medicine, 9, 63-74. https://doi.org/10.4236/cm.2018.92005