‘ Yang-Invigorating ’ Chinese Tonic Herbs Enhance Mitochondrial ATP Generation in H 9 c 2 Cardiomyocytes

‘Yang-invigorating’ Chinese tonic herbs have shown to enhance the myocardial mitochondrial ATP generation capacity in mice ex vivo. In the present study, we examined the effect of treatment with the methanol extract of ‘Yang-invigorating’ herbs on mitochondrial ATP generation capacity in H9c2 cardiomyocytes. The effect of ‘Yin-nourishing’ herbs was also investigated for comparison. The results indicated that all ‘Yang-invigorating’ Chinese tonic herbs dose-dependently enhanced the mitochondrial ATP generation capacity in H9c2 cardiomyocytes. Three out of nine ‘Yin-nourishing’ herbs produced a dose-dependent stimulatory effect on ATP generation, but to lesser extent than that of Yang herbs. Results obtained from activity-directed fractionation of the three most potent ‘Yang-invigorating’ herbs suggested that the ATP-stimulating ingredients were rather water insoluble and largely resided in the butanol fraction. In conclusion, ‘Yang-invigorating’ herbs invariably stimulated mitochondrial ATP generation capacity in H9c2 cardiomyocytes. The cell-based assay of ATP generation capacity may be used as pharmacological test for ‘Yang-invigorating’ Chinese tonic herbs.


Introduction
Chinese tonic herbs that can produce health-promoting action are used for the treatment of various patterns of deficiency in body function with respect to Yang, Yin, Qi, or Blood, and their combinations.These types of functional imbalance are viewed as sub-healthy conditions in modern medicine.Chinese tonic herbs are generally classified into four categories on the basis of their health-promoting actions: 'Yang-invigorating'; 'Yinnourishing'; 'Qi-invigorating' and 'Blood-enriching' [1].Of these four types of tonic herbs, the 'Qi-invigorating' and 'Blood-enriching' herbs are grouped under the 'Yang' family and 'Yin' family, respectively.While maintaining Yang and Yin in harmony is akin to attaining the homeostatic state in modern medicine, this phenomenon is aptly exemplified by the long-known antagonizing relationship between sympathetic and parasympathetic neural activities in the body.
According to traditional Chinese medicine theory, Yang is viewed as a manifestation of body function supported by various organs.A 'Yang-invigorating' action therefore involves the general up-regulation of cellular activities, particularly in the heart that plays a pivotal role in fueling the vital activities in all organs.As ATP, an energy-rich biomolecule, is universally used for energizing cellular activities, particularly in the energy demanding cardiomyocytes [2], we have proposed that the 'Yang-invigorating' action may be mediated by the enhancement of mitochondrial ATP generation [3].Recently, our laboratory has shown that treatment with the methanol extract of 'Yang-invigorating', but not 'Yinnourishing', herbs enhanced the myocardial ATP generation capacity in mice ex vivo [4].The stimulation of ATP generation was associated with an increased extent of mitochondrial electron transport [4].However, whether 'Yang-invigorating' herbs can produce the characteristic ATP stimulatory action in cultured cardiomyocytes is yet to be determined.In the present study, we investigated the effect of treatment with the methanol extract of 'Yang-invigorating' herbs on mitochondrial ATP generation capacity in H9c2 cardiomyocytes.Methanol extracts of 'Yin-nourishing' herbs were also tested for comparison.Three most active Yang herbs, namely, Herba Cistanches, Herba Cynomorii and Semen Cuscutae, were fractionated by differential solvent extraction with increasing polarity in order to characterize the physical property of the active ingredients.

Chinese Tonic Herbs
Dried Chinese herbs (see Table 1) used in the project were purchased from a local herbal dealer, Lee Koong Kee and voucher specimens were deposited in the Division of Life Science, Hong Kong University of Science and Technology, Hong Kong.For preliminary studies, herbs were extracted by methanol as previously described [5].Three of the most active herbs were further subjected to differential fractionation in succession by a set of four solvents, petroleum ether, ethylacetate, butan-1-ol and water, with increasing polarity.The pooled extracts were dried by evaporating the solvents in rotavaporator under heat and reduced pressure.Dried herbal extracts were stored at 4˚C until use.

Cell Culture
H9c2 cardiomyocytes, a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue and which exhibits many of the properties of skeletal muscle [6], was purchased from American Type Culture Collection.H9c2 cardiomyocytes were cultured as monolayers in Dulbecco's Modified Eagle Medium (Gibco BRL Life Technologies, Grand Island, NY), supplemented with 10% (v/v) fetal bovine serum, 100 IU/mL of penicillin (Sigma, St. Louis, MO), 100 µg/mL of streptomycin and 17 mM NaHCO 3 and were grown under an atmosphere of 5% (v/v) CO 2 in air at 37˚C.

Measurement of ATP Generation Capacity in Situ
H9c2 Cardiomyocytes were seeded at a density of 2.5 × 10 4 cells/well in 24-well tissue culture plates and were incubated in humidified incubator at 37˚C for 2 days to allow cell attachment and stable cell growth.After cell attachment, herbal extracts (dissolved in DMSO) were applied in the medium to achieve desired final concentrations (DMSO < 0.2%, v/v).After drug incubation, the ATP-generation capacity (ATP-GC) assay was performed.Culture medium was aspirated, and the cells were washed with phosphate-buffered saline (PBS).Digitonin (50 mg/mL) dissolved in incubation buffer (120 mM KCl, 5 mM KH 2 PO 4 , 2 mM EGTA, 10 mM HEPES, 0.1 mM MgCl 2 , 0.5% bovine serum albumin, pH 7.4) was added to permeabilize the cell membrane for the entry of substrates.Pyruvate (1.67 mM), malate (5 mM) and ADP (60 μM) were added for mitochondrial ATP generation in situ.Cellular ATP level was measured at increasing time intervals (0 to 15 min) following the addition of substrates.The ATP generation was terminated by the addition of 60 µL perchloric acid (30%, w/v).The reaction mixture was centrifuged at 540 × g for 20 minutes.An aliquot (120 µL) of the resultant supernatant was then mixed with 90 µL of 1.4M KHCO 3 for neutralization.Precipitate formed after neutralization was removed by centrifugation at 2150 × g for 10 min.The supernatant was subjected to measurement of ATP content by luciferase assay (ATPlite, Perkin Elmer, Boston, MA) as described [7].The ATP-GC of untreated cells was estimated by computing the area under the curve of the graph (AUC 1 ) plotting ATP generated (nmol/mg protein) against time and expressed in arbitrary unit.AUC 1 values of herbal extract-treated cells were normalized to respective mean control value from untreated samples and expressed as percent control.The area under the curve (AUC 2 ) of the graph plotting percent control against incubation time was computed and expressed in arbitrary unit.Results were expressed as percent control.

Protein Assay
Cell lysates were prepared by adding lysis buffer (0.1% (v/v) Triton X-100 in PBS).The protein concentration was determined by Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

Statistical Analysis
The intergroup difference was analyzed by student's t test, with P < 0.05 being regarded as statistical significant.

Results and Discussion
It is believed that the up-regulation of cellular activities by 'Yang-invigoration' in Chinese medicine requires an increased supply of ATP, which is in turn largely supported by mitochondrial oxidative phosphorylation.As shown in Figure 1, all of the 12 tested 'Yang-invigorating' herbs increased the ATP-GC in a concentrationdependent manner in H9c2 cardiomyocytes, with the extent of stimulation at the concentration of 300 µg/mL being 18-51%.Three out of the 9 tested 'Yin-nourishing herbs (Herba Eclipae, Herba Asparagi, Semen Prinsepiae) also dose-dependently enhanced the ATP-GC in H9c2 cardiomyocytes, but to a lesser extent, with the extent of stimulation at the concentration of 300 µg/mL being A recent study in our laboratory has demonstrated that 'Yang-invigorating', but not 'Yin-nourishing', Chinese tonic herbs can invariably enhance the myocardial mitochondrial ATP generation in mice ex vivo [4].In the present study, we extended the observation of this characteristic pharmacological action of 'Yang-invigorating' herbs in H9c2 cardiomyocytes.While all tested Yang herbs enhanced the mitochondrial ATP generation in H9c2 cardiomyocytes, one-third of the tested Yin herbs also showed moderate ATP stimulatory activity at the highest concentration tested.In this regard, the cell-based in situ assay for mitochondrial ATP generation capacity  seemed to be less selective than the previously described ex vivo assay model for Yang herbs, with the latter showing no stimulatory effect of Yin herbs on myocardial mitochondrial ATP generation [4].On the other hand, two Yin herbs, namely Radix Oryzae and Herba Dendrobii, were found to suppress the mitochondrial ATP generation in both ex vivo and cell-based in situ assays [4].While the discrepant observation between ex vivo and in situ assays for Yin herbs remains to be explained, the moderate stimulation of ATP generation in situ by Yin herbs may be related to the enhancement of cellular glutathione status (data not shown), which in turn can increase the functional ability of mitochondria [8].Interestingly, using the cell-based assay, the Herba Cistanches-induced enhancement of ATP generation was found to be suppressed by Fructus Ligustri (data not shown).This suggested the antagonistic relationship between Yang and Yin herbs in the regulation of mitochondrial ATP generation.Results from our previous and present studies suggest that the enhancement of mitochondrial ATP generation may serve as a pharmacological activity marker for 'Yang-invigorating' Chinese tonic herbs.In this regard, preliminary studies in our laboratory indicated that Chinese herbal products comprising 'Yang-invigorating' herbs were found to stimulate mitochondrial ATP-GC to varying extent in H9c2 cardiomyocytes.Results obtained from the activity-directed fractionation suggested that butanol fractions of Herba Cistanches and Herba Cynomorii contained relatively high content of ATP-stimulating active ingredients.The residence of active ingredients in relatively non-polar fractions of Semen Cuscutae might be explained by the unique characteristic of the herbs.The results indicated that the ATP-stimulating active ingredients of 'Yanginvigorating' herbs were relatively water-insoluble and may share similar structural characteristics.The measurement of ATP generation in H9c2 cardiomyocytes in situ using malate and pyruvate as substrates is an indirect measure of state 3 mitochondrial respiration [9].Under the present experimental conditions, the observation of changes in mitochondrial ATP generation by herbal pretreatment may be due to the increase in the activities of enzyme complexes in mitochondrial respiratory chain and/or the decrease in ATP consumption (or degradation) mediated by ATP-ase or other ATP-dependent enzymes.In this connection, an earlier report from our laboratory has shown that the enhancement of myocardial mitochondrial ATP generation capacity by Herba Cistanches treatment was associated with increases in complex I and III activities [10].On the other hand, the suppression of mitochondrial ATP generation capacity, as assessed by the present in situ assay, afforded by some Yin herbs may be related to the increase in ATP consumption.

Figures 1 .
Figures 1. Effects of Yang and Yin Chinese tonic herbs on mitochondrial ATP generation capacity in H9c2 cardiomyocytes.(a) 'Yang-invigorating'; (b)'Yin-nourishing'. (Cells were treated with herbal extracts at the indicated concentrations, as described in Materials and methods.# denotes assay concentrations of 40, 60 and 80 µg/mL.Mitochondrial ATP generation was measured in situ and the ATP generation capacity (ATP-GC) was estimated as described.The rate of ATP generation in untreated cells was 1.88 ± 0.16 (nmol/min/mg protein).Data were expressed in percent control (i.e.untreated), and values given are mean ± SD, with triplicate data.* P < 0.05; ** P < 0.005; *** P < 0.0001, when compared with the untreated control, using Student's t test.)

Table 2 . Effects of different fractions of Yang Chinese tonic herbs on mitochondrial ATP generation capacity in H9c2 car- diomyocytes.
Cells were treated with herbal extracts at the indicated concentrations, as described in Materials and methods.Mitochondrial ATP generation was measured in situ and the ATP generation capacity (ATP-GC) was estimated as described.Data were expressed in percent control (i.e.Untreated), and values given are means ± SD, n = 6.* Significantly different respective control groups (p < 0.05), using Student's t test.# Concentrations expressed as crude herb equivalence.