‘Yang-Invigorating’ Chinese Tonic Herbs Enhance Mitochondrial ATP Generation in H9c2 Cardiomyocytes

doi:10.4236/cm.2011.21001

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Chinese Medicine, 2011, 2, 1-5

doi:10.4236/cm.2011.21001 Published Online March 2011 (http://www.SciRP.org/journal/cm)

‘Yang-Invigorating’ Chinese Tonic Herbs Enhance

Mitochondrial ATP Generation in H9c2 Cardiomyocytes

Hoi Shan Wong, Hoi Yan Leung, Kam Min g Ko

Division of Life Science, Hong Kong University of Science & Technology

Clear Water Ba y, Hong Kong, China

E-mail: [email protected]

Received January 25, 2011; revised February 9, 2011; accepted February 10, 2011

Abstract

‘Yang-invigorating’ Chinese tonic herbs have shown to enhance the myocardial mitochondrial ATP genera-

tion 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 ca-

pacity in H9c2 cardiomyocytes. Three out of nine ‘Yin-nourishing’ herbs produced a dose-dependent stimu-

latory effect on ATP generation, but to lesser extent than that of Yang herbs. Results obtained from activi-

ty-directed fr actionat ion of the three most potent ‘Yan g-invigorating’ herbs suggeste d t hat t he 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 cardi-

omyocytes. The cell-based assay of ATP generation capacity may be used as pharmacological test for

‘Yang-invigorating’ Chinese tonic herbs.

Keywords: Yang, Yin, Chinese Medicine, ATP, Mitochondria, Cardiomyocytes

1. 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 condi-

tions i n modern med icine. Chinese toni c he rbs are gener-

ally classified into four categories on the basis of their

health-promoting actions: ‘Yang-invigorating’; ‘Yin-

nourishing’; ‘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 attain-

ing the homeostatic state in modern medicine, this phe-

nomenon is aptly exemplified by the long-known anta-

gonizing relationship between sympathetic and para-

sympathetic neural activities in the bod y.

According to traditional Chinese medicine theory,

Yang is viewed as a manifestation of body function sup-

ported 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 ene rgy-rich biomolecule, is universally used for ener-

gizing cellular activities, particularly in the energy de-

manding cardiomyocytes [2], we have proposed that the

‘Yang-invigorating’ action may be mediated by the en-

hancement of mitochondrial ATP generation [3]. Re-

cently, our laboratory has shown that treatment with the

methanol extract of ‘Yang-invigorating’, but not ‘Yin-

nourishing’, herbs enhanced the myocardial ATP genera-

tion capacit y 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 sti mulator y action in cu lt ured ca rdiomyocytes 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 gener-

ation capacity in H9c2 cardiomyocytes. Methanol ex-

tracts of ‘Yin-nourishing’ herbs were also tested for

comparison. Three most active Yang herbs, namely,

H. S. WONG ET AL.

Herba Cistanches, Herba Cynomorii and Semen Cuscu-

tae, were fractionated by differential solvent extraction

with increasing polarity in order to characterize the

physical property of the active ingredients.

2. Materials and Methods

2.1. 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 Divi-

sion of Life Science, Hong Kong University of Science

and Technology, Hong Kong. For preliminary studies,

herbs were extracted by methanol as previously de-

scribed [5]. Three of the most active herbs were further

subjected to differential fractionation in succession by a

set of four solvents, petroleum ether, ethylacetate, bu-

tan-1-ol and water, with increasing polarity. The pooled

extracts were dried by evaporating the solvents in rota-

vaporator under heat and reduced pressure. Dried herbal

extracts were stored at 4˚C until us e.

2.2. 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 mo-

nolayers in Dulbecco’s Modified Eagle Medium (Gibco

BRL Life Technologies, Grand Island, NY), supple-

mented with 10% (v/v) fetal bovine serum, 100 IU/mL of

penicillin (S igma , St. Louis, MO), 100 µg/mL of strep-

tomycin and 17 mM NaHCO3 and were grown under an

atmosphere of 5% (v/v) CO2 in air at 37˚C.

2.3. Measurement of ATP Generation Capacity

in Situ

H9c2 Cardiomyocytes were seeded at a density of 2.5 ×

104 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 concen-

trations (DMSO < 0.2%, v/v). After drug incubation, the

ATP-generation capacity (ATP-GC) assay was per-

formed. Culture medium was aspirated, and the cells

were washed with phosphate-buffered saline (PBS). Di-

gitonin (50 mg/mL) dissolved in incubation buffer (120

mM KC l, 5 mM KH2PO4, 2 mM E G TA, 1 0 mM HEPES,

0.1 mM MgCl2, 0.5% bovine serum albumin, pH 7.4)

was added to permeabilize the cell membrane for the

entry of substrates. Pyr uvate (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 ad-

dition of substrates. The ATP generation was terminated

by the addition of 60 µL p erchloric acid (30%, w/v ). The

reaction mixture was centrifuged at 540 × g for 20 mi-

nutes. An aliquot (120 µL) of the resultant supernatant

was then mixed with 90 µL of 1.4M KHCO3 for neutra-

lization. Precipitate formed after neutralization was re-

moved by centrifugation at 2150 × g for 10 min. The

supernatant was subjected to measurement of ATP con-

tent 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 (AUC1) plotting ATP generated (nmol/mg

protein) against time and expressed in arbitrary unit.

AUC1 values of herbal extract-treated cells were norma-

lized to respective mean control value from untreated

samples and expressed as percent control. The area under

the curve (AUC2) of the graph plotting percent control

against incubation time was computed and expressed in

arbitrary unit. Results were expressed as percent control.

2.4. 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).

2.5. Statistical Analysis

The intergroup difference was analyzed by student’s t

test, with P < 0.05 being regarded as statistical signifi-

cant.

3. 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 sup-

ported by mitochondrial oxidative phosphorylation. As

shown in Figure 1, all of the 12 tested ‘Yang-invigorat-

ing’ herbs increased the ATP-GC in a concentration-

dependent 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, b ut to a le sser extent, with the extent o f

stimulation at the concentration of 300 µg/mL being

H. S. WONG ET AL.

Table 1. Nomenclature of select ed Yang and Yi n Chine se tonic her bs .

Pharmaceutical Name Chines e Pin Yin Latin Botanical Name Family % Yield of

Metha nol Extract

Yang-invigorating

Radix Dipsaci Xu Duan Dipsacus japonicus Miq. Dipsacaceae 32.6

Rhizoma Ciboti i Gou Ji Cibo tium bar o m etz (L.) J. Sm. Dicksoniaceae 21.8

Rhizoma Curculi ginis Xian Mao Curculigo orchioides Gaertn. Amaryllidaceae 12.4

Herba Epimedii Yin Yang Huo Epimedium grandiflorum Morr. Berbendaceae 15.1

Herba Cynomorii Suo Yang Cynomorium songaricum Rupr. Cynomoriaceae 41.3

Herba Cis tanches Rou Cong Rong Cistanches salsa (C.A. Meyer) G. Beck. Orobanchaceae 33.6

Radix Morindae Ba Ji Tian Morinda officinalis How Rubiaceae 19.8

Semen Cuscuta e Tu Si Zi Cuscuta chinensis Lam. Convolvulaceae 5.3

Semen A l lii Jiu Zi Allium tuberosum Rottl. Liliaceae 8.3

Rhizoma Drynariae Gu Sui Bu Drynaria fortunei (Kunze) J. Sm . Polypodiaceae 2.1

Fructus Psoraleae Bu Gu Zhi Psoralea corylifolia L. Fabaceae (Leguminosae) 23.3

Cortex Eucommiae Du Zhong Euc ommia ulmoides Oliv. Eucommiaceae 15. 4

Yin-nourishing

Fructus Ligustri Nu Zhen Zi Ligustrum lucidum Ait. Oleaceae 25.7

Rad ix Oryzae Nuo Dao Gen Xu Oryza sativa L. Gramineae 1.3

Herba Dendrobii S hi H u Cao De ndr obium no bile L indl. Orchidaceae 16.8

Herba Eclip tae Han Lian Cao Ecliptae prostrata L. Compositae 4.5

Radi x Asparagi Tian Dong Asparagus cochinchinensis ( Lour. ) Merr. Liliaceae 56.3

Radi x Ophiopogon is Mai Dong Ophiopogon jap onicus (L. f.) Ker-Gawl. Liliaceae 25.8

Semen Prinsepi ae Rui Re n Prinsepia uniflora Batal. Rosaceae 9.5

Rhizoma Polygonati Yu Zhu Polygonat um odoratum (Mill.) Druce Liliaceae 38.3

Herba Pholidotae Shi Xian Tao Phol id o ta e c h inens is Lindl. Orchidaceae 25.0

14-27%. In contrast, 5 Yin herbs (Fructus Ligustri, Radix

Oryzae, Herba Dendrobii, Radix Ophiopogonii, Herba

Pholidotae) produced a dose-dependent suppression of

ATP-GC in H9c2 cardiomyocytes, with the degree of

inhibition at the concentration of 300 µg/mL (or 80

µg/mL for Fruct us Lig ustri) b eing 18-39%. Based on the

results obtained, three of the ‘Yang-invigorating’ herbs

(Herba Cistanches, Herba Cynomorii and Semen Cuscu-

tae) were selected for their high potency in stimulating

ATP generation and subjected to further investigations.

The three herbs were fractionated differentially by using

a set of four solvents (petroleum ether, ethylacetate, bu-

tanol and water) with increasing polarity in succession.

As shown in Table 2, butanol fractions of Herba Cis-

tanches and Herba Cynomorii increased the ATP-GC in

H9c2 cardiomyocytes, with the extent of stimulation

being 43% and 37% respectively. All four fractions of

Semen Cuscutae stimulated ATP-GC in H9c2 cardio-

myocytes, with the stimulation of ethylacetate fraction

being most potent (5 3%) among the m.

A rece nt stud y in our labor atory ha s demo nstrate d tha t

‘Yang-invigorating’, but not ‘Yin-nourishing’, Chinese

tonic herbs can invariably enhance the myocardial mito-

chondrial ATP generation in mice ex vivo [4]. In the

present study, we extended the observation of this cha-

racteristic 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

H. S. WONG ET AL.

(a)

(b)

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 w ere treated with herbal ex tracts at the indicated concentrations, as de-

scribed i n Materi al s and meth ods. # d enotes ass ay concentratio ns of 40, 60 and 80 µg/ mL. M itochondrial ATP generation was

measure d in situ a nd the A TP ge nerati on capac ity (ATP-GC) w as estimated as describ ed. The rate of ATP g eneration in un-

treated cel ls was 1 .88 ± 0.16 (nmol/min/ mg protei n). Data w ere ex pressed in per cent contr ol (i.e. untreate d), an d val ues g ive n

are mean ± SD, wi th tri plicat e dat a. * P < 0.05 ; ** P < 0 .00 5; *** P < 0. 0001 , when co mpare d wit h the untre ated co ntrol, us-

ing Student’s t test.)

Table 2. Effects of different fractions of Yang Chinese tonic herbs on mitochondrial ATP generation capacity in H9c2 car-

diomyocytes.

Petro leum eth er Eth ylacetate Butan-1-ol Aqueous

Herba Cis tanches (30 mg/mL)# 100.0 ± 18.0 90.0 ± 27.6 142.8* ± 13.2 99.6 ± 9.6

Herba Cynomorii (20 mg/mL) 95.7 ± 11.4 114.3 ± 4.3 137.1* ± 4.2 105.7 ± 5.7

Semen Cuscuta e (6 mg/mL) 131.3* ± 16.8 153.1* ± 4.0 145.2* ± 7.0 112.6* ± 3.9

Cells were treated with herbal extracts at the indicated concentrations, as described in Materials and methods. Mitochondrial ATP generati on 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 respe ctive control groups (p < 0.05), using Student’s t test. # Concentrations expressed as crude herb equivalence.

H. S. WONG ET AL.

seemed to be less selective than the previousl y described

ex vivo assay model for Yang herbs, with the latter

showing no stimulatory effect of Yin herbs on myocar-

dial 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 ex-

plained, 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-ind uced enhance ment of AT P generation was

found to be suppressed by Fructus Ligustri (data not

shown). This suggested the antagonistic relationship be-

tween Yang and Yin herbs in the regulation of mito-

chondrial AT P gene ration. Results from our p revious and

present studies suggest that the enhancement of mito-

chondrial ATP generation may serve as a pharmacologi-

cal activity marker for ‘Yang-invigo rat ing ’ Chine se tonic

herbs. In this regard, preliminary studies in our labora-

tory indicated that Chinese herbal products comprising

‘Yang-invigorating’ herbs were found to stimulate mito-

chondrial ATP-GC to varying extent in H9c2 cardio-

myocytes. Results obtained from the activity-directed

fractionation suggested that butanol fractions of Herba

Cistanches and Herba Cynomorii contained relatively

high co ntent 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 ‘Yang-

invigorating’ herbs were relatively water-insoluble and

may share similar structural characteristics.

The measurement of ATP generation in H9c2 cardi-

omyocytes in situ using malate and pyruvate as sub-

strates is an indirect measure of state 3 mitochondrial

respiration [9]. Under the present experimental condi-

tions, the observation of changes in mitochondrial ATP

generation by herbal pretreatment may be due to the in-

crease in the activities of enzyme complexes in mito-

chondrial 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 en-

hancement of myocardial mitochondrial ATP generation

capacity by Herba Cistanches treatment was associated

wit h increases in complex I and III activities [10]. On the

other hand, the suppression of mitochondrial ATP gener-

ation capacity, as assessed by the present in situ assay,

afforded by some Yin herbs may be related to the in-

crease in ATP consumption.

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