Herba Cistanche Extract Enhances Mitochondrial ATP Generation in Rat Hearts And H9c2 Cells

Contact: Audrey Hu audrey.hu@wecistanche.com

Hoi Yan Leung & Kam Ming Ko

To cite this article: Hoi Yan Leung & Kam Ming Ko (2008) Herba Cistanche Extract Enhances Mitochondrial ATP Generation in Rat Hearts and H9c2 Cells, Pharmaceutical Biology, 46:6, 418-424, DOI: 10.1080/13880200802055883

Abstract

To investigate the pharmacological basis of the “Yang- invigorating” action of Herbal Cistanche [the dried whole plant of Cistanche deserticola Y.C. Ma (Orobanchaceae)], in Chinese medicine, the effects of the methanol extract of Herba Cistanche on mitochondrial ATP generation capacity were examined using an ex vivo rat heart model and an in situ H9c2 cell assay. Treatment with Herba Cistanche extract increased the myocardial mitochondrial ATP generation capacity in a dose-dependent manner in rats, as assessed by in vitro measurement. The stimulation of ATP generation capacity was associated with a parallel enhancement in mitochondrial electron transport supported by pyruvate but not succinate. Herba Cistanche treatment also increased the myocardial mitochondrial complex I and complex III activities, with the extent of stimulation on complex I activity being larger. Herba Cistanche treatment produced a dose-and time-dependent increase in mitochondrial ATP generation capacity in H9c2 cells. The results indicate that Herba Cistanche treatment can increase mitochondrial ATP generation in rat hearts ex vivo and H9c2 cells in situ, possibly through enhancing oxidative phosphorylation.

Keywords: ATP, Cistanche deserticola, H9c2 cells, heart, mitochondria.

Cistanche deserticola Y.C. Ma

Introduction

“Yang invigoration,” which embodies the generalized enhancement of body function in Chinese medicine, involves the use of energy in driving various biochemical processes. In this regard, ATP, which is the universal currency of energy in fueling cellular function, is mainly generated through oxidative phosphorylation in mitochondria. We have postulated that “Yang invigorating” herbs possess the ability to increase mitochondrial ATP generation capacity (Ko et al., 2004). This is supported by our recent finding that “Yang- invigorating” but not “Yin-nourishing” Chinese tonifying herbs could enhance the myocardial ATP generation capacity in mouse hearts ex vivo (Ko et al., 2006). Herba Cistanche, the dried whole parasitic plant (excluding the flower) of Cistanche deserticola Y.C. Ma (Orobanchaceae), is classified as a ‘Yang-invigorating’ tonifying herb in traditional Chinese medicine (Chen, 1998), and it appears as the most popular ingredient in a number of Chinese patents formulations used for Yang-invigoration’. In China and Japan, Herba Cistanche is widely used for the treatment of a host of Yang deficiency symptoms. Pharmacological studies indicated that Herba Cistanche could scavenge free radicals (Xiong et al., 1996), produce sedative (Lu, 1998), antiaging (Lee et al., 1990), antinociceptive and anti-inflammatory effects (Lin et al., 2002), and enhance immune function (Wu et al., 2005). The main active ingredient of Herba Cistanche is phenylethanoid glycosides (Ouyang et al., 2003), which have been found to have antibacterial, antistress, and antioxidative properties (Xiong et al., 1998), as well as anti-apoptotic effects on cultured neurons (Tian & Pu, 2005). In the current study, in order to investigate the pharmacological basis of the Yang-invigorating action of Herba Cistanche, the effects of Herba Cistanche treatment on the ATP generation capacity in mitochondria isolated from rat hearts ex vivo and cultured H9c2 cardiomyocytes in situ were examined. To explore the biochemical mechanism underlying the ATP generation–enhancing action, the effects of Herba Cistanche treatment on mitochondrial electron transport and complex I–IV activities were also examined in rat hearts.

Cistanche deserticola extract

Materials and Methods

Chemicals, cell culture, and herbal materials ATP, ADP, and 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyl- tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Luciferase solution (ATPlite) was obtained from PerkinElmer (Boston, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL Life Technologies (Grand Island, NY, USA). H9c2 cell line was purchased from ATTC (Rockville, MD, USA). Herba Cistanche was supplied by a local herbal dealer (Lee Hoong Kee). The herb was authenticated by the supplier, and a voucher specimen (HKUSTY00301) was deposited in the Department of Biochemistry, Hong Kong University of Science & Technology (HKUST).

Herbal extraction

Herba Cistanche (100 g) was cut into small pieces and then extracted by heating under reflux in 300 mL methanol

at 65◦C for 2 h. The procedure was repeated twice. The pooled extract was dried by evaporating the solvent under reduced pressure, and the methanol extract of Herba Cistanche was obtained at a yield of 39% (w/w). Chemical analysis indicated that the methanol ex- tract of Herba Cistanche contained total saponins, total flavonoids, total lignans, and polysaccharides at concentrations of 1.62% (w/w), 0.08%, 0.15%, and 75.6%, respectively.

Animal care

Adult male Sprague-Dawley rats (8–10 weeks; 250–300g) were maintained under a 12-h dark/ light cycle in an air/humidity-controlled room at about 22◦C and allowed food and water ad libitum in the Animal Care Facilities

at HKUST. Experimental protocols were approved by the Research Practice Committee at HKUST.

Drug treatment

Animals were randomly divided into groups, with five to six animals in each. In the treatment groups, rats were intragastrically administered with the methanol extract of Herba Cistanche (dissolved/suspended in water) at increasing daily doses (0.031–0.5 g/kg) for 3 days. Control animals received water only. Twenty-four hours after the last dosing, the heart was obtained from phenobarbital-anesthetized animals and subjected to biochemical analysis.

Preparation of mitochondrial fractions and measurement of ATP generation capacity (ATP-GC) ex vivo

Heart left ventricular tissue samples were excised and rinsed with ice-cold isotonic buffer (0.32 M sucrose, 1 mM EDTA, 50 mM Tris/HCl, pH 7.4). Cardiac mitochondrial fractions were prepared by differential centrifugation in iso- tonic buffer at 4◦C. A 10% (w/v) cardiac homogenate was prepared by homogenizing the minced ventricular tissue with a Teflon-glass homogenizer at 4000 rpm for 20–30 complete strokes. The homogenate was centrifuged at 600 g for 10 min to remove nuclei and cell debris. The supernatant was then centrifuged at 8000 g for 30 min to sediment the mitochondria (Evan, 1992). The pellets were resuspended in 1 mL of the isotonic buffer and reconstituted the mitochondrial fractions. The mitochondrial ATP-GC of untreated animals was measured by the method of Leung et al. (2005).

Measurement of mitochondrial electron transport

The measurement of electron transport in isolated mitochondria, which is based on the reduction of MTT, was performed as described by Cohen et al. (1997). Mitochondria were prepared at a protein concentration of 1 mg/mL with an incubation buffer (250 mM sucrose, 50 mM HEPES, 10 mM KH2PO4, 2 mM MgCl2, 1 mM EGTA, pH 7.4). An aliquot (40 µL) of the mitochondria was mixed with 100 µL each of 15 mM pyruvate or 0.5 mM succinate and 0.42 mg/mL MTT. The reaction mixture was incubated at 37◦C for 10 min with gentle shaking. After the incubation, the reaction mixture was terminated by the addition of 100 µL lysis buffer (10%, w/v, sodium dodecyl sulfate, and 45% dimethylformamide, adjusted to pH 4.7 with glacial acetic acid). After standing for 5 min, absorbance readings of the reaction mixture were taken with a microtiter plate reader (Bio-Rad, Hercules, CA, USA) and reported as the difference between 570 nm and 630 nm. Data were expressed as a percentage of the mean value of the control (i.e., Herba Cistanche–untreated) group.

Cell culture

H9c2 cells, a permanent cell line derived from cardiac my- oblasts (Hescheler et al., 1991), were cultured as monolayers in DMEM supplemented with 10% (v/v) FBS. The medium contained glucose (4.5 g/L) and glutamine (4.5 mM), supplemented with NaHCO3 (17 mM), penicillin (100 IU/mL), and streptomycin (100 µg/mL). All cells were grown under an atmosphere of 5% (v/v) CO2 in air at 37◦C. The medium was replaced by fresh medium every 2 or 3 days. A stock of cells was grown in a 75 cm2 culture flask and split before confluence at a subcultivation ratio of 1:10. For ATP-GC assay, H9c2 cells were seeded at a density of 2.5 104 cells/well into a 24-well plate. After the cell attachment, Herba Cistanche extract (dissolved in phosphate-buffered saline; PBS) was applied in the medium, and the cells were incubated for increasing periods (2–16 h) of time. Control (untreated) cells were given the PBS only.

Measurement of ATP-GC in situ

After the indicated periods of incubation with Herba Cistanche extract at increasing concentrations (50–300 µg/mL), the ATP-GC assay was performed. The medium was aspirated and cells were treated with digitonin (50 µg/mL) in an incubation buffer (120 mM KCl, 5 mM KH2PO4, 2 mM EGTA, 10 mM HEPES, 0.1 mM MgCl2, 0.5% BSA, pH 7.4) for 3 min at 37◦C. After aspirating the digitonin, glutamate (5 mM), malate (5 mM), and ADP (60µM) were added to the cells for mitochondrial ATP generation, which was monitored at increasing time intervals ranging from 0 to 15 min. The reaction was terminated by the addition of 60 µL of perchloric acid (30%, w/v), and the reaction mixtures were then centrifuged at 600g for 10 min at 4◦C. An aliquot (120 µL) of the supernatant was mixed with 90 µL of 1.4 M KHCO3 for neutralization. The mixtures were centrifuged again at 600 g at 4◦C, and the supernatants were measured for ATP content as described earlier. The ATP-GC of the untreated cells was estimated by computing the area under the curve of the graph (AUC1) plotting ATP generated (nmol/mg protein) against time (0–15 min) and expressed in arbitrary units. For Herba Cistanche–treated cells, AUC1 values of increasing incubation times (3, 5, 7, 10, and 15 min) were normalized to a respective mean control value from untreated samples and expressed as percent control. Then, the area under the curve (AUC2) of the graph plotting percent control values against incubation time (3–15 min) was computed and ex-pressed in arbitrary units. Data of Herba Cistanche–treated groups were expressed as a percentage of the control from the equation:

[AUC2(HerbaCistanche − treated)/AUC2(untreated)]×100%.

Measurements of complex I–IV and citrate synthase activities

Mitochondrial electron transport chain (ETC) complex I– III activities was measured by spectrophotometric methods using complex-specific substrates (NADH for complex I, succinate for complex II, and decylubiquinol for complex III) as described (Grad & Lemire, 2004; Hsu et al., 2005; Mark et al., 2001). Complex IV activity was measured using a cytochrome c oxidase assay kit from Sigma. Mitochondrial citrate synthase activity was measured by the method of Srere (1969).

Protein assay

Protein concentrations of mitochondrial fractions and cell lysates were determined using a BioRad protein assay kit using bovine serum albumin as standard (0.038–0.600 mg/mL).

Statistical analysis

All data were expressed as the mean standard error of the mean (SEM). They were analyzed by one-way analysis of variance (ANOVA). Post hoc multiple comparisons were done with LSD. P values <0.05 were regarded as statistically significant.

Results

Effects of Herba Cistanche treatment on myocardial mitochondrial ATP-GC in rats

As shown in Figure 1, treatment with Herba Cistanche ex- tract at doses up to 0.5 g/kg increased myocardial mitochondrial ATP-GC in a dose-dependent manner in rats, with the degree of stimulation being 89% at a dose of 0.5 g/kg.

Effects of Herba Cistanche treatment on mitochondrial electron transport in rat hearts

The extent of electron transport in isolated mitochondria was measured by monitoring the reduction of MTT. The substrate used for the assay was either pyruvate or succinate. As shown in Figure 2a, Herba Cistanche treatment dose-dependently increased the extent of mitochondrial electron transport supported by pyruvate in rat hearts, with the optimal stimulation observable at doses of 0.5 g/kg. How- ever, no significant changes in the extent of mitochondrial electron transport supported by succinate were detected in Herba Cistanche–treated rat hearts (Fig. 2b).

Effects of Herba Cistanche treatment on mitochondrial respiratory complex activities in rat hearts

As shown in Figure 3, Herba Cistanche treatment at the optimal dose of 0.5 g/kg significantly increased complex I (34%) and complex III (22%) activities in rat hearts. The ac- tivity of citrate synthase, a key enzyme for the tricarboxylic acid cycle, was not affected by Herba Cistanche treatment (Fig. 3). Western blot analysis indicated that protein levels of complex I and III were not affected by Herba Cistanche treatment (data not shown).

Time course of Herba Cistanche-induced-changes in mitochondrial ATP-GC in H9c2 cells

Changes in ATP-GC were examined in Herba Cistanche– treated cells (50 and 150 µg/mL) following 2 to 16 h of incubation. As shown in Figure 4, Herba Cistanche treatment increased the ATP-GC in a time-dependent and biphasic manner in H9c2 cells, with the maximum stimulation (44% and 89%, respectively) observable after 8 h incubation for both drug concentrations. The ATP-GC then returned to untreated control values when the incubation time was ex- tended to 16 h. Earlier onset of stimulation occurred when the cells were treated with a higher concentration (150 µg/mL) of Herba Cistanche extract.

Concentration-dependence of Herba

Cistanche-induced increase in ATP-GC in H9c2 cells

H9c2 cells were treated with increasing concentrations (50– 300 µg/mL) of Herba Cistanche extract for 4 h. Figure 5 shows that Herba Cistanche treatment increased the ATP- GC in a concentration-dependent manner, with the maximum stimulation (82%) observable at the concentration of 300 µg/mL.

Cistanche extract increase in ATP production

Discussion

The upregulation of cellular activities for enhancing body function such as muscle contraction and immune responses by Yang invigoration necessitates the increase in the consumption of ATP, which is in turn supported by mitochondrial oxidative phosphorylation. In the current study, Herba Cistanche treatment was found to dose-dependently increase the mitochondrial ATP generation capacity ex vivo in rat hearts. This observation is consistent with our previous finding that among the Yang-invigorating Chinese tonifying herbs, Herba Cistanche treatment produced a relatively larger extent of stimulation on mitochondrial ATP generation in mouse hearts (Ko et al., 2006). The enhancement of mitochondrial ATP generation was paralleled by the increase in mitochondrial electron transport driven by pyruvate but not succinate, as assessed by the reduction of MTT. Results obtained from the kinetic measurement of mitochondrial complex I–IV indicated that activities of both complexes I and III were increased in Herba Cistanche–treated hearts, with the extent of stimulation on complex I activity being larger than that of complex III. As the activity of citrate synthase, a key enzyme of the tricarboxylic acid cycle, was not affected, the enhancement of mitochondrial ATP generation capacity by Herba Cistanche treatment may mainly be attributed to the stimulation of complex I and/or III activities. Most of the ATP requirements of the heart are met by mitochondria, which rely almost entirely on the complex I (NADH)-linked oxidation of fatty acid and glucose (Stanley et al., 1997; Taegtmeyer, 1998). Complex I, which exhibits a lower activity than do other respiratory complexes, is considered an important factor in the regulation of oxidative phosphorylation. Thus, the stimulation or inhibition of complex I activity can have a large impact on cardiac energetics. Though Western blot analysis did not reveal any changes in the protein level of these two complexes (data not shown), the increased enzyme activity may result from the changes in protein thiol status and/or lipid environment (Davey et al., 1998; Paradies et al., 2004).

Herba Cistanche extract could produce the ATP generation–enhancing effect in cultured H9c2 cells in situ. The relatively early onset of the stimulatory action produced by Herba Cistanche treatment, particularly at a high dose, is consistent with the observation from the ex vivo studies that indicated that the synthesis of complex I and III proteins is unlikely involved in the ATP generation–enhancing action. The demonstration of Herba Cistanche-induced enhancement in ATP generation in situ condition supports the notion that the increase in mitochondrial ATP generation capacity, as assessed by ex vivo measurement, may merely reflect a parallel effect produced by the drug treatment in vivo condition. The measurement of ATP generation in isolated mitochondria and cultured cells using malate and glutamate as substrates is an indirect measure of state 3 mitochondrial respiration (Chen et al., 2003). Using the same bioluminescent ATP assay, the rate of mitochondrial ATP generation in various tissues of untreated rats, as reported earlier in our laboratory (Leung et al., 2005), were in good agreement with the recently published data (Drew & Leeuwenburgh, 2003). The ATP generation capacity was estimated by a two-step integration of time-dependent changes in ATP level in both ex vivo and in situ assays. The observed drug-induced stimulatory effect was therefore magnified for the sake of comparison. The enhancement of ATP generation capacity by Herba Cistanche treatment in rat heart mitochondria ex vivo was paralleled by the stimulation of ATP generation in H9c2 cells in situ. Given the presence of ATP generation-enhancing activity in Yang-invigorating’ Chinese tonifying herbs, including Herba Cistanche, in mouse hearts ex vivo (Ko et al., 2006), the results suggest that the measurement of ATP generation capacity in H9c2 cells in situ may be used as a pharmaco- logical test for Yang-invigorating Chinese tonifying herbs or herbal formula.

In conclusion, treatment with Herba Cistanche extract could enhance the mitochondrial ATP generation capacity in rat hearts ex vivo and in H9c2 cells in situ. The findings support our postulation that the pharmacological basis of Yang-invigoration involves the enhancement of body functions through stimulating mitochondrial ATP production.

Herba Cistanche extract enhance the mitochondrial ATP generation capacity

References

Chen P (1998): Tonic Herbs. Advanced TCM Series. Beijing, China, Science Press, pp. 233–320.

Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ (2003): Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem 278: 36027– 36031.

Cohen G, Farooqui R, Kesler N (1997): Parkinson's disease: A new link between monoamine oxidase and mitochondrial electron flow. Proc Natl Acad Sci USA 94: 4890–4894.

Davey GP, Peuchen S, Clark JB (1998): Energy thresholds in brain mitochondria. Potential involvement in neurodegeneration. J Biol Chem 273: 12753–12757.

Drew B, Leeuwenburgh C (2003): Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction. Am J Physiol Regul Integr Comp Phys- iol 285: R1259–R1267.

Evan WH (1992): Isolation and characterization of membranes and cell organelles. In: Rickwood D, ed. Preparative Centrifugation: A Practical Approach. New York, Oxford University Press, pp. 223–270.

Grad LI, Lemire BD (2004): Mitochondrial complex I mutations in Caenorhabditis elegans produce cytochrome c oxidase deficiency, oxidative stress, and vitamin-responsive lactic acidosis. Human Mol Genetics 13: 303–314.

Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W, Schultz G (1991): Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.Circ Res 69: 1476–1486.

Hsu M, Srinivas B, Kumar J, Subramanian R, Andersen J (2005): Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via a NO-mediated pathway not involving peroxynitrite: Implications for Parkinson’s disease. J Neurochem 92: 1091– 1103.

Ko KM, Leon TYY, Mak DHF, Chiu PY, Du Y, Poon MKT (2006): A characteristic pharmacological action of ’Yang- invigorating’ Chinese tonifying herbs: Enhancement of myocardial ATP generation capacity. Phytomedicine 13: 636– 642.

Ko KM, Mak DH, Chiu PY, Poon MKT (2004): Pharmacological basis of ‘Yang-invigoration’ in Chinese medicine. Trends Pharmacol Sci 25: 3–6.

Lee CJ, Chuan S, Lee SL (1990): Study on the anti-aging effect of Cistanche deserticola Ma. Shanghai J Trad Chin Med 11: 22–23.

Lei L, Song ZH, Tu PF, Li YZ, Wu LJ, Chen FK (2001): Metabolic regulation of phenylethanoid glycosides from Herba cistanches in dogs’ gastrointestinal. Yao Xue Xue Bao 36: 432– 435

Leung HY, Chiu PY, Poon MKT, Ko KM (2005): A Yang- invigorating Chinese herbal formula enhances the mitochondrial functional ability and antioxidant capacity in various tissues of male and female rats. Rejuven Res 8: 238–247.

Lin LW, Hsieh MT, Tsai FH, Wang WH, Wu CR (2002): Antinociceptive and anti-inflammatory activity caused by Cistanche deserticola in rodents. J Ethnopharmacol 83: 177– 182.

Lu MC (1998): Studies on the sedative effect of Cistanche deserticola. J Ethnopharmacol 59: 161–165.

Mark A, Birch M, Douglass MT (2001): Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Meth Cell Biol 65: 97–117.

Ouyang J, Wang X, Zhao B, Yuan X, Wang Y (2003): Effects of rare earth elements on the growth of Cistanche deserticola cells and the production of phenylethanoid glycosides. J Biotech 102: 129–134.

Paradies G, Petrosillo G, Pistolese M, Di Venosa N, Federici A, Ruggiero FM (2004): Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: Involvement of reactive oxygen species and cardiolipin. Circ Res 94: 53– 59.

Srere PA (1969): Citrate synthase. Citric acid cycle. Meth Enzymol 13: 3–11.

Stanley WC, Lopaschuk GD, Hall JL, McCormack JG (1997): Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmaco- logical interventions. Cardiovasc Res 33: 243–257.

Taegtmeyer H (1998): Energy substrate metabolism as a target for pharmacotherapy in ischemic and reperfused heart muscle. Heart Metab 1: 5–9.

Tian XF, Pu XP (2005): Phenylethanoid glycosides from Cistanches salsa inhibit apoptosis induced by 1-methyl-4- phenylpyridinium ion in neurons. J Ethnopharmacol 97: 59–63.

Wu XM, Gao XM, Tsim KW, Tu PF (2005): An arabinogalactan isolated from the stems of Cistanche deserticola induces the proliferation of cultured lymphocytes. Int J Biol Macromol 37: 278–282.

Xiong Q, Hase K, Tezuka Y, Tani T, Namba T, Kadota S (1998): Hepatoprotective activity of phenylethanoids from Cistanche deserticola. Planta Med 64: 120– 125.

Xiong Q, Kadota S, Tani T, Namba T (1996): Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol Pharm Bull 19: 1580–1585.