Qi-Invigorating Chinese Tonic Herbs (Shens)

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Qi-Invigorating Chinese Tonic Herbs (Shens)
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  Chinese Medicine , 2012, 3, 101-105 http://dx.doi.org/10.4236/cm.2012.32016 Published Online June 2012 (http://www.SciRP.org/journal/cm) “Qi-Invigorating” Chinese Tonic Herbs ( Shens ) Stimulate Mitochondrial ATP Generation Capacity in H9c2 Cardiomyocytes in Situ  and Rat Hearts ex Vivo   Hoi Shan Wong, Weng Fat Cheung, Wai Ling Tang, Kam Ming Ko *   Division of Life Science, Hong Kong University of Science & Technology, Hong Kong, China Email: *  bcrko@ust.hk Received December 20, 2011; revised February 1, 2012; accepted February 23,   2012   ABSTRACT In the study, using ethanol extracts of  Renshen  ( Panax ginseng  C A Meyer),  Xiyangshen  ( Panax quinquefolius   L.) and  Dangshen  ( Codonopsis pilosulae ), we investigated the effect of these “Qi-invigorating” Chinese tonic herbs on mito- chondrial ATP generation capacity (ATP-GC) in H9c2 cardiomyocytes in situ  and rat hearts ex vivo . All three types of “ Shens ” stimulated mitochondrial ATP-GC, with  Renshen  being most potent. While a parallel enhancement in mito- chondrial ATP-GC was observed in  Renshen - and  Xiyangshen -pretreated rats,  Dangshen  treatment did not produce de- tectable effect ex vivo . The discrepancy between in situ  and ex vivo  assays for  Dangshen  may be attributed by its lim- ited oral-bioavailability to the heart. The tissue specific activity of Shens on mitochondrial ATP-GC may be explained  by the “Meridian Theory” in traditional Chinese medicine. Keywords:  Qi; Chinese Medicine; ATP; Mitochondria; Cardiomyocytes 1. Introduction “  Renshen ”, a Chinese “  pinyin ” for Radix Ginseng ( Panax ginseng  C A Meyer), has a long history of use in Chinese medicine for promoting health. Early pharmacological investigations have demonstrated the adaptogenic effect of  Renshen , which can enable the body to cope with various stress conditions [1]. Recent experimental studies have shown that  Renshen  or its ginsenosides produces anti-aging, anti-carcinogenic, anti-inflammatory and an-tioxidant actions [2-6]. According to the theory of Chi- nese medicine, Chinese tonic herbs can be classified into four functional categories, namely, “Yang-invigorating”, “Yin-nourishing”, “Qi-invigorating” and “Blood-enrich- ing”, with various “ Shens ” being categorized in the “Qi” family. Due to the popularity and therapeutic values of “Qi-invigorating” herbs, the investigation of biological activities and the underlying mechanisms in relation to “Qi-invigoration” is of great pharmacological interest. In this regard, a recent study has demonstrated the relation- ship between “Qi-invigorating” action and energy level in skeletal muscle isolated from the thigh of “Qi-invigo- rating” herb-treated rats [7]. However, the pharmacol- ogical basis of “Qi-invigorating” action has yet to be established. Previous experimental findings in our laboratory have demonstrated that all “Yang-invigorating” herbs are ca-  pable of enhancing mitochondrial ATP-GC in both cell and animal studies [8,9]. As a subcategory of “Yang- invigorating” herbs, “Qi-invigorating” herbs may also stimulate mitochondrial ATP generation capacity in various tissues. In the present study, using the ethanol extracts of  Renshen ,  Xiyangshen  ( Panax quinquefolius   L.) and  Dangshen ( Codonopsis pilosulae ), we investigated the effect of “Qi-invigorating” Chinese tonic herbs on mitochondrial ATP-GC using in situ  and ex vivo  assay systems. 2. Materials and Methods 2.1. Herbal Materials  Renshen ,  Xiyangshen  and  Dangshen  were purchased from a local herbal dealer (Lee Hoong Kee). The herbs were authenticated by the supplier and voucher speci- mens were deposited in the Division of Life Science, Hong Kong University of Science and Technology (HKUST). Individual herb was extracted by 95% ethanol under reflux for 2 h, as previously described [8], and obtained the ethanol extract. All the extracts were dried  by evaporating the solvent under reduced pressure and the dried extracts at varied yields (see Table 1 ) were stored at 4 ˚ C until use. * Corresponding author.   Copyright © 2012 SciRes. CM     H. S. WONG  ET AL .   102   Table 1. Yields of herbal extractions. Renshen Xiyangshen Dangshen Yield (%) 10.7 16.5 41.5 All herbal extracts were obtained by ethanol extraction under reflux for 2 h. 2.2. Cell Culture H9c2 cell, which is a subclone of the srcinal clonal cell line derived from embryonic BD1X rat heart tissue, was  purchased from American Tissue Culture Centre (ATCC). The cells were cultured as mono-layers in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL Life Technologies, Grand Island, NY, US), supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU/mL of  penicillin (Sigma, St. Louis, MO), 100 μ g/mL of strep- tomycin and 17 mM NaHCO 3 . All cells were grown un- der an atmosphere of 5% (v/v) CO 2  in air at 37 ˚ C. 2.3. Measurement of ATP-GC in Situ   H9c2 cardiomyocytes were seeded at a density of 2.5 × 10 4  cells/well into 24-well microtiter plates. After the cell attachment, cells were incubated with herbal extracts for 4 h at 37 ˚ C. Control group was given vehicle (DMSO) only. After the incubation, the ATP-GC assay was per- formed as previously described [8]. In brief, the removal of herbal extract-containing medium was followed by cell membrane permeabilization with digitonin (50 μ g/ mL) in an incubation buffer (120 mM KCl, 5 mM KH 2 PO 4 , 2 mM EGTA, 10 mM HEPES, 0.1 mM MgCl 2 , 0.5% BSA, pH 7.4). Pyruvate (15 mM), malate (5 mM) and ADP (10 μ M) were added for mitochondrial ATP generation at 37 ˚ C, which was monitored at increasing time intervals ranging from 0 to 15 min. The reaction was terminated by the addition of 60 μ L of perchloric acid (30%, w/v), and the reaction mixtures were then centrifuged at 540 × g  for 10 min at 4 ˚ C, and the super- natant were measured for ATP content by luciferase as- say (ATPlite, PerkinElmer Inc., MA). The mitochondrial ATP-GC of untreated control was estimated by computing the area under the curve of the graph (AUC 1 ) plotting ATP generated (nmol/mg protein) against time (0, 7.5 and 15 min) and expressed in arbi- trary unit. For herbal extract-treated samples, AUC 1  val- ues of increasing incubation times were normalized to a respective mean control value from untreated cells and expressed as percent control. The area under the curve (AUC 2 ) of the graph plotting percent control values against incubation time (7.5 and 15 min) was computed and expressed in arbitrary unit. Data of herbal extract- treated groups were expressed as percent control. The two-step data processing aims at minimizing the inter- animal and inter-assay variabilities under the present experimental conditions [10]. 2.4. Animal Care Female adult Sprague-Dawley rats (8-week old; 200 - 250 g) were maintained under a 12 h dark/light cycle at about 22 ˚ C and allowed water and food ad libitum . Ex-  perimental protocols were approved by the Research Practice Committee at HKUST. Animals were randomly divided into groups, with five animals in each. In the experiment, rats were administered intragastrically with herbal extracts at daily doses of 0.5 to 3 g/kg for 3 con- secutive days and were killed by decapitation 24 hours after the last dosing. Control animals were administered with the vehicle (water) only. Hearts were excised from control or herbal extract-pretreated rats, mitochondrial fractions were then isolated and subjected to the meas-urement of mitochondrial ATP generation capacity (ATP-GC) in vitro . 2.5. Preparation of Mitochondrial Fractions Myocardial mitochondrial fractions were prepared from heart tissue homogenates by differential centrifugation at 4 ˚ C. In brief, heart ventricular tissue samples were ex- cised and rinsed with ice-cold heart sucrose buffer (320 mM sucrose, 1 mM EDTA, 50 mM Tris-base, pH 7.4). A 10% (w/v) heart homogenate was prepared by homoge- nizing the minced ventricular tissue with a teflon-glass homogenizer at 4000 rpm for 30 min. The homogenate was centrifuged at 600 × g  for 10 min at 4 ˚ C to remove nuclei and cell debris. The supernatant was then centri- fuged at 9200 × g  for 30 min at 4 ˚ C to sediment the mi- tochondria. The pellets were resuspended in heart su- crose buffer and constituted the mitochondrial fractions. 2.6. Destruction of Contaminating ATP in Commercial ADP Preparation The contaminating ATP was removed by an enzymatic  procedure. To briefly describe, ATP was converted to ADP by reacting it with glucose at 25 ˚ C during a 18 h incubation in the reaction mixture (34 mM commercial ADP, 100 mM Tris-HCl buffer (pH 8.1), 2 mM MgCl 2 , 3.4 mM glucose, 1 mM NADH, 5.6 U hexokinase/mL, and 6 U glucose-6-phosphate dehydrogenase/mL). To destroy the formed NADPH, the solution was acidified with 0.1 volume of 1 M HCl and allowed to stand at 25 ˚ C for 10 min. The solution was then alkalinized by adding 0.017 volume of 6 M NaOH, and all enzymes were inactivated by heating the reaction mixture to 95 ˚ C for 15 min [11]. 2.7. Measurement of ATP-GC Aliquots (50 μ L) of mitochondrial fractions (1 mg pro- tein/mL) were mixed with 50 μ L of substrate solution (3 mM pyruvate and 3 mM malate) and 25 μ L of pretreated Copyright © 2012 SciRes. CM     H. S. WONG  ET AL .   103 ADP (30 mM) solution, and the reaction mixtures were incubated for increasing period of time (0, 10 and 20 min) at 37 ˚ C to allow mitochondrial ATP generation. The ATP content was then measured as described before. 2.8. Protein Assay Protein concentration was determined by Bio-Rad pro- tein assay kit (Bio-Rad, Hercules, CA), using bovine serum albumin as standard. 2.9. Statistical Analysis All data were expressed as mean ± standard error of the mean (SEM), unless otherwise specified. Data were ana- lyzed by one-way analysis of variance (one-way ANOVA) and inter-group difference was detected by Least Sig- nificant Difference (LSD) when  p  < 0.05. 3. Results Figure 1  shows that pretreatment with  Renshen  at con- centrations ranging from 50 to 300 μ g/mL significantly increased the mitochondrial ATP-GC in a concentration dependent manner in H9c2 cardiomyocytes, with the extent of stimulation being 42% at 300 μ g/mL.  Xiyang- shen  and  Dangshen  extracts significantly stimulated mi- tochondrial ATP-GC by 26% and 25%, respectively, at the concentration of 300 μ g/mL ( Figures 2  and 3 ). As shown in Figure 4 , pretreatment with  Renshen  ex- tract at daily doses of 0.5 and 1 g/kg significantly en- hanced the ATP-GC in mitochondria isolated from rat hearts in a dose-dependent manner, with the extent of Control 50 μ g/mL 150 μ g/mL 300 μ g/mL   Figure 1. The effect of  Renshen  extract on mitochondrial ATP-GC in H9c2 cardiomyocytes. Cells were pre-incubated with  Renshen  extract at concentrations of 50 to 300 μ g/mL for 4 h. Upon the removal of herbal extract-containing me- dium, the cells were subjected to the measurement of ATP- GC in situ . Data were expressed in percent control with respect to the untreated control. Values given are means ± SEM, with n = 3. *Significantly different from the control group. Control 50 μ g/mL 150 μ g/mL 300 μ g/mL   Figure 2. The effect of  Xiyangshen  extract on mitochondrial ATP-GC in H9c2 cardiomyocytes. Cells were pre-incubated with  Xiyangshen  extract at concentrations of 50 to 300 μ g/ mL for 4 h. Mitochondrial ATP-GC was measured as de- scribed in Figure 1. Data were expressed in percent control with respect to the untreated control. Values given are means ± SEM, with n = 3. *Significantly different from the control group.   Control 50 μ g/mL 150 μ g/mL 300 μ g/mL   Figure 3. The effect of  Dangshen  extract on mitochondrial ATP-GC in H9c2 cardiomyocytes. Cells were pre-incubated with  Dangshen  extract at concentrations of 50 to 300 μ g/mL for 4 h. Mitochondrial ATP-GC was measured as described previously. Data were expressed in percent control with respect to the untreated control. Values given are means ± SEM, with n = 3. *Significantly different from the control group. stimulation being 9.3% and 22.9%, respectively. Figure 5 shows that pretreatment with  Xiyangshen  extract at daily doses of 2 and 3 g/kg significantly increased mito- chondrial ATP-GC, with the extent of stimulation being 13% and leveled at the high dose. However,  Dangshen   pretreatment at daily doses up to 3 g/kg did not produce any detectable effect on ATP generation in mitochondria isolated from rat hearts ( Figure 6 ). 4. Discussion In the present study, we demonstrated that the ethanol Copyright © 2012 SciRes. CM     H. S. WONG  ET AL .   104   Figure 4. The effect of  Renshen  extract on ATP-GC in mi- tochondria isolated from rat hearts. Rats were orally ad- ministered with  Renshen  extract at daily doses of 0.5 and 1 g/kg for 3 consecutive days. Heart mitochondrial fractions were isolated and subjected to the measurement of ATP-GC ex vivo . Data were expressed in percent control with respect to the untreated control animals. Values given are means ± SEM, with n = 5. *Significantly different from the control group.   Figure 5. The effect of  Xiyangshen  extract on ATP-GC in mitochondria isolated from rat hearts. Rats were orally administered with  Xiyangshen  extract at daily doses of 2 and 3 g/kg for 3 consecutive days. ATP-GC of heart mito- chondria was measured as described in Figure 4. Data were expressed in percent control with respect to the untreated control animals. Values given are means ± SEM, with n = 5. *Significantly different from the control group.   extracts of all three types of “ Shens ” (namely,  Renshen ,  Xiyangshen  and  Dangshen ) increased mitochondrial ATP-GC in H9c2 cardiomyocytes, with  Renshen  being most potent among the tested “S hens ”. This finding, which is consistent with the observation in “Yang-in- vigorating” herbs [9], suggested that “Qi-invigorating” herbs can also stimulate mitochondrial ATP-GC in H9c2 cardiomyocytes. The stimulation of ATP-GC by  Renshen  and  Xiyangshen  in the cell-based in situ  assay was asso- ciated with a parallel enhancement of ATP-GC in mito- Figure 6. The effect of  Dangshen  extract on ATP-GC in mitochondria isolated from rat hearts. Rats were orally administered with  Dangshen  extract at daily doses ranging from 0.5 to 3 g/kg for 3 consecutive days. ATP-GC of heart mitochondria was measured as described previously. Data were expressed in percent control with respect to the un- treated control animals. Values given are means ± SEM, with n = 5. *Significantly different from the control group.   chondria isolated from  Renshen-  or  Xiyangshen -pre- treated rats, with the extent of stimulation by  Renshen   being larger than that of  Xiyangshen  at the same crude herb equivalent dose. However,  Dangshen  treatment did not cause any detectable change in ATP-GC in rat heart mitochondria. The discrepant observation between in situ  and ex vivo  assays for  Dangshen  on mitochondrial ATP- GC may be related to its limited oral bioavailability to the heart.  Dangshen  has been used as a milder and more eco- nomical substitute for  Renshen , and sometimes they are interchangeably used in many Chinese herbal formula- tions. However, a pharmacopoeia published in the Qing Dynasty differentiated  Dangshen  from the  Renshen  fam- ily, in which  Dangshen  belongs to the family of Cam-  panulaceae while  Renshen  and  Xiyangshen  are grouped under the family of Aralaceae [12]. Although  Dangshen  shares similar biological activities with  Renhsen  and  Xi-  yangshen , the tissue specificity varies in these two groups of herbs. It has been demonstrated that majority of the metabolites of  Renshen  and  Xiyangshen  could be found in the liver, heart and small intestine [1], while the action of  Dangshen  seemed to be limited to digestive, immune and respiratory systems [13-15]. Consistent with this,  Dangshen , as observed in the present study, pro- duced stimulatory effect on ATP-GC in H9c2 cardio- myocytes in situ  but not in rat hearts ex vivo . In the con- text of Chinese medicine, this finding may be explained  by the “Meridian Theory” (  Jingluo ), in which “Merid- ian” is the path that “Qi” (life energy) flows. According to the literature on materia medica ,  Renshen  and  Xi- yangshen  enter the “Meridians” of heart, spleen and lung Copyright © 2012 SciRes. CM   
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