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 mitochondrial 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 mitochondrial ATP-GC was observed in Renshenand Xiyangshen-pretreated rats, Dangshen treatment did not produce detectable effect ex vivo. The discrepancy between in situ and ex vivo assays for Dangshen may be attributed by its limited 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.

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 antioxidant actions [2-6]. According to the theory of Chinese medicine, Chinese tonic herbs can be classified into four functional categories, namely, “Yang-invigorating”, “Yin-nourishing”, “Qi-invigorating” and “Blood-enriching”, 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 relationship between “Qi-invigorating” action and energy level in skeletal muscle isolated from the thigh of “Qi-invigorating” herb-treated rats [7]. However, the pharmacological basis of “Qi-invigorating” action has yet to be established.

Previous experimental findings in our laboratory have demonstrated that all “Yang-invigorating” herbs are capable of enhancing mitochondrial ATP-GC in both cell and animal studies [8,9]. As a subcategory of “Yanginvigorating” 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 specimens 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.

2.2. Cell Culture

H9c2 cell, which is a subclone of the original 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 μg/mL of streptomycin and 17 mM NaHCO₃. All cells were grown under an atmosphere of 5% (v/v) CO₂ in air at 37˚C.

2.3. Measurement of ATP-GC in Situ

H9c2 cardiomyocytes were seeded at a density of 2.5 × 10⁴ 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 performed as previously described [8]. In brief, the removal of herbal extract-containing medium was followed by cell membrane permeabilization 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). Pyruvate (15 mM), malate (5 mM) and ADP (10 μM) 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 μL of perchloric acid (30%, w/v), and the reaction mixtures were then centrifuged at 540 × g for 10 min at 4˚C, and the supernatant were measured for ATP content by luciferase assay (ATPlite, PerkinElmer Inc., MA).

The mitochondrial ATP-GC of untreated control was estimated by computing the area under the curve of the graph (AUC₁) plotting ATP generated (nmol/mg protein) against time (0, 7.5 and 15 min) and expressed in arbitrary unit. For herbal extract-treated samples, AUC₁ values 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 (AUC₂) 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 extracttreated groups were expressed as percent control. The two-step data processing aims at minimizing the interanimal 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. Experimental 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 consecutive 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 measurement 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 excised 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 homogenizing 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 centrifuged at 9200 × g for 30 min at 4˚C to sediment the mitochondria. The pellets were resuspended in heart sucrose 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 MgCl₂, 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 μL) of mitochondrial fractions (1 mg protein/mL) were mixed with 50 μL of substrate solution (3 mM pyruvate and 3 mM malate) and 25 μL of pretreated 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 protein 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 analyzed by one-way analysis of variance (one-way ANOVA) and inter-group difference was detected by Least Significant Difference (LSD) when p < 0.05.

3. Results

Figure 1 shows that pretreatment with Renshen at concentrations ranging from 50 to 300 μg/mL significantly increased the mitochondrial ATP-GC in a concentration dependent manner in H9c2 cardiomyocytes, with the extent of stimulation being 42% at 300 μg/mL. Xiyangshen and Dangshen extracts significantly stimulated mitochondrial ATP-GC by 26% and 25%, respectively, at the concentration of 300 μg/mL (Figures 2 and 3).

As shown in Figure 4, pretreatment with Renshen extract at daily doses of 0.5 and 1 g/kg significantly enhanced the ATP-GC in mitochondria isolated from rat hearts in a dose-dependent manner, with the extent of

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 mitochondrial 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

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 “Shens”. This finding, which is consistent with the observation in “Yang-invigorating” 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 associated with a parallel enhancement of ATP-GC in mito-

chondria isolated from Renshenor Xiyangshen-pretreated 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 ATPGC may be related to its limited oral bioavailability to the heart.

Dangshen has been used as a milder and more economical substitute for Renshen, and sometimes they are interchangeably used in many Chinese herbal formulations. However, a pharmacopoeia published in the Qing Dynasty differentiated Dangshen from the Renshen family, in which Dangshen belongs to the family of Campanulaceae while Renshen and Xiyangshen are grouped under the family of Aralaceae [12]. Although Dangshen shares similar biological activities with Renhsen and Xiyangshen, 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, produced stimulatory effect on ATP-GC in H9c2 cardiomyocytes in situ but not in rat hearts ex vivo. In the context of Chinese medicine, this finding may be explained by the “Meridian Theory” (Jingluo), in which “Meridian” is the path that “Qi” (life energy) flows. According to the literature on materia medica, Renshen and Xiyangshen enter the “Meridians” of heart, spleen and lung after oral administration and then bring about the beneficial effects to the respective organs. Dangshen only enters the “Meridians” of spleen and lung, but not heart, which may be attributed to its inability to stimulate the ATP-GC in rat heart mitochondria.

In conclusion, the results obtained from the present study showed that the three tested “Qi-invigorating” Shens in Chinese medicine stimulated the ATP-GC in situ in the cell-based assay system. Interestingly, the “Qiinvigorating” Shens herbs may produce the beneficial action in a tissue specific manner. Further investigations should examine whether “Yang-invigorating” and “Qiinvigorating” Chinese tonic herbs can stimulate mitochondrial ATP-GC ex vivo in a tissue specific manner according to the “Meridian Theory” in Chinese medicine.

REFERENCES

NOTES

^*Corresponding author.