THESIS
2014
iv leaves, v-xxv, 203 pages : illustrations (some color) ; 30 cm
Abstract
Earlier studies in our laboratory have demonstrated that Cynomorii Herba can increase
mitochondrial adenosine triphosphate (ATP) generation capacity (ATP-GC), presumably by
enhancing mitochondrial electron transport, in both H9c2 cells and rat hearts, which is
characteristic of ‘Yang-invigoration’ in Chinese medicine. This thesis study aimed at (1)
identifying the active ingredient(s) in Cynomorii Herba and (2) further investigating its biological
activities and the underlying mechanism(s).
Utilizing the ATP-GC assay as an activity monitor, an ursolic acid (UA) -enriched active fraction
isolated from Cynomorii Herba ethanol extract, HCY2, was subjected to further investigation.
Pre-incubation of HCY2 or UA was found to increase ATP-GC, which may be caused by the
stimulation of...[
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Earlier studies in our laboratory have demonstrated that Cynomorii Herba can increase
mitochondrial adenosine triphosphate (ATP) generation capacity (ATP-GC), presumably by
enhancing mitochondrial electron transport, in both H9c2 cells and rat hearts, which is
characteristic of ‘Yang-invigoration’ in Chinese medicine. This thesis study aimed at (1)
identifying the active ingredient(s) in Cynomorii Herba and (2) further investigating its biological
activities and the underlying mechanism(s).
Utilizing the ATP-GC assay as an activity monitor, an ursolic acid (UA) -enriched active fraction
isolated from Cynomorii Herba ethanol extract, HCY2, was subjected to further investigation.
Pre-incubation of HCY2 or UA was found to increase ATP-GC, which may be caused by the
stimulation of mitochondrial electron transport secondary to increased fluidity of mitochondrial
inner membrane. It was hypothesized that the increase in mitochondrial reactive oxygen species
(ROS) production associated with the stimulation of mitochondrial electron transport chain could
trigger cellular responses including mitochondrial uncoupling and glutathione
reductase-catalyzed glutathione redox cycling, with resultant protection against oxidant injury.
To test this hypothesis, the effect of HCY2 or UA on mitochondrial ROS production was
examined. HCY2 or UA pre-incubation was found to increase mitochondrial ROS generation in
H9c2 cells. The suppression of mitochondrial ROS by antioxidant (dimethylthiourea, DMTU)
abrogated the HCY2 or UA-induced cellular responses and the associated cytoprotective effect.
Studies using specific inhibitors of reduced glutathione regeneration and uncoupling proteins
suggested that the HCY2/UA-induced mitochondrial uncoupling and glutathione redox cycling
play a determinant role in cytoprotection against oxidant injury.
Results obtained from animal studies indicated that long-term HCY2 or UA treatment invariably
protected against oxidative tissue damage in the rat models of myocardial ischemia/reperfusion
injury, carbon tetrachloride (CCl
4) hepatotoxicity and gentamicin nephrotoxicity. The tissue
protection was associated with the improvement in mitochondrial functional capacity, as assessed
by the measurement of ATP-GC, as well as the enhancement in glutathione redox status, with the
possible involvement of mitochondrial uncoupling.
As mitochondrial uncoupling has been considered as an attractive drug target for combating
obesity, the anti-obesity effect of HCY2 was also examined. Co-treatment with HCY2
significantly prevented body weight gain and reduced white fat pads in the mouse model of high
fat diet (HFD)-induced obesity. The weight reduction effect was associated with the
enhancement in mitochondrial uncoupling in skeletal muscle tissue. HCY2 treatment was also
found to ameliorate the HFD-induced hyperglycemia and dyslipidemia, suggestive of preventive
effect on metabolic syndrome and the associated health consequences. The signaling pathway
underlying HCY2/UA-induced weight reduction may be associated with the adenosine
monophosphate-activated protein kinase (AMPK)/ peroxisome proliferator-activated receptor-γ
coactivator-1α (PGC-1α) activation in skeletal muscle. The activation of AMPK/ PGC-1α further
increased the mitochondrial content and uncoupling protein 3 (UCP3) expression, and thus
regulated energy metabolism and mitochondrial biogenesis, eventually leading to weight loss in
mice.
In conclusion, HCY2, an UA-enriched fraction derived from Cynomorii Herba and its major
component, UA, could increase mitochondrial functional capacity and up-regulate
cellular/mitochondrial antioxidant status, thereby protecting against oxidant-induced
cellular/tissue injury. In addition, HCY2 treatment was found to prevent HFD-induced obesity
and the related metabolic syndrome. These beneficial effects produced by Cynomorii Herba are
likely mediated by mitochondrial uncoupling. The long-term intake of Cynomorii Herba
therefore offers a prospect for the prevention of aging and obesity.
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