T̠raditional U̠yghur m̠edicine (TUM; 維吾爾醫藥) has long been used to enhance physical condition and to treat diseases in northwest province of China. However, very few researches are focusing on the quality control and action mechanism of this herbal medicine. Here, Song Bu Li decoction (SBL; 松補力糖漿), the simplest TUM preparation, was analyzed. SBL contains only one herb, Nardostachyos Radix et Rhizoma (NRR, the root and rhizome of Nardostachys jatamansi DC). According to TUM theory, SBL can treat functional reduction of brain and heart caused by moist-cold and phlegm. The quality control parameters, as well as the biological efficacies of SBL have not been established. Therefore, we aim at setting up the criteria for preparation of SBL and reveal its biological mechanisms. The quality control parameters of NRR were constructed by measuring the amounts of nardosinone and total volatile oil, as well as the establishment of HPLC fingerprint chromatogram. Both water extract and volatile oil of NRR were chemically standardized. The volatile oil was analyzed by GC-MS: 14 compounds were identified, which accounted for ~80% of the total volatile oil. NRR water extract was standardized by HPLC fingerprint and the minimal amounts of ferulic acid and linarin. Here, we recommended a standardized NRR extract should contain at least 24.8 μg ferulic acid and 114.9 μg linarin in 1 g of dried NRR extract. A well-standardized SBL was prepared with a mixture of NRR extract and volatile oil in a ratio of 6 (NRR water extract; w): 1 (NRR volatile oil; v). These well-established quality control parameters guaranteed the quality of SBL to be consistent. For a comprehensive assessment of neuro-beneficial effects of SBL, different biological assays were employed, including neuroprotection and neuronal differentiation in PC12 neuronal cells. The pre-treatment of SBL protected cell against tBHP-induced cell death in a dose-dependent manner. In parallel, SBL suppressed intracellular r̠eactive o̠xygen s̠pecies (ROS) formation. Besides, a pARE-Luc reporter gene (an anti-oxidant responsive element) was transfected into PC12 cells. The treatment of SBL increased the transcriptional activity of pARE-Luc in dose-dependent manner. In parallel, the treatment of SBL also elevated the key anti-oxidant stress proteins, which included glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase (NQO1), glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunits. In cultured PC12cells, the expression of neurofilament, a protein marker for neuronal differentiation, was markedly induced by the applied herbal extract. Moreover, the nerve growth factor (NGF)-induced neurite outgrowth in cultured PC12 cells was significantly potentiated by the co-treatment of SBL. The involvement of cAMP-dependent kinase in SBL-induced neuronal differentiation was investigated; the pre-treatment of the kinase inhibitor, H89, abolished the expression of neurofilament proteins. In cultured astrocytes, the treatment of SBL significantly induced neurotrophic factor expression. For the molecular mechanism, the pre-treatment of H89 significantly attenuated SBL-induced neurotrophic factor expression. It was demonstrated the involvement of cAMP-dependent kinase in this induction. This effect of SBL on astrocytes might be helpful for the growth of neuron, and which could be a potential treatment for neurotrophic factor insufficient disorders. For the cardiovascular effects, human umbilical vein endothelium (HUVEC) cells and H9c2 cardiomyocytes were applied here. The treatment of SBL inhibited the ADP-induced platelet aggregation. In HUVEC cells, SBL induced production of nitric oxide (NO). Here, volatile oil from NRR exhibited robust effect in an induction of NO production; however, the NRR water extract did not. In parallel, the treatment of the volatile oil induced the phosphorylation of endothelial nitric oxide synthase (eNOS), which triggered vasodilation and reduced platelet aggregation. The treatment of NRR volatile oil activated the phosphorylation of Akt and increased intracellular calcium level. Pre-treatment of LY294002 and BAPTA-AM abolished the Akt phosphorylation and intracellular calcium increment, respectively. These results therefore suggested that Akt and intracellular calcium were involved in volatile oil-induced NO induction. For the cardioprotective effect, SBL protected H9c2 cells from hypoxia/reoxygenation-induced apoptosis through suppress ROS formation and caspase 3 activity. NRR volatile oil was further investigated for its anti-oxidant mechanism. In cultured H9c2 cells, application of NRR volatile oil exhibited strong potency in preventing tBHP-induced cell death and accumulation of ROS in a concentration-dependent manner. In addition, the application of volatile oil stimulated the gene expressions of anti-oxidant enzymes, which was mediated by the transcriptional activation of ARE. The induced genes were NQO1, GST, GCLM, GCLC. In addition, the volatile oil of NRR activated the phosphorylation of Akt in cultured H9c2 cells. The treatment of LY294002, an Akt inhibitor, significantly inhibited the volatile oil-mediated ARE transcriptional activity, as well as the cell protective effect of NRR oil. Taken together, the present results indicated that SBL might exert its neuro-beneficial and cardiovascular effects by (i) inducing neuronal differentiation; (ii) enhancing neurotrophic factor expression; (iii) increasing NO production; and (iv) anti-oxidation effect against oxidative stress.
| Date of Award | 2014 |
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| Original language | English |
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| Awarding Institution | - The Hong Kong University of Science and Technology
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