Investigation On Toxicity And Of Attenuated Methods Aconitum Brachypoaum Diels In Vivo And In Vitro

Aconitum brachypodum Diels belong to the genus of Aconitum(Family Ranunculaceae). Its root has been used as a traditional medicine of China and its chemical composition is mainly alkaloids which has anti-inflammatory and anti-nociceptive effect. Aconitum brachypodum Diels are mainly used for clinical treatment of neuropathic pain, rheumatism, joint pain, trauma, fractures and post-operative pain and advanced cancer pain. Because of its toxicity, the current research was mainly focused on the mechanism of its toxicity. However, to date, no detoxication strategy is available to completely eliminate the toxicity of aconitum plants. The present study will help to find new natural products to reduce the toxcity of Aconitum brachypodum Diels.Neurotoxicity detoxification methods and mechanisms of Aconitum brachypodum Diels have been studied to find natural antidote to reduce its toxicity.Baicalin, glycyrrhetinic acid and D1 were used to detoxify PC12 cells induced by total alkaloids of aconitum brachypodum diel(CFA) and the detoxification mechanism was studied. Moreover, Water decoction of Veratrilla baillonii Franch was also used to reduce the acute toxicity on mice induced by CFA. Thus the study is of high theoretical and practical significance.In this study, we explored the cytotoxicity and apoptosis on PC12 cells induced by CFA, as well as the release of intracelluler calcium, dopamine(DA) and reactive oxygen species(ROS). The cellular neurotoxicity of CFA on PC12 cell was estimated and assessed by MTT assay. The production of DA was measured by ELISA method. Moreover, the mitochondrial membrane potential(??m), the intracellular reactive oxygen species(ROS) and calcium were detected by flow cytometry, respectively. The experimental results showed that CFA could significantly reduce the viability of PC12 cells. ROS and intracellular calcium concentration was increased after CFA incubation. Baicalin and glycyrrhetinic acid could improve cell survival by inhibiting intracellular ROS as well as release of calcium ions, reducing mitochondrial damage, so as to achieve the role of detoxification. The results indicated that CFA cause oxidative stress in dopaminergic nerve via inhibiting the release of dopamine, which would lead to mitochondrial dysfunction, neurotoxicity and lastly induce cell apoptosis.Through consuling literature material and on-the-spot investigation, it was found that veratrilla baillonii Franch are used by Lisu and Naxi nationalities in Yunnan Province to reduce the toxicity of CFA. The toxicological profiles of CFA were evaluated on Qunming mice. To clarify the detoxication mechanism of the water decoction of veratrilla baillonii Franch(WVBF) on the CFA induced toxicity, the animal's physiological as well as pathological biochemistry parameters were assessed and used as the markers for the toxicity. Moreover, 1H nuclear magnetic resonance(NMR) based metabolomics approach was adopted to estimate the toxic and detoxic effects of CFA and WVBF, respectively. The results showed that at doses of 20–62.5?mg·kg-1, retching, hyperventilation, hypoactivity, scratching mouth, diaphoresis, dribbling, diarrhea, protopsis, writhing and even hyperspasmia were observed. In most of the cases, death occurred within 10-30 min for the groups that received 20–62.5?mg·kg-1 of CFA. WVBF(25-200 mg/kg) could attenuate all the acute toxicity induced by 40 mg/kg of CFA. The number of death as well as the toxic symptoms were reduced after oral gavage, demonstrating the detoxification effects of WVBF on the CFA-induced acute toxicity. Furthermore, the integral values of different groups were compared and showed that WVBF treatment could properly regulate the concentration alterations for some metabolites in the brain of mice with the CFA-induced toxicity. CFA treatment brain regions analyzed were metabolically altered. Notably, total creatine and taurine, the metabolites involved in bioenergetics and synaptic efficiency, respectively, were up-regulated in brain tissues, suggesting the injuries of brain. WVBF could markedly attenuate the increases of the above metabolites.Based on previous research, The detoxication mechanism of D1(A mixture of two kinds of monomers of veratrilla baillonii Franch) on CFA induced toxicity was clarifyed. The viability of PC12 cells were observed, the content of dehydrogenase(LDH) and the expression quantity of Bcl-2 were detected, too. The cellular neurotoxicity of CFA on PC12 cells was estimated and assessed by MTT assay. The production of LDH was measured by LDH Assay Kit. Western-blotting method was used to detect the expression quantity of Bcl-2. The results showed that D1 can significantly enhance cell viability of PC12 cells, reduce the content of LDH, and increased expression quantity of Bcl-2. It indicates that D1 can reduce apoptosis of PC12 cell. Therefore, we hypothesized that the detoxification of D1 is related to Bcl-2.The results showed that low doses of CFA displayed little toxicity on PC12 cells. However, long term of high doses exposure to CFA could lead to significant cell damage, mainly including cell apoptosis and necrosis, increasing intracellular ROS, mitochondria dysfunction and redox imbalance. Pre-incubation of baicalin and glycyrrhetic acid could both decrease the cytotoxicity of PC12 induced by CFA. WVBF treatment could properly regulate the concentration alterations for some metabolites in the brain of mice with the CFA-induced toxicity. CFA treatment brain regions analyzed were metabolically altered. Notably, total creatine and taurine, the metabolites involved in bioenergetics and synaptic efficiency, respectively, were up-regulated in brain tissues, suggesting the injuries of brain. D1 could significantly enhance cell viability of PC12, reduce the release of LDH, and increase expression quantity of Bcl-2. It indicates that D1 can reduced apoptosis of PC12 cell.