Extraction, Separation And Pharmacological Activities Of Alkaloids In Aconitum Tanguticum

Aconitum tanguticum was the dried grass of Ranunculaceae Aconitum plant A. tanguticum, traditional Tibetan medicine was mainly used for heat in liver, gallbladder heat, lung heat, hepatitis, pneumonia, gastroenteritis, influenza, fever caused by infectious disease, and achieved good therapeutic effect for a long time ago.The main medicinal ingredients included alkaloids, glycosides, volatile oil and etc. Effects of alkaloids were anti-inflammatory, analgesic, antipyretic, sedative, immunosuppressive, anti-tumor, Anti hypertension, dilate blood vessels and other effects. In this study, A. tanguticum main medicine effective component extracted by 90% ethanol, Yield was 17.65%, and extraction separation with different polar organic solvent, to observe the inhibitory effect on non specific impatient chronic inflammation and analgesic effect of ethanol extract(EE), petroleum ether(PE), ethyl acetate part(EA), chloroform fraction(CH), n-butanol fraction(BU) and water soluble part(WA). The results showed that the anti-inflammatory and analgesic effects of Tibetan medicine A. tanguticum was clearly, anti-inflammatory and analgesic effects of alcohol extract was stronger, preliminary screening discovery chloroform separation part was the major functional group of anti-inflammatory and analgesic substances, other components also had relatively weak anti-inflammatory and analgesic activity.On the basis of pharmacodynamic substance with anti-inflammatory and analgesic effects of A. tanguticum was mainly concentrated chloroform extraction part. In this research, the total alkaloids of A. tanguticum(TAA) was extracted, researchs on quality control and their qualitative and quantitative analyses were underway to establish a method for determination of the content of TAA, The results show that contains alkaloids(aconitine in dollars) was 0.3323%. Using Box-Behnken Design with the content of total alkaloids and ester-alkaloids, based on the single factor experiment, preferably extraction conditions of TAA were the temperature of 85℃, extracting 1.5h, and 90% ethanol. Under the optimum process conditions, the experiment showed good reproducibility, the extraction process was stable and feasible. So the new suitable technology of extraction for TAA was found.Datas showed that A. tanguticum can cause acute toxicity. To study acute toxicity on mice by the oral ingestion or intraperitoneal injection, the experiments were operated. The results showed LD50 of intragastric administration TAA was 0.7456g/kg, LD50 of intraperitoneal injection TAA was 0.3358g/kg, the research confirmed that TAA had certain toxicity by gavage and intraperitoneal injection on mice, and the toxic target organ were the autonomic nervous system and motor nervous system. Toxic reactions happen faster and short duration. Further pharmacodynamic study found acute non-specific inflammation reaction and chronic inflammatory hyperplasia were obviously inhibited, and analgesic effect was exist, these activity showed a dose-dependent relationship.Acute lung injury(ALI) model was copied by lipopoly saccharide(LPS)-induced in rats, the Pa O2 and Pa O2/Fi O2 values, lung wet/dry(W/D) weight ratio and histological changes in lung tissue were measured. The cell counts, protein concentration, tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1β(IL-1β) in bronchoalveolar lavage fluid(BALF), myeloperoxidase(MPO) activity in lung tissue was determined at 6, 12 or 24 h after LPS treatment. In addition, the NF-κ B activation in lung tissue was analyzed by western blot. The results showed TAA significantly reduced the lung W/D ratio and increased the value of Pa O2 or Pa O2/Fi O2 at 6, 12 or 24 h after LPS challenge. TAA also reduced the total protein concentration and the number of total cells, neutrophils or lymphocytes in BALF. In addition, TAA decreased MPO activity in the lung and attenuated histological changes in the lung. Furthermore, TAA inhibited the concentration of TNF-α, IL-6 and IL-1β in BALF at 6, 12 or 24 h after LPS treatment. Further study demonstrated that TAA significantly inhibited NF-κ B activation in lung tissue. The current study proved that TAA exhibited a potent protective effect on LPS-induced ALI in rats through its anti-inflammatory activity.Acute hemorrhagic enteritis(AHE) model was copied by methotrexate(MET)-induced in rats, the TNF-α, NO DAO, MPO activity and histological changes in ileum tissue were measured at 3 or 5 day after MET treatment. In addition, the ICAM-1 activation in ileum tissue was analyzed by Immunohistochemistry. The results showed TAA significantly decreased ICAM-1 activity in the ileum and attenuated histological changes in the ileum Furthermore, TAA inhibited the concentration of TNF-α and NO in serum at 3 or 5day after MET treatment. Further study demonstrated that TAA significantly inhibited DAO and MPO activation in ileum tissue. The current study proved that TAA exhibited a potent protective effect on MET-induced AHE in rats through its anti-inflammatory activity.The common pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Candida albicans were chosen as the research object, the results showed that G+ bacteria and G- bacteria were inhibited obviously, but no antibacterial effect on Candida albicans in the range of 1.0~10.0mg/m L of TAA. Furthermore, the strongest antibacterial activity on Staphylococcus aureus and the weakest antibacterial effect on Escherichia coli were found in the study. In vitro anti-inflammatory results showed TAA can inhibit the macrophage by LPS-induced released NO and IL-1, and reduce the inflammatory injury effectively, TAA can also significantly enhance the phagocytic function of macrophages in the range of 60-240mg/L, the results further proved that the anti-inflammatory effects of TAA was real.