The Activity And Mechanism Of Aconite On Diabetic Peripheral Neuropathy And The Identification Of Aconitine Components

Diabetic peripheral neuropathy(DPN)is the most prevalent complication of diabetes.Radix Aconite Lateralis Preparata(Fuzi)is a well-known traditional Chinese medicinal herb and often used for the treatment of DPN in the form of Chinese herbal compound.In order to study the effect and mechanism of traditional Chinese medicine,the anti-DPN bioactive constituents and mechanisms of the single tradition Chinese medicine herb,Fuzi,were explored in this paper at greater length and this work would provide certain theory baisi for the DPN treatment of Fuzi.Additionally,given that the compounds with the same chemical structure types often have the similar regulations of MS fragmentation,the regulations of fragmentation of Aconitum alkaloids were summarized.Then based on these rules and HPLC-MSn,the Aconitum alkaloids of other Chinese materia medica or its prepared medicine from the same genus were identified rapidly and the date is helpful for the further study of the chemical components of Chinese herbals.The content and the main results of the study are as follows:1 The protective effects of Fuzi aqueous extract(FZE)on streptozotocin-induced diabetic rats and high glucose-induced Schwann CellsIn vivo,Male Sprague Dawley rats(n=36)were randomly divided into control,DPN(after 6 weeks induction by streptozotocin),FZE 1.75 g/kg,FZE 3.50 g/kg,FZE 7.00 g/kg,and methylcobalamin groups.Two-week treatments were carried out and and blood glucose and weight were monitored every week in the process.After the two week treatment,nerve conduction velocity and paw withdrawal latency were measured.The results from animal experiments showed that FZE was effective in accelerating nerve conduction velocity and shortening paw withdrawal latency in diabetic rats,although it could not well improved the higher blood glucose and lower weight caused by dibetes.In vitro,the Schwann cells were grouped according to exposure:normal glucose,normal glucose plus mannitol,high glucose,and HG plus different concentrations of FZE(0.1?g/mL,1.0?g/mL,and 10.0?g/mL).Oxygen free radicals and apoptosis were evaluated through DCFH2DA,DHE and annexin-PE/7-AAD assay,respectively.The ROS levels and apoptotic ratio were increased by the high glucose stimulation.And after treatment for 48h,FZE was also found to protect Schwann cells against high glucose injury by the means of decreasing the apoptotic ratio,superoxide anion and peroxide level.Therefore,FZE has a protective effect in diabetic neuropathic rats,which is probably achieved by the anti-apoptotic and anti-oxidant effect of FZE on Schwann cells2.The extraction and separation of the alkaloids and polysaccharides from FZEThrough repeated column chromatography and preparative chromatography,6 alkaloid monomers were obtained.The alkaloids were songorine,neoline,fuziline,14-benzoylaconine,14-benzoylmesaconine,14-benzoylhypaconine.Fuzi polysaccharides(FPS)were obtained by hot water extraction and alcohol sedimentation and deproteinization from FZE.The content of polysaccharide is 70.24%determined by Phenol-sulfuric acid method and the method of pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone(PMP)coupled with electrospray ionisation mass spectrometry(HPLC/ESI-MSn)was used to analyse the monosaccharide composition.Nine monosaccharides were detected and seven of them were identified and fucose was first reported.What's more,A characteristic fragment ion m/z 215 was found,which is helpful to confirm the number of monosaccharides.3 The screening of active constituents from alkaloids and FPS based on the oxidative stress mechanism and the primary investigation of its mechanisms.Similarly,the antioxidant activity of alkaloids(0.1?g/mL,1.0?g/mL,10?g/mL and 100?g/mL)and FPS(1.0?g/mL,10?g/mL and 100?g/mL)were evaluated via the detection of intracellular ROS and determination of apoptotic cells.The results showed that neoline could reduce the level of ROS without dose-dependent relationship and benzoylaconine,only high concentrations,presented the antioxidant effect.However,FPS could significantly decrease superoxide anion and peroxide level in a dose-dependent manner.Simultaneously,the apoptosis level could recover to the normal level.These suggested that the alkaloids had a weaker antioxidant effect and Fuzi polysaccharides could be determined as the active constituents by the detection of intracellular ROSTo clarify the mechanism underlying the inhibitory effect of FPS on ROS production,we detected the expression of enzymes including superoxide dismutase(SOD),catalase(CAT),and NADPH oxidase-1(Nox1),which were directly related to the formation and removal of ROS.Simultaneously,peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1?),a transcriptional coactivator required for the induction of many ROS-detoxifying enzymes and mitochondrial biogenesis and AMP-activated protein kinase(AMPK),an essential upstream mediator of PGC-la,were investigated.FPS could significantly decrease superoxide anion and peroxide level in a dose-dependent manner.Simultaneously,the apoptosis level could recover to the normal level.Additionally,FBS significantly upregulated SOD,CAT and PGC-la protein level but downregulated Noxl protein level.Moreover,FPS could also increase AMPK activation significantly.AMPK-PGC-la pathway plays an important role in the protective effect of FBS against high glucose-induced cell damage.4 The rapid identification of of Aconitum alkaloidsIn the process of researching Aconitum alkaloids,Mass spectroscopy(MS)was employed to deduce the regulations of fragmentation of Aconitum alkaloids,which displayed a characteristic behavior of loss of CO(28u),CH3COOH(60u),CH3OH(32u),H2O(18u)and C6H5COOH(122u).According to the regulations and HPLC-MSn,42 alkaloids were found in A.pendulum.Among them,38 compounds were identified and 29 alkaloids were reported for the first time for this herb.In addition,by the similar method,the changes of components between the raw and processed Radix Aconitum kusnezoffii,which were stored at 110? for 8 h,were explored.32 components of the crude RAK were discovered,among which,26 alkaloids were tentatively identified.Meanwhile,24 ingredients were tentatively identified in the processed RAK decoction pieces,among which,20 alkaloids were determined.By comparison,7 diester diterpenoid alkaloids all disappeared after processing and 11 new constituents yield were mostly the monoester or amino alcohol type alkaloids.Therefore,the new processing method was proved to have a good effect of decreasing toxicity from the point of view of chemical component change.