Studies On Preparation Method And Application Of The Monoester Aconitium Total Alkaloids

Aconitum alkaloids possess several pharmacological effects,such as anti-inflammation,analgesia,anticancer,cardiac and so on,is the main medicinal component of the genus aconitum?Aconiti Radix,Aconiti kusnezoffii Radix,etc.?.Besides,it is toxic and mainly has bad effect on circulatory system,heart and central nervous system.Aconitum alkaloids belongs to diterpenoid alkaloids,which are usually divided into diestertype and monoester aconitines.The three most common diestertypes aconitines are aconitine,hypaconitine and mesaconitine aconitine,which belong to effective components and toxic components.Hence,the medicinal herbs of aconite must be processed strictly before being used,during the process the three diester aconitine was hydrolyzed off the C-8 acetyl and became the corresponding monoester aconitine?benzoylaconine,benzoylmesaconine and benzoylhypaconine?,or further hydrolysis off C-14 benzene formyl to forming the corresponding aconitum original alkali?aconine,mesaconine and hypaconine?.The common processing include steaming,boiling,sand pressing,adding auxiliary materials and so on.Research of scholars both at home and abroad show that the monoester type aconitine still has the same pharmacological effects as diester aconitines.Herein,the preparation method and application research of monoester aconitine have aroused people's attention.The contents of monoester alkaloids is extremely low in the genus aconitum,and they are usually prepared by hydrolysis from the diestertype alkaloids.In this paper,hydrolysis method and comparative research on acute toxicity and antitumor activity between diestertype alkaloids and monoester alkaloids have been studied firstly.Then,the method for the preparation of the empladstrum with monoester type aconitine alkaloids was studied and the HPLC method for the determination of benzoylmesaconine and benzoylhypaconine was established.Firstly,the total alkaloids are extracted from the medicinal materials of Aconiti Radix by using ethyl acetate-ammonia as the extracting solution.Taking all kinds of influence factors into consideration,the final optimal experiment conditions have been established.The dired powder was soaked in ethyl acetate?contain ammonium hydroxide 2%?for 5 h and then the thin layer chromatography was used to detect the alkaloids until no signal output.After the extract is condensed,the pH value was adjusted to 1² by 0.5%HCl solution and then extraction by ethyl acetate.The pH value of water layer was adjusted to 11¹2 by 0.5%NaOH solution and the process of extraction above mentioned were repeated again.Finally,the diestertype aconitium total alkaloids was obtained.Then,the conversion rate of benzoyl aconitine?BM?and benzoyl aconitine?BH?was used as the investigation index,the degradation conditions of the total alkaloids from the total alkaloid of the aconitum as raw material were established by single factor investigation and orthogonal test.The optimal degradation condition was determined as using 55%?v/v,mL/mL?aqueous solution of 1,4-dioxane as solvent,ratio of liquid to material is 16:1?v/w,mL/g?,and then heating reflux set at 165?for 6 h.Under this condition,the conversion rate ofbenzoylmesaconine was 68.9%and the conversion rate of benzoylhypaconine was 58.5%.This method has the advantages of simple,fast and high conversion rate.Furthermore,the comparative researches about acute toxicity and antitumor activity for monoester and diestertype aconitium total alkaloids have been tested in this experiment.The acute toxicity experiment of mice results showed that the median lethal dose(LD₅₀)of diestertype aconitium total alkaloids was 26.2 mg/kg,belongs to the high toxic substaces.However,the median lethal dose(LD₅₀)for monoester aconitium total alkaloids is 775.8 mg/kg,belonging to the low toxic substances,of which the toxicity reduced to about one thirty of the original.Using CCK-8 cytotoxic activity test,the incremental inhibition effects of total alkaloid and the monoester aconitium total alkaloids on HepG2 cells and A549 of lung cancer were investigated.The experimental results showed that the total alkaloids of aconitum had a certain inhibitory effect on HepG2 and A549 cells,and the IC₅₀0 for HepG2 of hepatocellular carcinoma cells and A549 of lung cancer cells A549 are 14.5?g·mL^-11 and 45.0?g·mL^-1,respectively.The monoester aconitium total alkaloids were inhibitory to HepG2 and A549 at the concentration of 50?g/mL.The inhibitory effect of the monoester aconitium total alkaloids on HepG2 and A549 in tumor cells was not stronger than the diestertype aconitium total alkaloids.Literatures show that the monoester aconitium total alkaloids have better anti-inflammatory and analgesic effect.In this paper,a kind of empladstrum that possesses performance of anti-inflammatory and analgesic has been prepaered by using the monoester aconitium total alkaloids.We discussed its preparation technology and established the effective composition content determination method.The preparation method of this emplastrum is that the water phase?monoester aconite class total alkaloid ethanol solution?and the oil phase?glycerin,sodium propionate,dihydroxyaluminum aminoacetate,EDTA disodium salt,titanium pigment and tartaric acid?in the vacuum state mixed stirring,then through coating.Finally,the emplastrum could be obtained by slicing.The contents determination of BM and BH by HPLC method have been studied and some conditions were described as follows.Methylene chloride-ammonia water were used as extraction solvent,and then the BM and BH could be obtained by heating reflux method.Besides,the content determination could betestedbyusinghighperformanceliquidchromatography?HPLC?.Chromatographic column Acchrom Tnature-C18?4.6 mm*250 mm,and 5?m?column,column temperature 35.0?,the flow rate is 1.0 mL/min,the detection wavelength is 235 nm,the mobile phase is methanol-water?33:67??including 5mmol·L^-11 ammonium acetate and 0.2%acetic acid?.Methodology study results show that the BM and BH exhibits good linear relationship between the peak area?S?and the sample quantity?m?within 0.2?g¹0?g,and the detection limits were 1.2×10^-2?g and 1.3×10^-2?g.The RSDs of precision,repeatability and stability for two components are less than 2%,the average recovery rate are 97.14%?RSD 1.15%?and 96.87%?RSD 1.13%?,respectively.The method has the advantages of high sensitivity,high accuracy and good precision.In conclusion,this paper established a new method to synthesize the monoester aconitium total alkaloids.By contrasting the toxicity and antitumor activity between the diestertype aconitium total alkaloids and the monoester aconitium total alkaloids,the preparation technology has been established,which made the product into emplastrum,and the quantitative determination of BM and BH by HPLC method.In this paper,the research results establish the foundation for the utilization of the monoester aconitium total alkaloids and provide scientific basis for its other applied researches.