| Objective:To establish the extraction process of free amino acids in the Peganum harmala lerb, determine the free amino acids in the Peganum harmala herb in different seasons and lifferent parts in the same season by HPLC, and analysis the correlation between amino cids content accumulation characteristics of the Peganum harmala herb in different seasons and different parts in the same season, and it’s alkaloids and flavonoids content accumulation characteristics, which provide a material basis for the biosynthetic nechanism and transformation of the structure and the biomimetic synthesis of the ilkaloids in the Peganum harmala herb. On basis of amino acids content accumulation characteristics analysis, to investigate the influence of amino acids on vasicine’s acute toxicity and main pharmacokinetic properties. Methods:1) The Peganum harmala herb of different seasons and different parts in the same season was collected to establish a quantitative method by using HPLC for the determination of amino acids in the Peganum larmala herb.2) The total content of free amino acids was used as index, the single-factor test and the orthogonal test were adopted to optimize the extraction conditions of different extraction methods, and then selected a better extraction program of amino acids in the Peganum harmala herb.3) The HPLC method was used to analysis the content of amino icids in the Peganum harmala herb in different seasons and different parts in the same season, and analysed the correlation between amino acids content accumulation characteristics of the Peganum harmala herb in different seasons and different parts in the same season, and it’s alkaloids and flavonoids content accumulation characteristics.4) The JD50 of vasicine and intervention administrating amino acids before oral administration of the vasicine were determined to investigate the influence of amino acids on vasicine’s acute toxicity.5) The content of vasicine by oral gavage and the content of intervention administrating amino acids before oral administration of the vasicine in vivo were determined and compared. The pharmacokinetics parameters were calculated by pharmacokinetics software PK Solution 2.0 to investigate the influence of amino acids on vasicine’s process in vivo.Results:1) The determination of amino acids in the Peganum larmala herb was performed on VP-ODS column (4.6x250mm,5μm) and mobile phase consisted of acetonitrile-ammonium acetate buffer solution (PH6.5). The column emperature was 40℃. The flow rate was 1.0 mL/min and the UV detective wavelength was set at 254 nm. The simultaneous determination of amino acids in the Peganum larmala herb could be used as quality control method. The results of amino acids of inearity, stability, reproducibility and precisions were good. The recoveries were )8%～102%.2) Reflux extraction method was suitable for amino acids in the Peganum narmala herb. The process conditions for the reflux extraction method were as following: 60% ethanol was selected as the extraction solvent and solvent multiple is 50, water bath inder 70℃ for 1.5 h.3) The amino acids in the Peganum harmala herb in different seasons ind different parts in the same season were different, and the amino acids content ccumulation characteristics were relevant to the flavonoids and alkaloids of the Peganum larmala. The amino acids in the above-ground parts of the Peganum harmala mainly ncluded proline, phenylalanine, leucine, serine, glycine, methionine and so on. The amino acids in the root mainly included lysine, proline, serine, phenylalanine, arginine and so on. The amino acids in the seed of the Peganum harmala herb mainly included ysine, phenylalanine, serine, glycine, proline and so on. The amino acids in the Peganum larmala herb in different parts’high content were the seed, root and the above-ground )arts in order. The contents of amino acids in the Peganum harmala herb in different seasons were different, and the highest was in July. The contents of the roline, phenylalanine, arginine and leucine in the above-ground parts of the Peganum larmala in different seasons were increased gradually from May, reached a maximum in fune, after July gradually reduced, and small fluctuations from September to November. The contents of the methionine were increased gradually from May, reached a maximum in September, decreased slightly in October, and increased slightly in November. The contents of the serine, tryptophan, glycine, valine, isoleucine, lysine and alanine were educed gradually with the change of content of the month. The results showed that the henylalanine may take part in the metabolic pathways of phenylpropanoid compounds to ecome the deacetylpeganetin and the peganetin in the Peganum harmala by the enzymatic eaction. The serine, tryptophan, methionine, isoleucine, leucine, alanine and lysine ydrochloride might take part in the process of secondary metabolism of the harmaline. The serine, tryptophan, glycine, valine, arginine and proline may take part in the process of secondary metabolism of the harmine.4) The LD50 of vasicine after oral administration in Kunming mice was 308.25mg·kg-1, with 95% confidence interval of 268.13-354.36 mg·kg-1. The LD50 of intervention administrating amino acids before oral administration of vasicine in Kunming mice was 376.94mg·kg-1, with 95% confidence interval of 330.04～430.52 mg·kg-1. The results showed that the amino acids in the Peganum harmala herb could reduce the toxicity of vasicine.5) For the group with oral administration of vasicine, the acquired parameters were:Cmax was 2897.86 ng/mL, t1/2Ke was 146.98 min, MRT was 271.27 min, AUC(0-t) was 701.90μg·min/mL. For the group with intervention administrating amino acids before oral administration of vasicine, the acquired parameters were:Cmax was 2216.85 ng/mL, t1/2Ke was 114.60 min, MRT was 177.378 min, AUC(0-t) vas 508.74μg·min/mL. The results showed that the amino acids in the Peganum harmala erb had a influence on vasicine’s pharmacokinetics parameters.Conclusion:Established he method of qualitative and quantitative is precise and stable. The optimized reflux extraction method is stable, feasible, and reasonable, which can be used to extract the mino acids in the Peganum harmala herb in lab. The results also show that, the contents and types of amino acids in the Peganum harmala herb are correlated with the different seasons and different parts as well as the content accumulation characteristics of the alkaloids and flavones in the Peganum harmala herb. The amino acids in the Peganum harmala herb can reduce the toxicity of vasicine and have a influence on vasicine’s pharmacokinetics parameters. |
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