| Objects: Aconitum soongaricum Stapf. is exclusively distributed in the northern region of Xinjiang, China. The A. soongaricum Stapf. dried tuber can be utilized as medicine. A. soongaricum Stapf. is a medicinal plant with toxic, and it is as well the primary detrimental grass threatening Xinjiang grassland in recent years. Those toxic plants can also become valuable medicine if we make good use of them. Therefore, it is hypothesized that A. soongaricum Stapf. has certain phylogenetic relationship with medicinal materials of Aconitum tuber; it is also hypothesized and that A. soongaricum Stapf. has similar medicinal value as medicinal materials of Aconitum tuber such as analgesic, anti-inflammatory and immune regulations; processing of A. soongaricum Stapf. was studied for reducing the toxicity. In this study, root alkaloid monomer component was extracted, isolated and toxicity analyzed after a comprehensive understanding of medicinal value of A. soongaricum; this lays a foundation for utilizing A. soongaricum as substitution materials of Aconiti Radix or Aconiti kusnezoffii Radix, and for establishing the legal medicine quality standards of A. soongaricum. Methods: 1. Random amplified polymorphic DNA(RAPD) and inter-simple sequence repeat(ISSR) molecular labeling techniques were performed to analyze the DNA molecular phylogenic relationships of A. soongaricum with Aconiti Radix and Aconiti kusnezoffii Radix; 2. HPLC was applied to investigate the chemical phylogenic relationships of A. soongaricum with Aconiti Radix and Aconiti kusnezoffii Radix through a combination of similarity analysis, cluster analysis and principal component analysis; 3.(1) A. soongaricum was processed with 3 types of common aconitum medicine processing methods, and thealkaloid chemical component variation of crude and processed A. Sanitarium was compared with HPLC method.(2) Acute toxicity, animal analgesic, anti-inflammatory, immune-modulatory experiments were utilized to compare the pharmacological effects variation of crude and processed A. soongaricum;(3) Rank correlation statistical method was applied to analyze the correlation between chemical compositions and pharmacological effect of crude and processed A. soongaricum, and the attenuating method of A. soongaricum was confirmed with the understanding of toxicity of processed A. soongaricum product; 4.(1) Solvent extraction and separation method was used to extract and evaluate the alkaloid monomer composition of A. soongaricum.(2) Cell culture in vitro was introduced to investigate the toxicity of alkaloid monomer composition on rat cardiac myocytes. Results: 15 RAPD primers were screened out from 120 random primers and 15 ISSR primers were screened out from 100 random primers. Both types of screened primers were utilized for Aconitum sample analyzing. According to the RAPD and ISSR analyzing, the average coefficients of gene differentiation(GST) were 0.4358 and 0.5005 respectively. The differences of genetic differentiation among 3 kinds of Acontium were comparably significant, which indicates that the gene flow(Nm) is low. The estimates of gene flow(Nm = 0.6473 and 0.4991 respectively) as well illustrate significant difference; 15 samples were classified into 3 types: the first type was Aconiti kusnezoffii Radix and A.soongaricum, the second type was Aconiti Radix and the third type was Aconitum leucostomum Worosch. The result suggests that A.soongaricum and Aconiti kusnezoffii Radix have a closer DNA molecular phylogeny relationship; 2. HPLC fingerprint of A.soongaricum alkaloid composition was established, and 5 characteristic peaks were collected, which were(1)benzoylaconine,(2)benzoylhypacoitine,(3)unnamed compound-2,(4) aconitine and(5) unnamed compound-4 respectively; results of similarity analysis, cluster analysis and principal component analysis indicate that A. soongaricum has a closer chemical relationship with Aconiti kusnezoffii Radix; 3.(1) After processing of A. soongaricum, the content of diester-type alkaloids( aconitine) was declined while monoester-type alkaloid(benzoylaconine, benzoylhypacoitine, benzoylmesaconie)content was increased; this suggests that the A.soongaricum attenuating mechanism is the diester-type alkaloids from raw product was hydrolyzed into low-toxic monoester-type alkaloid;(2) The result of acute toxicity experiment has preliminary illustrated that the toxicity of A.soongaricum has attenuated after processing; the processed product possesses pharmacological effects of analgesic and anti-inflammation, therefore it can regulate immune functions in two-wayas well as curing rheumatic diseases;(3) Results of Rank correlation analysis show that the alkaloid content in A. soongaricum raw materials and processed products have a higher correlation with the anti-inflammatory efficacy index, and a certain dose-dependent relationship was shown. Water bath processed product has better performances than other 2 products in aspects of both acute toxicity and pharmacological activities. The effective dosage of water bath processed product was 3.82 g/person/day, which is assemble to clinical dosage of Aconiti kusnezoffii Radix in Chinese pharmacopoeia; diester aconitine and monoester aconitine type contents of water bath processed product meet the Aconiti kusnezoffii Radix.content limits of Chinese pharmacopoeia, hence “water bath processing” was utilized as attenuating method during A. soongaricum processing.(4) 5 monomeric compounds were extracted and evaluated, which are: aconitine(1), Songorine(2), 16, 17-dihydro-12β, 16β-epoxynapelline(3), 12-epinapellin(4) and deoxyaconitine(5). Aconitine and deoxyaconitine were aconitine-type C19 diterpenoid alkaloids, and they were the main active ingredients(analgesic and anti-inflammation) and also the main toxic ingredients in A. soongaricum. Songorine, 16, 17-dihydro-12β, 16β-epoxynapelline, 12-epinapellin are all aconitine-type C20 diterpenoid alkaloids of napellin;(2) Both 12-epinapellin and A. soongaricum aconitine have certain toxicity on cardiomyocytes, and their IC50 were 726.80μg/m L and 766.11μg/m L respectively, which were lower than acoitione(IC50 was 562.06μg/m L). Conclusions: 1. This paper has primarily investigated the phylogeny relationships between A.soongaricum and legal standard-Aconitum medicinal material, and it is clear that A. soongaricum and Aconiti kusnezoffii Radix have a closer DNA molecular and chemical phylogeny relationships. This provides scientific evidences to replace Aconiti kusnezoffii Radix with A. soongaricum and to utilize as a new medicinal resource. 2. A. Soongarium possesses pharmacological activities of Aconitum medicinal material( analgesic and anti-inflammatory effects), and the toxicity can be attenuated with water bath processing. It has special advantage in curing rheumatoid disease commonly in Xinjiang, thus it possesses good medicinal values; 3. 5 A. soongaricum alkaloid monomer compositions have been extracted and evaluated, which lays a solid foundation for establishment of legal standard of medicinal materials; 4. The subacute and long term toxicity of A. soongaricum processed product, the change pathway of alkaloid monomer during hydrolyzing and the toxicity of hydrolysates are still remained for further studies. |
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