Study On The Chemical Composition And Preliminary Quality Standard Of Anchusa Italica Retz

Objective: To study the chemical composition and set up the preliminary quality standard of Anchusa italica Retz.(AIR) which may be help to describe the the substance basis of the pharmacological activity and provide a scientific basis for further development and utilization. Methods: 1) The system pre-test of chemical composition of the petroleum ether extracts and ethanol extracts, acid ethanol extracts and water extracts from AIR upper part was carried out. Different polar extracts of 60% ethanol extract of AIR were prepared, which include the petroleum ether extracts, ethyl acetate extracts, n-butanol extracts. Silica gel column chromatography, reversed phase silica gel column chromatography and other modern separation technology were used to separate the monomer compound from ethyl acetate extracts. Structural elucidation of compounds was carried out according to the physical and chemical properties and spectroscopic data. 2)Silica gel G plate was used to indentify daucosterol in AIR, developing agent was the mixture of ether, ethyl acetat, acetone and formic acid(5:3:1:1), 10% sulfuric acid in ethanol was selected as chromogenic agent, the silica gel G plate was detected at 366 nm. Silica gel G plate was used to indentify nigaichigoside in AIR, developing agent was the mixture of trichloromethane, methanol and water(18:8:1),and 10% sulfuric acid in ethanol was selected as chromogenic agent, the silica gel G plate was detected at natural light. According to Chinese Pharmacopoeia, the mass of total ash, acid insoluble ash, water extracts, alcohol extracts, arsenic salt contents and heavy metals contents of 10 batches AIR upper parts were checked. Reversed-phase high performance liquid chromatography(RP-HPLC) was used to establish the methods of content determination of rutin in AIR. The RP-HPLC analysis was carried out on Kromasil C18 column(250×4.6mm, 5μm), gradient elution was performed by mobile phase of acetonitrile-0.2% phosphoric acid solution, the flow rate was 1.0ml/min and the detection wavelength was set at 354 nm, the column temperature was kept at 30℃, sample volume is 20μl. Results: 1) The pre-test results of chemical composition suggest that AIR may contain steroids, triterpenes, volatile oil, phenols, tannins, flavonoids, coumarins and lactones, alkaloids, carbohydrates, amino acids, protein, saponin compounds. 7 compounds were isolated and identified, they were β-sitosterol acetate(Ⅰ), β-sitosterol(Ⅱ), nigaichigoside(Ⅲ), rutin(Ⅳ), kaempferol(Ⅴ), caffeic acid(Ⅵ) and daucosterol(Ⅶ). 2) The method for the identification of daucosterol and nigaichigoside in AIR was established, which effectively carry out the TLC identification of AIR. The water content is not more than 11.00% of the dry weight of AIR, the total ash content is not more than 14.00% of the dry weight of AIR, acid insoluble ash content is not more than 3.00% of the dry weight of AIR, water extract content content is not less than 13.00% of the dry weight of AIR, alcohol extract content content is not less than 5.00% of the dry weight of AIR, Heavy metal content is not more than 10 ppm, arsenic content is not more than 2 ppm. There was a linear relationship between concentration of rutin and peak area in the range of 1.64 ~52.55 μg·ml-1, the average recovery of different concentration was 98.01%, 98.87% and 99.45% respectively. The RSD of stability tests and repeatability tests were less than 2.0%. Rutin content range of 10 batches AIR is 1.2997~1.3511 mg·g-1. Conclusion: 7 compounds are isolated from AIR for the first time. The qualitative, inspection and quantitative analysis methods, which are in line with the requirements of methodology, can be used to effectively control the quality of AIR. The results of the study lay the foundation of improvement of the quality standard of AIR.