| Pueraria radix, the root of Pueraria lobota (Willd.) Ohw, is used as an important Chinese traditional medicine for thousands of years in China. There are many ingredients in puerariae radix, including isoflavonoids, carbohydrates, starch, Ca and K etc. Among them, isoflavonoids are the most active ingredients, and puerarin was the most abundant component in tatol isoflavonoids in the extract of Pueraria radix. As for puerarin, because of its especially important medical function, it has been clinically used for treating coronary heart disease, myocardial infarction, hypertension and potential diabetes etc. With the continuous demands and extensive applications of puerarin in commercially dietary supplements, cosmetics and medicine, the study on extraction of puerarin and screening inherent antioxidants in the isoflavonoid extract and interaction modes of puerarin with DNA have become to be of prime importance. Therefore, this thesis contains the following there parts:(1) The acidified microwave-assisted extraction of puerarinUsing the Puerariae radix as the material, acidified microwave-assisted extraction combined with HPLC-UV analysis was used to extract and determine puerarin in Puerariae radix. In order to show the superiority of acidified microwave-assisted extraction, other extraction methods including marinated extraction, reflux heating, acidified ultrasonic extraction and primary microwave-assisted extraction were compared. Taking the extraction ratio of puerarin as the index, single factor test and orthogonal test were made to optimize the extraction conditions. The results showed that the primary and secondary order of factors influencing extraction efficiency were as follows:concentration of hydrochloric acid> extraction temperature> concentration of ethanol. The optimum extraction conditions through orthogonal experiment were that the concentration of HCl was 0.4 mol L-1, concentration of ethanol was 80% (v/v), extraction time was 3 min and reaction temperature was 60℃.The calibration curve for puerarin was linear with the correlation coefficients of 0.9996. The recovery of puerarin was in the range from 96.3% to 103.6%, and the RSD was 3.2%(n=3).(2) Screening of antioxidative components in the total isoflavonoidsPuerariae radix (PR) is a Chinese traditional medicine for the treatment of coronary heart disease, myocardial infarction and hypertension. Among the complex constituents in the crude extract of PR, puerarin and daidzin and daidzein, which have shown strong antioxidant activities, were screened and identified by HPLC technique coupled with MS for the first time. Antioxidant capacities of the identified compounds were evaluated by three different assays:DPPH-radical scavenging assay and Fe2+-metal chelating assay and HO-radical scavenging assay. Results showed that daidzein possessed a strong free radical scavenging activity and metal chelating activity. Puerarin exhibited a good DPPH-free redical scavenging activity, while its metal chelating capacity was relatively weak. By contrast, the antioxidant capacity of daidzin was relatively much weaker. The proposed method might have alternative applications for food preservation and health supplement.(3) Study on the interaction modes between puerarin with sperm DNAPuerarin is one of the major isoflavonoid compounds in Pueraria lobota (Wild) which is an important Chinese traditional herbal medicine. Puerarin can suppresses myocardial infarction, hypertension and potential diabetes. In order to understand the pharmacology of puerarin, in our present study, the interaction between puerarin and salmon sperm DNA was investigated by means of fluorescence spectroscopy, UV-vis spectrophotometry and synchronous fluorescence spectroscopy with the use of Neutral Red (NR) dye. The obtained data indicated that the fluorescence quenching of puerarin by DNA was static processes through formation of a nonfluorescence complex and the interaction modes between puerarin and DNA were the integration of electrostatic attraction and the intercalation binding. The binding constant (KA) for puerarin-DNA was 1.99×105 M-1 and the binding site (n) was about 1.28. It seems that one molecule of puerarin can bind DNA tightly. In addition, the linear concentration range of puerarin fluorescence and the effects of pH, incubation time on puerarin fluorescence were also discussed in detail. |
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