Studies On Quality Assessment Of Coreopsis Tinctoria Flos

The flower of Coreopsis tinctoria Nutt. has some effects on lowering blood pressure, regulation of blood glucose and blood lipids. But the lack of quality evaluation method and quality standards restrict the development and application of Coreopsis Tinctoria Flos. This paper provides theoretical foundation for the research of quality standards for Coreopsis Tinctoria Flos through pharmacognostic indentification, determination of active ingredients and establishment of HPLC fingerprint analysis method. The following four topics were discussed in the thesis. Methods:1. Pharmacognostic study: Collected 20 batches of Coreopsis Tinctoria Flos for macroseopic and microscopic indentification. Chlorogenic acid and luteolin as the index components in Coreopsis Tinctoria Flos were indentified qualitatively by thin layer chromatography:(1)Polyamid membrane chromatography was adopted for indentification of chlorogenic acid with 36% acetic acid as developing system and examined under 365nm; and(2)Slica gel G plates was adopted for indentification of luteolin with Cyclohexane: ethyl acetate: formic acid(3:5:0.5)as developing system, aluminium chloride solution as chromogenic reagent, examining under 365 nm.2. Determination of total flavonoids: To optimize ultrasound extraction process of total flavonoids from Coreopsis Tinctoria Flos. With the extract of total flavonoids as index, effects of ethanol concentration, solid-liquid ratio, ultrasonic time and temperature on extraction process were investigated by single factor tests and Box-Behnken response surface methodology. Total flavonoid contents of their extract were determined.3. Determinnation of chlorogenic acid and luteolin by HPLC: Agilent TC-C18(4.6mm×250mm, 5 μm)were adopted, with acetonitrile-0.1% phosphoric as the mobie phase at the flow rate of 1.0 m L/min. The colum temperature was set at 30°C, and the UV detection wavelength were 328 and 350 nm. The contents of chlorogenic acid and luteolin in 20 batchs of Coreopsis Tinctoria Flos were determined simultaneously by HPLC.4. Analysis of HPLC fingerprint of Coreopsis Tinctoria Flos: The fingerprint of Coreopsis Tinctoria Flos was established using HPLC. The fingerprint common pattern and similarity calculation were established using traditional Chinese medicine chromatographic fingerprint evaluation system(version A). The PLS-DA model was establish using SIMCA-P 11.0 software to evaluate the differences in the quality of Coreopsis Tinctoria Flos from Xinjiang and Yunnan areas. Results:1. Medicinal parts of Coreopsis tinctoria Nutt. is capitulum. Iigulate flower round with 8 yellow petals, the base or the lower part is red brown. Tubular flower is violet brown. Involucral bracts has two layer and the theinner layer is longer than outer. In the powder of the herb, infinitive stomata, pollen tube, fibre, corolla parenchyma cells, epidermal cells and secretory canal could be seen. TLC result showed that the herbs all dispayed spots with chlorogenic acid and luteolin standard. It indicated that tall the 20 samples had chlorogenic acid and luteolin.2. Optimum ultrasound extraction process was as following: ultrasonicly extracted 34 min with 40 times of 65% ethanol to the herb at 62°C. The content of total flavonoids from different areas was determined to be 177.06 and 254.85 mg/g.3. The results showed that the calibration curve for chlorogenic acid was linear in the range of 9.9~99.0 μg/m L with regression of: y=13402x-652.38(R2=0.9998), the average recovery was 100.66%(n=6), and RSD was 1.79%; The calibration curve for luteolin was linear in the range of 0.97 ~ 19.40μg/m L with regression of: y=27343x+4401.2(R2=0.9999), the average recovery was 98.89%(n=6)and RSD was 1.71%. The proposed method is sensitive, accurate and reproducible, it can be used to determine chlorogenic acid and luteolin in Coreopsis Tinctoria Flos. The contents of chlorogenic acid and luteolin among producing areas. The content of the componets were in the range of 0.93~3.91mg/g, 0.61~1.32mg/g, respectively.4. The fingerprint consisted of 17 common peaks. Two components were identified as chlorogenic acid and luteolin. Through further similarity analysis, the similarity calculation results, ranged from 0.942~0.992, showed high similarity in the 20 batches of samples. By combining the method Score plot of PLS-DA we found that the 20 samples of Coreopsis Tinctoria Flos could be clearly classified into two groups as Xinjiang and Yunnan species, their qulity are obvious different. The PLS-DA variable important for the projection plot and loading plot indicated that 11 markers were the most important contribution to classification. It shows that 11 markers can be effective as the indicators for quliaty control of Coreopsis Tinctoria Flos.