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       【摘要】 
       ObjectiveTo establish a spectrophotometric method for determination of chlorogenic acid in Eucommia Ulmoides leaves.Methods The complex chromogenic reaction of chlorogenic acid and Al3+ was applied for the determination with visible spectrophotometer at 530 nm after chlorogenic acid was extracted by thin-layer chromatography. ResultsThe working curve for the determination of chlorogenic acid solution was linear in the range of 1.7×10-4～1.0×10-2g·L-1. The corresponding equation of linear regression was Y=0.003 92 X－0.0067，r=0.9995. The recovery rates of the leaves were 98% and 101% in autumn and summer, respectively. ConclusionThis method is simple, practical and accurate. It meets the requirement for the daily determination in the lab and industry.
       【关键词】  Eucommia ulmoides leaves Chlorogenic acid Complex chromogenic reaction Visible spectrophotometry Thin-layer chromatography
       Eucommia ulmoides is an economic plant which is peculiar in China. Its medicinal value has long been noted. It was listed as a top grade of traditional Chinese medicine in Shen Nong Ben Cao Jing more than 2000 years ago. Eucommia has a lot of medicinal functions［1,2］ such as lowering blood pressure, restoring liver and kidney, strengthening bones and muscles, inhibiting tumor, having anti-aging activity, and so on. It has been approved that the leaves and bark of Eucommia contain similar chemical substances, of which chlorogenic acid is one of the main components contributing to the pharmacological action［3,4］ . Therefore, compared with other Chinese medicinal herbs such as honeysuckle and Eucommia bark, Eucommia leaf is a cheaper and more reasonable raw material to extract chlorogenic acid.
       
       The analytical methods to determine the concentration of chlorogenic acid include thin layer chromatography［5］, derivative polarography［6］, ultraviolet spectrophotometry, chemoiluminescence analysis, gas chromatography, high-performance liquid chromatography［7~10］, and so on. The quantitative analysis of chlorogenic acid often requires special analytical equipments which are not only expensive but also low efficient for the daily operation in the lab. Therefore, it is desirable to find a fast and accurate analytical method with high sensitivity to measure chlorogenic acid, in which only the conventional apparatus and basic operation are needed.
       
       In this work, the chromogenic complexing reaction of Al3+ and chlorogenic acid was applied to determine chlorogenic acid in Eucommia leaves by visible spectrophotometry. The principle for analysis method is that chlorogenic acid and Al3+ can form a stable mauve complex under alkaline conditions, which can show the characteristic absorption spectrum between wavelength 440 nm and 620 nm. The structures of chlorogenic acid and its complex with Al3+ are illustrated in Fig.1.
       Fg 1  The gas chromatography chromatogram of sample 1 essential oil （略）
       1  Materials and Methods
       1.1  Reagents and solutionsAll reagents were of analytical grade and were used as received. Double-distilled water was used in the present work. Standard chlorogenic acid solution 2×10-2g·L-1 was prepared by dissolving 0.0100g of chlorogenic acid in 500 ml of 95% ethanol solution.
       1.2  Measurement of the absorption spectrum of standard chlorogenic acid solutionA solution was prepared by adding standard chlorogenic acid solution into a 10 ml measuring flask, into which 0.5ml 5% NaNO2 and 0.5 ml 10% Al(NO3)3  were charged simultaneously. Then, 5 minutes later after the addition, 2 ml 1.0 mol/L NaOH was added. Finally, 95% ethanol solution was used to make the solution volume constant. Absorption spectrum of this solution was measured with a 721 visible spectrophotometer (made in Shanghai No.3 Analytical Instrument Company). Meanwhile, 95% ethanol solution was selected as background solution.
       1.3  Determination of chlorogenic acid in Eucommia leaves10.0 g Eucommia leaves and 40% ethanol solution were mixed at ratio 1:14 by weight. With vigorous stirring, the mixture was heated to refluxing at 80℃ for 150 minutes. After cooled to room temperature, the slurry was filtered. The filtration containing chlorogenic acid was collected. The remained leaves were treated by the same procedure for two more times.
       
       In thin layer chromatographic analysis, fluorescence phenomenon could be monitored by ultraviolet analytical instrument. It was observed that the blue fluorescence of chlorogenic acid in the third extracting solution was very dim, which indicated that the third extracting solution had very low concentration of chlorogenic acid. This result confirmed that our extraction procedure was extremely efficient. Then the solutions from the first and the second extraction were combined and condensed to about 200 ml. 100 μl of the condense was spread onto a 20 cm×10 cm thin-layer plate which had silica gel G at 2 cm-width ribbon shape. Then the plate was developed by the solvent mixture of ethyl acetate, methanol and water (mixed at ratio 10∶4∶3 by volume) as mobile phase. A spot of standard chlorogenic acid solution was used as development control. When a clear chlorogenic acid fluorescent band was observed under Ultra-Violet light, the plate was moved out, and air dried. The silica gel which lay in the absorption band was scraped off and put it in 50ml conical flask, and then it was washed with 10ml 80% ethanol solution for 30 minutes. After filtration, the filtrate was diluted to 20ml by 80 % ethanol. This solution was ready to be used in chlorogenic acid measurement.
       
       1.0，2.0，3.0，4.0，5.0 ml of the washing solution was measured and added to 10ml of the constant volume by water, respectively. Absorption of these samples was measured by the method mentioned in 1.2, and then content of chlorogenic acid in Eucommia leaves could be calculated according to the relationship between absorbance and concentration.
       2  Experimental Results
       2.1  Absorption spectrum of coloring complex The absorbance of standard chlorogenic acid solution of certain concentration was measured according to the method mentioned in 1.2 and the absorption spectroscopy was obtained. As a result from the spectroscopy, the maximum absorption wavelength was 530 nm, so it was selected as the characteristic wavelength during the measurement.
       2.2  Working curve for the determination of chlorogenic acid solution After the above tests, the determination conditions were optimized. Under the selected conditions, the working curve for chlorogenic acid solution determination was obtained by establishing a linear correlation between chlorogenic acid solution concentration and absorbance. It could be concluded therefor that the correlation was linear in the range of 1.7×10-4～1.0×10-2g·L-1 for chlorogenic acid solution with the regression equation of A = 0.00392 C－0.0067 (r=0.999 5), and the detection limit was calculated as 0.19 mg·L-1.
       2.3  The accuracy and recovery rate10 g Eucommia leaves in summer and 10 g Eucommia leaves in autumn were measured respectively according to the method mentioned in 1.3 to determine the content of chlorogenic acid, then relative standard deviation (RSD) was also calculated.
       
       5.0 ml condensed extracting solution and the standard chlorogenic acid solution with the expected volume were mixed and then added to 10 ml with water, 100 μl of the mixed solution was taken out and measured according to the procedure mentioned in 1.3. Accordingly the content of chlorogenic acid in the extracting solution after the standard chlorogenic acid solution was added could be calculated. And the recovery rate（R%）was calculated according to the following formula. The overall results were shown in Tab.1.
       Tab.1  The concentration of chlorogenic acid in practical samples（略）
       (where R% is recovery rate, CA,0 represents the content of chlorogenic acid in extracting solution after standard solution was added, CA and C0 represent the content of chlorogenic acid in extracting solution and the content of chlorogenic acid in added standard solution, respectively. RSD% represents the relative standard deviation.)
       3  Discussions
       3.1  Selection of developing solvent in thin layer chromatographyAccording to the comparison among developing solvents in thin layer chromatography of different component and different volume proportion, we found that ethyl acetate- methyl alcohol- water mixed with a volume ratio of 10:4:3 had the best separation efficiency, it could separate chlorogenic acid from the interferences completely with  the retention factor (Rf value) being 0.65.
       3.2  Selection of chromogenic solventDistilled water and 95% ethanol were chosen as the chromogenic complexing reaction solvent respectively. The result showed that there was not significant difference between the initial absorbances of the two coloring solutions under the same determination condition, but the absorbance of  95% ethanol solution could stay longer, implying that ethanol could make the coloring complex more stable. In our experiment, the color stability of ethanol solution can last up to 1 hour, which could completely meet the analysis requirement.
       3.3  Selection of the dosage of chromogenic agentChlorogenic acid could be completely turned into stable mauve complex in which center ion was Al3+ on the condition that the dosage of 5% NaNO2 and 10% Al(NO3)3 added into the solution were both 5ml, and that the concentration of chlorogenic acid is within 1.0×10-2g·L-1. Here NaNO2 was used as reducing agent to avoid the oxidation of chlorogenic acid, and Al(NO3)3 was used as chromogenic agent. Its optimal dosage of chromogenic agent was 5 ml. The absorbance tended to be lower when the dosage was less, whereas absorbance did not change obviously as chromogenic agent was more than 5 ml.
       3.4  Selection of chromogenic reaction timeThe time of complex attaining stable was 5，3，1 min at the temperature of 25℃，35℃，50℃, respectively. It could be seen that the chromogenic reaction time decreased obviously as the temperature increased. Because the chromogenic reaction could complete in shorter time at the indoor temperature, 5 minutes was chosen as the optimized chromogenic reaction time.
       4  Conclusion
          
       Experimental results confirmed that the maximum absorption wavelength of the coloring complex was 530 nm, the regression equation of the standard curve was: A= 0.00392 C－0.0067 (r=0.999 5), and the correlation is linear in the range of 1.7×10-4～1.0×10-2g·L-1 for chlorogenic acid solution, the detection limit was calculated as 0.19 mg·L-1. Chlorogenic acid can form stable mauve complex with Al3+ under alkaline conditions, and produce characteristic absorption spectrum in the meantime. The determination of chlorogenic acid in Eucommia leaves with a 721 visible spectrophotometer was simple, practical, accurate and with high sensitivity. This method can meet the requirement for the daily analysis and measurement in the lab and industry.
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