Study On Large-scale Preparation Of High Purity Chlorogenic Acid From Helianthus Tuberosus L. Leaves

Chlorogenic acid (3-O-ceffeoylquinic acid,3-CQA), a kind of important memberof phenolic family, has received considerable attention recently because of its widespectrum of pharmacological properties including antioxygenation, anticancer,anti-inflammatory, hypoglycaemic and hepatoprotective effect.3-CQA has beenwidely applied in healthcare industry, food industry and cosmetics industry. In thispaper, we have proved that there is high content of chlorogenic acid in Helianthustuberosus leaves. It is a very practical topic to research the large-scale preparation ofhigh purity3-CQA from Helianthus tuberosus leaves.In this paper, a simple, scientific, stable and reliable technology route ofextraction, separation and purification of3-CQA from H. tuberosus leaves wasdesigned rigorously and rationally. And the scale-up experiment was also conducted.The main results were shown as follows:(1) The quantitative analysis method of3-CQA was established by using highperformance liquid chromatography (HPLC). The detecting conditions were listed asfollows: column: Hypersil BDS C-18(4.6×250mm，5μm); flow rate:1mL/min;Detection wavelength:327nm; injection volume:10μL; column temperature:30℃;mobile phase:0.1%trifluoroacetic acid in redistilled water (A) and acetonitrile (B).Methodology validations were performed for this method. The results demonstratedthat the HPLC method was a fast and reliable method for quantitative analysis of3-CQA in H. tuberosus leaves.(2) The distribution of3-CQA in the different parts of H. tuberosus and theinfluence of varieties, salinity, collecting time on3-CQA content in H. tuberosusleaves were investigated. The results showed that the content of3-CQA in H.tuberosus leaves was the highest. Collecting time existed great influence on thecontent of3-CQA in H. tuberosus leaves, and October could be chosen as the mostsuitable time to collect H. tuberosus leaves. Different cultivars of H. tuberosusshowed different salinity tolerance. NanYu might be the more suitable cultivar thatadapted well to salt-alkaline soils type.（3）The extraction process of3-CQA from H. tuberosus leaves was optimizedby the single factor experiments and the orthogonal design. The optimal conditionswere:60%methanol as the extraction solvent, material-liquid ratio of1:25g/mL,extraction temperature60℃, repeated twice and0.5h for each cycle. The technological process was simple, steady and effective. Meanwhile the yield of3-CQA was as high as94%.(4) ADS-21was selected as a suitable resin for separating3-CQA. Theadsorption data of ADS-21at25℃fitted best to the Langmuir isotherm model andpseudo-second-order kinetic model. Some process parameters including concentration,pH value, volume of sample solution and concentration, volume of ethanol wereoptimized. The optimal enrichment and separation conditions for3-CQA withADS-21resin were established as follows:①For adsorption: concentration of3-CQA in sample solution1.6mg/mL; pH2; feed volume7BV; flow rate2BV/h;temperature25℃.②For desorption:60%ethanol3BV, then70%ethanol9BV;flow rate2BV/h. After one run treatment with ADS-21resin, the content of3-CQA inthe product was increased5.42-fold from12.0%to65.2%, with a recovery yield of94%.（5）Active carbon method was applied for the decoloration of the3-CQAcoarseproduct. The best technical conditions were summarized as follows: active carbon wasadded into the coarse product sample solution with the material-liquid ratio of1:20(g:mL), and the adsorption temperature and time were50℃and30min,respectively. Under this condition, the best decolorizing property was obtained withthe least loss of3-CQA.(6) The further refining process of3-CQA crude product with polyamide columnchromatography was investigated. The best technical conditions were summarized asfollows: firstly, the3-CQA crude sample solution was concentrated under vacuum.Then, the adsorption process was conducted by loading6BV sample solution into thepretreated polyamide column. After reaching adsorptive equilibration, theadsorbate-laden column was eluted with6BV60%ethanol-water solution. As a result,the desorption ratio and purity of3-CQA in the product were95%and96,respectively.(7) The scale-up tests of the extraction, separation and purification process of3-CQA from H. tuberosus leaves were performed. The results showed that H.tuberosus leaves could be used as a new resource to extract3-CQA and the processdeveloped in this work was feasible and helpful for industrial utilization.