| Kudingchahas, one kind of herbal tea, is rich in alkaloid, flavonoids, and amino acids, resulting in many beneficial functions such as cardiovascular, antioxidant, antiobesity, antidiabetic, anti-inflammatory, neuroprotective and hepatoprotective effects. In China, the most common kudingcha categories consumed are from 2 genera in two families, i.e., the genus Ilex in the family Aquifoliaceae and the genus Ligustrum in the family Oleaceae. In genus Ilex, the leaves of Ilex kudingcha and Ilex latifolia have often been used as the materials for production of kudingcha. Recently, there are some reports about its chemical compositions and pharmaceutical functions. However, much attention has been paid on the biological functions, mechanism and some bioactive compounds such as flavonoids, triterpenes and triterpenoid saponins in crude extracts. Therefore, the first purpose of the present work was to study the determination, extraction, purification, identification and antioxidant activity of polyphenols in kudingcha made from Ilex kudingcha C.J. Tseng or Ilex latifolia. The second one was to investigate the extraction, purification, preliminary characterization and antioxidant activity of polysaccharides of kudingcha made from I. latifolia.1. Determination of the total polyphenol content and antioxidant activity of I. kudingchaA quantitative method for the determination of the total content of polyphenols (TPC) in kudincha was studied by Folin-Ciocalteu colorimetry (FC method) with chlorogenic acid as standard. The results showed that the TPC of kudincha could be well calculated according to their colorimetric absorption at 747 nm by applying Folin-Ciocalteu reagent 3.0 ml and saturated Na2CO34.5 ml at 30℃for 30 min, and the linear range of standard curve was 20.0～100.0 mg/L. Stability, precision, repetition and recovery rate were in the range of 0.887～1.416% by RSD (relative standard deviation). There are not any differences for the results obtained by FC method and ferrous tartrate colourimetry.The hot water extract (crude extract) of kudingcha made from I. kudincha C.J. Tseng and its four fractions of chloroform, ethyl acetate, n-butanol, and water were prepared. The TPC and the antioxidant activities of the extracts were determined by Folin-Ciocalteu method and DPPH assay, FRAP assay, and TEAC assay, respectively. The results showed that kudingcha extracts contained high TPC and showed potent antioxidant activity. The antioxidant activities of crude extract and its fractions decreased in the order of ethyl acetate fraction>butanol fraction> crude extract> water fraction> chloroform fraction according to the DPPH assay and FRAP assay, which were the same with the exception of the rank order of water fraction and chloroform fraction obtained from TEAC assay. Satisfactory correlations between TPC and antioxidant activity and between measuring methods of antioxidant activity were observed. It suggests that the polyphenols in the extracts are largely responsible for the antioxidant activities.The contents of polyphenol and flavonoid and the antioxidant activities in vitro of kudingcha crude extracts between I. latifolia and I. kudingcha C.J. Tseng were determined. The results showed that the crude extract of kudingcha from I. kudingcha C.J. Tseng with higher TPC showed higher antioxidant activity than that of I, latifolia with higher flavonoid content. In addition, the correlations between evaluation methods for antioxidant activities were good (R>0.8478), especially that between DPPH assay and ABTS assay (R 0.9967).2. Optimization of extraction conditions for phenol antioxidants from kudingchaThe TPC of extract varied greatly with the solvents used (methanol, ethanol and water). Statistical analysis showed that significant differences were existed among ethanol, methanol and water (P<0.05) as solvent. It has demonstrated that ethanol solution was the most effective in extracting phenolic compounds from kudingcha.The effects of three factors of ethanol concentration, extraction temperature and time on the extraction of polyphenols were investigated to determine the appropriate experimental ranges for central composite design. As results, ethanol concentration 70%, extraction temperature 70℃and extraction time 60 min were chosen as the central points. Based on results of single-factor experiments, Design-Expert 7.1.3 software was then employed to determine the optimal combination of extraction variables for the extraction of polyphenolic antioxidants with the total polyphenol content and IC50 of scavenging activity on DPPH free radical as the response values. Through optimization, the optimal extraction conditions with ethanol solution as solvent were as follows:ethanol concentration 70.5%, extraction temperature 73.9℃and time 61.2 min for TPC (235.24 mg chlorogenic acid/g); ethanol concentration 70.2%, extraction temperature 70.6℃and time 60.4 min for potent scavenging activity on DPPH free radical (IC50480.87μg/mL). While for water as a solvent, the optimal conditions were extraction temperature 91.2℃and time 35.7 min for TPC (198.45 mg chlorogenic acid/g) and extraction temperature 92.2℃and time 35.7 min for potent DPPH free radical scavenging activity (744.74μg/mL). Similar parameters were obtained for extraction with higher TPC and lower IC50 of scavenging activity on DPPH free radical when using either ethanol or water as solvent. The results suggested that the same optimal conditions could be used to get kudingcha extract with higher TPC and lower IC50 value of scavenging activity on DPPH free radical with ethanol or water as solvent.In order to validate the adequacy of the model equations, ten verification experiments were carried out under various extraction conditions (five of which were the optimized conditions, while the other five were randomly chosen). The results indicated that the experimental values were in good agreement with the predicted ones, and also suggested that the regression models were accurate and adequate for the extraction of polyphenol antioxidants from kudingcha.3. Isolation and structure elucidation of phenolic compounds in kudingchaThe polyphenolic substances in kudincha and green tea (Yuhua greent tea) were firstly analyzed by high-performance liquid chromatography with diode-array detector (HPLC-DAD). It was found that the polyphenolic substances in kudincha might be quite different from those in green tea made from Camellia sinensis L. In addition, the polyphenolic substances in kudincha made from I. Kudincha C.J. Tseng might be quite different quite from those in kudingcha made from Oleaceae.The main phenolic compounds in green tea were gallic acid, galloylquinic acid, EC, EGC, ECG and EGCG through the analysis of HPLC-DAD and HPLC-mass spectroscopy combined with verification experiments of standard compounds in tea. In similar manner, caffeoylquinic acid (CQA) derivatives such as 3-CQA,4-CQA,5-CQA (chlorogenic acid), 3,4-diCQA,3,5-diCQA, and 4,5-diCQA were suggested for the main phenolic compounds of kudingcha made from I. Kudincha C.J. Tseng, while ligupurpuroside A, ligurobustoside N, ligupurpuroside B, ligupurpuroside B and osmanthuside B6 for kudingcha made from Oleaceae.Five compounds were isolated from the extract of kudingcha made from I. Kudincha C.J. Tseng by semi-preparative HPLC. They were characterized as hydroxytyrosol glucoside,5-CQA,3,4-diCQA,3,5-diCQA, and 4,5-diCQA, respectively, by HPLC-DAD, HPLC-MS and NMR.4. Extraction, purification and antioxidant activity of polysaccharides from kudingcha made from I. latifoliaKudingcha made from I. latifolia was defatted with 85% ethanol, and then extracted with hot water, decolored by S-8 macroporous resin, precipitated by ethanol, and dried to afford crude polysaccharide (ILPS). The crude ILPS was purified by DEAE-52 cellulose anion-exchange chromatography, resulting in four purified polysaccharides as ILPS-1, ILPS-2, ILPS-3 and ILPS-4. Furthermore, the crude and purified ILPS were preliminary characterized by GC and FT-IR. As results, it has been demonstrated that the crude ILPS and its purified fractions were polysaccharides by FT-IR analysis. Differences in the contents of protein, uronic acid and sulfate were found for crude ILPS and its purified fractions. And rhamnose, arabinose and galactose were found the main monosaccharide compositions for crudeILPS, ILPS-1, ILPS-2 and ILPS-3. Finally, the antioxidant activities in vitro of the crude ILPS and its purified fractions were evaluated by determinations of scavenging activities on DPPH free radicals, superoxide anion radicals (O2.), hydroxyl radicals (.OH), ferric reducing-antioxidant power and chelating capacity. The results indicated that ILPS exhibited a strong antioxidant activity, and the antioxidant activity was significantly correlated with its concentration. |
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