Studies On Extraction And Separation Of Antioxidant Constituents From Areca Nut

In order to study the antioxidant activity compounds of areca nut, Hai nan areca nut was taken as the research object, using different extraction methods with in vitro antioxidant activity, the antioxidant activity compounds were researched about the separation activity and structure by HPLC and NMR analysis. The definite work was as follows:1. Compare the content and activity of antioxidant compounds in the different parts of areca nut.The antioxidant activities of areca (Areca catectu L.) flower, husk and seed extracts were evaluated using three complementary in vitro assays:inhibition of DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals, inhibition of hydroxyl radicals and reducing power system. The EC50 values were calculated for all the methods in order to evaluate the antioxidant efficiency of areca extracts. The polyphenol and flavonoid contents were also analyzed. The seed of areca nut has the best antioxidant properties, presenting much lower EC50 values for DPPH radicals scavenging activity, hydroxyl radicals scavenging activity and reducing power(0.409mg/mL,3.575mg/mL and 0.188mg/mL, respectively). Furthermore, the highest polyphenols and flavonoids contents were found from areca seed extracts.2. Choose the best extraction solvent for the antioxidant compounds in the areca nut seed.The areca nut seed was chosed to be the research objects, ethyl acetate,70% ethanol and water were used as extraction solvents, three extracts were got from areca nut seed. Antioxidant activities of different extracts were evaluated by three different methods, such as DPPH radical scavenging assay, potassium ferricyanide reduction method and ABTS radical scavenging assay. The results showed that the reducing power, DPPH and ABTS radicals scavenging activity of 70% ethanol extract were stronger than those of water extract and ethyl acetate extract. The antioxidant activity of each extract which was correlated with the composition and contents of phenolic compounds, became stronger with the increasing of concentration.3. Optimize the extraction process of total phonel in the areca nut seed by reflux extraction.Optimization of the technology for extracting total phenol by reflux extraction from areca nut seed was carried out. On the basis of one factor tests, the method of response surface analysis with 3 factors of extracting temperature, time and solvent-material ratio on the content of total phenol was adopted. The optimal extracting conditions were as follows:extraction temperature,58℃; extraction time, 4h; solvent-material ratio,47(mL/g). The predicted value and measured value of total phenol was 148.09mg/g and 146.63mg/g, respectively. The results indicate that the obtained mode developed by response surface methodology is credible and could be used for practical prediction. The experiments of antioxidant activity showed that the areca nut seed extract exhibited strong antioxidant activities by the DPPH and ABTS radical scavenging assay, the EC50were 145.62μg/mL and 139.38ug/mL, respectively. The same result was obtained in the reducing power system.4. Optimize the extraction process of total phonel in the areca nut seed by ultrasonic extraction.Using ultrasonic extraction and on the basis of one factor tests, the method of response surface analysis with 3 factors of extracting temperature, time and solvent-material ratio on the content of total phenol was adopted. The optimal extracting conditions were as follows:extraction temperature,58℃; extraction time, 42min; solvent-material ratio,53(mL/g). The predicted value and measured value of total phenol was 164.74mg/g and 160.95mg/g, respectively. The experiments of antioxidant activity showed that the areca nut seed extract showed strong antioxidant activities by the DPPH and ABTS radical scavenging assay, the activities were becoming stronger with the increasing of concentration. Compared with the antioxidant activities of reflux extraction and ultrasonic extraction, we can found that the extract which was extracted by ultrasonic extraction was stronger than reflux extraction.5. The separation and structure analysis of ethanol extract from areca nut seed.The extract which was extracted by ultrasonic extraction was separated by different solvents, and the ethyl acetate part showed the strongest antioxidant activity by ABTS radical scavenging assay. Then the ethyl acetate part was separated by chromatography, five constituents(Fr-1-Fr-5) were obtained and Fr-4 showed the strongest antioxidant activity. Through the TLC and HPLC analysis, we found that Fr-4 was a highly purified compound. The structure of Fr-4 was determined by NMR analysis, with the reference we confirmed the compound of Fr-4 was Epicatechin.Innovation:This thesis researched the distribution of antioxidant compounds in the different parts of areca nut, and the antioxidant activity of extracts has the positive correlation to the content of total phenol. Response surface analysis was used to optimize the extraction process of total phenol from the areca nut seed for the first time, and epicatechin was determined as the strongest antioxidant activity compound in the areca nut at last.This work was supported in part by grants from National Key Technologies R&D Program of P. R. China (2007BAD76B03).