| Objective:With the Ch.P(2015 edition) contains five kinds of fruit drugs of Rosaceae(Rubus chingii Hu, Rosa laevigata Michx, Crataegus pinnatifida Bge. Prunus mume(Sieb.)Sieb.et Zucc. and Chaenomeles speciosa(Sweet)Nakai) as experimental objects,using different solvents and different solvent extraction to explore the antioxidant activity and the antioxidant activity of different medicinal ingredients group of polarity distribution of the five kinds of fruit drugs. To establish a TLC-bioautography method which can rapidly represent the main antioxidant activities of these drugs. In order to analysis the mainly polyphenols, establishing HPLC related methods. This research provide theoretical and experimental basis of the antioxidant activities about these fruit drugs.Methods:1. Antioxidant activity evaluation system is established to select the extraction solvents,and to determine the composition of the active parts.The study comprehensive evaluation of the extraction solvent is by radical scavenging effect and oxidation reduction method. Using DPPH, ABTS, Fenton and FRAP methods to determine the Chaenomeles speciosa(Sweet)Nakai) of different extraction parts of free radicals and Fe3+ removal effect. On the basis of precvious experiments, selectiong DPPH and FRAP methods to choose extraction solvent for the other four kind of fruit drugs. In the comprehensive of experiment results, the antioxidant activity of different extacts about drugs were compared.2. F-C method was used to determined the content of polyphenol and analysis the correlation between the antioxidant activity and the content of polyphenol.The total polyphenol content was determined by F-C of different drug extracts and different extraction parts. Doing correlation analysis between different drug extracts and the antioxidant activity of these extraction parts to explore the relationship about them.3. The TLC-bioautography tese is established to observe the active components of different extraction parts of the five fruit drugs.The study uses the TLC-bioautography test to observe the polyphenol components of different extraction parts of the five fruit drugs, and to determine the main components of polyphenols in extraction parts.4. To establish HPLC method to determine the contents which have strong antioxidant activity of the ethyl acetate parts of the five fruit drugs.HPLC method was used to determine the compositon which have strong antioxidant activity in the ethyl acetate extract, and to further determine the polyphenol components.In combination with antioxidant activity, compare the commonness and characteristics of the five kinds of fruit drugs, and the relationship between different polyphenol components and antioxidant activities.Results:1. Using methanol, ethanol and acetone as extraction solvent for the study. The result showed that in the methods of DPPH, ABTS, Fenton and FRAP, methanol extracts showed significant antioxidant activity and with each sample concentration increased antioxidant capacity increase gradually; the ethyl acetate and n-butanol extractions have strong antioxidant activity than petroleum extraction of the alcohol of five drugs.2. In the correlation analysis of total polyphenols and antioxidant activity, the correlation coefficient between the methanol extract polyphenol content and DPPH method was 0.871, the correlation coefficient between the methanol extract polyphenol content and FRAP method was 0.966, and the correlation between them were highly correlated with significant correlation; the correlation coefficient between different extraction parts of polyphenol content and DPPH method was 0.507, the correlation coefficient between different extraction parts of polyphenol content and FRAP method was 0.878, both of which were moderately correlated with highly correlated. The experimental results show that the correlation of polyphenold content and antioxidant activity is good.3. According to the result of TLC-bioautography, the different polarity parts of the five fruit drugs all have radical scavenging capacity; compared with the control article, the content of polyphenols was detected. In addition to tiliroside outside, the selected control articles all have ability to scavenge DPPH free radical. The components of polyphenols is gallic acid in Rubus chingii, the catechin and protocatechuic acid in Rosa laevigata, the catechin, protocatechuic acid and chlorogenic acid in Crataegus pinnatifida, the chlorogenic acid in Prunus mume, the protocatechuic acid and chlorogenic acid in Chaenomeles speciosa.4. HPLC test results showed that the ethyl acetate parts of the five drugs had more total polyphenol content, and the polyphenols were not identical. The polyphenols were gallic acid, ellagic acid and tiliroside in Rubus chingii. The polyphenols were gallic acid,protocatechuic acid, catechin, ellagic acid and tiliroside in Rosa laevigata.Protocatechuic acid, chlorogenic acid, epicatechin, hyperin, rutin and quercetin were be tested in Crataegus pinnatifida. Protocatechuic acid, chlorogenic acid, caffeic acid,4-Hydroxybenzoic and quercetin were be tested in Prunus mume. Gallic acid,Protocatechuic acid, chlorogenic acid, 4-Hydroxybenzoic, rutin and quercetin were be tested in Chaenomeles speciosa. Among them, protocatechuic acid is common composition in four drugs(including Rosa laevigata, Crataegus pinnatifida, Prunus mume and Chaenomeles speciosa), gallic acid is common composition in three drugs(including Rubus chingii,Rosa laevigata, and Chaenomeles speciosa), chlorogenic acid and quercetin is tested in three drugs(including Crataegus pinnatifida, Prunus mume and Chaenomeles speciosa), rutin was in two drugs(including Crataegus pinnatifida and Chaenomeles speciosa), 4-Hydroxybenzoic was tested in two drugs(including Prunus mume and Chaenomeles speciosa). Caffic acid was only detected in Prunus mume, catechin was only detected in Rosa laevigata, epicatechin and hyperoside were only detected in Crataegus pinnatifida. Ellagic acid and tiliroside were detected in Rubus chingii and Rosa laevigata.Conclusion:The extracts of methanol of the five fruit drugs all showed great antioxidant activity, and the antioxidant active components mainly existed in the extract of ethyl acetate and n-butyl alcohol. The antioxidant activity and polyphenol content have good correlation in the five fruit drugs. To establish a high performance liquid chromatography(HPLC) method and the results showed that even though the polyphenol components of the ethyl acetate parts were similar, each parts had its own characteristics. |
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