Studies On Flavoids And Essential Oils And Biological Activities Of Flavoids And Essential Oils In PCR

Pericarpium Citri Reticulatae (PCR) is the dried ripe peel of Citrus reticulata Blanco and its cultivars, which has been acknowledged by the People's Republic of China pharmacopoeia, and has been used in traditional Chinese herbal medicines for a long time. Citrus reticulata 'Chachi', mainly produced in Xinhui county of Guangdong Province in China, is regarded as a genuine PCR. It has been applied in foods and drugs due to the effectiveness of regulating qi (energy), normalizing the function of spleen and stomach, resolving phlegm, and so on. This study was established in the systemic discussion of flavonoids and essential oil from PCR 'Chachi'. The flavonoids were isolated and identified, and its content as well as the antioxidative activity from different harvested time and different storage time of PCR 'Chachi' and different cultivars of citrus peels were analyzed. The antioxidant activity of flavonoid monomers, antioxidative synergy between them were analyzed, and further research work was carried out on the antioxidant synergy when organic acids and amino acids were added to PCR 'Chachi' flavonoids model, repectively. In addtion, the chemical compositions, antioxidant and antimicrobial activity of the essential oil from different storage time of PCR 'Chachi' were studied. These researches would give references for the validity of parlance, "the older the better" and "genuineness".The major results are as follows:1. Isolation and identification of flavonoids in PCR 'Chachi'. A scavenging DPPH free radical method was used to evaluate the antioxidant activity of different polarity extract and the content of total flavonoids in PCR 'Chachi', and the results showed that the part of EtOAc extract was the best. HPLC-DAD-ESI/MS was used to determmine the flavonoids compounds in the part of EtOAc extract. Eight flavonoids were separated and identified which were vicenin-2, hesperidin,3',4',5,7,8-Pentamethoxyflavone, 3',4',5,6,7-Pentamethoxyflavone, nobiletin,4',5,7,8-tetramethoxyflavone, heptamethoxyflavone and tangeretin. In addition, six compounds were obtained from the part of EtOAc using conventional extraction and separation. Then, these flavonoids were identified by physicochemical character and spectral (1H-NMR,13C-NMR and MS) methods. The compounds were hesperidin, nobiletin, tangeretin,5-O-desmethyl nobiletin, 4',5,7,8-tetramethoxyflavone and 3',4',5,7,8-Pentamethoxyflavone. 2. Analysis of flavonoids at different storage time, different harvested time of PCR 'Chachi' and 11 varieties of citrus peels. The isolated flavonoids were selected as chemical standards and quantitative analysis by HPLC was established. Then, the method was applied to determine the content of flavonoids at different storage time, different harvested time PCR 'Chachi' and 11 varieties of citrus peels. The results showed that the total content of the flavonoids decreased with the duration of storage time, and the content of the flavonoids and hesperidin significantly decreased (p<0.05) with harvested time. The flavonoids in PCR 'Chachi' and 11 varieties of citrus peels were quite different.3. Analysis of antioxidant activity at different storage time, different harvested time of PCR 'Chachi' and 11 varieties of citrus peels. The antioxidant activity were analyzed by DPPH, FRAP and ABTS, and the relationship between flavonoids and antioxidant activity was studied. The results showed that the antioxidant activity of PCR 'Chachi' decreased with the duration of storage time, there were strong correlation between the total content of the flavonoids and the antioxidant activity tested by DPPH and ABTS methods. The antioxidant activity decreased with harvested time, and there were strong correlation between the total content of the flavonoids and the antioxidant activity tested by DPPH,ABTS and FRAP methods. The antioxidant activity in PCR 'Chachi' and 11 varieties of citrus peels were quite different, in which Nanfengju showed the highest antioxidant activity. The correlation coefficient (r) between the antioxidant activity and the total flavonoid contents of 11 varieties of citrus peels were weak. The DPPH radical-scavenging activity and the total flavonoid contents showed a correlation (r= 0.5044).4. Analysis of the antioxidant activity of flavonoid monomers, antioxidative synergy between them, and further research work were carried out on the antioxidative synergy when organic acids and amino acids were added to PCR 'Chachi' flavonoids model, repectively. The result showed that hesperidin had the highest antioxidant activity, followed by 5-O-desmethyl nobiletin. For ABTS, these interactions tended to have an antagonistic effect except 4',5,7,8-tetramethoxyflavone and 5-O-desmethyl nobiletin. Noticeable decrease (p<0.05) in antioxidant activity was found when nobiletin mixed with another flavonoid. For FRAP, these interactions showed significant synergy (p<0.05) and hesperidin paired with other flavonoids was the best. The addition of organic acids and amino acids to PCR 'Chachi' flavonoids model mainly were antagonistic effects while His and Arg showed synergistic effects.5. Analysis of PCR'Chachi'essential oil at different storage times. Yields rates of the essential oil of PCRs by Clevenger-type apparatus and modified Clevenger-type apparatus was compared, and modified Clevenger-type apparatus was better (p<0.05). The volatile components of PCR 'Chachi' essential oil were determined by GC-MS. The results showed that 53 volatile components were detected, of these compounds, including 26 terpenes,10 alcohols,10 aldehydes,3 esters,2 ketones and 1 phenol. D-limonene was determined as the major component accounting for 68.2-76.1% in PCR essential oil. In addition, (3-myrcene, y-terpinene, a-pinene, terpinolene andβ-pinene were analyzed to be relatively high. There were 53,48,46,47 and 45 volatile compounds identified in Chachi 2008, Chachi 2004, Chachi 2001, Chachi 1998 and Chachi 1994, respectively. The volatile compounds of essential oil reduced with the duration of storage time, and the content of D-limonene andβ-myrcene decreased while the content of a-pinene andβ-pinene increased.6. Analysis of antioxidant and antimicrobial activity of PCR 'Chachi' essential oil at different storage times. The antioxidant activity of PCR 'Chachi' essential oil was carried out by DPPH, ABTS and FRAP methods, and the antimicrobial activity was analyzed by agar disc diffusion and MIC. The results showed that the antioxidant capacity of PCR 'Chachi' essential oil varied considerably with the storage time while the reducing power by FRAP increased. Among the 11 tested microbes, the essential oil possessed effective antimicrobial activity against Gram-positive bacteria and fungi. There were no obvious difference detected in the antimicrobial activity of PCR 'Chachi' essential oil at different storage times.