摘要
陈皮(Pericarpium Citri Reticultae)是芸香科植物橘及其栽培变种的干燥果皮,作为我国传统药材收载于《中国药典》。主产于广东新会的茶枝柑是陈皮道地药材,因其具有理气健脾、燥湿化痰等功效而广泛应用于食品和药品中。本文立足于对广陈皮中的黄酮类化合物和挥发油两大类活性成分进行系统探讨,对广陈皮中黄酮类化合物进行分离纯化和结构鉴定,对比分析不同贮藏年份、不同采收期的广陈皮和不同柑橘皮品种中黄酮类化合物组分和含量差异及其抗氧化活性差异,并研究6种黄酮单体的抗氧化活性、黄酮单体两两组合及有机酸、氨基酸分别与广陈皮黄酮模拟体系之间的抗氧化协同作用。同时,对不同贮藏年份广陈皮挥发油的成分进行分析鉴定,并比较各挥发油的抗氧化活性和抑菌活性差异。为“陈久者良”和“道地性”提供理论支持。
主要研究结果如下：
1.广陈皮中黄酮类化合物的分离纯化和结构鉴定。比较了广陈皮不同极性部位总黄酮含量及DPPH自由基清除效果,筛选出乙酸乙酯部总黄酮含量最高且活性最强。采用液相色谱-质谱联用技术对广陈皮乙酸乙酯部含有的黄酮类化合物进行分析,初步鉴定出其中的8种黄酮类化合物,分别为：葡萄糖基芹菜素、橙皮苷、3',4',5,7,8-五甲氧基黄酮、3',4',5,6,7-五甲氧基黄酮、川陈皮素、4',5,7,8-四甲氧基黄酮、3,5,6,7,8,3',4'-七甲氧基黄酮和橘皮素。同时,采用传统分离纯化方法对乙酸乙酯部进行分离,得到6个化合物,经理化鉴定和光谱解析(1H-NMR、13C-NMR、MS)鉴定其均为黄酮类化合物,分别为：橙皮苷、川陈皮素、橘皮素、5-羟基-6,7,8,3',4'-五甲氧基黄酮、4',5,7,8-四甲氧基黄酮和3',4',5,7,8-五甲氧基黄酮。
2.不同贮藏年份、不同采收期广陈皮和不同品种柑橘皮黄酮含量的比较。以分离出的6种黄酮类化合物为标准品,建立HPLC定量分析广陈皮黄酮类化合物方法。分别分析了不同贮藏年份广陈皮、不同采收期广陈皮和不同品种柑橘皮中主要黄酮类化合物含量。结果表明,随贮藏时间的增加,广陈皮中各主要黄酮类化合物总量呈减少趋势；主要黄酮类化合物总含量和橙皮苷含量随采收期推迟而显著减少(p<0.05)；不同品种柑橘皮主要黄酮类化合物含量差异较大。
3.不同贮藏年份、不同采收期广陈皮和不同品种柑橘皮抗氧化活性的比较。采用DPPH法、FRAP法和ABTS法分别分析比较了不同贮藏年份、不同采收期广陈皮以及不同品种柑橘皮的抗氧化活性,并探讨黄酮类化合物含量与抗氧化性之间的相关性。结果表明,广陈皮抗氧化能力随贮藏时间增加总体呈下降趋势,不同贮藏年份广陈皮的总黄酮含量与DPPH自由基清除能力和ABTS自由基清除能力呈正相关,而与FRAP铁还原能力相关性不明显。不同采收期广陈皮抗氧化能力为：青皮>微红皮>大红皮。不同采收期广陈皮的总黄酮含量与DPPH自由基清除能力、ABTS自由基清除能力和FRAP铁还原能力呈正相关性。不同品种柑橘皮抗氧化能力存在一定的差异,南丰桔的抗氧化能力最强。对不同品种柑橘皮来说,其总黄酮含量和DPPH自由基清除能力、ABTS自由基清除能力和FRAP铁还原能力之间的相关性都不强,其中,总黄酮含量和DPPH自由基清除能力之间的相关系数最大,为0.5044。
4.广陈皮黄酮类化合物体外抗氧化活性协同效应。采用ABTS法和FRAP法测定了广陈皮中6种黄酮化合物单体的抗氧化活性,分析了单体化合物两两之间、5种有机酸和20种氨基酸分别与广陈皮黄酮模拟体系之间的抗氧化协同效应。结果表明,6种黄酮类化合物中,橙皮苷的抗氧化活性最强,其次是5-羟基-6,7,8,3’,4’-五甲氧基黄酮。单体化合物两两组合时,ABTS值除4’,5,7,8-四甲氧基黄酮和5-羟基-6,7,8,3’,4’-五甲氧基黄酮两两混合表现为协同效应外,其它4种单体化合物均表现为拮抗效应,当川陈皮素与其他化合物混合时拮抗效应较理论值显著增强(p<0.05)；FRAP值各单体均表现出显著的协同效应(p<0.05),其中橙皮苷的协同效应最强。各有机酸和氨基酸在广陈皮黄酮类化合物模拟体系中主要表现为拮抗效应,仅组氨酸和精氨酸显示出协同效应。
5.广陈皮挥发油成分分析比较。比较Clevenger法和改进的Clevenger法提取广陈皮挥发油的出油率,结果表明,采用改进的Clevenger法可显著提高广陈皮挥发油出油率(p<0.05)。采用GC-MS分析不同贮藏年份广陈皮挥发油的成分及含量变化,结果表明,不同贮藏年份广陈皮挥发油中共检测出53种成分,萜烯烃类化合物26种、醇类化合物10种、醛类化合物有10种、酯类化合物3种、酮类化合物2种和酚类化合物1种。萜烯烃类是挥发油的主要成分,D-柠檬烯、p-月桂烯、γ-松油烯、α-蒎烯、β-蒎烯和异松油烯是萜烯烃类中最主要的成分。D-柠檬烯的含量最高,占挥发油中挥发性物质总量的68.2%-76.1%。
从Chachi 2008, Chachi 2004, Chachi 2001, Chachi 1998, Chachi 1994挥发油中分别检测出53,48,46,47和45种挥发性物质。随着贮藏年份的增加,挥发性物质减少。其中,D-柠檬烯和p-月桂烯含量逐渐降低,而α-蒎烯和β-蒎烯含量逐渐增加。
6.不同贮藏年份广陈皮挥发油抗氧化活性和抑菌活性比较。采用DPPH法、ABTS法和FRAP法分析不同贮藏年份广陈皮挥发油的体外抗氧化活性,采用滤纸片平板扩散法和测定最小抑菌浓度来确定挥发油的抑菌能力。结果显示,不同贮藏年份广陈皮挥发油抗氧化活性变化较大,仅FRAP值是随着贮藏时间的增加而增加的。广陈皮挥发油对11种供试菌种有不同程度的抑制作用,对革兰氏阳性菌及真菌的抑制效果更好。不同贮藏年份广陈皮挥发油的抑菌效果差异不大。
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.
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