Tropical citrus antioxidants and catabolism of phenolics in green tea, coffee, cocoa and orange juice

Citrus fruits are of special interest as they accumulate large amounts of flavonoids and are consumed in substantial quantities worldwide. Despite extensive research on citrus flavonoids, many compounds remain unidentified in tropical citrus species. High performance liquid chromatography with mass spectrometry and electrospray ionisation (HPLC-MS–ESI) has proved to be a powerful tool for flavonoid characterisation. This study describes how HPLC-MS-ESI and HPLC with a photodiode array detector (PDA) was used to identify and quantify flavonoids in the tropical species Citrus microcarpa, Citrus hystrix, Citrus medica var. 1 and 2, Citrus suhuiensis and Citrus medica var. sarcodactylis. Among the major compounds detected were apigenin-6-8-di-C-glucopyranoside, apigenin-8-C-glucoside-2′-rhamnoside, phloretin-3′,5′-di-C-glucopyranoside, diosmetin-7-O-neohesperidoside, hesperetin-7-O-rutinoside, diosmetin-7-O-rutinoside and hesperetin-7-O-neohesperidoside. Most of the C-glycosylated flavones and dihydrochalcone have not been found previously in tropical citrus. C. microcarpa contained a high amount of phloretin-3′,5′-di-C-glucopyranoside that was shown to possess a high Trolox Equivalent Antioxidant Ratio (TEAR) value due to its 2,4,6-trihydroxyacetophenone structure. In general, most of tropical citrus flavanones were neohesperidoside conjugates, which are responsible for the bitter taste of the fruits and juices. Tropical citrus essential oils were extracted by steam distillation (SD) and simultaneous distillation extraction (SDE) and analysed using gas chromatography-mass spectrometry (GC-MS). More than 40 compounds were identified and the major component in almost all citrus fruits and leaves was R–(+)-limonene. Citronellal was detected and was highest in C. hystrix leaf (87.8%). The radical-scavenging activity of each oil was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. Essential oils obtained from all citrus fruits showed very high radical-scavenging activity against the DPPH radical, well in excess that in leaves. The antioxidant activities of standard flavour compounds were also determined. The antioxidant activities of essential oils of selected tropical citrus fruits were attributed mainly to the presence of gamma-terpinene and terpinolene. These compounds showed high free radical-scavenging activity against DPPH radicals and were more active than vitamin E analogue. The colonic bacterial contribution to the catabolism of the flavanoids in a range of beverages was tested by comparing the urine profile of intact adults and adults with ileostomies and also using in vitro fermentation studies. Analysis of catabolic products i.e. free phenolic acids in human urine and faecal slurries following ingestion and supplementation of green tea and green tea catechins was conducted using GC-MS. Pyrogallol, pyrocatechol, 4-hydroxyphenylacetic acid, (-)5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, (-)-5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone, 3-(3-hydroxyphenyl)propionic acid and 5-(3,4-dihydroxyphenyl)-gamma-valeric acid were among catabolites produced during a 4-48 h fermentation of green tea catechins. In total, 58.4% of (-)-epigallocatechin gallate, 25.2% of (-)-epigallocatechin and 50.1% of (-)-epicatechin were catabolised to phenolic acids in the human faecal slurries in the presence of glucose. Hippuric acid and 4-hydroxyphenylacetic acid were found to be abundant after drinking green tea, but did not appear to be major phenolic acids produced from the catabolism of green tea catechins. Calculation based on the levels of selected phenolic acids excreted in urine of healthy volunteers after drinking green tea indicated excretion was equivalent to 23.8% of flavan-3-ols intake. The mean 0-24 h excretion of 163 ± 48 μmole phenolic acid for healthy volunteers who drank green tea was significantly higher (p<0.05) than the excretion of phenolic acids by volunteers who drank water (20 ± 3 μmole). Analysis of phenolic acids in brewed green tea was also carried out in this study. Gallic acid and p-coumaric acid were by far the most abundant phenolic acids present both in free and conjugate forms, but the amounts were small compared to the quantities of phenolic acids excreted in urine 0-24 h after drinking green tea. This study also describes the pharmacokinetics study of urinary excretion of phenolic acids in human volunteers (with and without a colon) after drinking instant coffee. All volunteers displayed a substantial increase in excretion of 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, dihydroferulic acid, ferulic acid and 3-hydroxyhippuric acid 8-24 h after drinking coffee. The mean amount of urinary phenolic acids excreted after drinking coffee was 134 ± 43 μmole, which was equivalent to 29.4% of chlorogenic acid intake and significantly higher (p<0.05) than phenolic acids excreted when water was consumed instead coffee. The influence of the food matrixes (yoghurt and milk) on the catabolism of polyphenols is described in this study. 3-Methoxy 4-hydroxyphenylhydracrylic acid, 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, hippuric acid, 3-hydroxyhippuric acid, 3-dihydroxyphenylacetic acid and dihydroferulic acid were found in human urine after drinking orange juice enriched with hesperetin-7-O-rutinoside. The total 0-24 h urinary excretion of flavanone-derived phenolic acids increased by a statistically significant nine-fold (p