Citrus paradisi and Citrus sinensis flavonoids: Their influence in the defence mechanism against Penicillium digitatum

Citrus peel is rich in flavanone glycosides and polymethoxyflavones. In view of their importance for industrial application as well as for their pharmacological properties, their content was analyzed in the mature fruits of several Citrus paradisi (grapefruit) and Citrus sinensis (orange) varieties, with a view to select the most interesting for isolation. The results shows that the Star Ruby grapefruit and the Sanguinelli orange stand out for their high contents of naringin and hesperidin, respectively. The presence of the polymethoxyflavones nobiletin, heptamethoxyflavone and tangeretin, could be ascertained in all the grapefruit varieties analysed. Higher polymethoxyflavone levels were recorded in orange, with Valencia Late showing the greatest nobiletin, sinensetin and tangeretin contents and Navelate the highest heptamethoxyflavone levels. An in vitro study revealed that these compounds acted as antifungal agents against Penicillium digitatum, the polymethoxyflavones being more active than the flavanones in this respect. The possible participation of these phenolic compounds in the defence mechanism of Citrus against P. digitatum is discussed.     

Determination of the change of flavonoid components as the defence materials of Citrus unshiu Marc. fruit peel against Penicillium digitatum by liquid chromatography coupled with tandem mass spectrometry

A healthy fruit peel of Citrus unshiu Marc. and one infected by Penicillium digitatum were analysed for flavonoids via high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) in the positive mode with selected ion monitoring (SIM). Among 16 flavonoid components characterised in C. unshiu Marc., four flavanones and nine flavones were identified for the first time. The identified compounds were quantified by HPLC–UV. To investigate the function of the flavonoids as defence materials, the flavonoid content change of the fruit peel inoculated with P. digitatum was monitored by HPLC. The flavonoid concentration in the infected fruit peel decreased initially after the infection and then gradually increased before finally progressively decreasing.     

Differentiation of juices from clementine ( Citrus clementina ), clementine-hybrids and satsuma ( Citrus unshiu ) cultivars by statistical multivariate discriminant analysis of their flavanone-7- O -glycosides and fully methoxylated flavones content as determined by liquid chromatography

Mandarin juices from three pure clementine (Citrus clementina Hort. ex Tan.) cultivars: Clemenules, Hernandine and Marisol, three clementine-hybrids: Nova (clementine×tangelo Orlando), Ortanique (clementine×orange) and Fortune (clementine×mandarin Dancy) and one satsuma (Citrus unshiu Marc) cultivar: Clauselline have been analyzed for simultaneous determination of their flavanone-7-O-glycosides (FGs) and fully methoxylated flavones (FMFs) content, using high-performance liquid chromatography. Statistical multivariate discriminant analysis from the FGs concentration data showed that its discriminant power was rather poor, leading to overlapping between some groups of the assayed cultivars. By contrast, the discriminant power from the FMFs concentration data was very high and only two concentration ratios were needed as discriminant parameters to significantly differentiate between all the assayed cultivars. Moreover, the values of the discriminant parameters were almost independent of the location of the orchards and the maturity stage of the fruits.     

Citrus phenylpropanoids and defence against pathogens. Part II: Gene expression and metabolite accumulation in the response of fruits to Penicillium digitatum infection

The effect of infection of Citrus sinensis (var. Navelina) fruits with Penicillium digitatum was studied at gene expression and metabolite levels. In this study, expression of genes involved in the phenylpropanoid pathway was studied in the flavedo (outer coloured part of the peel) and albedo (inner white part) in response to pathogen infection. Results of the time-course experiment showed that maximal expression of 10 out of 17 phenylpropanoid genes analysed occurred at 48 h post-inoculation, when decay symptoms started to appear, and mRNA levels either kept constant or decreased after 72 h post-inoculation. To further investigate the putative involvement of the phenylpropanoid pathway in the defence of citrus fruit, changes in the metabolic profile of both tissues infected with P. digitatum was studied by means of HPLC-PDA-FD. Metabolite accumulation levels along the time course suggest that flavanones, flavones, polymethoxylated flavones and scoparone are induced in citrus fruit in response to P. digitatum infection, although with different trends depending on the tissue.     

Isolation of hesperidin from peels of thinned Citrus unshiu fruits by microwave-assisted extraction

Simultaneous extraction by microwave-irradiation and crystallisation were performed in the same pot of solvent of 70% (v/v) aqueous ethanol for isolation of hesperidin from thinned immature fruit peels of Citrus unshiu as refining of Citrus waste biomass. The hesperidin content in immature fruits peels was about 3.2-fold higher than that of mature fruit. After microwave-assisted extraction (MAE), the yield of hesperidin reached 58.6 mg/g, which was comparable to the amount obtained after extraction using DMSO:methanol (1:1, v/v) as a solvent for 30 min at room temperature. Heating temperature and time for isolation of hesperidin crystallites were optimised as 140 °C and 8 min by using response surface methodology. Under this optimal condition, 86.8% (47.7 mg/g) of total hesperidin was isolable by MAE and low-temperature storage (5 °C, 24 h).     

Total,cumulative fast-kinetics and cumulative slow-kinetics antiradical activities of juices from clementine (<Emphasis Type="Italic">Citrus clementina</Emphasis>), clementine-hybrids and satsuma (<Emphasis Type="Italic">Citrus unshiu</Emphasis>) cultivars and their utility as discriminant variables

Mandarin juices from three pure clementine (Citrus clementina Hort. ex Tan.) cultivars: Clemenules, Hernandine and Marisol, two clementine-hybrid cultivars: Nova (clementine×tangelo Orlando) and Fortune (clementine× mandarin Dancy), one mandarin-hybrid cultivar: Ortanique (mandarin×orange) and one satsuma (Citrus unshiu Marc) cultivar: Clauselline have been analysed for determination of total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activities. Total antiradical activity followed the order: pure clementine and Nova> Ortanique>Fortune and Clauselline. Cumulative fast-kinetics antiradical activity was the major contributor to total antiradical activity, accounting for about 87% as average. Ascorbic acid content was the major contributor to cumulative fast-kinetics antiradical activity, accounting for about 93.5% as average. A weak correlation (r ²=0.151) between total flavanone-7-O-glycosides (FGs, Narirutin, Hesperidin and Didymin) content and cumulative slow-kinetics antiradical activity was observed. However, ascorbic acid and total FGs contents showed a significant negative correlation (r ²=0.658). By using the cumulative fast-kinetics and slow-kinetics antiradical activities as variables, all the assayed juices were significantly discriminated (<0.05) by statistical multivariate discriminant analysis.     

Antimicrobial activity of acid-hydrolyzed Citrus unshiu peel extract in milk

Citrus fruit (Citrus unshiu) peels were extracted with hot water and then acid-hydrolyzed using hydrochloric acid. Antimicrobial activities of acid-hydrolyzed Citrus unshiu peel extract were evaluated against pathogenic bacteria, including Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes. Antilisterial effect was also determined by adding extracts at 1, 2, and 4% to whole, low-fat, and skim milk. The cell numbers of B. cereus, Staph. aureus, and L. monocytogenes cultures treated with acid-hydrolyzed extract for 12 h at 35°C were reduced from about 8 log cfu/mL to <1 log cfu/mL. Bacillus cereus was more sensitive to acid-hydrolyzed Citrus unshiu peel extract than were the other bacteria. The addition of 4% acid-hydrolyzed Citrus unshiu extracts to all types of milk inhibited the growth of L. monocytogenes within 1 d of storage at 4°C. The results indicated that Citrus unshiu peel extracts, after acid hydrolysis, effectively inhibited the growth of pathogenic bacteria. These findings indicate that acid hydrolysis of Citrus unshiu peel facilitates its use as a natural antimicrobial agent for food products.     

Exploring the phytochemical content and the antioxidant potential of Citrus fruits grown in Cyprus

The physicochemical and phytochemical profile as well as the antioxidant properties of the most important Citrus fruits grown in Cyprus [orange (Citrus sinensis, cv. ‘Valencia’), grapefruit (Citrus paradisi, cvs. ‘White Marsh’, ‘Star Ruby’, ‘Rio Red’) and an interspecific hybrid (Citrus reticulata x Citrus sinensis, cv. ‘Mandora’)] were determined. ‘Valencia’ fruit exhibited the highest phenolic, flavonoid and ascorbic acid content, as well as a higher antioxidant potency, followed by ‘Mandora’ fruit and the three grapefruit cultivars. HPLC indicated significant diversity on flavanones among the examined fruits, while LC-MS analysis revealed the presence of hydroxycinnamic acid derivatives, polymethoxyflavones and a furocoumarin. Overall, this study provides supporting evidence for the superiority of ‘Valencia’ orange fruit as an excellent source of bioactive compounds. In addition, to the best of our knowledge, this is the first study thoroughly describing the phytochemical profile of ‘Mandora’ fruit, an interspecific hybrid cultivated in Cyprus.     

The methoxyflavones in Citrus reticulata Blanco cv. ponkan and their antiproliferative activity against cancer cells

The major polymethoxyflavones in the fruit (ponkan) peels of Citrus reticulata Blanco cv. ponkan were identified as isosinensetin, sinensetin, nobiletin and tetramethyl-o-scutellarein by a combined separation using high-speed countercurrent chromatography and preparative high performance liquid chromatography, and structure elucidation by electrospray ionisation mass spectrometry (ESI-MS) and ¹H and ¹³C nuclear magnetic resonance (NMR). The antiproliferative activity of the four compounds against four cancer cell lines (A549, HL-60, MCF-7 and HO8910) showed that isosinensetin had a lower IC₅₀ value for MCF-7 and HO8910 cancer cell lines. Determination of polymethoxyflavones in ponkan peels from different cultivation regions displayed relatively steady contents of the four compounds and a higher content of isosinensetin, which suggested that ponkan peels are excellent sources of functional polymethoxyflavones that may help prevent female cancers, such as ovarian cancer and breast cancer.     

Simultaneous determination of polymethoxyflavones in Citrus species,Kiyomi tangor and Satsuma mandarin,by high performance liquid chromatography

The content of polymethoxyflavones (PMFs) in Kiyomi tangor (Citrus unshiu Marcov. forma miyagawa-wase × C. sinensis) and Satsuma mandarin (C. unshiu Marcov. forma miyagawa-wase) was determined by HPLC/UV. The major PMFs of Kiyomi tangor were determined as 3,6,7,4′-tetramethoxyflavone in the peels (10.1 mg/g) and leaves (9.2 mg/g), and 3-hydroxy-5,6,7,4′-tetramethoxyflavone in the stems (1.5 mg/g). The major PMFs of Satsuma mandarin were determined as 5,6,7,8,4′-pentamethoxyflavone in the peels (2.2 mg/g) and leaves (1.6 mg/g), and 5,6,7,3′,4′-pentamethoxyflavone in the stems (1.4 mg/g). Large amounts of Citrus by-products can ultimately provide a cheap and convenient source of PMFs.     

Odour gradients and patterns in volatile emission of different plant parts and developing fruits of grapefruit (Citrus paradisi L.)

The volatiles spontaneously emitted in vivo by different plant parts of grapefruit (Citrus paradisi Macf., Rutaceae) were collected by solid phase micro-extraction (SPME) during the whole vegetative cycle of the plant and characterised by GC–MS to verify their involvement in entomophilous pollination, a controversial topic in the biology of this species. Furthermore, the essential oils obtained by expression of the pericarp from unripe and ripe fruits were studied. Altogether 127 compounds were identified, accounting from 82.1% to 99.9% of the whole volatiles. The main constituents detected were mono- and sesquiterpenes, with limonene (0.5–95.2%), linalool (0.2–52.5%), sabinene (0.5–42.5%), myrcene (0.2–15.4%), and β-caryophyllene (0.3–41.0%) as the most represented ones.     

Enhanced extraction of flavanones hesperidin and narirutin from Citrus unshiu peel using subcritical water

Flavanones including hesperidin and narirutin constitute the majority of the flavonoids that occur naturally in citrus fruits. The main purpose of this study was to extract valuable natural flavanones from agricultural by-products such as citrus peels using subcritical water extraction (SWE). Thus, the application of SWE to extraction of flavanones hesperidin and narirutin from Citrus unshiu peel was evaluated, and the effect of key operating conditions was determined by varying the extraction temperature (110–200 °C) and time (5–20 min) under high pressure (100 ± 10 atm). The maximum yields of hesperidin (72 ± 5 mg/g C. unshiu peel) and narirutin (11.7 ± 0.8 mg/g C. unshiu peel) were obtained at an extraction temperature of 160 °C for an extraction time of only 10 min. These yields accounted for approximately 99% of the total amount of these flavanones in the original material. The SWE was compared with three conventional extraction methods in terms of the extraction time and recovery yields for hesperidin and narirutin. The hesperidin yield by SWE was more than 1.9-, 3.2-, and 34.2-fold higher than those obtained by extraction methods using ethanol, methanol, or hot water, respectively, and the narirutin yield was more than 1.2-, 1.5-, and 3.7-fold higher.     

Determination of flavonoids in a Citrus fruit extract by LC–DAD and LC–MS

Flavonoids are a group of polyphenolic compounds with health-related properties. Citrus fruits are rich in flavonoids and their extracts are being used as functional ingredients for several industrial products. A new high performance liquid chromatography technique with an UV photodiode-array detector was used to analyze flavonoids of an extract of Citrus species. To our knowledge this is the first study that reports isoquercitrin presence at a level of 77.3 mg/100 g in a sample made of Citrus fruits; four other flavonoids were quantified as rutin (326.59 mg/100 g), naringin (338.36 mg/100 g), quercetin (96.35 mg/100 g) and naringenin (2.35 mg/100 g). Identification was confirmed by a liquid chromatography mass spectrometer system. Method validation was achieved, providing an analytical technique that can be used to detect trace amounts of these compounds in Citrus extracts with an extremely rapid sample preparation.     

Efficacy of killer yeasts in the biological control of Penicillium digitatum on Tarocco orange fruits (Citrus sinensis)

Killer Saccharomyces cerevisiae and Wickerhamomyces anomalus yeast strains were tested as biocontrol agents against Penicillium digitatum, one the most important causes of postharvest decay in orange fruits.     

Essential Oils of New Pigmented Citrus Hybrids: Citrus sinensis L. Osbeck x C clementina Hort. ex Tanaka

The chemical composition of the essential oils of 2 new pigmented Citrus hybrids (Omo‐6 and Omo‐12) was studied by gas chromatography‐mass spectrometry (GC‐MS). The hybrids were obtained by crossbreeding the orange Citrus sinensis (L.) Osbeck cultivar‘Moro’ and clementine Citrus clementina Hort. ex Tanaka cultivar‘Oroval’. Seventy‐eight components were fully characterized and grouped in five classes (monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes, aliphatic alcohols and ethers, and aliphatic aldehydes) for an easier comparison of the oils with parents and those of 2 previously analyzed hybrids (Omo‐15 and Omo‐31) from the same crossbreeding procedure.     

Bioactive compounds and antioxidant capacities in different edible tissues of citrus fruit of four species

Different edible tissues of citrus fruit, namely juice sacs (JS), segment membrane (SM), and segment (Seg), of four species, were examined for contents of bioactive compounds and total antioxidant capacities (TAC) by ferric reducing antioxidant power (FRAP) assay. Two flavanones (naringin and hesperidin) were identified by HPLC; hesperidin accounted for 18.5–38.5% of the total phenolics in the species Citrus unshiu, Citrus reticulata, and Citrus sinensis, while naringin was only found in Citrus changshanensis and it accounted for 53.7% of the total phenolics in SM of this species. In SM of all selected species, the contents of phenolic compounds and TAC were significantly higher than those in JS and Seg. Highest total phenolics, total flavonoids, naringin, and TAC were found in SM of C. changshanensis, while the highest carotenoid content was found in JS of C. reticulata. The contribution of vitamin C to TAC ranged from 26.9% to 45.9% in JS and Seg of all selected species. In SM, however, a high contribution from hesperidin was observed in C. unshiu (54.0%), C. sinensis (46.7%) and C. reticulata (30.0%). The results indicated that SM of citrus fruit were high in contents of bioactive compounds and TAC; it is thus recommended to consume citrus fruit with all edible tissues rather than juice or JS alone.     

Preparation of a polymethoxyflavone-rich fraction (PRF) of Citrus sunki Hort. ex Tanaka and its antiproliferative effects

The peel of Citrus sunki is a rich source of polymethoxyflavones (PMFs). In this study, a PMF-rich fraction (PRF) was obtained from the peels of C. sunki by hot-water extraction and subsequent fractionation with n-hexane. Six PMFs and two demethylated PMFs were isolated by semi-preparative HPLC, and their structures were identified by ¹H NMR and ¹³C NMR. To define the synergistic effects of PRF, the PRF and its eight individual components were assessed for antiproliferative activity using HL-60 cells. The PRF showed greater growth inhibitory activity (IC₅₀ value = 25.14 μg/ml), compared with its individual components. Flow cytometric analysis confirmed that the PRF significantly increased the percentage of sub-G₁ cells (30.1% at 25 μg/ml). Our study provides an efficient method for preparing a PRF for use as a functional food ingredient.     

Effect of technological processes and storage on flavonoids content and total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activities of citrus juices

Juices from the mandarin Clemenules (Citrus clementina Hort. ex Tan.), the tangor Ortanique (Citrus reticulata Blanco × Citrus sinensis Osb.) and the sweet orange Valencia Late (Citrus sinensis) have been industrially squeezed, pasteurized, concentrated and stored under refrigeration (4 °C) and at room temperature (20 °C). After each process, the flavanone-7-O-glycosides (FGs) and fully methoxylated flavones (FMFs) contents as well as total, cumulative fast-kinetics and cumulative slow-kinetics antiradical activities were determined and compared with those from the corresponding fresh hand-squeezed juices. Neither industrial-squeezing, nor pasteurization or concentration significantly affected FGs and FMFs contents and antiradical activities of assayed juices. Storage caused a slight decrease of the FMFs contents but a significant reduction of both soluble hesperidin contents and cumulative fast-kinetics antiradical activities in all assayed juices. These decreases were dependent on storage temperature. Characteristic values of the varietal characterization parameters, which are derived from the FMFs contents and antiradical activities of fresh hand-squeezed juices, held valid for industrially squeezed, pasteurized and concentrated juices. After storage, however, only the FMFs-derived varietal characterization parameters and cumulative slow-kinetics antiradical activity remained valid for the resulting juices.     

Antioxidant capacity and hydrophilic phytochemicals in commercially grown native Australian fruits

Hydrophilic phytochemicals and antioxidant capacities of eight commercially grown native Australian fruits were determined. Kakadu plum (Terminalia ferdinandiana) contained a 6-fold higher level of total phenolic compounds and quandong (Santalum acuminatum) a 1.9-fold higher level of total phenolic compounds (TP, Folin–Ciocalteu assay) than blueberry (Vaccinum sp., cv. Biloxi). Both fruits displayed superior oxygen radical-scavenging capacity (ORAC-H assay) that was, respectively 4.1-fold and 6.5-fold of that of blueberry. The total reducing capacity (TRC; ferric reducing antioxidant power (FRAP) assay) of Kakadu plum and quandong exceeded the TRC of blueberry, respectively, 13.1- and 2.3-times. The primary sources of antioxidant capacities in the evaluated fruits were phenolic acids (benzoic and cinnamic) and flavonoids (flavonols, flavanones and anthocyanins) tentatively detected by liquid chromatography–mass spectrometry (LC–PDA–MS/MS). A high level of vitamin C was recorded for Kakadu plum and Australian citrus fruits. The major organic acids detected were citric and malic acid.