Comparative study of flavonoid and scoparone accumulation in different Citrus species and their susceptibility to Penicillium digitatum

Fungi of the genus Penicillium are responsible for substantial post-harvest losses in Citrus fruits. The results obtained following artificial inoculation of Citrus fruits with Penicillium digitatum showed that the degree of fungal development depended on the Citrus species. Thus, the mature fruit of Citrus paradisi were more susceptible to this fungus than the mature fruit of Citrus limon, Citrus sinensis,Citrus clementina Hort. ex Tan., and Citrus unshiu (mak) Marc. The results point to an inverse correlation between the degree of susceptibility of Citrus species to this fungus and the flavanone content – hesperidin in C. sinensis, C. clementina Hort. ex Tan. and C. unshiu (mak) Marc.; naringin in C. paradisi and the flavanone hesperidin and the flavone diosmin in C. limon. Thus, in C. sinensis,C. clementina Hort. ex Tan and C. unshiu (mak) Marc. the highest levels of the polymethoxyflavones, sinensetin, tangeretin, heptamethoxyflavone and nobiletin, were observed in the least susceptible varieties and viceversa. In the case of C. paradisi, no significant differences were detected in the polymethoxyflavone levels between varieties, while in C. limon, polymethoxyflavones were hardly detectable. The production of scoparone was observed in all the species and varieties studied after inoculation with the fungus, especially in C. limon fruits. Based on the evidence, it seems that flavanones, flavones, polymethoxyflavones (phytoanticipins) and scoparone (a phytoalexin) may well be involved in the defence mechanisms of Citrus fruits against P. digitatum. Depending on the Citrus species in question, the relative participation of one group of secondary compounds or another may vary.     

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.     

Studies on the antioxidant potential of flavones of Allium vineale isolated from its water-soluble fraction

The aim of this work was to examine the chemical constituents and antioxidant potential of water-soluble fractions from the commonly consumed vegetable, Allium vineale. The water-soluble fraction, containing phenolic compounds, was extracted with ethyl acetate to obtain flavonoids which were separated and purified by repeated column chromatography over Sephadex LH-20, RP C18 and silica gel. The isolated compounds were identified according to their physicochemical properties and spectral data (UV, HPLC–TOF/MS, ¹H NMR, ¹³C NMR and 2D NMR). Three flavonoids were isolated and identified as chrysoeriol-7-O-[2″-O-E-feruloyl]-β-d-glucoside (1), chrysoeriol (2), and isorhamnetin-3-β-d-glucoside (3). Antioxidant studies of the aqueous extract and three isolated compounds, 1, 2, 3, were undertaken and they were found to have significant antioxidant activity. Antioxidant activities were evaluated for total antioxidant activity by the ferric thiocyanate method, ferric ion (Fe³⁺) reducing antioxidant power assay (FRAP), ferrous ion (Fe²⁺) metal chelating activity, and DPPH free radical-scavenging activity. The water-soluble ethyl acetate and methanol extraction methods were also compared using HPLC–TOF/MS.     

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 flavonoid level variation in onion (Allium cepa L.) infected by Fusarium oxysporum using liquid chromatography–tandem mass spectrometry

In order to evaluate the flavonoid level variation in an onion (Allium cepa L.) infected by Fusarium oxysporum, the bulbs of a healthy onion and of an infected one were analysed for flavonoids via high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS). Among eleven flavonoids characterised, isorhamnetin 4′-O-galactoside (8) was identified in an onion for the first time. When the healthy bulb was inoculated with the fungus, the two quercetin derivatives (4 and 7) and the two isorhamnetin derivatives (5 and 9) underwent concentration changes typical for the defense materials against pathogens. The yellow granules that were accumulated on the abaxial epidermal cell layers after 8 days of inoculation were confirmed as quercetin (10) and isorhamnetin (11). It was deduced that they were produced from flavonoids 4, 5, 7 and 9 by hydrolysis enzyme of the fungus.     

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.     

Comparative analyses of seeds of wild fruits of Rubus and Sambucus species from Southern Italy: Fatty acid composition of the oil,total phenolic content,antioxidant and anti-inflammatory properties of the methanolic extracts

Fruit seeds are byproducts from fruit processing. Characterisation of the bioactive compounds present in seeds and evaluation of their potential biological properties is therefore of particular importance in view of a possible valorisation of seeds as a source of health beneficial components. In this work, we have analysed the seeds of Sambucus and Rubus species in order to identify their bioactive components and to determine the antioxidant and anti-inflammatory activities of the extracts. We first analysed their oil content, in order to assess the fatty acid profile and tocopherol content. Moreover, the methanolic extracts of the seeds were analysed for their total phenolic contents and antioxidant capacities. Polyphenols were identified by HPLC–ESI–MS/MS analysis. Furthermore, extracts were evaluated for their inhibitory effects on the production of LPS-induced inflammatory mediators (NO, CCL-20) in RAW 264.7 cells.     

Total flavan glycoside from Abacopteris penangiana rhizomes and its acid hydrolysate: Characterisation and anti-benign prostatic hyperplasia potential

This study was conducted to characterise the flavonoid components of total flavan glycoside from Abacopteris penangiana rhizomes (TFA) and its acid hydrolysate (AHT) through HPLC–DAD–ESI–MS/MS analysis, and to investigate the hypothesis that TFA and AHT exhibit anti-benign prostatic hyperplasia (BPH) potential in castrated rats with testosterone-induced BPH. HPLC–MS/MS analysis indicated that TFA is rich in flavan-4-ol glycosides and AHT mainly contains 3-deoxygenated anthocyanidin. After 4 weeks of administration, TFA and AHT successfully decreased the prostate index and prostate specific antigen plasma concentrations in the rats. Histoarchitectural improvement in the prostate gland was also observed. Reduced dihydrotestosterone, VEGF, bFGF, EGF, and KGF levels were observed both in TFA- and AHT-treated rats. Furthermore, the prostatic expression of Blc-2 was inhibited, whereas that of Bax and p53 was activated by TFA and AHT. In conclusion, TFA and AHT have anti-BPH properties. Hence, plants with flavan glycosides have potential use in the treatment of BPH.     

Bioassay-guided isolation and identification of antifungal components from propolis against Penicillium italicum

The present study was aimed at identification of antifungal components against Penicillium italicum from Chinese propolis with bioassay-guided fractionation technique. Propolis ethanolic extract (PEE) was separated and purified by liquid–liquid extraction and thin layer chromatography (TLC) and the most active band was subjected to HPLC–MS/MS to identify the antifungal compounds. The results showed PEE and its fractions had strong antifungal activity against P. italicum. Among the fractions of PEE partitioned by petroleum ether, ethyl acetate, n-butanol and water, ethyl acetate fraction (E-Fr) exhibited the most effective activity against P. italicum. Further bioautographic TLC assay showed Band I, with Rf value of 0.70, had an inhibitive zone, which showed the strongest antifungal activity and completely inhibited the growth of P.italicum at 200 mg/L. Bioactive components found in Band I were further identified as pinobanksin, pinocembrine, chrysin and galangin. This study exhibited Chinese propolis and its main flavonoids was potential natural alternatives for the control of citrus blue mould caused by P.italicum.     

Quality and antioxidant activity detection of Crataegus leaves using on-line high-performance liquid chromatography with diode array detector coupled to chemiluminescence detection

An on-line high-performance liquid chromatography–diode array detector combined with chemiluminescence detection (HPLC–DAD–CL) method was developed to evaluate the antioxidant activity and quality of Crataegus leaves derived from different species and habitats. The chromatographic fingerprint and active profiles of Crataegus leaves were simultaneously achieved by HPLC–DAD–CL. Main antioxidants in Crataegus leaves were identified by HPLC–DAD–CL and HPLC/ESI/MS. Data analysis based on the total antioxidant activities of 10 batches of samples indicated that there were obvious differences among samples from different species and habitats. Good precision and reproducibility were obtained for DAD and CL detection with relative standard deviations less than 1.0% for retention time and 4.0% for intra and inter-day variations. The results suggested that the on-line method might provide potential information for the identification and quantitation of active markers, which were directly associated with the quality of herbal medicines.     

Quali-quantitative analysis of flavonoids of Cornus mas L. (Cornaceae) fruits

The methanol extract obtained from the ripe fruits of Cornus mas L. (Cornaceae) have been phytochemically studied. On the basis of HPLC–PDA–MS/MSⁿ analysis eight compounds have been identified as quercetin, kaempferol, and aromadendrin glycosilated derivatives. Three major compounds have been also isolated by Sephadex LH-20 column chromatography followed by HPLC and characterised by NMR spectroscopy. Moreover, LC–PDA–MS analysis of the red pigment revealed the presence of three anthocyanins. The quantitative analysis of all compounds was reported.     

Phenolic acid profiles of mangosteen fruits (Garcinia mangostana)

The composition of phenolic acids in various parts of mangosteen fruit (Garcinia mangostana) was determined by GC and MS. The total content of phenolic acids, identified by GC-FID ranged from 265.7 ± 12.7 (aril) to 5027.7 ± 188.0 (peel) mg per kg of dry matter of sample. Ten phenolic acids were identified in mangosteen fruit. Of these, protocatechuic acid was the major phenolic acid in the peel and rind, while p-hydroxybenzoic acid was the predominant phenolic acid in the aril. m-Hydroxybenzoic acid was detected only in the peel, while 3,4-dihydroxymandelic was present only in the rind. The phenolic acids liberated from esters and glycosidic bonds were the major fractions of phenolic acids in mangosteen fruit.     

LC–DAD–MS (ESI+) analysis of the phenolic content of Sorbus domestica fruits in relation to their maturity stage

The aim of this study was the detailed qualitative and quantitative analysis of the phenolic content of several extracts and fractions of Sorbus domestica fruits. The analysis was realized by LC–DAD–MS (ESI+). Twenty-four different extracts and fractions of five different maturity stages of the fruit were analyzed for comparison reasons in order to determine the most beneficial for health type of consuming. Sixty-two different phenolics were identified. There were significant qualitative and quantitative differentiations in the phenolic content among the different types of the fruits. All categories were rich in benzoic, phenylpropanoic and cinnamoylquinic acids and derivatives. Unripe fruit categories were also rich in flavonoids, while well matured fruit categories had a low content of flavonoids. Fruit pulp, which was proved to be a strong antioxidant according to previous research work, contained very low amounts of both acids and flavonoids, but its phenolic content was highly qualitatively differentiated from the other categories.     

Chemical composition and antioxidant activities of Myrtus communis L. berries extracts

Myrtle (Myrtus communis L.) berries extracts were prepared with solvents at different polarity (water, ethanol, and ethyl acetate) and analysed using different in vitro tests in order to evaluate their antioxidant properties. Antiradical and total antioxidant activities were measured with DPPH and FRAP tests, respectively. Their ability to protect biological molecules was assessed using the cholesterol and LDL oxidation assays. In addition, phenolic compounds and unsaturated fatty acids composition was analysed by HPLC–DAD and HPLC–MS/MS. Ethanol and water extracts showed the highest amount of extracted compounds, but the highest antiradical and antioxidant activities were found in ethanol and ethyl acetate extracts. These extracts were also the ones with the highest content of phenolic compounds. In addition, our results showed a highly significant correlation between the amount of total phenols and antiradical (R² = 0.9993) or antioxidant activities (R² = 0.9985) in these extracts. HPLC–DAD and HPLC–MS analyses showed significant quantitative and qualitative differences among these three extracts. The ethyl acetate extract had the highest protective effect in assays of thermal (140 °C) cholesterol degradation and Cu²⁺-mediated LDL oxidation, inhibiting the reduction of polyunsaturated fatty acids and cholesterol, and the increase of their oxidative products. These results suggest that because of these properties, myrtle berries could be used in dietary supplements preparations or as food additives.     

Polyphenolic profiles detected in the ripe berries of Vitis vinifera germplasm

Polyphenolic profiles in the berry samples of 344 European grape (Vitis vinifera) cultivars were evaluated for two consecutive years. These cultivars represent a diverse collection of V. vinifera germplasm maintained at the USDA-Agricultural Research Service Vitis Clonal Repository in Davis of California, USA. A total of 36 polyphenolic compounds, including 16 anthocyanins, 6 flavonols, 6 flavanols, 6 hydroxycinnamic acids and 2 hydroxybenzoic acids, were identified via HPLC–MS and quantified by HPLC–DAD. The mean contents for anthocyanins, flavanols, flavonols, hydroxycinnamic acids and hydroxybenzoic acids were 0.946 (coloured cultivars), 0.147, 0.043, 0.195 and 0.016 mg g⁻¹ FW, respectively. On average, wine grapes had higher concentrations than had table grapes for all of these compounds except hydroxycinnamic acids. Berry colours affected the total contents of anthocyanins, but not others. Positive correlations (0.151–0.535) were found among these groups of compounds. As expected, these groups of compounds were all negatively correlated with berry weight.     

Distribution of quercetin-3,4′-O-diglucoside,quercetin-4′-O-monoglucoside,and quercetin in different parts of the onion bulb (Allium cepa L.) influenced by genotype

Flavonoids have gained much attention because of their proposed positive effects for human health. Onions are a rich source of flavonoids, consisting mainly of the major flavonols quercetin-3,4′-O-diglucoside (QDG) and quercetin-4′-O-monoglucoside (QMG) in the bulb and the aglycone quercetin in the outer scales. In this study, distribution of these three flavonoids was determined in 16 onion cultivars (Allium cepa) using HPLC–DAD. Three different parts of the onion bulb, the inner layers, the middle layers and the outer scales were analysed. The analysis showed varietal differences in the selected onion cultivars. The cultivars with the highest total flavonoid content were the red skinned ‘Red Baron’ and the yellow skinned cultivars ‘Ailsa Craig’ and ‘Prilep’. The distribution of the total flavonoid content in the different parts of the onion bulb showed the following order: middle layers > outer scales > inner layers. In the inner layers QDG was the major flavonoid, while in the middle layers QDG and QMG were in equal amounts. In the outer scales quercetin was the major flavonoid prior to QMG.     

Phytochemicals and antioxidant activity of different fruit fractions (peel,pulp, aril and seed) of Thai gac (Momordica cochinchinensis Spreng)

Three fractions (peel, pulp and aril) of gac fruit (Momordica cochinchinensis Spreng) were investigated for their phytochemicals (lycopene, beta-carotene, lutein and phenolic compounds) and their antioxidant activity. The results showed that the aril had the highest contents for both lycopene and beta-carotene, whilst peel (yellow) contained the highest amount of lutein. Two major phenolic acid groups: hydroxybenzoic acids and hydroxycinnamic were identified and quantified. Gallic acid and p-hydroxybenzoic acid were found in all fractions. Ferulic acid and p-hydroxybenzoic acid were most evident in pulp. Myricetin was the only flavonoid found in all fractions. Apigenin was the most predominant flavonoid in pulp (red), whereas rutin and luteolin gave the highest content in aril. The extracts of different fractions exhibited different levels of antioxidant activity in the systems tested. The aril extract showed the highest FRAP value. The greatest antioxidant activities of peel and pulp extracts were at immature stage, whereas those in the seed extracts increased from mature stage to ripe stage. The contents of total phenolic and total flavonoid in peel and pulp decreased during the fruit development stage (immature > ripe fruit) and subsequently displayed lower antioxidant capacity, except for the seed.     

Chemical characterisation of anthocyanins in tamarillo (Solanum betaceum Cav.) and Andes berry (Rubus glaucus Benth.) fruits

The anthocyanin composition of tamarillo (Solanum betaceum Cav., red variety) and Andes berry (Rubus glaucus Benth.) was determined by HPLC–PDA and HPLC–ESIMS. From the anthocyanin-rich extracts (AREs), pure compounds (1–7) were obtained by MLCCC (multilayer countercurrent chromatography) and further preparative HPLC, and their unequivocal structures were obtained by 1D and 2D NMR analyses. The new anthocyanin delphinidin 3-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside-3′-O-β-d-glucopyranoside, as well as the known cyanidin-3-O-rutinoside, pelargonidin-3-O-rutinoside, and delphinidin-3-O-rutinoside were identified as constituents of tamarillo fruit. Although the anthocyanin composition of Andes berry had been reported before in the literature, the unequivocal structure elucidation of the major compound, cyanidin-3-O-α-l-rhamnopyranosyl-(1 → 6)-O-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside, was achieved for the first time.     

Utilisation of orange peel in the production of α-terpineol by Penicillium digitatum (NRRL 1202)

The use of orange peel oil in the biotransformation of d-limonene was investigated. The physicochemical properties of cold-pressed orange peel oil, used in this investigation were determined to define its identity. The chemical composition of orange peel oil was determined by using GC/MS. Monoterpene compounds amounted to 98.0%, followed by aldehdyde components 1.09%. The main component of orange peel oil was d-limonene, which represented 96.1%, of the total content. A strain of Penicillium digitatum NRRL 1202 was used to carry out the biotransformation of d-limonene to α-terpineol. Two different media, malt yeast broth (MYB) and malt extract broth (MEB) were used. It was found that the highest bioconversion of d-limonene into α-terpineol was obtained by using MYB medium (pH 6.1).