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.     

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).     

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.     

Chemical and aroma profiles of yuzu (Citrus junos) peel oils of different cultivars

The essential oils of six different yuzu cultivars, Kumon (KUM), Nagano (NAG), Yasu (YAS), Jimoto (JIM), Komatsu Sadao (KOS) and Komatsu Koichi (KOK), were extracted by cold-pressing method. A total of 69 compounds of the six samples were identified. Application of GC-olfactometry and aroma extraction dilution analysis technique in three-fold stepwise dilution of the neat oil for all samples indicated eight odourants with the highest flavour dilution (FD) values. Those were limonene, α-pinene, α- and β-phellandrene, myrcene, γ-terpinene, (E)-β-farnesene and linalool. ‘KOS’ was differentiated from the other oil samples by showing the highest number of components having yuzu-like odour notes and also from the PCA analysis of the FD-factor values. This is the first time the aroma characteristics of yuzu essential oils of specified cultivars were investigated.     

Antioxidative and anti-inflammatory properties of Citrus sulcata extracts

Citrus sulcata was subjected to ultrasound, high-pressure, and Soxhlet extractions. The antioxidant and anti-inflammatory activities of the extracts were evaluated qualitatively and quantitatively. The antioxidant content of the peel extract was twice that of the fruit extract. The quantitative analysis showed that the narirutin and hesperidin contents in the peel extracts were 8.8 and 7.5 mg/100 g, respectively. These extracts had a total phenolic content of 112.22 ± 2.89 gallic acid equivalent (GAE) mg/100 g, a total flavonoid content of 54.09 ± 1.01 rutin equivalent (RE) mg/100 g, DPPH radical scavenging activity of 46%, and antioxidant activity of 213.25 ± 2.82 μM of Trolox equivalents (TEAC). C. sulcata extracts could prevent hydrogen peroxide (H₂O₂)-induced oxidative stress, reduce expression of the inflammatory markers nuclear Factor kappa B (NFκB) and phosphorylated IκBα (p-IκBα) in A549 human lung carcinoma cells, and inhibit Lipopolysaccharide (LPS)-induced THP-1 monocyte differentiation to an extent of 85%.     

Characterisation of calamansi (Citrus microcarpa). Part I: Volatiles,aromatic profiles and phenolic acids in the peel

Volatile compounds in the peel of calamansi (Citrus microcarpa) from Malaysia, the Philippines and Vietnam were extracted with dichloromethane and hexane, and then analysed by gas chromatography–mass spectroscopy/flame ionisation detector. Seventy-nine compounds representing >98% of the volatiles were identified. Across the three geographical sources, a relatively small proportion of potent oxygenated compounds was significantly different, exemplified by the highest amount of methyl N-methylanthranilate in Malaysian calamansi peel. Principal component analysis and canonical discriminant analysis were applied to interpret the complex volatile compounds in the calamansi peel extracts, and to verify the discrimination among the different origins. In addition, four common hydroxycinnamic acids (caffeic, p-coumaric, ferulic and sinapic acids) were determined in the methanolic extracts of calamansi peel using ultra-fast liquid chromatography coupled to photodiode array detector. The Philippines calamansi peel contained the highest amount of total phenolic acids. In addition, p-Coumaric acid was the dominant free phenolic acids, whereas ferulic acid was the main bound phenolic acid.     

Inhibitory effect of sour orange (Citrus aurantium) juice on Salmonella Typhimurium and Listeria monocytogenes

The sour orange (Citrus aurantium) juice is commonly used as flavoring and acidifying agent for vegetable salads and appetizers in Turkey. It was aimed to determine the survival and growth pattern of Salmonella Typhimurium and Listeria monocytogenes in sour orange juice. Different concentrations of neutralized and un-neutralized juice samples were inoculated with each of the test microorganisms (∼6 log CFU/mL) separately and then incubated at 4 °C and 37 °C for seven days. It was detected both of the test microorganisms could survive and even grow in neutralized juice samples at 37 °C for two days. However, none of them could survive at the end of seventh day of incubation at 37 °C. Low incubation temperature (+4 °C) increased the survival of the tested microorganisms. Also, it was detected that L. monocytogenes were less resistant to the variable conditions than S. Typhimurium. It was concluded that the antimicrobial effect of sour orange juice mainly depends on the low pH value of the product. However, incubation time and temperature are also effective on the survival of the tested pathogens.     

Chemical composition and biological activity of Citrus jambhiri Lush

The fresh peel of Citrus jambhiri was extracted with aqueous methanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. The chemical structure of the isolated compounds was then identified by MS and NMR. Column chromatography of the petroleum fraction resulted in the isolation of nobiletin, 5-O-demethylnobiletin, tangeretin, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and a mixture of β-sitosterol and stigmasterol. The chloroform fraction afforded 6-demethoxynobiletin, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, limonin and nomilin. The flavonoid glycosides naringin, hesperidin and neohesperidin were isolated from the ethyl acetate fraction. The chemical structure of the isolated compounds was established by MS and NMR (APT, COSY, HSQC, HMBC, and NOESY). LC–ESI-MS analysis of the ethyl acetate fraction afforded eight flavonoid glycosides, while the dichloromethane fraction of the defatted seeds contained seven limonoid aglycones. The chloroform fraction exerted the strongest DPPH^∗ free radical scavenging activity in comparison to other fractions. The petroleum fraction showed a significant inhibition of lipoxygenase indicating an anti-inflammatory action (IC₅₀ 29 ± 1 μg/mL). Some of the isolated polymethoxyflavones exhibited strong cytotoxicity against COS7, HeLa and Caco-2 cell lines.     

Antimicrobial activity of binary combinations of natural and synthetic phenolic antioxidants against Enterococcus faecalis

This study investigated the antimicrobial activity of 3 natural (thymol, carvacrol, and gallic acid) and 2 synthetic [butylated hydroxyanisole (BHA) and octyl gallate] phenolic compounds, individually and in binary combinations, on 4 dairy isolates of Enterococcus faecalis with different virulence factors (β-hemolytic, gelatinase, or trypsin activities; acquired resistance to erythromycin or tetracycline; and natural resistance to gentamicin). A checkerboard technique and a microdilution standardized method were used. All compounds individually tested exhibited antimicrobial activity against E. faecalis, with minimal inhibitory concentrations (MIC) ranging from 30 μg/mL (octyl gallate) to 3,150 μg/mL (gallic acid), although no significant differences were detected among strains to each phenolic compound. Carvacrol in combination with thymol or gallic acid, and gallic acid combined with octyl gallate showed partial synergistic inhibition of all E. faecalis strains. The most effective combinations were thymol + carvacrol and gallic acid + octyl gallate, as the MIC for each of these compounds was reduced by 67 to 75% compared with their respective individual MIC. These results highlight the possibility of using combinations of these phenolic compounds to inhibit the growth of potential virulent or spoilage E. faecalis strains by reducing the total amount of additives used in dairy foods.     

Antioxidant and antidiabetic activity of Dangyuja (Citrus grandis Osbeck) extract treated with Aspergillus saitoi

Dangyuja is a citrus fruit of Cheju Island in Korea, which is known to have a high content of flavanone glycosides, such as naringin and neohesperidin. Flavanone glycosides of Dangyuja (Citrus grandis Osbeck) extract were converted into their aglycones by naringinase and hesperidinase, and into their hydroxylated forms by Aspergillus saitoi.Dangyuja extract treated with A. saitoi had significantly higher antioxidant and antidiabetic activity than both its glycosides and aglycones in oxygen radical absorption capacity (ORAC) and supercoiled DNA strand scission assay, rat intestinal α-glucosidase and porcine pancreatic α-amylase inhibition, suggesting that the fermentation of flavanone aglycones of Dangyuja extract with A. saitoi can increase antioxidant and antidiabetic activity, compared to their glycosides and aglycones. This result suggests that Dangyuja extract prepared with enzymatic and microbial treatments could be used for the development of pharmaceutical foods to control the blood glucose level of diabetic patients by inhibiting α-amylase and α-glucosidase in the intestinal tract.     

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.     

Isolation and structural characterisation of pectin from endocarp of Citrus depressa

A polysaccharide was extracted with hot HCl from the endocarp of Citrus depressa grown in Okinawa, Japan. The yield was 4.1% on a fresh weight basis. Composition of the polysaccharide was 89.3% total carbohydrate, 79.2% uronic acid, 4.1% ash and 8.8% moisture. The degree of methoxylation was estimated to be 66.2%. The polysaccharide was composed of d-GalA, d-Gal, l-Ara and l-Rha in the ratio of 100:10.3:1.53:0.94, respectively. The molecular mass was estimated to be approximately 4.1 × 10⁴. NMR spectra indicated that the polysaccharide was mainly composed of (1→4)-linked α-d-GalA and (1→4)-linked β-d-Gal. Methylation analysis results identified 1→2 and 1→2,4 linked Rha, 1→4, 1→3, 1→6 and 1→3,6 linked Gal. The results indicated that the polysaccharide was a pectin, which was relatively simpler than other pectins. The polysaccharide was classified as high-methoxyl pectin. The pectin turned into a gel by the generalised method.     

Antioxidant potentials of buntan pumelo (Citrus grandis Osbeck) and its ethanolic and acetified fermentation products

The antioxidant potentials of buntan pumelo (Citrus grandis Osbeck) and its fermented products were determined. The essential oil from peel had higher total phenolic (214 mM) and flavonoid (134 mg QE/g of dried material) contents than those of different solvent extracts from fruit pulp. In addition, DPPH free radical-scavenging activity and ferric-reducing antioxidant power values determined for the essential oil were 26.1 ± 1.2% and 2.3 ± 0.3 mM, respectively, which were significantly higher than those of various fruit pulp extracts. The ethanol-fermented products of pumelo juice had higher total phenolic and flavonoid contents than those of fresh juice. For maintenance of the substantial antioxidant properties of pumelo products, ethanol-fermented juice rather than fresh or acetate-fermented juice is recommended. Through correlation analysis, the phenolic compounds in the fermented pumelo products were found to be the major contributors to the free radical-scavenging activity and ferric-reducing power.     

Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India

The essential oil (EO) isolated by hydro-distillation from the peel of fully matured ripen fruits of Citrus reticulata Blanco were analyzed by GC and GC-MS. Thirty seven different components were identified constituting approximately ≥99% of the oil. The major components were limonene (46.7%), geranial (19.0%), neral (14.5%), geranyl acetate (3.9%), geraniol (3.5%), β-caryophyllene (2.6%), nerol (2.3%), neryl acetate (1.1%) etc. The antifungal activity of the oil was tested by poisoned food (PF) technique and the volatile activity (VA) assay against five plant pathogenic fungi viz Alternaria alternata (Aa), Rhizoctonia solani (Rs), Curvularia lunata (Cl), Fusarium oxysporum (Fo) and Helminthosporium oryzae (Ho). The oil showed better activity in VA assay. The Minimum inhibitory concentration (MIC) for Aa, Rs and Cl was 0.2 ml/100 ml whereas >0.2 ml/100 ml for Fo and Ho in PF technique. Fungal sporulation was also completely inhibited at 2 ml/100 ml of the oil except for Cl and Ho, which was only 0.5% (±0.5) and 0.25% (±0.25) respectively as compared to control.     

Characterisation of calamansi (Citrus microcarpa). Part II: Volatiles,physicochemical properties and non-volatiles in the juice

Calamansi juices from three countries (Malaysia, the Philippines and Vietnam) were characterised through measuring volatiles, physicochemical properties and non-volatiles (sugars, organic acids and phenolic acids). The volatile components of manually squeezed calamansi juices were extracted using dichloromethane and headspace solid-phase microextraction, and then analysed using gas chromatography–mass spectrometry/flame ionisation detector, respectively. A total of 60 volatile compounds were identified. The results indicated that the Vietnam calamansi juice contained the highest amount of volatiles. Two principal components obtained from principal component analysis (PCA) represented 89.65% of the cumulative total variations of the volatiles. Among the non-volatile components, these three calamansi juices could be, to some extent, differentiated according to fructose and glucose concentrations. Hence, this study of calamansi juices could lead to a better understanding of calamansi fruits.     

Evaluation of the antioxidant activity of extracts from buntan (Citrus grandis Osbeck) fruit tissues

The goal of the present work was to evaluate the antioxidant properties of buntan (Citrus grandis Osbeck) using various solvents, such as n-hexane, ethyl acetate (EtOAc), butanol and methanol. The antioxidant activities of crude extracts were evaluated by using the free radical scavenging β-carotene assay and total polyphenol. Ethyl acetate extracts of falvedo exhibited high antioxidative activities, followed by albedo and segment membrane extracts. Chromatography separation of EtOAc extract of flavedo using a silica gel column, yielded six fractions (A, B, C, D, E and F) using gradient elution with benzene and acetone (19:1, 14:1, 9:1, 5:1, 1:1 and 0:l). Among them, two fractions (C and D) showed strong antioxidant activities using the free radical scavenging activity (DPPH) antioxidant assay. These two fractions were further purified using silica gel column chromatography and preparative TLC. Their extracts could well be useful to prevent oxidation in fruit juices and essential oil food products as well as for health supplements. Identification of the responsible components is underway.     

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.     

Isolation and characterization of pigment from Cinnamomum burmannii' peel

Cinnamomum burmannii is cultivated for use as a spice, and as an ornamental tree. With the aim to develop a natural colorant for use in cosmetics and food additives from a new source, in the present study, pigment derived from C. burmannii' peel (CBP) was isolated by alkaline extraction, acid hydrolysis, repeated precipitation and purification by Sephadex G-75 with a total yield of 0.06 g/100 g (wet weight basis). The color values of the pigment was E_1 cm^1% 278 nm = 65.21. Physical and chemical properties revealed that CBP presses similar properties as most natural pigment. It was scarcely soluble in both water and all common organic solvents, and was soluble only in alkaline aqueous and DMSO. It was stable under ultraviolet light or room-light, stable in the range of 25-100 °C, relatively stable in alkaline aqueous and reducer, but was bleached by strong oxidants (KMnO₄, H₂O₂ and NaOCl). Sodium benzoic acid, tartaric acid, table salt and cane sugar affected it slightly. Spectroscopic analysis of CBP in relation with its structure was also discussed. This is the first report on the characterization of pigment obtained from C. burmannii 'peel.     

Antimicrobial activity of extract and two alkaloids from traditional Chinese medicinal plant Stephania dielsiana

Two alkaloids, stephanine and crebanine, were isolated from tubers of the traditional Chinese medicinal plant Stephania dielsiana, using an activity-directed isolation method, and inhibitory activity of methanol extract, stephanine and crebanine against ten animal pathogenic bacteria and eight plant pathogenic fungi was evaluated in vitro. The results showed that extract from S. dielsiana exhibited high inhibitory activity against five gram-positive and four gram-negative animal pathogenic bacteria, with MIC values of 0.625–7.5 g/l; stephanine and crebanine had high inhibitory activity against gram-positive animal pathogenic bacteria, with MIC values of 0.078–0.312 g/l, but low inhibitory activity against gram-negative animal pathogenic bacteria. Methanol extract, stephanine and crebanine also inhibited hyphal growth of the plant pathogens Cercospora kaki, Gymnosporangium haraeanum, Pyricularia oryzae, Rhizoctonia solani and Colletotrichum graminicola, and spores germination of Thielaviopsis paradoxa, Fusarium oxysporum f. sp. niveum, Sphaceloma fawcettii and G. haraeanum.     

Induction of apoptosis in U937 human leukaemia cells by the hexane fraction of an extract of immature Citrus grandis Osbeck fruits

The antiproliferative effect of an immature Citrus grandis Osbeck fruit extract was investigated using U937 human leukaemia cells. Maximum cytotoxicity was observed using the hexane fraction (HF) of the extract. Cell death was dose-dependent (IC₅₀ = ca. 60 μg/ml) and was characterised by chromatin condensation, apoptotic body formation, and DNA fragmentation. The induction of apoptosis was confirmed by caspase-3 activity assays and by immunoblotting using antibodies against Bcl-2, Bax, poly(ADP-ribose) polymerase (PARP), caspase-9, and caspase-3. The molecular mechanism underlying HF-induced apoptosis in U937 cells may involve a mitochondria-mediated signalling pathway, as demonstrated by an increase in the Bax/Bcl-2 expression ratio. Analyses of the HF by gas chromatography (GC) and GC-mass spectrometry (MS) tentatively identified 19 compounds, including γ-sitosterol (17.5%), 7-methoxy-8-(2-oxo-3-methylbutyl) coumarin (6.8%), stigmasterol (3.8%), and campesterol (3.4%). Together, our results provide the first evidence that the HF of an immature C. grandis Osbeck fruit extract induces apoptosis in U937 cells.