Antioxidant and antibacterial activities of Artemisia absinthium and Citrus paradisi extracts repress viability of aggressive liver cancer cell line.

BACKGROUND: Numerous reports show that herbal medicines can be utilized in the treatment of different liver disorders. In this study, antioxidant, antibacterial, and anticancer activities of individual as well as combined 80% ethanolic extracts of Artemisia absinthium leaves and Citrus paradisi peels were investigated. METHODS AND RESULTS: Values of total phenolic contents (TPC), total flavonoid contents (TFC), DPPH-radical scavenging activity, and ferric reducing antioxidant power (FRAP) were measured to explore the antioxidant capacity. To assess antibacterial activity, four bacterial strains (Escherichia coli, Staphylococcus aureus, Salmonella enterica, and Klebsiella pneumoniae) were used. Anticancer activity was assessed on Huh-7 (liver cancer) and Vero (non-cancerous) cell lines. FRAP activity of combined plants extract was higher as compared to their individual effect; the trend did not hold in the case of DPPH-radical scavenging activity. Antibacterial activity of combined extracts by disk diffusion method was observed only against E.coli. MTT results indicated that both plants had a cytotoxic effect on Huh-7 cell line but did not show any effect on Vero cell line. Our data showed a strong negative correlation between the amount of TPC, TFC, & DPPH radicals-scavenging activity and viability of Huh-7 cell line.However, no effect was shown on the non-cancerous cell line. CONCLUSION: The ethanolic extracts of Artemisia absinthium leaves and Citrus paradisi peels can be used against liver cancer because of their antioxidant, antibacterial, and anticancer activities.

Synergistic effect of carboxymethylcellulose and Cryptococcus laurentii on suppressing green mould of postharvest grapefruit and its mechanism.

The synergistic effects of carboxymethylcellulose (CMC) combined with Cryptococcus laurentii FRUC DJ1 were studied on controlling green mould resulting from Penicillium digitatum in grapefruit fruit. The results indicate that both C. laurentii and the CMC treatment suppressed P. digitatum conidia germination. In addition, C. laurentii growth in vitro was not affected by low CMC concentrations, nevertheless, the biofilm of C. laurentii was enhanced. Compared with the control fruit, the grapefruit had a lower green mould in all treatments. Significantly synergistic effects were caused by combining C. laurentii and CMC on minimum decay incidence and lesion diameter. Combined treatment induced defence enzyme activities, including chitinase, ß-1,3-glucanase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase, together with disease tolerance-associated total phenol. Also, this combination inhibited the pathogen growth by adhered to the hyphae and reduced its infection in fruit wounds. Moreover, the commercial quality parameters in the combined treatment of C. laurentii and CMC, including weight loss, total soluble solids, ascorbic acid, and titratable acidity, were superior to single treatment. The combination of C. laurentii and CMC can not only control postharvest decay but also maintain fruit qualities. Thus, it can be used in grapefruit for commercial purposes.

Variation in Key Flavonoid Biosynthetic Enzymes and Phytochemicals in 'Rio Red' Grapefruit (Citrus paradisi Macf.) during Fruit Development.

In the current study, the phytochemical contents and expression of genes involved in flavonoid biosynthesis in Rio Red grapefruit were studied at different developmental and maturity stages for the first time. Grapefruit were harvested in June, August, November, January, and April and analyzed for the levels of carotenoids, vitamin C, limonoids, flavonoids, and furocoumarins by HPLC. In addition, genes encoding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and 1,2-rhamnosyltransferase (2RT) were isolated, and their expression in grapefruit juice vesicles was studied. Fruit maturity had significant influence on the expression of the genes, with PAL, CHS, and CHI having higher expression in immature fruits (June), whereas 2RT expression was higher in mature fruits (November and January). The levels of flavonoids (except naringin and poncirin), vitamin C, and furocoumarins gradually decreased from June to April. Furthermore, limonin levels sharply decreased in January. Lycopene decreased whereas ß-carotene gradually increased with fruit maturity. Naringin did not exactly follow the pattern of 2RT or of PAL, CHS, and CHI expression, indicating that the four genes may have complementary effects on the level of naringin. Nevertheless, of the marketable fruit stages, early-season grapefruits harvested in November contained more beneficial phytochemicals as compared to mid- and late-season fruits harvested in January and April, respectively.

Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi.

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.

Analysis of ethylene biosynthesis and perception during postharvest cold storage of Marsh and Star Ruby grapefruits.

Grapefruits are among the citrus species more sensitive to cold and develop chilling injury symptoms during prolonged postharvest storage at temperatures lower than 8 ℃-10 ℃. The plant hormone ethylene has been described either to protect or potentiate chilling injury development in citrus whereas little is known about transcriptional regulation of ethylene biosynthesis, perception and response during cold storage and how the hormone is regulating its own perception and signaling cascade. Then, the objective of the present study was to explore the transcriptional changes in the expression of ethylene biosynthesis, receptors and response genes during cold storage of the white Marsh and the red Star Ruby grapefruits. The effect of the ethylene action inhibitor, 1-MCP, was evaluated to investigate the involvement of ethylene in the regulation of the genes of its own biosynthesis and perception pathway. Ethylene production was very low at the harvest time in fruits of both varieties and experienced only minor changes during storage. By contrast, inhibition of ethylene perception by 1-MCP markedly induced ethylene production, and this increase was highly stimulated during shelf-life at 20 ℃, as well as transcription of ACS and ACO. These results support the auto-inhibitory regulation of ethylene in grapefruits, which acts mainly at the transcriptional level of ACS and ACO genes. Moreover, ethylene receptor1 and ethylene receptor3 were induced by cold while no clear role of ethylene was observed in the induction of ethylene receptors. However, ethylene appears to be implicated in the transcriptional regulation of ERFs both under cold storage and shelf-life.

De novo synthesis of high-value plant sesquiterpenoids in yeast.

Terpenoids comprise a structurally diverse group of natural products. Despite various and important uses of terpenoids (e.g., flavors, drugs, and nutraceuticals), most of them are, however, still extracted from plant sources, which suffer from high cost and low yield. Alternatively, terpenoids can be produced in microbes using their biosynthetic genes. With the explosion of sequence data, many genes for terpenoid metabolism can be characterized by biochemical approaches and used for the microbial production of terpenoids. However, substrates for in vitro studies of terpene synthases are costly, and the enzymatic synthesis of terpenoids in vitro using recombinant enzymes is insufficient to meet the chemical characterization need. Here, we describe the use of engineered yeast (EPY300) to evaluate in vivo production of sesquiterpenoids. Two sesquiterpene synthase genes (for valencene and 5-epi-aristolochene synthases) were expressed in EPY300 in native and N-terminal thioredoxin fusion forms. By using the thioredoxin fusion, valencene biosynthesis was slightly decreased; however, the production of 5-epi-aristolochene was increased by 10-fold, producing 420 µg mL(-1) of 5-epi-aristolochene. Accordingly, the thioredoxin-fused 5-epi-aristolochene was coexpressed with 5-epi-aristolochene dihydroxylase (cytochrome P450 monooxygenase) and its reductase in EPY300. This combinatorial expression yielded hydroxylated sesquiterpene, capsidiol, at ~250 µg mL(-1). Detailed experimental procedures and other considerations for this work are given.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco.

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Characterization and developmental expression of genes encoding the early carotenoid biosynthetic enzymes in Citrus paradisi Macf.

In the present study, the full-length cDNA sequences of PSY, PDS, and ZDS, encoding the early carotenoid biosynthetic enzymes in the carotenoid pathway of grapefruit (Citrus paradisi), were isolated and characterized for the first time. CpPSY contained a 1311-bp open reading frame (ORF) encoding a polypeptide of 436 amino acids, CpPDS contained a 1659-bp ORF encoding a polypeptide of 552 amino acids, and CpZDS contained a 1713-bp ORF encoding a polypeptide of 570 amino acids. Phylogenetic analysis indicated that CpPSY shares homology with PSYs from Citrus, tomato, pepper, Arabidopsis, and the monocot PSY1 group, while CpPDS and CpZDS are most closely related to orthologs from Citrus and tomato. Expression analysis revealed fluctuations in CpPSY, CpPDS, and CpZDS transcript abundance and a non-coordinated regulation between the former and the two latter genes during fruit development in albedo and juice vesicles of white ('Duncan') and red ('Flame') grapefruits. A 3× higher upregulation of CpPSY expression in juice vesicles of red-fleshed 'Flame' as compared to white-fruited 'Duncan' was observed in the middle stages of fruit development, which correlates with the well documented accumulation pattern of lycopene in red grapefruit. Together with previous data, our results suggest that the primary mechanism controlling lycopene accumulation in red grapefruit involves the transcriptional upregulation of CpPSY, which controls the flux into the carotenoid pathway, and the downregulated expression of CpLCYB2, which controls the step of cyclization of lycopene in chromoplasts during fruit ripening. A correlation between CpPSY expression and fruit color evolution in red grapefruit is demonstrated.

Expression and phylogenetic analysis of two new lycopene ß-cyclases from Citrus paradisi.

Two new lycopene ß-cyclases (LCYBs) were cloned and characterized from grapefruit (Citrus paradisi Macf.). During fruit ripening, CpLCYB1 expression did not show significant differences between 'Flame' (red flesh) and 'Marsh' (white flesh), and was much lower than CpLCYB2 and nearly constant; however, CpLCYB2 expression dramatically changed in a similar tendency in the pulp of both grapefruit cultivars, but the relative abundance of mRNA in 'Flame' was significantly lower than in 'Marsh'. Phylogenetically and structurally, CpLCYB1 was a chloroplast-specific member and CpLCYB2 a chromoplast-specific member, the two subfamilies of all the LCYB genes. An intron was found in the 5'-untranslated region of CpLCYB1 and in two other Citrus LCYB1 genes (CcLCYB1 and CsLCYB1-2), resulting in an extra 20 amino acids, compared with all the other LCYB1s. It suggested that a different genomic event, in addition to gene duplication, has contributed to the evolution of these LCYB genes, and likewise, the change of their functions.

Effects of dense phase carbon dioxide pasteurization on the physical and quality attributes of a red grapefruit juice.

Red grapefruit juice was treated with continuous dense phase carbon dioxide (DPCD) equipment to inactivate yeasts and molds and total aerobic microorganisms. A central composite design was used with pressure (13.8, 24.1, and 34.5 MPa) and residence time (5, 7, and 9 min) as variables at constant temperature (40 degrees C), and CO(2) level (5.7%) after experimentally measuring CO(2) solubility in the juice. Five log reduction for yeasts and molds and total aerobic microorganisms occurred at 34.5 MPa and 7 min of treatment. A storage study was performed on the fresh juice DPCD treated at these conditions. degrees Brix, pH, titratable acidity (TA), pectinesterase (PE) inactivation, cloud, color, hue tint and color density, total phenolics, antioxidant capacity, and ascorbic acid were measured after the treatment and during 6 wk storage at 4 degrees C. During storage, the DPCD-treated juice showed no growth of total aerobic microorganisms and yeasts and molds. Cloud increased (91%) while percent PE inactivation was partial (69.17%). No significant (alpha= 0.05) differences were detected between treated and untreated samples for degrees Brix, pH, and TA. Treated juice had higher lightness and redness and lower yellowness. No significant differences (alpha= 0.05) were detected for the hue tint values while the color density value was higher for the treated samples compared to the untreated. The treatment and the storage did not affect the total phenolic content of the juice. Slight differences were detected for the ascorbic acid content and the antioxidant capacity. The experimental results showed evidence that the treatment can maintain the physical and quality attributes of the juice, extending its shelf life and safety.

Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (K(m)(app)=67 microM; V(max)=20.45 pKat/microg), kaempferol (K(m)(app)=12 microM; V(max)=11.63 pKat/microg), and myricetin (K(m)(app)=33 microM; V(max)=12.21 pKat/microg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu(2+), Fe(2+), and Zn(2+) as well as UDP (K(i)(app)=69.5 microM), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.

Potentiation of the antiinflammatory effect of Anacardium occidentale (Linn.) stem-bark aqueous extract by grapefruit juice.

In an attempt to scientifically appraise some of the ethnomedical uses of Anacardium occidentale Linn. (family: Anacardiaceae), the present study was undertaken to examine the antiinflammatory effect of the plant's stem-bark aqueous extract in rats. Young adult male Wistar rats weighing 250-300 g were used. The antiinflammatory effect of A. occidentale stem-bark aqueous extract alone and in combination with grapefruit (Citrus paradisi Macf.) juice was investigated on fresh egg albumin-induced rat paw edema. Like diclofenac (100 mg/kg p.o.), aqueous extract of A. occidentale stem-bark (800 mg/kg p.o.) produced time-related, sustained and significant reduction (p < 0.05-0.001) of the fresh egg albumin-induced acute inflammation of the rat hind paw. However, the antiinflammatory effect of the plant extract was found to be approximately 8-15 times less than that of diclofenac. Coadministration of grapefruit juice (5 ml/kg p.o.) with A. occidentale stem-bark aqueous extract (800 mg/kg p.o.) or diclofenac (100 mg/kg p.o.) significantly potentiated (p < 0.05-0.001) the antiinflammatory effects of the crude plant extract and diclofenac on fresh egg albumin-induced rat paw edema. Although A. occidentale stem-bark aqueous extract is less potent than diclofenac as an antiinflammatory agent, the results of this experimental animal study indicate that the plant extract possesses antiinflammatory activity, and thus lend pharmacological support to the folkloric use of the plant in the management and/or control of arthritis and other inflammatory conditions among the Yoruba-speaking people of western Nigeria.

Flavanone 3-hydroxylase expression in Citrus paradisi and Petunia hybrida seedlings.

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.