Effects of Various Temperatures and pH Values on the Extraction Yield of Phenolics from Litchi Fruit Pericarp Tissue and the Antioxidant Activity of the Extracted Anthocyanins

Litchi fruit pericarp tissue is considered an important source of dietary phenolics. This study consisted of two experiments. The first was conducted to examine the effects of various extraction temperatures (30, 40, 50, 60, 70 and 80 °C) and pH values (2, 3, 4, 5 and 6) on the extraction yield of phenolics from litchi fruit pericarp. Extraction was most efficient at pH 4.0, while an extraction temperature of 60 °C was the best in terms of the combined extraction yield of phenolics and the stability of the extracted litchi anthocyanins. The second experiment was carried out to further evaluate the effects of various temperatures (25, 35, 45, 55 and 65 °C) and pH values (1, 3, 5 and 7) on the total antioxidant ability and scavenging activities of DPPH radicals, hydroxyl radical and superoxide anion of the extracted anthocyanins. The results indicated that use of 45–60 °C or pH 3–4 exhibited a relatively high antioxidant activity. The study will help improve extraction yield of phenolics from litchi fruit pericarp and promote better utilization of the extracted litchi anthocyanins as antioxidants

Crocins with high levels of sugar conjugation contribute to the yellow colours of early-spring flowering

Crocus sativus is the source of saffron spice, the processed stigma which accumulates glucosylated apocarotenoids known as crocins. Crocins are found in the stigmas of other Crocuses, determining the colourations observed from pale yellow to dark red. By contrast, tepals in Crocus species display a wider diversity of colours which range from purple, blue, yellow to white. In this study, we investigated whether the contribution of crocins to colour extends from stigmas to the tepals of yellow Crocus species. Tepals from seven species were analysed by UPLC-PDA and ESI-Q-TOF-MS/MS revealing for the first time the presence of highly glucosylated crocins in this tissue. beta-carotene was found to be the precursor of these crocins and some of them were found to contain rhamnose, never before reported. When crocin profiles from tepals were compared with those from stigmas, clear differences were found, including the presence of new apocarotenoids in stigmas. Furthermore, each species showed a characteristic profile which was not correlated with the phylogenetic relationship among species. While gene expression analysis in tepals of genes involved in carotenoid metabolism showed that phytoene synthase was a key enzyme in apocarotenoid biosynthesis in tepals. Expression of a crocetin glucosyltransferase, previously identified in saffron, was detected in all the samples. The presence of crocins in tepals is compatible with the role of chromophores to attract pollinators. The identification of tepals as new sources of crocins is of special interest given their wide range of applications in medicine, cosmetics and colouring industries.The laboratory is supported by the Spanish Ministerio de Ciencia e Innovacion (BIO2009-07803) and participates in the IBERCAROT network (112RT0445). Dr. Ahrazem was funded by FPCYTA through the INCRECYT Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Rubio-Moraga, A.; Ahrazem, O.; Rambla Nebot, JL.; Granell Richart, A.; Gómez Gómez, L. (2013). Crocins with high levels of sugar conjugation contribute to the yellow colours of early-spring flowering. PLoS ONE. 8(9):71946-71946. https://doi.org/10.1371/journal.pone.0071946S719467194689Auldridge, M. E., McCarty, D. R., & Klee, H. J. (2006). Plant carotenoid cleavage oxygenases and their apocarotenoid products. Current Opinion in Plant Biology, 9(3), 315-321. doi:10.1016/j.pbi.2006.03.005AKIYAMA, K. (2007). Chemical Identification and Functional Analysis of Apocarotenoids Involved in the Development of Arbuscular Mycorrhizal Symbiosis. Bioscience, Biotechnology, and Biochemistry, 71(6), 1405-1414. doi:10.1271/bbb.70023Lendzemo, V. W., Kuyper, T. W., Matusova, R., Bouwmeester, H. J., & Ast, A. V. (2007). Colonization by Arbuscular Mycorrhizal Fungi of Sorghum Leads to Reduced Germination and Subsequent Attachment and Emergence ofStriga hermonthica. Plant Signaling & Behavior, 2(1), 58-62. doi:10.4161/psb.2.1.3884Gomez-Roldan, V., Fermas, S., Brewer, P. B., Puech-Pagès, V., Dun, E. A., Pillot, J.-P., … Rochange, S. F. (2008). Strigolactone inhibition of shoot branching. Nature, 455(7210), 189-194. doi:10.1038/nature07271Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., … Yamaguchi, S. (2008). Inhibition of shoot branching by new terpenoid plant hormones. Nature, 455(7210), 195-200. doi:10.1038/nature07272Jella, P., Rouseff, R., Goodner, K., & Widmer, W. (1998). Determination of Key Flavor Components in Methylene Chloride Extracts from Processed Grapefruit Juice. Journal of Agricultural and Food Chemistry, 46(1), 242-247. doi:10.1021/jf9702149Pfander, H., & Schurtenberger, H. (1982). Biosynthesis of C20-carotenoids in Crocus sativus. Phytochemistry, 21(5), 1039-1042. doi:10.1016/s0031-9422(00)82412-7Bathaie, S. Z., & Mousavi, S. Z. (2010). New Applications and Mechanisms of Action of Saffron and its Important Ingredients. Critical Reviews in Food Science and Nutrition, 50(8), 761-786. doi:10.1080/10408390902773003Abdullaev, F. I., & Espinosa-Aguirre, J. J. (2004). Biomedical properties of saffron and its potential use in cancer therapy and chemoprevention trials. Cancer Detection and Prevention, 28(6), 426-432. doi:10.1016/j.cdp.2004.09.002Zhang Z, Wang CZ, Wen XD, Shoyama Y, Yuan CS (2013) Role of saffron and its constituents on cancer chemoprevention. Pharm Biol.Schmidt, M., Betti, G., & Hensel, A. (2007). Saffron in phytotherapy: Pharmacology and clinical uses. Wiener Medizinische Wochenschrift, 157(13-14), 315-319. doi:10.1007/s10354-007-0428-4Howes, M.-J. R., & Perry, E. (2011). The Role of Phytochemicals in the Treatment and Prevention of Dementia. Drugs & Aging, 28(6), 439-468. doi:10.2165/11591310-000000000-00000Castillo, R., Fernández, J.-A., & Gómez-Gómez, L. (2005). Implications of Carotenoid Biosynthetic Genes in Apocarotenoid Formation during the Stigma Development of Crocus sativus and Its Closer Relatives. Plant Physiology, 139(2), 674-689. doi:10.1104/pp.105.067827Moraga, Á. R., Rambla, J. L., Ahrazem, O., Granell, A., & Gómez-Gómez, L. (2009). Metabolite and target transcript analyses during Crocus sativus stigma development. Phytochemistry, 70(8), 1009-1016. doi:10.1016/j.phytochem.2009.04.022Rubio-Moraga, A., Trapero, A., Ahrazem, O., & Gómez-Gómez, L. (2010). Crocins transport in Crocus sativus: The long road from a senescent stigma to a newborn corm. Phytochemistry, 71(13), 1506-1513. doi:10.1016/j.phytochem.2010.05.026Moraga, A. R., Nohales, P. F., P�rez, J. A. F., & G�mez-G�mez, L. (2004). Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas. Planta, 219(6), 955-966. doi:10.1007/s00425-004-1299-1Harpke, D., Meng, S., Rutten, T., Kerndorff, H., & Blattner, F. R. (2013). Phylogeny of Crocus (Iridaceae) based on one chloroplast and two nuclear loci: Ancient hybridization and chromosome number evolution. Molecular Phylogenetics and Evolution, 66(3), 617-627. doi:10.1016/j.ympev.2012.10.007Mathew B (1982) The crocus - A revision of the Genus crocus; Batsford B, editor. London.Nørbæk, R., Nielsen, K., & Kondo, T. (2002). Anthocyanins from flowers of Cichorium intybus. Phytochemistry, 60(4), 357-359. doi:10.1016/s0031-9422(02)00055-9Zhu, C., Bai, C., Sanahuja, G., Yuan, D., Farré, G., Naqvi, S., … Christou, P. (2010). The regulation of carotenoid pigmentation in flowers. Archives of Biochemistry and Biophysics, 504(1), 132-141. doi:10.1016/j.abb.2010.07.028OHMIYA, A. (2011). Diversity of Carotenoid Composition in Flower Petals. Japan Agricultural Research Quarterly: JARQ, 45(2), 163-171. doi:10.6090/jarq.45.163KISHIMOTO, S., MAOKA, T., SUMITOMO, K., & OHMIYA, A. (2005). Analysis of Carotenoid Composition in Petals of Calendula (Calendula officinalisL.). Bioscience, Biotechnology, and Biochemistry, 69(11), 2122-2128. doi:10.1271/bbb.69.2122Ohmiya, A., Kishimoto, S., Aida, R., Yoshioka, S., & Sumitomo, K. (2006). Carotenoid Cleavage Dioxygenase (CmCCD4a) Contributes to White Color Formation in Chrysanthemum Petals. Plant Physiology, 142(3), 1193-1201. doi:10.1104/pp.106.087130Ohmiya, A., Sumitomo, K., & Aida, R. (2009). «Yellow Jimba»: Suppression of Carotenoid Cleavage Dioxygenase (CmCCD4a) Expression Turns White Chrysanthemum Petals Yellow. Journal of the Japanese Society for Horticultural Science, 78(4), 450-455. doi:10.2503/jjshs1.78.450Brandi, F., Bar, E., Mourgues, F., Horváth, G., Turcsi, E., Giuliano, G., … Rosati, C. (2011). Study of «Redhaven» peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism. BMC Plant Biology, 11(1), 24. doi:10.1186/1471-2229-11-24Campbell, R., Ducreux, L. J. M., Morris, W. L., Morris, J. A., Suttle, J. C., Ramsay, G., … Taylor, M. A. (2010). The Metabolic and Developmental Roles of Carotenoid Cleavage Dioxygenase4 from Potato. Plant Physiology, 154(2), 656-664. doi:10.1104/pp.110.158733Ahrazem, O., Rubio-Moraga, A., Lopez, R. C., & Gomez-Gomez, L. (2009). The expression of a chromoplast-specific lycopene beta cyclase gene is involved in the high production of saffron’s apocarotenoid precursors. Journal of Experimental Botany, 61(1), 105-119. doi:10.1093/jxb/erp283Ahrazem, O., Rubio-Moraga, A., Trapero, A., & Gomez-Gomez, L. (2011). Developmental and stress regulation of gene expression for a 9-cis-epoxycarotenoid dioxygenase, CstNCED, isolated from Crocus sativus stigmas. Journal of Experimental Botany, 63(2), 681-694. doi:10.1093/jxb/err293Moraga, Á., Mozos, A., Ahrazem, O., & Gómez-Gómez, L. (2009). Cloning and characterization of a glucosyltransferase from Crocus sativus stigmas involved in flavonoid glucosylation. BMC Plant Biology, 9(1), 109. doi:10.1186/1471-2229-9-109Tarantilis, P. A., Tsoupras, G., & Polissiou, M. (1995). Determination of saffron (Crocus sativus L.) components in crude plant extract using high-performance liquid chromatography-UV-visible photodiode-array detection-mass spectrometry. Journal of Chromatography A, 699(1-2), 107-118. doi:10.1016/0021-9673(95)00044-nWalter, M. H., Fester, T., & Strack, D. (2000). Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the «yellow pigment» and other apocarotenoids. The Plant Journal, 21(6), 571-578. doi:10.1046/j.1365-313x.2000.00708.xGómez-Miranda, B., Rupérez, P., & Leal, J. A. (1981). Changes in chemical composition during germination ofbotrytis cinerea sclerotia. Current Microbiology, 6(4), 243-246. doi:10.1007/bf01566981Cooper, C. M., Davies, N. W., & Menary, R. C. (2003). C-27 Apocarotenoids in the Flowers ofBoronia megastigma(Nees). Journal of Agricultural and Food Chemistry, 51(8), 2384-2389. doi:10.1021/jf026007cFloss, D. S., Schliemann, W., Schmidt, J., Strack, D., & Walter, M. H. (2008). RNA Interference-Mediated Repression of MtCCD1 in Mycorrhizal Roots of Medicago truncatula Causes Accumulation of C27 Apocarotenoids, Shedding Light on the Functional Role of CCD1. Plant Physiology, 148(3), 1267-1282. doi:10.1104/pp.108.125062Fester, T., Schmidt, D., Lohse, S., Walter, M., Giuliano, G., Bramley, P., … Strack, D. (2002). Stimulation of carotenoid metabolism in arbuscular mycorrhizal roots. Planta, 216(1), 148-154. doi:10.1007/s00425-002-0917-zKlingner, A., Bothe, H., Wray, V., & Marner, F.-J. (1995). Identification of a yellow pigment formed in maize roots upon mycorrhizal colonization. Phytochemistry, 38(1), 53-55. doi:10.1016/0031-9422(94)00538-5Rychener, M., Bigler, P., & Pfander, H. (1984). Isolierung und Strukturaufkl�rung von Neapolitanose (O-?-D-Glucopyranosyl-(1?2)-O-[?-D-glucopyranosyl-(1?6)]-(D-glucose), einem neuen Trisaccharid aus den Stempeln von Gartenkrokussen (Crocus neapolitanus var.). Helvetica Chimica Acta, 67(2), 386-391. doi:10.1002/hlca.19840670205Lu, S., Van Eck, J., Zhou, X., Lopez, A. B., O’Halloran, D. M., Cosman, K. M., … Li, L. (2006). The Cauliflower Or Gene Encodes a DnaJ Cysteine-Rich Domain-Containing Protein That Mediates High Levels of β-Carotene Accumulation. The Plant Cell, 18(12), 3594-3605. doi:10.1105/tpc.106.046417Rubio, A., Rambla, J. L., Santaella, M., Gómez, M. D., Orzaez, D., Granell, A., & Gómez-Gómez, L. (2008). Cytosolic and Plastoglobule-targeted Carotenoid Dioxygenases fromCrocus sativusAre Both Involved in β-Ionone Release. Journal of Biological Chemistry, 283(36), 24816-24825. doi:10.1074/jbc.m804000200Dufresne, C., Cormier, F., & Dorion, S. (1997). In VitroFormation of Crocetin Glucosyl Esters byCrocus sativusCallus Extract. Planta Medica, 63(02), 150-153. doi:10.1055/s-2006-957633Wakelin, A. M., Lister, C. E., & Conner, A. J. (2003). Inheritance and Biochemistry of Pollen Pigmentation in California Poppy (Eschscholzia californica Cham.). International Journal of Plant Sciences, 164(6), 867-875. doi:10.1086/378825Cooper, C. M., Davies, N. W., & Menary, R. C. (2009). Changes in Some Carotenoids and Apocarotenoids during Flower Development in Boronia megastigma (Nees). Journal of Agricultural and Food Chemistry, 57(4), 1513-1520. doi:10.1021/jf802610pPfister, S., Meyer, P., Steck, A., & Pfander, H. (1996). Isolation and Structure Elucidation of Carotenoid−Glycosyl Esters in Gardenia Fruits (Gardenia jasminoidesEllis) and Saffron (CrocussativusLinne). Journal of Agricultural and Food Chemistry, 44(9), 2612-2615. doi:10.1021/jf950713eDufresne, C., Cormier, F., Dorion, S., Niggli, U. A., Pfister, S., & Pfander, H. (1999). Glycosylation of encapsulated crocetin by a Crocus sativus L. cell culture. Enzyme and Microbial Technology, 24(8-9), 453-462. doi:10.1016/s0141-0229(98)00143-4Lundmark, M., Hurry, V., & Lapointe, L. (2009). Low temperature maximizes growth of Crocus vernus (L.) Hill via changes in carbon partitioning and corm development. Journal of Experimental Botany, 60(7), 2203-2213. doi:10.1093/jxb/erp103Schliemann, W., Schmidt, J., Nimtz, M., Wray, V., Fester, T., & Strack, D. (2006). Accumulation of apocarotenoids in mycorrhizal roots of Ornithogalum umbellatum. Phytochemistry, 67(12), 1196-1205. doi:10.1016/j.phytochem.2006.05.005Gómez-Gómez L, Moraga-Rubio A, Ahrazem O (2010) Understanding Carotenoid Metabolism in Saffron Stigmas: Unravelling Aroma and Colour Formation. In: Teixeira da Silva JA, editor. Functional Plant Science adn Biotechnology United Kingdon: GLOBAL SCIENCE BOOKS. pp.56–63.Schwartz, S. H., Qin, X., & Zeevaart, J. A. D. (2001). Characterization of a Novel Carotenoid Cleavage Dioxygenase from Plants. Journal of Biological Chemistry, 276(27), 25208-25211. doi:10.1074/jbc.m102146200Ilg, A., Yu, Q., Schaub, P., Beyer, P., & Al-Babili, S. (2010). Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta. Planta, 232(3), 691-699. doi:10.1007/s00425-010-1205-yAlmeida, E. R. A., & Cerdá-Olmedo, E. (2008). Gene expression in the regulation of carotene biosynthesis in Phycomyces. Current Genetics, 53(3), 129-137. doi:10.1007/s00294-007-0170-xKachanovsky, D. E., Filler, S., Isaacson, T., & Hirschberg, J. (2012). Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids. Proceedings of the National Academy of Sciences, 109(46), 19021-19026. doi:10.1073/pnas.1214808109Walter, M. H., Floss, D. S., & Strack, D. (2010). Apocarotenoids: hormones, mycorrhizal metabolites and aroma volatiles. Planta, 232(1), 1-17. doi:10.1007/s00425-010-1156-3GIACCIO, M. (2004). Crocetin from Saffron: An Active Component of an Ancient Spice. Critical Reviews in Food Science and Nutrition, 44(3), 155-172. doi:10.1080/10408690490441433Hosseinzadeh, H., & Nassiri-Asl, M. (2012). Avicenna’s (Ibn Sina) the Canon of Medicine and Saffron (Crocus sativus): A Review. Phytotherapy Research, 27(4), 475-483. doi:10.1002/ptr.4784Ochiai, T., Shimeno, H., Mishima, K., Iwasaki, K., Fujiwara, M., Tanaka, H., … Soeda, S. (2007). Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo. Biochimica et Biophysica Acta (BBA) - General Subjects, 1770(4), 578-584. doi:10.1016/j.bbagen.2006.11.01

Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana: A factor of potential importance for auxin–cytokinin-regulated development

One of the most long-lived models in plant science is the belief that the long-distance transport and ratio of two plant hormones, auxin and cytokinin, at the site of action control major developmental events such as apical dominance. We have used in vivo deuterium labeling and mass spectrometry to investigate the dynamics of homeostatic cross talk between the two plant hormones. Interestingly, auxin mediates a very rapid negative control of the cytokinin pool by mainly suppressing the biosynthesis via the isopentenyladenosine-5′-monophosphate-independent pathway. In contrast, the effect of cytokinin overproduction on the entire auxin pool in the plant was slower, indicating that this most likely is mediated through altered development. In addition, we were able to confirm that the lateral root meristems are likely to be the main sites of isopentenyladenosine-5′-monophosphate-dependent cytokinin synthesis, and that the aerial tissue of the plant surprisingly also was a significant source of cytokinin biosynthesis. Our demonstration of shoot-localized synthesis, together with data demonstrating that auxin imposes a very rapid regulation of cytokinin biosynthesis, illustrates that the two hormones can interact also on the metabolic level in controlling plant development, and that the aerial part of the plant has the capacity to synthesize its own cytokinin independent of long-range transport from the root system