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The effects of 1-MCP in cyclodextrin-based nanosponges to improve the vase life of Dianthus caryophyllus cut flowers
Postharvest Biology and Technology 59 (2011) 200–205
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
The effects of 1-MCP in cyclodextrin-based nanosponges to improve the vase life of Dianthus caryophyllus cut flowers Ludovica Seglie a,∗, Katia Martina b, Marco Devecchi a, Carlo Roggero d, Francesco Trotta c, Valentina Scariot a a
Department of Agronomy, Forest and Land Management, Faculty of Agriculture, University of Turin, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy Department of Pharmaceutical Science and Technology, University of Turin, Via Pietro Giuria 9, 10125 Turin, Italy c Department of Chemistry IFM, University of Turin, Via Pietro Giuria 7, 10125 Turin, Italy d Sea Marconi Technologies S. a. s., Via Ungheria 20, 10093 Collegno (TO), Italy b
a r t i c l e
i n f o
Article history: Received 8 April 2010 Accepted 15 August 2010 Keywords: Carnation Ethylene antagonist Longevity Nanocarriers Senescence
a b s t r a c t The present research investigated the effects of a non-volatile formulation of 1-methylcyclopropene (1-MCP) embedded in different cyclodextrin (CD)-based nanosponges (NSs) to extend the postharvest longevity of an ethylene-sensitive carnation cultivar. Cut flowers of Dianthus caryophyllus L. ‘Idra di Muraglia’ were treated with ␣- and -CD-based nanosponge-1-MCP complexes (␣- and -NS complexes) in tap water to achieve two different concentrations of active ingredient (0.25 and 0.5 L L−1 ). Treated flowers were compared to cut stems exposed to equivalent concentrations of volatile 1-MCP as well as a tap water control with or without pure ␣- and -NS. Identical nanoporous compounds were applied by perfusion to yield a total of 15 treatments. Twenty-four hours after the treatments were applied, the cut flowers were exposed to exogenous ethylene (1 ± 0.2 L L−1 ) for 24 h. The postharvest carnation flower and leaf quality in addition to ethylene production levels were determined daily (beginning 24 h after treatment). None of the ␣-NS complex applications statistically improved the vase life of cut flowers; however, -NS complexes were effective in preventing senescence, reducing ethylene production (measured at nearly nil after 11 d), and maintaining original petal color longer. These results were particularly strong at the lowest concentration (0.25 L L−1 ) of -NS complex. Overall, this method promoted cut flower longevity (loss of ornamental value after 14.7 d; complete damage at day 18.5) better than the commercial 1-MCP gaseous application method. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Vase life is the main determinant of ornamental cut flower commercial value (Nukui et al., 2004). Exposure to ethylene can reduce flower longevity by causing undesirable physiological disorders to vegetative and flowering organs. Ethylene-mediated premature floral senescence has been noted in several species, such as Pelargonium and Begonia (Brown, 1997). Many species have been shown to wilt in response to ethylene, including orchids (Phalaenopsis), Hibiscus (C¸elikel and Reid, 2002a), and carnation (Diathus caryophyllus) (Woltering and van Doorn, 1988; Serek et al., 1995a,b). In other species, such as Antirrhinum majus, Rosa hybrida (Serek et al., 1995a), and wax flower (Chamelaucium uncinatum) (Macnish et al.,
Abbreviations: CD, cyclodextrin; 1-MCP, 1-methylcyclopropene; NS complex, CD-based-nanosponge-1-MCP complex; VS, visual check for symptoms of senescence variation. ∗ Corresponding author. Tel.: +39 011 6708789, fax: +39 011 6708798. E-mail address: [email protected] (L. Seglie). 0925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2010.08.012
2000), abscission of petals or flowers when ethylene was present has been demonstrated. The negative effects of ethylene can be significantly delayed by treatment with inhibitors of ethylene action, such as silver thiosulfate (STS) (Veen, 1979), 2,5-norbornadiene (2,5-NBD) (Sisler et al., 1983; Wang and Woodson, 1989), and 1-methylcyclopropene (1-MCP) (Serek et al., 1995b, 2006a). Particularly, 1-MCP shows several advantages, as it is a non-toxic cyclic olefin that competes with ethylene at the receptor level (Sisler et al., 1996). Moreover, many studies of carnation cut flowers have reported 1-MCP to be effective at preventing ethylene responses even at very low concentrations, in the range of nL L−1 (Sisler and Serek, 2003). However, the gaseous nature of 1-MCP leads to several treatment difficulties: (i) plant material must be maintained in enclosed areas to prevent gas leakage which can be commercially difficult; (ii) some ornamental species require continuous or repeated applications (Serek and Sisler, 2005; Serek et al., 2006b); (iii) the action of commercial 1-MCP appears to be strongly reduced by treatment temperature (0–5 ◦ C) and by the presence of exogenous ethylene (C¸elikel and Reid, 2002b; Reid and C¸elikel, 2008). The development
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of compounds to inhibit ethylene action, in a non-volatile formulation, would be advantageous. Sisler et al. (2001, 2009), Sisler and Serek (2003) and Seglie et al. (2010) treated novel cyclopropenes as salts, showing very positive qualities when applied as a spray, but with limited efficacy when applied in solution. Recently, nanosponges have been synthesized from natural derivatives of starch (cyclodextrins or CDs) (Cavalli et al., 2006). These nano-sized colloidal carriers contain -CDs as buildingblocks, linked with carbonate groups to form a highly cross-linked network. The cyclodextrin-based nanosponge (NS) structures contain both CD lypophilic cavities and carbonate bridges, leading to a network of hydrophilic channels (Trotta and Cavalli, 2009). They show a highly branched structure and, as a polymeric conjugate, cooperation among several CD units may increase the stability of the inclusion complex with bioactive molecules. Their main feature is their tunable polarity. Nanosponges are solid, obtainable in crystalline form, insoluble in water and in common organic solvents, non-toxic, porous, stable above 300 ◦ C, and may be used to encapsulate, carry, and/or release a great variety of substances. They facilitate gradual release of different compounds over extended times, increasing their bioavailability (Vyas et al., 2008; Trotta and Cavalli, 2009). The initial use of NSs was mainly for removal of persistent organic pollutants in water purification (POPs) (Li and Ma, 1999; Arkas et al., 2006). Recently, NSs have been tested in the chemical–pharmaceutical field, in which they have been shown to enhance overall drug pharmaco-kinetics by increasing their availability at the target site (Loftsson and Olafsson, 1998; Swaminathan et al., 2007). In floriculture, NSs are relatively unexplored. They have been developed and proposed to deliver the anti-ethylene compound 1-MCP to improve cut flower vase life. Specifically, the present study evaluated the effectiveness of a non-volatile form of 1-MCP within different NSs (Trotta et al. ultrasound-assisted synthesis of cyclodextrin-based nanosponges patent WO2006/002814) to extend the postharvest longevity of an ethylene-sensitive carnation cultivar.
2. Materials and methods 2.1. Synthesis and characterization of 1-methylcyclopropene embedded in cyclodextrin-based nanosponges Commercially available reagents and solvents were used without further purification. Native ␣- and -cyclodextrins were kindly provided by Wacker Chemie (Munich, Germany). GC analyses were carried out on a Shimadzu-14B gas chromatograph equipped with a flame ionization detector; GC–MS analyses were conducted on an Agilent Technologies 6850 Network GC System with 5973 Network Mass Selective Detector (Santa Clara, California). Nanosponges were prepared as reported by Trotta and Cavalli (2009). The 1-MCP was synthesized from 2-methyl-3-chloro-propene by a modified procedure of Fisher and Applequist (1965). The gaseous 1-MCP was distilled into a flask, kept in a refrigerated bath, containing a suspension of NSs in buffer solution (0.2 M acetic acid, 0.2 M sodium acetate). The aqueous suspension of NSs and 1MCP was left overnight in the refrigerator, then filtered and dried to obtain a powder. To confirm the presence of 1-MCP contained within NS, an aliquot was treated with 1-(trimethyl silyloxy)-1,3butadiene in tetrahydrofuran (THF), and then the mixture was stirred for 1 h at room temperature. One hundred microlitres of this solution were diluted with 1 mL of diethyl ether; the Diels Alder adduct was then analyzed by gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR) (McEwen et
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al., 1996; Keeler, 2005). The final concentration of 1-MCP included in NSs was quantified as 6% using a calibration curve. 2.2. Plant materials Untreated ethylene-sensitive flowers of a model species for senescence studies (D. caryophyllus L. ‘Idra di Muraglia’, white flower) were grown in standard greenhouse conditions (20 ± 2 ◦ C day/night temperature and 60% relative humidity) in Sanremo, Liguria, Italy (latitude 43◦ 49 9.12 N, longitude 7◦ 45 43.92 E). Cut stems at commercial maturity (sepals standing vertically, petal colors and stems very vivid) were assigned to treatments within 24 h of arrival in the postharvest laboratory of the Department of Agronomy, Forest and Land Management at the University of Turin. Stems were kept in a standard vase life room at 20 ± 2◦ C, 60% RH, and 46 mol m−2 s−1 cool white light (meter model HT307; HT, Faenza, Italy) for 12 h per day. The experimental conditions were intended to reproduce interior home conditions. 2.3. Experimental trials Ten stems (each 30 cm long) were used per treatment. Each experiment was performed at least twice. All the cut flowers were re-cut, labeled, and placed in tap water. ␣- and -CD-based nanosponge-1-MCP complexes (␣- and NS; 6% a.i.) were weighed to obtain two different concentrations of active ingredient (0.25 and 0.5 L L−1 ), and applied to cut flowers both suspended in the tap water and by perfusion. Efficiency of these treatments was compared to equivalent concentrations of volatile 1-MCP (3.3% a.i., SmartFreshTM , AgroFresh Inc., USA), to pure ␣- and -NS, either in vase suspension or sprayed, and to tap water. Twenty-four hours after treatment, cut flowers were enclosed in an air tight cabinet (112 L) and exposed to exogenous ethylene (1 ± 0.2 L L−1 ) for 24 h. 2.4. Data collection and statistical analysis Every day, we evaluated the postharvest performance of the flowers according to the following factors: visual check for symptoms of variation in senescence (VS), petal color, leaf chlorophyll content (SPAD), and endogenous ethylene production. Visual senescence was rated according to a rating scale from 0 to 2, in which 0 = no visible senescence, 1 = initial senescence, 2 = complete senescence. The loss of ornamental value of flowers was considered when they reached level 1 on the scale (Seglie et al., 2010). Petal color (L*, a*, b* space) was measured on flowers using a Spectrophotometer CM-2600 (Konica Minolta Sensing Inc., Osaka, Japan). Chroma (C*) and hue angle (h◦ ) were calculated according to Onozaky et al. (1999). Chlorophyll content was indirectly measured in leaves through a Chlorophyll Meter SPAD-502 instrument (Konica Minolta Sensing Inc., Osaka, Japan). Beginning 24 h after treatment, ethylene production was measured daily by keeping a single stem in air tight tubes (1 L) containing 300 mL of tap water. The ethylene concentration was monitored by a digital Agilent Technologies gas chromatograph, 6890N Network GC system (Santa Clara, California). The gas carrier was N2 at 40 mL min−1 , and column temperature was 60 ◦ C. For each treatment three samples were considered. Photo-documentation at different stages of senescence was carried out. Data were registered until all the cut flowers appeared completely damaged (day 19), after which they were analyzed statistically using SPSS software Inc. (Chicago, United States). The analysis of variance (ANOVA) was established through the
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Fig. 1. Longevity (in days) of Dianthus caryophyllus L. ‘Idra di Muraglia’ cut flowers, placed in tap water, treated with: ␣- and -NS complex (0.25 and 0.5 L L−1 1-MCP), either sprayed (sp) or in vase suspension (su); and gaseous 1-MCP (0.25 and 0.5 L L−1 , for 6 h). Tap water with or without pure ␣- and -NS, either sprayed or in vase suspension, was used as controls. Twenty-four hours after treatment, plant material was exposed to exogenous ethylene (1 ± 0.2 L L−1 , for 24 h). Number of days to reach the stage of initial senescence (level 1) and complete senescence (level 2) were considered. *Mean separation within columns by the Ryan–Einot–Gabriel–Welsch’s multiple stepdown F (REGW-F) test, P ≤ 0.001.
Ryan–Einot–Gabriel–Welsch’s multiple stepdown F (REGW-F) test (P < 0.05). 3. Results 3.1. Symptoms of senescence variation A visual check for symptoms of senescence variation (Fig. 1) failed to show any difference among cut flowers kept in tap water, with or without NSs. Among the 1-MCP applications, the -NS complex showed the best results. Specifically, this complex was more effective in a tap water suspension than as a spray application. The lowest -NS complex concentration (0.25 L L−1 ) prevented flower wilting (initial senescence at 14.7 d; complete senescence at 18.5 d) better than the highest concentration (0.5 L L−1 ; level 1, 12 d; level 2, 15.7 d). Furthermore, this treatment (0.25 L L−1 -NS complex) was even more effective than the 1-MCP gaseous application at both 0.25 L L−1 (level 1, average vase life 9.0 d; level 2, average vase life 10.5 d), and 0.5 L L−1 (level 1, 10.5 d; level 2, 13.8 d) concentrations (Fig. 2). None of the ␣-NS complex applications statistically improved the vase life of cut flowers, compared to the control in tap water (level 1, average longevity 2.8 d; and completely damaged after 5.2 d).
3.2. Flower and leaf color Petal color started to show significant differences in L*, C*, and h◦ values among the treatments after five days from the start of the experiment (Table 1). The results were strictly correlated with VS. The lowest concentration of -NS complex suspended in tap water (0.25 L L−1 ) maintained the nearly pure color of carnation petals better. L*, C*, and h◦ were not statistically different among flowers supplied with the highest -NS complex concentration suspended in tap water, or treated with both doses of gaseous 1-MCP. Both concentrations of -NS complex perfusion statistically improved the L*, C*, and h◦ of petals (white petal color), compared to the controls in tap water with or without pure ␣- and -NS, and to the ␣-NS complex treatments. No appreciable differences were detected in L*, C*, and h◦ among the control flowers nor among the flowers treated with ␣-NS complex at either concentrations. SPAD values for measuring the chlorophyll content in leaves are not shown as no statistical differences were noted among treatments.
3.3. Endogenous ethylene production Fig. 3 shows differences in ethylene production among carnation cut flowers that underwent the 15 treatments. Generally,
Fig. 2. D. caryophyllus ‘Idra di Muraglia’ flowers treated with 0.25 L L−1 -NS complex suspended in tap water, compared to 0.5 L L−1 1-MCP for 6 h and the control in tap water, after 9 d.
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Table 1 Effects on color variation of 1-MCP, included or not in ␣- and -NS, both sprayed (sp) and suspended in tap water (su), compared to commercial gaseous application, and to control flowers kept in tap water with and withheld pure ␣- and -NS. The L* (lightness), C* (chroma), and h◦ (hue angle) values were measured after five days in petals of D. caryophyllus ‘Idra di Muraglia’. Treatments
5th day L*
Tap water 1-MCP (0.25 L L−1 ) 1-MCP (0.5 L L−1 ) Pure ␣-NS Pure -NS ␣-NS complex (0.25 L L−1 ) ␣-NS complex (0.5 L L−1 ) -NS complex (0.25 L L−1 ) -NS complex (0.5 L L−1 ) A
81.4 e 88.9 b 89.7 b sp 81.6 e 81.8 e 82.8 de 82.7 de 86.1 c 86.7 c
h◦
C* A
19.1 a 13.5 d 12.5 d su 82.0 e 81.9 e 83.7 d 83.6 d 91.1 a 89.3 b
sp 19.1 a 18.9 a 18.0 ab 18.2 ab 15.5 c 15.0 c
121.2 a 114.2 dc 112.7 d su 18.7 a 18.8 a 17.6 b 16.9 b 10.3 e 12.2 d
sp 119.3 a 120.3 a 118.8 ab 118.5 ab 115.9 c 115.2 c
su 119.4 a 119.6 a 117.9 b 117.8 b 110.5 e 113.2 d
Mean separation within columns by the Ryan–Einot–Gabriel–Welsch’s multiple stepdown F (REGW-F) test, P ≤ 0.001.
Fig. 3. Mean values of the endogenous ethylene production in Dianthus caryophyllus ‘Idra di Muraglia’ cut flowers placed in tap water, treated with: ␣- and -NS complex (0.25 and 0.5 L L−1 1-MCP), either sprayed (sp) or in vase suspension (su); and gaseous 1-MCP (0.25 and 0.5 L L−1 , for 6 h). Tap water with or without pure ␣- and -NS, either sprayed or in vase suspension, was used as controls.
the control flowers produced ethylene within 24 h, and peaked at day 8 with a range of 0.55–0.63 L L−1 . While the two application methods and concentrations of ␣-NS complex slowed the endogenous ethylene production, after 15 d they did not reduce the final level (range of 0.57–0.63 L L−1 ), compared to the controls (0.63–0.68 L L−1 ). After 15 d, almost no ethylene production was detected in flowers treated with either low or high concentrations of 1-MCP (0.07 and 0.13 L L−1 ), and of -NS complex in tap water (0.04 and 0.08 L L−1 , respectively). A low level of endogenous ethylene was also measured in flowers sprayed with both doses of -NS complex (0.21 and 0.18 L L−1 , respectively).
4. Discussion In floriculture, cut flowers are a very important economic resource in the world. Carnation is one of the most popular species and it is considered as a model in studies on postharvest preservation. Since there was no previous information about NS activity in flowers, we evaluated both the adsorption and the possible phytotoxicity of two types of NS (␣ and ) on cut stems.
Both CDs (Mäkelä et al., 1987) and NSs (diameter 517.7 ± 12.4 nm; Swaminathan et al., 2010) cause the violet color of a phenolphthalein dye solution to disappear, a property that allowed us to follow the flow of the NSs through the xylem vessels (38–70 m) until their presence was confirmed to be at the top of the stems (data not shown). This combined application and perfusion method (also commonly used in postharvest) were adopted for this investigation. The results showed that application of the NS suspended in tap water was more effective than spray application. The reduced effect of the latter could be attributed to slow adsorption of molecules through petal and leaf tissues, as previously detected in banana by Sisler et al. (2009). Moreover, the delivery of 1-MCP from the adsorbed -NS complex may prolong the bio-availability of the complex as compared to the sprayed applications and to gaseous 1-MCP. Different types of NS could have different effects on guest molecules. The binding of guest molecules within the host NS is not fixed or permanent; rather, it is a dynamic equilibrium. Binding strength depends on how well the host–guest complex fits together and on specific local interactions between surface atoms (Del Valle, 2004). When guest 1-MCP is temporarily locked
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or caged within the host cavity, it brings about beneficial or negative modifications of the guest molecule, according to the ability of a NS to form a proper inclusion complex. For this reason, we tested ␣- and -NS that have the same size, but differ with respect to their interior molecule reticulations, internal cavity diameters, volumes, and bonding constants (Del Valle, 2004). In this study, -NS complexes outperformed the ␣-NS complexes. Since the ␣-NS complex contained similar amounts of 1-MCP, its reduced effect on cut flower longevity could be attributed to a stronger bonding constant or to low kinetics of delivery. To assess the potential for phytotoxicity, treatments with pure ␣- and -NS were compared to controls in tap water. As no differences in aesthetic or longevity performances were detected, the results showed that the complexes were not toxic and were totally colourless and odourless for flowers, which is consistent with observations in several pharmaceutical applications of natural CD and their derivatives (Irie and Uekama, 1997; Thompson, 1997). Moreover, the spray application of pure ␣- and -NS did not favor the development of any particular stains on the petal surface. This research into anti-ethylene activity has been supported by the work of others who have shown that many ethylene-sensitive cultivars of D. caryophyllus, such as ‘Sandra’ and ‘White Sim’, react well to 1-MCP treatments (Sisler et al., 1986, 1996; Reid and C¸elikel, 2008). Senescence in carnation cut flowers usually occurs in a few days (2.3) (Serek et al., 1995a), but application of 1-MCP allowed longevity to increase to 12–15 d (Sisler and Serek, 1997). In our study, performed by perfusion, the highest dose of -NS complex was as effective as the lowest concentration of gaseous 1-MCP. Suspended in tap water, the lowest concentration of the -NS complex outperformed the highest concentration of gaseous 1-MCP, increasing the longevity of carnation cut flowers until day 18.5. The efficacy of this complex was confirmed by flower color results, showing that it could also favor the maintenance of the original white color of petals, whether suspended or sprayed, longer than in the controls. In conclusion, a -NS complex, supplied in the conservation solution, was a very effective substitute for gaseous 1-MCP, preventing pigment degradation in petals, and reducing endogenous ethylene production. The use of NSs to carry an anti-ethylene substance could provide a continuous supply of the active ingredient over an extended time, reducing the usual high concentration associated with common practice, which would allow for the potential to lower production costs. The present study represents the first application of NS in an important economic field, such as floriculture. Future commercial use of nanosponges to extend the vase life of cut flowers, and, eventually, of ornamental potted plants, may require further investigation on different -NS reticulations to evaluate optimization. The use of ␣-NS complexes could also be broadened for transporting other preservative compounds. Acknowledgements The authors gratefully acknowledge the entire staff of the SeaMarconi Technologies S. a. s., for their support, AgroFresh Inc. (Rohm and Haas) for providing SmartFreshTM , and Ms. Joan Leonard for language improvements. This research was funded by the Italian Ministry of Education, University and Research (Miur) – PRIN 2007TNTWH7. References Arkas, M., Allabashi, R., Tsiourvas, D., Mattausch, E.M., Perfler, R., 2006. Organic/inorganic hybrid filters based on dendritic and cyclodextrin “nanosponges” for the removal of organic pollutants from water. Environ. Sci. Technol. 40, 2771–2777. Brown, K.M., 1997. Ethylene and abscission. Physiol. Plantarum 100, 567–576.
Cavalli, R., Trotta, F., Tumiatti, W., 2006. Cyclodextrin-based nanosponges for drug delivery. J. Incl. Phenom. Macrocycl. Chem. 56, 209–213. C¸elikel, F.G., Reid, M.S., 2002a. Postharvest handling of stock (Matthiola incana). HortScience 37, 144–147. C¸elikel, F.G., Reid, M.S., 2002b. An alternative model for the inhibition of ethylene action by 1-methylcyclopropene (1-MCP). In: Mencarelli, F., Serra, G. (Eds.), Tecniche postraccolta dei prodotti ortoflorofrutticoli. ARSIA, Florence, Italy. Del Valle, E.M.M., 2004. Cyclodextrins and their uses: a review. Process Biochem. 39, 1033–1046. Fisher, F., Applequist, D.E., 1965. Synthesis of 1-methylcyclopropene. J. Org. Chem. 30, 2089–2090. Irie, T., Uekama, K., 1997. Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation. J. Pharm. Sci. 86, 147–162. Keeler, J., 2005. Understanding NMR Spectroscopy. John Wiley & Sons Inc., Hoboken, NJ, USA. Li, D., Ma, M., 1999. Nanoporous polymers: new nanosponge absorbent media. Filtr. Separat. 36, 26–28. Loftsson, T., Olafsson, J.H., 1998. Cyclodextrins: new drug delivery systems in dermatology. Int. J. Dermatol. 37, 241–246. Macnish, A.J., Joyce, D.C., Hofman, P.J., Simons, D.H., Reid, M.S., 2000. 1Methylcyclopropene treatment efficacy in preventing ethylene perception in banana fruit and grevillea and waxflowers. Aust. J. Exp. Agr. 40, 471–481. Mäkelä, M., Korpela, T., Laakso, S., 1987. Colorimetric determination of beta cyclodextrin: two assay modifications based on molecular complexation of phenolphthalein. J. Biochem. Biophys. Methods 14, 85–92. McEwen, C.N., Kitson, F.G., Larsen, B.S., 1996. Gas Chromatography and Mass Spectrometry: A Practical Guide. Academic Press, Boston, Massachusetts, USA. Nukui, H., Kudo, S., Yamashita, A., Satoh, S., 2004. Repressed ethylene production in the gynoecium of long-lasting flowers of the carnation ‘White Candle’: role of gynoecium in carnation flower senescence. J. Exp. Bot. 55, 641– 650. Onozaky, T., Mato, M., Shibata, M., Ikeda, H., 1999. Differences in flower colour and pigment composition among white carnation (Dianthus caryophyllus L.) cultivars. Sci. Hortic. 82, 103–111. Reid, M.S., C¸elikel, F.G., 2008. Use of 1-methylcyclopropene in ornamentals: carnations as a model system for understanding mode of action. HortScience 43, 95–98. Seglie, L., Sisler, E.C., Mibus, H., Serek, M., 2010. Use of a non-volatile 1-MCP formulation, N,N-dipropil(1-cyclopropenylmethyl)amine, for improvement of postharvest quality of ornamental crops. Postharvest Biol. Technol. 56, 117–122. Serek, M., Sisler, E.C., Reid, M.S., 1995a. Effects of 1-MCP on the vase life and ethylene response of cut flowers. Plant Growth Regul. 16, 93–97. Serek, M., Sisler, E.C., Reid, M.S., 1995b. 1-Methylcyclopropene, a novel gaseous inhibitor of ethylene action, improves the vase life of fruits, cut flowers and potted plants. Acta Hortic. 394, 337–346. Serek, M., Sisler, E.C., 2005. Impact of 1-MCP on postharvest quality of ornamentals. In: APEC Symp. Quality Management of Postharvest System Proceedings, pp. 121–128. Serek, M., Woltering, E.J., Sisler, E.C., Frello, S., Sriskandarajah, S., 2006a. Controlling ethylene at the receptor level. Biotechnol. Adv. 24, 368–381. Serek, M., Sisler, E.C., Frello, S., Sriskandarajah, S., 2006b. Postharvest technologies for extending the shelf life of ornamental crops. Int. J. Postharvest Technol. Inn. 1, 69–75. Sisler, E.C., Reid, M.S., Fujino, D.W., 1983. Investigation of the model of action of ethylene in carnation senescence. Acta Hortic. 141, 229–234. Sisler, E.C., Reid, M.S., Yang, S.F., 1986. Effect of antagonists of ethylene action on binding of ethylene in cut carnations. Plant Growth Regul. 4, 213– 218. Sisler, E.C., Dupille, E., Serek, M., 1996. Effect of 1-methylcyclopropene and methylenecyclopropane on ethylene binding and ethylene action on cut carnations. Plant Growth Regul. 18, 79–86. Sisler, E.C., Serek, M., 1997. Inhibitors of ethylene responses in plants at the receptor level: recent developments. Physiol. Plantarum 100, 577–582. Sisler, E.C., Serek, M., Roh, K.A., Goren, R., 2001. The effect of chemical structure on the antagonism by cyclopropenes of ethylene responses in banana. Plant Growth Regul. 33, 107–110. Sisler, E.C., Serek, M., 2003. Compounds interacting with the ethylene receptor in plants. Plant Biol. 5, 473–480. Sisler, E.C., Goren, R., Apelbaum, A., Serek, M., 2009. The effect of dialkylamine compounds and related derivatives of 1-methylcyclopropene in counteracting ethylene responses in banana fruit. Postharvest Biol. Technol. 51, 43– 48. Swaminathan, S., Vavia, P.R., Trotta, F., Torne, S., 2007. Formulation of betacyclodextrin based nanosponges of itraconazole. J. Incl. Phenom. Macrocycl. Chem. 57, 89–94. Swaminathan, S., Pastero, L., Serpe, L., Trotta, F., Vavia, P., Aquilano, D., Trotta, M., Zara, G.P., Cavalli, R., 2010. Cyclodextrin-based nanosponges encapsulating camptothecin: physicochemical characterization, stability and cytotoxicity. Eur. J. Pharm. Biopharm. 74, 193–201. Thompson, D.O., 1997. Cyclodextrins-enabling excipients: their present and future use in pharmaceuticals. Crit. Rev. Ther. Drug Carrier Syst. 14, 1– 104.
L. Seglie et al. / Postharvest Biology and Technology 59 (2011) 200–205 Trotta, F., Cavalli, R., 2009. Characterization and application of new hyper-crosslinked cyclodextrins. Compos. Interface 166, 39–48. Veen, H., 1979. Effects of silver on ethylene synthesis and action in cut carnations. Planta 145, 467–470. Vyas, A., Saraf, S., Saraf, S., 2008. Cyclodextrin based novel drug delivery systems. J. Incl. Phenom. Macrocycl. Chem. 62, 23–42.
205
Wang, H., Woodson, W.R., 1989. Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene. Plant Physiol. 89, 434–438. Woltering, E.J., van Doorn, W.G., 1988. Role of ethylene in senescence of petalsmorphological and taxonomical relationship. J. Exp. Bot. 39, 1605–1616.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
The effects of 1-MCP in cyclodextrin-based nanosponges to improve the vase life of Dianthus caryophyllus cut flowers Ludovica Seglie a,∗, Katia Martina b, Marco Devecchi a, Carlo Roggero d, Francesco Trotta c, Valentina Scariot a a
Department of Agronomy, Forest and Land Management, Faculty of Agriculture, University of Turin, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy Department of Pharmaceutical Science and Technology, University of Turin, Via Pietro Giuria 9, 10125 Turin, Italy c Department of Chemistry IFM, University of Turin, Via Pietro Giuria 7, 10125 Turin, Italy d Sea Marconi Technologies S. a. s., Via Ungheria 20, 10093 Collegno (TO), Italy b
a r t i c l e
i n f o
Article history: Received 8 April 2010 Accepted 15 August 2010 Keywords: Carnation Ethylene antagonist Longevity Nanocarriers Senescence
a b s t r a c t The present research investigated the effects of a non-volatile formulation of 1-methylcyclopropene (1-MCP) embedded in different cyclodextrin (CD)-based nanosponges (NSs) to extend the postharvest longevity of an ethylene-sensitive carnation cultivar. Cut flowers of Dianthus caryophyllus L. ‘Idra di Muraglia’ were treated with ␣- and -CD-based nanosponge-1-MCP complexes (␣- and -NS complexes) in tap water to achieve two different concentrations of active ingredient (0.25 and 0.5 L L−1 ). Treated flowers were compared to cut stems exposed to equivalent concentrations of volatile 1-MCP as well as a tap water control with or without pure ␣- and -NS. Identical nanoporous compounds were applied by perfusion to yield a total of 15 treatments. Twenty-four hours after the treatments were applied, the cut flowers were exposed to exogenous ethylene (1 ± 0.2 L L−1 ) for 24 h. The postharvest carnation flower and leaf quality in addition to ethylene production levels were determined daily (beginning 24 h after treatment). None of the ␣-NS complex applications statistically improved the vase life of cut flowers; however, -NS complexes were effective in preventing senescence, reducing ethylene production (measured at nearly nil after 11 d), and maintaining original petal color longer. These results were particularly strong at the lowest concentration (0.25 L L−1 ) of -NS complex. Overall, this method promoted cut flower longevity (loss of ornamental value after 14.7 d; complete damage at day 18.5) better than the commercial 1-MCP gaseous application method. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Vase life is the main determinant of ornamental cut flower commercial value (Nukui et al., 2004). Exposure to ethylene can reduce flower longevity by causing undesirable physiological disorders to vegetative and flowering organs. Ethylene-mediated premature floral senescence has been noted in several species, such as Pelargonium and Begonia (Brown, 1997). Many species have been shown to wilt in response to ethylene, including orchids (Phalaenopsis), Hibiscus (C¸elikel and Reid, 2002a), and carnation (Diathus caryophyllus) (Woltering and van Doorn, 1988; Serek et al., 1995a,b). In other species, such as Antirrhinum majus, Rosa hybrida (Serek et al., 1995a), and wax flower (Chamelaucium uncinatum) (Macnish et al.,
Abbreviations: CD, cyclodextrin; 1-MCP, 1-methylcyclopropene; NS complex, CD-based-nanosponge-1-MCP complex; VS, visual check for symptoms of senescence variation. ∗ Corresponding author. Tel.: +39 011 6708789, fax: +39 011 6708798. E-mail address: [email protected] (L. Seglie). 0925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2010.08.012
2000), abscission of petals or flowers when ethylene was present has been demonstrated. The negative effects of ethylene can be significantly delayed by treatment with inhibitors of ethylene action, such as silver thiosulfate (STS) (Veen, 1979), 2,5-norbornadiene (2,5-NBD) (Sisler et al., 1983; Wang and Woodson, 1989), and 1-methylcyclopropene (1-MCP) (Serek et al., 1995b, 2006a). Particularly, 1-MCP shows several advantages, as it is a non-toxic cyclic olefin that competes with ethylene at the receptor level (Sisler et al., 1996). Moreover, many studies of carnation cut flowers have reported 1-MCP to be effective at preventing ethylene responses even at very low concentrations, in the range of nL L−1 (Sisler and Serek, 2003). However, the gaseous nature of 1-MCP leads to several treatment difficulties: (i) plant material must be maintained in enclosed areas to prevent gas leakage which can be commercially difficult; (ii) some ornamental species require continuous or repeated applications (Serek and Sisler, 2005; Serek et al., 2006b); (iii) the action of commercial 1-MCP appears to be strongly reduced by treatment temperature (0–5 ◦ C) and by the presence of exogenous ethylene (C¸elikel and Reid, 2002b; Reid and C¸elikel, 2008). The development
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of compounds to inhibit ethylene action, in a non-volatile formulation, would be advantageous. Sisler et al. (2001, 2009), Sisler and Serek (2003) and Seglie et al. (2010) treated novel cyclopropenes as salts, showing very positive qualities when applied as a spray, but with limited efficacy when applied in solution. Recently, nanosponges have been synthesized from natural derivatives of starch (cyclodextrins or CDs) (Cavalli et al., 2006). These nano-sized colloidal carriers contain -CDs as buildingblocks, linked with carbonate groups to form a highly cross-linked network. The cyclodextrin-based nanosponge (NS) structures contain both CD lypophilic cavities and carbonate bridges, leading to a network of hydrophilic channels (Trotta and Cavalli, 2009). They show a highly branched structure and, as a polymeric conjugate, cooperation among several CD units may increase the stability of the inclusion complex with bioactive molecules. Their main feature is their tunable polarity. Nanosponges are solid, obtainable in crystalline form, insoluble in water and in common organic solvents, non-toxic, porous, stable above 300 ◦ C, and may be used to encapsulate, carry, and/or release a great variety of substances. They facilitate gradual release of different compounds over extended times, increasing their bioavailability (Vyas et al., 2008; Trotta and Cavalli, 2009). The initial use of NSs was mainly for removal of persistent organic pollutants in water purification (POPs) (Li and Ma, 1999; Arkas et al., 2006). Recently, NSs have been tested in the chemical–pharmaceutical field, in which they have been shown to enhance overall drug pharmaco-kinetics by increasing their availability at the target site (Loftsson and Olafsson, 1998; Swaminathan et al., 2007). In floriculture, NSs are relatively unexplored. They have been developed and proposed to deliver the anti-ethylene compound 1-MCP to improve cut flower vase life. Specifically, the present study evaluated the effectiveness of a non-volatile form of 1-MCP within different NSs (Trotta et al. ultrasound-assisted synthesis of cyclodextrin-based nanosponges patent WO2006/002814) to extend the postharvest longevity of an ethylene-sensitive carnation cultivar.
2. Materials and methods 2.1. Synthesis and characterization of 1-methylcyclopropene embedded in cyclodextrin-based nanosponges Commercially available reagents and solvents were used without further purification. Native ␣- and -cyclodextrins were kindly provided by Wacker Chemie (Munich, Germany). GC analyses were carried out on a Shimadzu-14B gas chromatograph equipped with a flame ionization detector; GC–MS analyses were conducted on an Agilent Technologies 6850 Network GC System with 5973 Network Mass Selective Detector (Santa Clara, California). Nanosponges were prepared as reported by Trotta and Cavalli (2009). The 1-MCP was synthesized from 2-methyl-3-chloro-propene by a modified procedure of Fisher and Applequist (1965). The gaseous 1-MCP was distilled into a flask, kept in a refrigerated bath, containing a suspension of NSs in buffer solution (0.2 M acetic acid, 0.2 M sodium acetate). The aqueous suspension of NSs and 1MCP was left overnight in the refrigerator, then filtered and dried to obtain a powder. To confirm the presence of 1-MCP contained within NS, an aliquot was treated with 1-(trimethyl silyloxy)-1,3butadiene in tetrahydrofuran (THF), and then the mixture was stirred for 1 h at room temperature. One hundred microlitres of this solution were diluted with 1 mL of diethyl ether; the Diels Alder adduct was then analyzed by gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR) (McEwen et
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al., 1996; Keeler, 2005). The final concentration of 1-MCP included in NSs was quantified as 6% using a calibration curve. 2.2. Plant materials Untreated ethylene-sensitive flowers of a model species for senescence studies (D. caryophyllus L. ‘Idra di Muraglia’, white flower) were grown in standard greenhouse conditions (20 ± 2 ◦ C day/night temperature and 60% relative humidity) in Sanremo, Liguria, Italy (latitude 43◦ 49 9.12 N, longitude 7◦ 45 43.92 E). Cut stems at commercial maturity (sepals standing vertically, petal colors and stems very vivid) were assigned to treatments within 24 h of arrival in the postharvest laboratory of the Department of Agronomy, Forest and Land Management at the University of Turin. Stems were kept in a standard vase life room at 20 ± 2◦ C, 60% RH, and 46 mol m−2 s−1 cool white light (meter model HT307; HT, Faenza, Italy) for 12 h per day. The experimental conditions were intended to reproduce interior home conditions. 2.3. Experimental trials Ten stems (each 30 cm long) were used per treatment. Each experiment was performed at least twice. All the cut flowers were re-cut, labeled, and placed in tap water. ␣- and -CD-based nanosponge-1-MCP complexes (␣- and NS; 6% a.i.) were weighed to obtain two different concentrations of active ingredient (0.25 and 0.5 L L−1 ), and applied to cut flowers both suspended in the tap water and by perfusion. Efficiency of these treatments was compared to equivalent concentrations of volatile 1-MCP (3.3% a.i., SmartFreshTM , AgroFresh Inc., USA), to pure ␣- and -NS, either in vase suspension or sprayed, and to tap water. Twenty-four hours after treatment, cut flowers were enclosed in an air tight cabinet (112 L) and exposed to exogenous ethylene (1 ± 0.2 L L−1 ) for 24 h. 2.4. Data collection and statistical analysis Every day, we evaluated the postharvest performance of the flowers according to the following factors: visual check for symptoms of variation in senescence (VS), petal color, leaf chlorophyll content (SPAD), and endogenous ethylene production. Visual senescence was rated according to a rating scale from 0 to 2, in which 0 = no visible senescence, 1 = initial senescence, 2 = complete senescence. The loss of ornamental value of flowers was considered when they reached level 1 on the scale (Seglie et al., 2010). Petal color (L*, a*, b* space) was measured on flowers using a Spectrophotometer CM-2600 (Konica Minolta Sensing Inc., Osaka, Japan). Chroma (C*) and hue angle (h◦ ) were calculated according to Onozaky et al. (1999). Chlorophyll content was indirectly measured in leaves through a Chlorophyll Meter SPAD-502 instrument (Konica Minolta Sensing Inc., Osaka, Japan). Beginning 24 h after treatment, ethylene production was measured daily by keeping a single stem in air tight tubes (1 L) containing 300 mL of tap water. The ethylene concentration was monitored by a digital Agilent Technologies gas chromatograph, 6890N Network GC system (Santa Clara, California). The gas carrier was N2 at 40 mL min−1 , and column temperature was 60 ◦ C. For each treatment three samples were considered. Photo-documentation at different stages of senescence was carried out. Data were registered until all the cut flowers appeared completely damaged (day 19), after which they were analyzed statistically using SPSS software Inc. (Chicago, United States). The analysis of variance (ANOVA) was established through the
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Fig. 1. Longevity (in days) of Dianthus caryophyllus L. ‘Idra di Muraglia’ cut flowers, placed in tap water, treated with: ␣- and -NS complex (0.25 and 0.5 L L−1 1-MCP), either sprayed (sp) or in vase suspension (su); and gaseous 1-MCP (0.25 and 0.5 L L−1 , for 6 h). Tap water with or without pure ␣- and -NS, either sprayed or in vase suspension, was used as controls. Twenty-four hours after treatment, plant material was exposed to exogenous ethylene (1 ± 0.2 L L−1 , for 24 h). Number of days to reach the stage of initial senescence (level 1) and complete senescence (level 2) were considered. *Mean separation within columns by the Ryan–Einot–Gabriel–Welsch’s multiple stepdown F (REGW-F) test, P ≤ 0.001.
Ryan–Einot–Gabriel–Welsch’s multiple stepdown F (REGW-F) test (P < 0.05). 3. Results 3.1. Symptoms of senescence variation A visual check for symptoms of senescence variation (Fig. 1) failed to show any difference among cut flowers kept in tap water, with or without NSs. Among the 1-MCP applications, the -NS complex showed the best results. Specifically, this complex was more effective in a tap water suspension than as a spray application. The lowest -NS complex concentration (0.25 L L−1 ) prevented flower wilting (initial senescence at 14.7 d; complete senescence at 18.5 d) better than the highest concentration (0.5 L L−1 ; level 1, 12 d; level 2, 15.7 d). Furthermore, this treatment (0.25 L L−1 -NS complex) was even more effective than the 1-MCP gaseous application at both 0.25 L L−1 (level 1, average vase life 9.0 d; level 2, average vase life 10.5 d), and 0.5 L L−1 (level 1, 10.5 d; level 2, 13.8 d) concentrations (Fig. 2). None of the ␣-NS complex applications statistically improved the vase life of cut flowers, compared to the control in tap water (level 1, average longevity 2.8 d; and completely damaged after 5.2 d).
3.2. Flower and leaf color Petal color started to show significant differences in L*, C*, and h◦ values among the treatments after five days from the start of the experiment (Table 1). The results were strictly correlated with VS. The lowest concentration of -NS complex suspended in tap water (0.25 L L−1 ) maintained the nearly pure color of carnation petals better. L*, C*, and h◦ were not statistically different among flowers supplied with the highest -NS complex concentration suspended in tap water, or treated with both doses of gaseous 1-MCP. Both concentrations of -NS complex perfusion statistically improved the L*, C*, and h◦ of petals (white petal color), compared to the controls in tap water with or without pure ␣- and -NS, and to the ␣-NS complex treatments. No appreciable differences were detected in L*, C*, and h◦ among the control flowers nor among the flowers treated with ␣-NS complex at either concentrations. SPAD values for measuring the chlorophyll content in leaves are not shown as no statistical differences were noted among treatments.
3.3. Endogenous ethylene production Fig. 3 shows differences in ethylene production among carnation cut flowers that underwent the 15 treatments. Generally,
Fig. 2. D. caryophyllus ‘Idra di Muraglia’ flowers treated with 0.25 L L−1 -NS complex suspended in tap water, compared to 0.5 L L−1 1-MCP for 6 h and the control in tap water, after 9 d.
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Table 1 Effects on color variation of 1-MCP, included or not in ␣- and -NS, both sprayed (sp) and suspended in tap water (su), compared to commercial gaseous application, and to control flowers kept in tap water with and withheld pure ␣- and -NS. The L* (lightness), C* (chroma), and h◦ (hue angle) values were measured after five days in petals of D. caryophyllus ‘Idra di Muraglia’. Treatments
5th day L*
Tap water 1-MCP (0.25 L L−1 ) 1-MCP (0.5 L L−1 ) Pure ␣-NS Pure -NS ␣-NS complex (0.25 L L−1 ) ␣-NS complex (0.5 L L−1 ) -NS complex (0.25 L L−1 ) -NS complex (0.5 L L−1 ) A
81.4 e 88.9 b 89.7 b sp 81.6 e 81.8 e 82.8 de 82.7 de 86.1 c 86.7 c
h◦
C* A
19.1 a 13.5 d 12.5 d su 82.0 e 81.9 e 83.7 d 83.6 d 91.1 a 89.3 b
sp 19.1 a 18.9 a 18.0 ab 18.2 ab 15.5 c 15.0 c
121.2 a 114.2 dc 112.7 d su 18.7 a 18.8 a 17.6 b 16.9 b 10.3 e 12.2 d
sp 119.3 a 120.3 a 118.8 ab 118.5 ab 115.9 c 115.2 c
su 119.4 a 119.6 a 117.9 b 117.8 b 110.5 e 113.2 d
Mean separation within columns by the Ryan–Einot–Gabriel–Welsch’s multiple stepdown F (REGW-F) test, P ≤ 0.001.
Fig. 3. Mean values of the endogenous ethylene production in Dianthus caryophyllus ‘Idra di Muraglia’ cut flowers placed in tap water, treated with: ␣- and -NS complex (0.25 and 0.5 L L−1 1-MCP), either sprayed (sp) or in vase suspension (su); and gaseous 1-MCP (0.25 and 0.5 L L−1 , for 6 h). Tap water with or without pure ␣- and -NS, either sprayed or in vase suspension, was used as controls.
the control flowers produced ethylene within 24 h, and peaked at day 8 with a range of 0.55–0.63 L L−1 . While the two application methods and concentrations of ␣-NS complex slowed the endogenous ethylene production, after 15 d they did not reduce the final level (range of 0.57–0.63 L L−1 ), compared to the controls (0.63–0.68 L L−1 ). After 15 d, almost no ethylene production was detected in flowers treated with either low or high concentrations of 1-MCP (0.07 and 0.13 L L−1 ), and of -NS complex in tap water (0.04 and 0.08 L L−1 , respectively). A low level of endogenous ethylene was also measured in flowers sprayed with both doses of -NS complex (0.21 and 0.18 L L−1 , respectively).
4. Discussion In floriculture, cut flowers are a very important economic resource in the world. Carnation is one of the most popular species and it is considered as a model in studies on postharvest preservation. Since there was no previous information about NS activity in flowers, we evaluated both the adsorption and the possible phytotoxicity of two types of NS (␣ and ) on cut stems.
Both CDs (Mäkelä et al., 1987) and NSs (diameter 517.7 ± 12.4 nm; Swaminathan et al., 2010) cause the violet color of a phenolphthalein dye solution to disappear, a property that allowed us to follow the flow of the NSs through the xylem vessels (38–70 m) until their presence was confirmed to be at the top of the stems (data not shown). This combined application and perfusion method (also commonly used in postharvest) were adopted for this investigation. The results showed that application of the NS suspended in tap water was more effective than spray application. The reduced effect of the latter could be attributed to slow adsorption of molecules through petal and leaf tissues, as previously detected in banana by Sisler et al. (2009). Moreover, the delivery of 1-MCP from the adsorbed -NS complex may prolong the bio-availability of the complex as compared to the sprayed applications and to gaseous 1-MCP. Different types of NS could have different effects on guest molecules. The binding of guest molecules within the host NS is not fixed or permanent; rather, it is a dynamic equilibrium. Binding strength depends on how well the host–guest complex fits together and on specific local interactions between surface atoms (Del Valle, 2004). When guest 1-MCP is temporarily locked
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or caged within the host cavity, it brings about beneficial or negative modifications of the guest molecule, according to the ability of a NS to form a proper inclusion complex. For this reason, we tested ␣- and -NS that have the same size, but differ with respect to their interior molecule reticulations, internal cavity diameters, volumes, and bonding constants (Del Valle, 2004). In this study, -NS complexes outperformed the ␣-NS complexes. Since the ␣-NS complex contained similar amounts of 1-MCP, its reduced effect on cut flower longevity could be attributed to a stronger bonding constant or to low kinetics of delivery. To assess the potential for phytotoxicity, treatments with pure ␣- and -NS were compared to controls in tap water. As no differences in aesthetic or longevity performances were detected, the results showed that the complexes were not toxic and were totally colourless and odourless for flowers, which is consistent with observations in several pharmaceutical applications of natural CD and their derivatives (Irie and Uekama, 1997; Thompson, 1997). Moreover, the spray application of pure ␣- and -NS did not favor the development of any particular stains on the petal surface. This research into anti-ethylene activity has been supported by the work of others who have shown that many ethylene-sensitive cultivars of D. caryophyllus, such as ‘Sandra’ and ‘White Sim’, react well to 1-MCP treatments (Sisler et al., 1986, 1996; Reid and C¸elikel, 2008). Senescence in carnation cut flowers usually occurs in a few days (2.3) (Serek et al., 1995a), but application of 1-MCP allowed longevity to increase to 12–15 d (Sisler and Serek, 1997). In our study, performed by perfusion, the highest dose of -NS complex was as effective as the lowest concentration of gaseous 1-MCP. Suspended in tap water, the lowest concentration of the -NS complex outperformed the highest concentration of gaseous 1-MCP, increasing the longevity of carnation cut flowers until day 18.5. The efficacy of this complex was confirmed by flower color results, showing that it could also favor the maintenance of the original white color of petals, whether suspended or sprayed, longer than in the controls. In conclusion, a -NS complex, supplied in the conservation solution, was a very effective substitute for gaseous 1-MCP, preventing pigment degradation in petals, and reducing endogenous ethylene production. The use of NSs to carry an anti-ethylene substance could provide a continuous supply of the active ingredient over an extended time, reducing the usual high concentration associated with common practice, which would allow for the potential to lower production costs. The present study represents the first application of NS in an important economic field, such as floriculture. Future commercial use of nanosponges to extend the vase life of cut flowers, and, eventually, of ornamental potted plants, may require further investigation on different -NS reticulations to evaluate optimization. The use of ␣-NS complexes could also be broadened for transporting other preservative compounds. Acknowledgements The authors gratefully acknowledge the entire staff of the SeaMarconi Technologies S. a. s., for their support, AgroFresh Inc. (Rohm and Haas) for providing SmartFreshTM , and Ms. Joan Leonard for language improvements. This research was funded by the Italian Ministry of Education, University and Research (Miur) – PRIN 2007TNTWH7. References Arkas, M., Allabashi, R., Tsiourvas, D., Mattausch, E.M., Perfler, R., 2006. Organic/inorganic hybrid filters based on dendritic and cyclodextrin “nanosponges” for the removal of organic pollutants from water. Environ. Sci. Technol. 40, 2771–2777. Brown, K.M., 1997. Ethylene and abscission. Physiol. Plantarum 100, 567–576.
Cavalli, R., Trotta, F., Tumiatti, W., 2006. Cyclodextrin-based nanosponges for drug delivery. J. Incl. Phenom. Macrocycl. Chem. 56, 209–213. C¸elikel, F.G., Reid, M.S., 2002a. Postharvest handling of stock (Matthiola incana). HortScience 37, 144–147. C¸elikel, F.G., Reid, M.S., 2002b. An alternative model for the inhibition of ethylene action by 1-methylcyclopropene (1-MCP). In: Mencarelli, F., Serra, G. (Eds.), Tecniche postraccolta dei prodotti ortoflorofrutticoli. ARSIA, Florence, Italy. Del Valle, E.M.M., 2004. Cyclodextrins and their uses: a review. Process Biochem. 39, 1033–1046. Fisher, F., Applequist, D.E., 1965. Synthesis of 1-methylcyclopropene. J. Org. Chem. 30, 2089–2090. Irie, T., Uekama, K., 1997. Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation. J. Pharm. Sci. 86, 147–162. Keeler, J., 2005. Understanding NMR Spectroscopy. John Wiley & Sons Inc., Hoboken, NJ, USA. Li, D., Ma, M., 1999. Nanoporous polymers: new nanosponge absorbent media. Filtr. Separat. 36, 26–28. Loftsson, T., Olafsson, J.H., 1998. Cyclodextrins: new drug delivery systems in dermatology. Int. J. Dermatol. 37, 241–246. Macnish, A.J., Joyce, D.C., Hofman, P.J., Simons, D.H., Reid, M.S., 2000. 1Methylcyclopropene treatment efficacy in preventing ethylene perception in banana fruit and grevillea and waxflowers. Aust. J. Exp. Agr. 40, 471–481. Mäkelä, M., Korpela, T., Laakso, S., 1987. Colorimetric determination of beta cyclodextrin: two assay modifications based on molecular complexation of phenolphthalein. J. Biochem. Biophys. Methods 14, 85–92. McEwen, C.N., Kitson, F.G., Larsen, B.S., 1996. Gas Chromatography and Mass Spectrometry: A Practical Guide. Academic Press, Boston, Massachusetts, USA. Nukui, H., Kudo, S., Yamashita, A., Satoh, S., 2004. Repressed ethylene production in the gynoecium of long-lasting flowers of the carnation ‘White Candle’: role of gynoecium in carnation flower senescence. J. Exp. Bot. 55, 641– 650. Onozaky, T., Mato, M., Shibata, M., Ikeda, H., 1999. Differences in flower colour and pigment composition among white carnation (Dianthus caryophyllus L.) cultivars. Sci. Hortic. 82, 103–111. Reid, M.S., C¸elikel, F.G., 2008. Use of 1-methylcyclopropene in ornamentals: carnations as a model system for understanding mode of action. HortScience 43, 95–98. Seglie, L., Sisler, E.C., Mibus, H., Serek, M., 2010. Use of a non-volatile 1-MCP formulation, N,N-dipropil(1-cyclopropenylmethyl)amine, for improvement of postharvest quality of ornamental crops. Postharvest Biol. Technol. 56, 117–122. Serek, M., Sisler, E.C., Reid, M.S., 1995a. Effects of 1-MCP on the vase life and ethylene response of cut flowers. Plant Growth Regul. 16, 93–97. Serek, M., Sisler, E.C., Reid, M.S., 1995b. 1-Methylcyclopropene, a novel gaseous inhibitor of ethylene action, improves the vase life of fruits, cut flowers and potted plants. Acta Hortic. 394, 337–346. Serek, M., Sisler, E.C., 2005. Impact of 1-MCP on postharvest quality of ornamentals. In: APEC Symp. Quality Management of Postharvest System Proceedings, pp. 121–128. Serek, M., Woltering, E.J., Sisler, E.C., Frello, S., Sriskandarajah, S., 2006a. Controlling ethylene at the receptor level. Biotechnol. Adv. 24, 368–381. Serek, M., Sisler, E.C., Frello, S., Sriskandarajah, S., 2006b. Postharvest technologies for extending the shelf life of ornamental crops. Int. J. Postharvest Technol. Inn. 1, 69–75. Sisler, E.C., Reid, M.S., Fujino, D.W., 1983. Investigation of the model of action of ethylene in carnation senescence. Acta Hortic. 141, 229–234. Sisler, E.C., Reid, M.S., Yang, S.F., 1986. Effect of antagonists of ethylene action on binding of ethylene in cut carnations. Plant Growth Regul. 4, 213– 218. Sisler, E.C., Dupille, E., Serek, M., 1996. Effect of 1-methylcyclopropene and methylenecyclopropane on ethylene binding and ethylene action on cut carnations. Plant Growth Regul. 18, 79–86. Sisler, E.C., Serek, M., 1997. Inhibitors of ethylene responses in plants at the receptor level: recent developments. Physiol. Plantarum 100, 577–582. Sisler, E.C., Serek, M., Roh, K.A., Goren, R., 2001. The effect of chemical structure on the antagonism by cyclopropenes of ethylene responses in banana. Plant Growth Regul. 33, 107–110. Sisler, E.C., Serek, M., 2003. Compounds interacting with the ethylene receptor in plants. Plant Biol. 5, 473–480. Sisler, E.C., Goren, R., Apelbaum, A., Serek, M., 2009. The effect of dialkylamine compounds and related derivatives of 1-methylcyclopropene in counteracting ethylene responses in banana fruit. Postharvest Biol. Technol. 51, 43– 48. Swaminathan, S., Vavia, P.R., Trotta, F., Torne, S., 2007. Formulation of betacyclodextrin based nanosponges of itraconazole. J. Incl. Phenom. Macrocycl. Chem. 57, 89–94. Swaminathan, S., Pastero, L., Serpe, L., Trotta, F., Vavia, P., Aquilano, D., Trotta, M., Zara, G.P., Cavalli, R., 2010. Cyclodextrin-based nanosponges encapsulating camptothecin: physicochemical characterization, stability and cytotoxicity. Eur. J. Pharm. Biopharm. 74, 193–201. Thompson, D.O., 1997. Cyclodextrins-enabling excipients: their present and future use in pharmaceuticals. Crit. Rev. Ther. Drug Carrier Syst. 14, 1– 104.
L. Seglie et al. / Postharvest Biology and Technology 59 (2011) 200–205 Trotta, F., Cavalli, R., 2009. Characterization and application of new hyper-crosslinked cyclodextrins. Compos. Interface 166, 39–48. Veen, H., 1979. Effects of silver on ethylene synthesis and action in cut carnations. Planta 145, 467–470. Vyas, A., Saraf, S., Saraf, S., 2008. Cyclodextrin based novel drug delivery systems. J. Incl. Phenom. Macrocycl. Chem. 62, 23–42.
205
Wang, H., Woodson, W.R., 1989. Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene. Plant Physiol. 89, 434–438. Woltering, E.J., van Doorn, W.G., 1988. Role of ethylene in senescence of petalsmorphological and taxonomical relationship. J. Exp. Bot. 39, 1605–1616.