Tetrahydrocurcumin encapsulation in cyclodextrins for water solubility improvement: synthesis, characterization and antifungal activity as a new biofungicide
The solubility of the scarcely water-soluble molecule tetrahydrocurcumin (THC), a promising curcumin derivative to be used as biopesticide, was enhanced through the formation of inclusion complexes with different cyclodextrins (CDs). Randomly methylated β-cyclodextrin (MeβCD) gave the best results among the different CDs whose size, substituent or structure was changing. Differential scanning calorimetry as well as 1H- and 2D-Nuclear Magnetic Resonance (NMR) proved the inclusion of THC in MeβCD.
Highlights
Methylated β-cyclodextrin enhanced 100 times tetrahydrocurcumin water-solubility.
Stoichiometry of THC/MeβCD complex is 1:1.
DSC, 1H- and 2D-NMR revealed THC complexation.
No anti-fungal activity for MeβCD and THC/MeβCD.
Polymerization of MeβCD allowed anti-fungal properties.
Rotating-frame Overhauser effect spectroscopy NMR especially illustrated specific interactions of aromatic protons of THC and protons located inside the CD cavity. The complex formation between THC and MeβCD was studied using the Higuchi and Connor method, giving an association constant of 591 M−1. THC-loaded MeβCD did not show any growth inhibition of the target fungus Fusarium graminearum. However, THC-loaded MeβCD polymers exhibited 25 % inhibition of the fungal growth, thus making them promising material for solvent-free, aqueous and bio-based fungicide formulations.
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Introduction
Plants are subject to attacks by fungi, nematodes and insects, which generate yield losses and several species in the genus Fusarium are generally considered the most important in Europe as regards crop contamination. Among them, Fusarium graminearum can infect cereals such as maize, wheat or barley, leading to yield decreases up to 50 % (Shah et al., 2017) but also the potential production of mycotoxins, that can cause health problems to humans and animals. Nowadays, pest control strategies and particularly regarding Fusarium species mainly rely on synthetic fungicides, which could be harmful to human health and detrimental to the environment (Gupta, 2011; Smart, 2003). As mentioned by Kumar and Singh (2015), the entry of synthetic pesticides into the food chain coupled with their bioaccumulation triggers several unforeseen consequences. It has become important now to develop alternative strategies to overcome the drawbacks of synthetic pesticides. Therefore, scientific research has shifted towards the use of sustainable and renewable products, exhibiting significant efficacy while being less harmful to people and environment friendly.
Phenolic compounds belong to one of the most studied classes of natural active substances to be potentially used in biopesticide formulations. They are present in almost all plants and possess antioxidant properties (Dai, & Mumper, 2010). Essential oils (Avanço et al., 2017) or cereal extracts (Heidtmann-Bemvenuti, Tralamazza, Jorge Ferreira, & Corrêa, Badiale-Furlong, 2016) containing phenolic derivatives are already used to control fungal development. Pure phenolic compounds such as carvacrol, thymol and o-cresol have also shown ability to reduce the mycelium development (Teodoro, Ellepola, Seneviratne, & Koga-Ito, 2015; Wang et al., 2019). In this respect tetrahydrocurcumin (THC), obtained by hydrogenation of curcumin, one of the curcuminoids present in the rhizomes of Curcuma longa L. commonly called turmeric, has shown promising results for biopesticide formulations (Coma, Portes, Gardrat, Richard-Forget, & Castellan, 2011). THC consists of two guaiacyl subunits linked by a C7 aliphatic chain bearing a β-diketone group (THC, Fig. 1 A), and exhibits antioxidant, antifungal and antimycotoxinogenic properties. Unfortunately, its very low water-solubility strongly limits its potential applications in water-based formulations. Many dispersion techniques to increase the solubility of phenolic compounds are reported in the literature (Zhang, Xing, Zhao, & Ma, 2018.) and among others, encapsulation techniques such as spray-drying, solvent evaporation, freeze-drying, and liposome formation are attractive (Fang, & Bhandari, 2010; Munin, & Edwards-Lévy, 2011). Because of their natural origin and their GRAS status (Braga, 2019), cyclodextrins (CDs) are particularly suitable carrier materials for such hydrophobic active agents (Cheirsilp, & Rakmai, 2017; Del Valle, 2004; Nardello-Rataj, & Leclercq, 2014; Pinho, Grootveld, Soares, & Henriquez, 2014). Briefly, CDs are biopolymers commonly composed of 6,7 or 8 β-linked glucose units, with a funnel-shaped structure composed of an external hydrophilic shell and an internal hydrophobic cavity (Fig. 1 B). The hydrophilic part makes them water-soluble whereas the lipophilic inside confers them the ability to trap hydrophobic molecules. CDs have been scarcely used in agriculture (Morillo, 2006) and inclusion complex systems have been yet reported to increase the solubility of poor water-soluble active agents (Singh et al., 2020) thus allowing a reduction of the applied dose (Morillo, 2006). For instance, CDs have been used to encapsulate synthetic and harmful fungicides like carbendazim (Wang et al., 2017) or chlorothalonil (Gao et al. 2019) and two plant extracts, carvacrol and linalool (Campos et al., 2018). All obtained inclusion complexes have been more efficient to control pest, leading to a reduction of the minimum active concentration.
The aim of the current work was to investigate native and modified CDs to increase the apparent water solubility of THC in order to create new antifungal bio-based complexes potentially used for biopesticide applications.
The impact of some CD structures on the enhancement of the THC solubility was firstly investigated to select the most appropriate cyclodextrin. Described for the first time, THC-loaded MeβCD complexes were then deeply characterized in terms of physico-chemical properties by using differential scanning calorimetry, NMR spectroscopy and scanning electron microscopy. Finally, the bioactive properties of some THC-loaded cyclodextrins were compared to free THC against a model strain of F. graminearum.
Materials
When the abbreviation CD is used, it refers either to the word “cyclodextrin” or to any type of cyclodextrins in this study (βCD, γCD, MeβCD, Poly-βCD and Poly-MeβCD).
β-Cyclodextrin Kleptose® (βCD) and Randomly-methylated-β-cyclodextrin Kleptose Crysmeb® (MeβCD) were provided by Roquette (Batch E0002, Lestrem, France). Citric acid (CTR), sodium dihydrogen hypophosphite (NaH2PO2•H2O) and sodium carbonate (Na2CO3) used in the synthesis of polymer of βCD (Poly-βCD) were supplied from Sigma Aldrich (Saint-Quentin Fallavier, France).
MeβCD degree of substitution was 0.5, leading to a molecular mass of 1184 g/mol. γ-Cyclodextrin (γCD) was purchased from TCI Europe. FTIR and DSC spectra of CDs are available in supplementry information (Fig. S1 and Fig. S2).
Anionic water-soluble polymer of βCD (Poly-βCD) and MeβCD (Poly-MeβCD) were previously synthetized according to Junthip, Tabary, Leclercq, & Martel (2015) by using citric acid as crosslinking agent, sodium hypophosphite as catalyst and CD in molar ratio 59/40/1 and 61/40/1 for Poly-βCD and Poly-MeβCD, respectively. Briefly, after water removal, the solid mixture was cured at 140°C during 30 min under vacuum. Water was then added, the resulting suspension filtered and the filtrate dialyzed during 72 h against water through 6–8 kDa membranes (SPECTRAPOR 1, Spectrumlabs). Finally, the water soluble anionic CD polymer was recovered after freeze drying. The number average molar mass (Mn) measured by aqueous size exclusion chromatography (SEC) were 11,800 g/mol and 15,600 g/mol, respectively for Poly-βCD and Poly-MeβCD (Polydispersity =1.7), using a DIONEX Ultimate 3000 equipped with a DAWN HELEOS II multi-angle light scattering and an OPTILAB rEX differential refractometer (4 columns Shodex OH-Pak 30 cm, flow rate = 0.5 mL/min, concentration of polymer = 3 g/L, mobile phase = 0.1 M NaNO3 and NaN3, dn/dc = 0.139). Based on the integrations values of H1 (CD) and citrate methylene signals (CH2-CTR) by proton NMR studies, Poly-βCD and Poly-MeβCD were estimated to contain 72.0 wt% in βCD moieties and 63.4 wt% in MeβCD moieties respectively. This percentage was employed to calculate the concentration of CD cavities. FTIR spectra of polymers are available in supplementary information (Fig. S3) and DSC spectra in the work of Tabary et al. (2016).
Tetrahydrocurcumin Tetrapure (THC) was purchased from Sabinsa Corporation SabiWhite® (USA).
Potato Dextrose Agar (PDA) medium (potato starch 4g/L, dextrose 20g/L, agar 15g/L) was provided from Biokar. For the Carboxymethylcellulose medium (CMC medium, CMC low viscosity 15 g/L; Yeast extract 1 g/L; MgSO4; 7H2O 0.5 g/L; NH4NO3 1 g/L; KH2PO4 1 g/L), products were provided by Aldrich (Saint-QuentinFallavier, France) and all salts used in media were in analytical grade.
The strain CBS 185.32 of F. graminearum belonging to INRAe MycSA laboratory collection (Centre INRA de Nouvelle-Aquitaine Bordeaux UR1264 MycSA, INRAe, Villenave d’Ornon, France) was used as the targeted strain for antifungal studies.
Article information: Anne Loron, Christian Gardrat, Nicolas Tabary, Bernard Martel, Véronique Coma. Tetrahydrocurcumin encapsulation in cyclodextrins for water solubility improvement: synthesis, characterization and antifungal activity as a new biofungicide, Carbohydrate Polymer Technologies and Applications, 2021. https://doi.org/10.1016/j.carpta.2021.100113.