Article pubs.acs.org/JAFC
Preparation and Characterization of Azadirachtin AlginateBiosorbent Based Formulations: Water Release Kinetics and Photodegradation Study Francisco Flores-Céspedes, Gerardo P. Martínez-Domínguez, Matilde Villafranca-Sánchez, and Manuel Fernández-Pérez* Department of Chemistry and Physics, Research Centre for Agricultural and Food Biotechnology (BITAL), University of Almería, Agrifood Campus of International Excellence, CeiA3, E-04120 Almería, Spain ABSTRACT: The botanical insecticide azadirachtin was incorporated in alginate-based granules to obtain controlled release formulations (CRFs). The basic formulation [sodium alginate (1.47%) − azadirachtin (0.28%) − water] was modified by the addition of biosorbents, obtaining homogeneous hybrid hydrogels with high azadirachtin entrapment efficiency. The effect on azadirachtin release rate caused by the incorporation of biosorbents such as lignin, humic acid, and olive pomace in alginate formulation was studied by immersion of the granules in water under static conditions. The addition of the biosorbents to the basic alginate formulation reduces the rate of release because the lignin-based formulation produces a slower release. Photodegradation experiments showed the potential of the prepared formulations in protecting azadirachtin against simulated sunlight, thus improving its stability. The results showed that formulation prepared with lignin provided extended protection. Therefore, this study provides a new procedure to encapsulate the botanical insecticide azadirachtin, improving its delivery and photostability. KEYWORDS: alginate hydrogels, azadirachtin, controlled release, photostability, biosorbents
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INTRODUCTION The use of biopesticides including microbial pesticides, entomopathogenic nematodes, baculoviruses, natural plantderived pesticides, and insect pheromones as crop protectionagents has increased not only due to their low environmental impact and their low toxicity in mammals but also due to the increasing resistance that pests show to synthetic pesticides.1,2 Among these substances, neem seeds (Azadirachta indica A. Juss) products are one of the more important natural plantderived pesticides, which have influence over 200 species of insects, many of which are resistant to synthetic pesticides. The major insecticidal component in neem products is azadirachtin. Moreover azadirachtin, a powerful insect antifeedant and growth-regulating substance with exceptional low residual power, has been isolated from the seed kernels of the neem tree.3 However, the use of azadirachtin-based pesticides may be limited by the acid and base sensitivity of the compound and its susceptibility to photodegradation due to the presence of lightabsorbing moieties,4,5 which limits their use in agriculture. Many attempts to stabilize azadirachtin have been researched. The mixing of Aza-A with UV light absorbers can enhance its photostability to a moderate degree.5 New formulation technologies (ingredients and processes) must be developed to provide enhanced protection for azadirachtin, controlling also its release. A suitable technology for this area can be the application of polymeric materials to obtain controlled release formulations (CRFs). In CRFs, active ingredients are trapped, that is, they are integrated into a polymer matrix, where they are dissolved or linked to it physically or chemically.6 The main objectives of controlled release technology are the protection of the active ingredient and the © XXXX American Chemical Society
regulation of its supply to the specified target. Previously, urea formaldehyde cross-linked by starch, guar gum, and urethane foams,7,8 poly(ε-caprolactone)9 and sodium alginate,10 have been used to encapsulate azadirachtin. The release of the active ingredient was dependent on the type of matrix and its swelling ability. Also, a polymer network based on poly(vinyl alcohol) (PVA) and sodium alginate as well as glutaraldehyde as a crosslinking agent was prepared to enhance the stability of azadirachtin.11 In this study, as a novelty, biosorbents such as kraft lignin, olive pomace, and acid humic have been incorporated as modifying agents in azadirachtin alginate-based CRFs. Modifying agents such as natural and activated clays, activated carbon, and anthracite have been previously used by the authors in the preparation of synthetic pesticides in alginate-based CRFs12−14 to increase the encapsulation and to reach a better control on release profiles of active ingredients. The use of biosorbents, obtained from agricultural wastes and byproducts, can not only reduce a large quantity of solid waste but also be very attractive due to their low investment cost, simplicity of design and operation, together with a remarkable interaction capacity by organic molecules.15 Besides, the biosorbents can provide protection to natural pesticides from sunlight degradation in environments where high temperatures and lengthy exposures to sunlight are very normal, such as greenhouses, indoor environments, or crop fields.16 Although applications of biosorbents in Received: July 15, 2015 Revised: September 3, 2015 Accepted: September 7, 2015
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DOI: 10.1021/acs.jafc.5b03255 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry water treatment to removal organic pollutants and heavy metals have been extensively tested,17,18 studies on the use of biosorbents in the preparation of CRFs have emerged in recent years.19,20 The main objective of this research was the development of azadirachtin alginate-based CRFs using biosorbents as modifying agents not only to control the release rate of azadirachtin but also to stabilize this botanical insecticide against degradation. To achieve this objective, the following tasks were performed: (i) preparation of azadirachtin alginate-based formulations using biosorbents like kraft lignin, olive pomace, and humic acid, (ii) characterization of prepared formulations, and (iii) evaluation of azadirachtin formulations through kinetic and photodegradation studies.
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(total surface acidity) − (carboxylic groups) = phenolic hydroxyl groups (meq g −1) These data are given in Table 1. The organic matter content of lignin, humic acid, and olive pomace was determined by the Walkley-Black method.23 These data are given in Table 1. Preparation of the Azadirachtin CRFs. The prepared CRFs were based on the gelling properties of the alginate in the presence of divalent cations. It was made up of formulations in water containing different percentages of technical grade azadirachtin (Az), sodium alginate (A), lignin (L), humic acid (H), and olive pomace (O) (shown in Table 2). These mixtures were vigorously stirred for 1 h.
Table 2. Percentage (by Weight) of the Components of CR Formulations Containing Azadirachtin
MATERIALS AND METHODS
formulationa azadirachtin
Materials. Azadirachtin from a neem tree (17.7%) was provided from The Indian Neem Tree Company, Mumbai, India. The chemical structure of azadirachtin is shown in Figure 1.
AAz AOAz AHAz ALAz
0.28 1.17 1.17 1.17
alginate 1.47 1.40 1.40 1.40
lignin
humic acid
olive pomace 4.75
4.75 4.75
water 98.25 92.69 92.69 92.69
a
AAz: alginate-azadirachtin. AOAz: alginate-olive pomace-azadirachtin. AHAz: alginate-humic acid-azadirachtin. ALAz: alginate-lignin-azadirachtin.
The alginate mixtures (100 g) were dropwise added to a 250 mL gellant bath of 0.25 M CaCl2, as was described previously.14 The resulting beads were allowed to gel in the 0.25 M CaCl2 solution for a total of 20 min, then they were filtered and dried first at room temperature and then in an oven (40 °C) to constant weight. CRF granules were maintained in an oven at controlled environment (40 °C) before experiments were carried out. The resulting products are labeled in the text as AAz, ALAz, AHAz, and AOAz. Azadirachtin CRFs Characterization. Determination of Azadirachtin Content in CRFs. The concentration of azadirachtin in the CRFs was determined by dissolving 20 mg of the dry granules in a 0.03 M tripolyphosphate solution (5 mL). This solution was placed in stoppered conical flasks and shaken in a thermostatic bath at 25 °C ± 0.1 °C for 2 h. After that, 80 mL of a methanol/water mixture (80:20 v/ v) was added, and the solutions were left in the thermostatic bath at the same conditions for 24 h. The resulting extract was made up to 100 mL with methanol/water mixture (80:20 v/v) and filtered using 0.20 μm syringe nylon filters (Millex GN, Millipore Co., Billerica, MA USA). The insecticide concentration was determined by HPLC, using a Beckman Coulter Inc., System Gold HPLC (Fullerton, CA, USA) equipped with a diode-array detector and 32 Karat data station. HPLC operating conditions to analyze azadirachtin were described previously by other authors.24−26 The mobile phase was an acetonitrile−water mixture 40:60, the flow rate was 1 mL/min, and the column used was a Nova-Pak C18 4 μm 3.9 × 150 mm supplied by Waters Assoc. (Massachusetts, USA). The analysis was performed at the wavelength of 217 nm by injecting a volume of 50 μL. External standard calibration was used, and three replicates were carried out for each formulation. Mean Granule Size and Average Granule Mass. The average diameter of CR granules was determined using a Stereoscopic Zoom Microscope from Nikon, model SMZ1000, provided with a camera
Figure 1. Chemical structure of azadirachtin.
Alginic acid was used as sodium salt obtained from Macrocystis pyrifera, and calcium chloride (97%) and sodium tripolyphospate (90%) were provided by Sigma-Aldrich Co LLC. The lignin used in this study was a commercially available pine kraft lignin, Indulin AT (Westvaco Corp., Charleston, SC, USA). The olive pomace used in this study was obtained from a three phases extraction procedure on olive oil by the company Almazara de Felix S.C.A. The humic acid was provided by the company Sigma-Aldrich Co., LLC. Chemical Analysis of Modifying Agents. The elemental analyses of lignin, humic acid, and olive pomace were performed for C, H, N, O, and S in an elemental analyzer from Elementar, model Vario Micro CHNS-O. All of these characteristics of the modifying agents are shown in Table 1. The total surface acidity (R-COOH, Ar-COOH, and Ar-OH groups) of humic acid and olive pomace was determined using the Schnitzer and Gupta method.21 The total amount of carboxylic groups was determined using the method proposed by Schnitzer and Wright.21,22 The total amount of phenolic hydroxyl groups was determined following the method proposed by Schnitzer,22 that is
Table 1. Physicochemical Characteristics of Modifying Agents elemental analysis
total surface acidity
organic matter content
(%)
(meq g−1)
(%)
modifying agent
C
S
H
N
O
total acidity
−COOH groups
−OH groups
O.C.
O.M
olive pomace humic acid lignin
53.91 45.28 62.54
