Phyto-Saponins as a Natural Adjuvant for Delivery of Agromaterials through Plant Cuticle Membranes BISHNU P. CHAPAGAIN AND ZEEV WIESMAN* The Phyto-Lipid Biotechnology Laboratory, Department of Biotechnology Engineering, The Institutes for Applied Research, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel With growing use of synthetic adjuvants in modern agriculture, their impacts on the environment are being questioned. In a search for an environmentally safe phyto-adjuvant, we have investigated natural glycosidic saponin for delivery of agromaterials through plant cuticle membranes. Four saponin preparations from Quillaja saponaria bark (QE), obtained from Sigma-Aldrich, and Balanites aegyptiaca fruit mesocarp (ME), kernel (KE), and root (RE), isolated and characterized in our laboratory, were used for testing the delivery of [ 14 C]-2,4-dichlorophenoxyacetic acid (2,4-D) across isolated intact astomatous adaxial Citrus grandis leaf cuticle membranes (CMs). The results showed that both Q. saponaria and B. aegyptiaca saponin preparations enhanced delivery of 2,4-D through CMs. Among the saponin preparations, ME exhibited a significantly higher level of delivery of 2,4-D with a concentration effect (2% being the highest). Transmission electron microscope (TEM) and dynamic light scattering (DLS) characterization of these saponin preparations in aqueous solution clearly demonstrated the formation of nanoscale vesicles. Various possibilities for a natural amphiphatic phyto-saponin as a delivery adjuvant through CMs are discussed. KEYWORDS: Phyto-saponins; cuticle membrane; phytoadjuvant; nanovesicles; Balanites aegyptiaca; Quillaja saponaria INTRODUCTION Plant cuticle is a thin (<0.1-10 μm) continuous layer or membrane of predominantly lipid material that covers the entire external surface of plants, including leaves of the higher plants, and forms the interface between the foliage and the atmosphere (1). The main function of the plant cuticle is to minimize water loss from plants when stomata are closed and to protect the plant against physical, chemical, and biological attack; however, cuticle remains the main barrier to the penetration of compounds applied to the foliage or ground (2). The outer surface of the cuticle is covered by epicuticular wax which can occur in many forms, from amorphous to crystalline deposits, and consists of complex mixtures of long-chain aliphatic and cyclic components (3). Plant cuticles also contain non-lipid constituents such as polysaccharides and phenolics. It has been reported that various phytosterols such as pentacylic triterpenols and Δ 5 -sterols are found in the cuticular membrane in leaves and rhizomes (4). The epicuticular wax layer of the cuticle is the main barrier to penetration of applied material because of its hydrophobic nature; this layer must be penetrated before any applied agromaterial can enter living cells. In the past few decades, the use of agricultural adjuvants and/ or surfactants has become common practice in foliar application to enhance the delivery of agromaterials to the inner tissue of the plant through the cuticular layer (5). Although the term “adjuvant” has numerous meanings and has been the source of unending confusion, it is commonly understood that agricultural adjuvants are any materials other than water that are added to the agromaterials to increase their efficiency when applied to the plants (6). The major category of agricultural adjuvants is the oil-based group. Historically, these have been of mineral origin, but these are gradually being superseded by vegetable oils and their derivatives. The second major category of adjuvants consists of surfactants (surface-active agents). These include a wide range of materials, which can be organic or inorganic in nature. Other chemical categories include various polymers, film-forming materials, and inorganic salts (7). Among the surfactants, silicone-based nonionic surfactants are the most commonly recommended and used adjuvants. Although these surfactant-type adjuvants increase the diffusive mobility of agromaterials across the cuticle, thereby increasing the penetration potential, since these adjuvants were originally designed for herbicides, severe necrotic damage to the treated leaves is commonly encountered when using these surfactants with foliar nutrients (8). Although adjuvants are generally considered “inert” or essentially nonhazardous, the main reason behind nondisclosure of most ingredients is the manufacturers’ reticence (9). The long- term fates of most adjuvants in soils and elsewhere in the environment are also largely unknown because of the lack of * To whom correspondence should be addressed: Phyto-Lipid Biotech- nology Laboratory, Department of Biotechnology Engineering, The Institutes for Applied Research, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel. Telephone and fax: 972-8-647-7184. E-mail: wiesman@bgu.ac.il. J. Agric. Food Chem. 2006, 54, 6277-6285 6277 10.1021/jf060591y CCC: $33.50 © 2006 American Chemical Society Published on Web 07/28/2006