Prenyloxycoumarins with antioxidant and lipoxygenase inhibitory activity: Synthesis, bioactivity evaluation and encapsulation in biodegradable poly (lactic acid) nanoparticles

Oxyprenylated natural products (isopentenyloxy-, geranyloxyand the less spread farnesyloxycompounds and their biosynthetic derivatives) represent a family of secondary metabolites that have been considered for years just as biosynthetic intermediates of C-prenylated derivatives. Prenyloxycoumarins are secondary metabolites commonly present in plants belonging to the families of Rutaceae and Umbelliferae. Several of these coumarins were shown to possess valuable pharmacological properties. Among them, auraptene (7geranyloxycoumarin) which is isolated from the peel of citrus fruit has been reported to have chemopreventive effects on chemically induced carcinogenesis and possess antioxidant activity. In this work, the synthesis of the natural product auraptene as well as its analogue, 4-methyl-7geranyloxycoumarin will be presented. The compounds were evaluated in vitro for their antioxidant activity as well as for their ability to inhibit soybean lipoxygenase, as an indication of their potential anti-inflammatory activity. In addition, the compounds were encapsulated in biodegradable poly (lactic acid) (PLA) nanoparticles and this was achieved via the emulsification-solvent evaporation technique. Size, polydispersity index and ζpotential of the nanoparticles were measured by Dynamic Light Scattering method whereas the Encapsulation Efficiency (EE) was determined indirectly, using UV-Vis spectroscopy. INTRODUCTION Coumarins are a class of compounds widely encountered in nature which possess numerous therapeutic prospects. Among the biological activities which have been attributed to simple coumarins and their analogues is the antimicrobial activity, antiviral activity, antitumor, enzyme inhibitory activity, anti-inflammatory, antioxidant and antithrombotic activity. [1] Specifically, the alkylation of aromatic secondary metabolites plays an important role in the biosynthesis of many molecules with significant biological activities. The first example of a prenylated secondary metabolite is auraptene or 7-geranyloxy-coumarin which was isolated in 1930 from the plant Citrus aurantium L. (Rutaceae) and its structure was assigned by Kariyone and Matsuno (Figure 1). [2] Figure 1. The structure of auraptene The pharmacological actions of auraptene have been mentioned in literature since 1991 [3] and up to date 300 oxy-prenylated derivatives, which have been synthesized or isolated, exhibit interesting biological properties. 10ο ΠΑΝΕΛΛΗΝΙΟ ΕΠΙΣΤΗΜΟΝΙΚΟ ΣΥΝΕΔΡΙΟ ΧΗΜΙΚΗΣ ΜΗΧΑΝΙΚΗΣ, ΠΑΤΡΑ, 4-6 ΙΟΥΝΙΟΥ, 2015. Auraptene is a chemoprotective agent against skin, tongue, oesophagus and colon carcinogenesis in rodents [4] and also, prevents the mutation of tumor cells, accelerating the action of metabolism and some enzymes, such as glutathione S-transferase. [5] In addition to these properties, the anti-inflammatory activity of auraptene has been proven in vivo and in vitro. [6] Nanoencapsulation is of particular interest in the field of drug delivery, through particulate systems which operate as "carriers" of some bioactive compounds. These nanoparticles can provide protection against unwanted degradation, photoand air-oxidation as well as modifying the aqueous solubility of compounds. [7] Poly (lactic acid) (PLA) is a polymer with many important advantages, such as the renewability, biocompatibility, biodegradation and it is ideal for the encapsulation of hydrophobic compounds. [8] As a continuation of our studies toward the synthesis of bioactive coumarin analogues, [9], [10] we report here the synthesis and evaluation of antioxidant and anti-inflammatory activity of the natural product auraptene (3) and its 4-methylated analogue (4). EXPERIMENTAL (a) Synthesis of compounds 3 and 4 The synthesis of two geranyloxy-coumarins 3 and 4 was carried out by Williamson’s ether reaction, using the commercially available 7-hydroxy-coumarin (umbelliferone) (1) or the synthesized 4-methyl-7-hydroxycoumarin (2) as starting materials. The appropriate coumarin reacts with geranyl bromide (Scheme 1) in a basic environment, to provide coumarins 3 and 4 in good yields and high purity. The structures of the synthesized compounds were identified using NMR spectroscopy. Scheme 1. The synthetic route for 7-geranyloxy-coumarins 3 and 4 (b) Encapsulation of compounds 1-4 in PLA nanoparticles The produced geranyloxy-coumarins 3 and 4 were encapsulated in PLA nanoparticles via the emulsificationsolvent evaporation technique, which forms simple emulsions and it is appropriate for hydrophobic compounds. According to this method, the polymer was dissolved in a volatile organic solvent, such as acetone, and was mixed with the solution of the compound which will be encapsulated. The new solution was emulsified in an aqueous phase containing an emulsifier (in this case poly(vinyl alcohol), PVA) which does not dissolve the polymer. The organic solvent was then evaporated under mild conditions (shaker or rotary evaporator at room 10ο ΠΑΝΕΛΛΗΝΙΟ ΕΠΙΣΤΗΜΟΝΙΚΟ ΣΥΝΕΔΡΙΟ ΧΗΜΙΚΗΣ ΜΗΧΑΝΙΚΗΣ, ΠΑΤΡΑ, 4-6 ΙΟΥΝΙΟΥ, 2015. temperature) and the particles are collected by filtration or centrifugation and redispersed in distilled water. (c) Evaluation of biological activity The antioxidant activity of coumarins 1-4 was evaluated by the DPPH assay and their ability to inhibit lipid peroxidation of linoleic acid induced by the thermal free radical producer AAPH. Moreover, the ability of compounds 1-4 to inhibit the activity of soybean lipoxygenase was evaluated as an indication of their antiinflammatory activity. RESULTS AND DISCUSSION In the present work, the natural product 7-geranyloxy-coumarin (auraptene) (3) and its 4-methyl analogue (4) were synthesized and evaluated for their antioxidant and soybean lipoxygenase inhibitory activity. The results of in vitro biological testing of compounds 1-4 are shown in Table 1. Table 1. In vitro antioxidant and lipoxygenase inhibitory activities of compounds 1-4 Compound DPPH radical scavenging ability (%100 μM) 20/60 min Inihibition of lipid peroxidation induced by AAPH (% 100 μM) Inhibition of soybean lipoxygenase (% 100 μM) Umbelliferone (1) 1/2 92 100 4-methyl-umbelliferone (2) No/no 93 No Auraptene (3) 26/6 52 44 4-methyl-auraptene (4) 21/No 20 78 Nordihydroguaiaretic acid (NDGA) 93 86