ABSTRACT
Taxifolin has a plethora of therapeutic activities and is currently isolated from the stem bark of the tree Larix gmelinni (Dahurian larch). It is a flavonoid of high commercial interest for its use in supplements or in antioxidant-rich functional foods. However, its poor stability and low bioavailability hinder the use of flavonoid in nutritional or pharmaceutical formulations. In this work, taxifolin isolated from the seeds of Mimusops balata, was evaluated by in silico stability prediction studies and in vitro forced degradation studies (acid and alkaline hydrolysis, oxidation, visible/UV radiation, dry/humid heating) monitored by high performance liquid chromatography with ultraviolet detection (HPLC-UV) and ultrahigh performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS). The in silico stability prediction studies indicated the most susceptible regions in the molecule to nucleophilic and electrophilic attacks, as well as the sites susceptible to oxidation. The in vitro forced degradation tests were in agreement with the in silico stability prediction, indicating that taxifolin is extremely unstable (class 1) under alkaline hydrolysis. In addition, taxifolin thermal degradation was increased by humidity. On the other hand, with respect to photosensitivity, taxifolin can be classified as class 4 (stable). Moreover, the alkaline degradation products were characterized by UPLC-ESI-MS/MS as dimers of taxifolin. These results enabled an understanding of the intrinsic lability of taxifolin, contributing to the development of stability-indicating methods, and of appropriate drug release systems, with the aims of preserving its stability and improving its bioavailability.
ABSTRACT
INTRODUCTION: Eugenia umbelliflora fruits are an important source of phloroglucinols, as eugenial C and eugenial D, related to antimicrobial activity against Staphylococcus aureus. However, for the establishment of new antimicrobial substances, it is essential to know their stability profile, in view of driving the administration route and the release system development. METHODOLOGY: The in silico approaches, based on the Fukui indices and bond dissociation analysis, were performed. Eugenial C and eugenial D, isolated from the green fruits of E. umbelliflora, with purity > 90%, were submitted to stress degradation including: acid (0.5 mM hydrochloric acid) and alkaline (0.5 mM sodium hydroxide) hydrolysis, and oxidation (0.25% hydrogen peroxide), in different periods, monitoring by high-performance liquid chromatography with ultraviolet detector (HPLC-UV). Eugenial C was also submitted to UV-visible radiation (2,400 lux/h) and dry/humid heating (40°C, 75% relative humidity). RESULTS: In silico studies indicated that both molecules have regions of high susceptibility to nucleophilic and electrophilic attack as well as sites likely to suffer auto-oxidation. Under in vitro tests, both phloroglucinols proved to be very unstable under hydrolysis (eugenial C and D were degraded 23.8% and 89.0% in acid and 78.4% and 97.8% in alkaline conditions, respectively) and oxidation (eugenial C and D degraded 31.9% and 28.6%, respectively), both during 5 min. Eugenial C degraded 12.6% and 63.8% under dry and humid heat, respectively, without photosensitivity. CONCLUSION: The in vitro stress tests monitored by HPLC-UV were in agreement with in silico degradation prediction. Phloroglucinols could be unstable if administered by oral route and also under environmental conditions demanding a protective release system.
