Available online 23 October 2015
Highlights
- •
- Phenolic compounds from sage and savoury extracts are entrapped in SLN.
- •
- SLN loaded with savoury extracts showed higher antioxidant activity.
- •
- The digestion of SLN is affected by the type of wax used at production.
- •
- At the small intestine all SLNs showed ca. 100% release of rosmarinic acid.
- •
- Witepsol SLNs showed to be the most stable vehicles for sage and savoury extracts.
ABSTRACT
Solid
lipid nanoparticles (SLN) can be used as vehicles for phenolic
compounds rich extracts. In the present work two types of waxes —
witepsol and carnauba were tested for the first time in the production
of solid lipid nanoparticles (WSLN and CSLN, respectively) loaded with
sage and savoury extracts. Physical characterization and association
efficiencies calculation were performed. Discrimination of loaded
phenolic compounds from each extract was made using HPLC assays.
Antioxidant activities of SLN were characterized using two different
methods — ABTS and ORAC. Finally, the phenolic compound release profile
from SLN and stability when exposed to simulated gastrointestinal tract
(GIT) conditions were also evaluated. Different phenolic compounds from
sage and savoury extracts were entrapped in SLN. The highest antioxidant
activity was obtained for the SLN loaded with savoury extract. Stomach
simulated condition provokes a partial release of rosmarinic acid from
SLN, whereas at small intestine simulation step, all SLN showed a
release of ca. 100%. Witepsol SLN were the ones that best maintained
their physical integrity during digestion, showing to be the most stable
vehicles for sage and savoury extracts. These SLN show to be suitable
for the production of food functional ingredients bearing antioxidant
activity.
Keywords
- SLN;
- Herbal extracts;
- Digestion;
- Antioxidant activity;
- Release profiles
1. Introduction
Sage and savoury (common English names for Salvia sp. and Satureja montana,
respectively) are herbs often used in Mediterranean traditional
medicines. These herbs are widely applied as seasoning, but also have
been used as anti-diarrheic, digestive aid, wound-healing,
anti-inflammatory, anti-insomnia and anti-hypertensive vectors; some of
these biological activities have been accounted for the presence of
rosmarinic acid (RA) ( Gião et al., 2009 and Giao et al., 2010).
Extraction of antioxidants (mainly phenolic compounds) from these herbs
can be easily achieved using boiling water and powdered plant material.
The majority of the phenolic compounds in Salvia species are
derivatives of caffeic acid, which is the building block of a variety of
plant metabolites. Caffeic acid plays a central role in the
biochemistry of the Lamiaceae plants, and occurs mainly in a dimer form
as RA ( Kamatou, Viljoen, & Steenkamp, 2010). Savoury on the other hand, contains rutin and RA (ca. 10 and 4%, respectively) ( Gião et al., 2009).
The use of herbal extracts can reduce the production costs of
antioxidant nano/microparticles and also the toxicological risk
associated with the use of pure or synthetic compounds.
These
extracts have a variety of compound types and with different stability
behaviours when incorporated in a food compound or when at
gastrointestinal tract (GIT) conditions. The loss of part of the extract
constituents can result on bioactivity decreases. Some of these
compounds also have low bioavailability, and are poorly absorbed either
due to their large molecular size and poor lipid solubility and cannot
be absorb by passive diffusion (Manach, Williamson, Morand, Scalbert, & Rémésy, 2005).
In phyto-formulation research, it is believe that the development of
nanodosage forms of phyto-compounds could overpass these disadvantages (Nunes et al., 2015).
Hence,
the development of a system that can protect these compounds and
maintain them stable or even that improve their bioavailability was
sought. The systems proposed here are at nanoscale and have lipidic
nature. For their formulation and for oral administration destination,
lipid, emulsifier and water are required as essential components and all
need to hold a generally recognized as safe (GRAS) status (Olbrich et al., 2002 and Severino et al., 2011).
Encapsulation of pure RA using witepsol and carnauba waxes was already
performed elsewhere. These systems had mean diameters between 270 and
1000 nm, with ca. 99% of association efficiencies, and demonstrated to
be highly stable (Madureira et al., 2015).
At the time, the best formulations were selected as the ones having
1.0% (w/v) lipid, 2.0% (v/v) polysorbate 80 and 0.15 mg/mL RA.
Thus,
this research describes the production of SLN loaded with extracts from
sage and savoury herbs using these two lipid matrices. These SLN were
characterized in terms of their physical and morphological
characterization and association efficiencies. The antioxidant
activities were determined using two different methods based on single
electron transfer (SET) –
2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), and
hydrogen atom transfer reaction (HAT) – ORAC assays. Stability of SLN
and phenolic compound release at a dialysis bag and when exposed to a
simulated gastrointestinal tract (GIT) were also evaluated, in what
concerns the release of phenolic compounds and physical properties of
SLN stabilities. These nanoparticles could be further used as functional
ingredients bearing antioxidant activity, for incorporation in several
food matrices.
2. Material and methods
The major steps of the experimental work performed are schematically presented in Fig. 1.
- Fig. 1.Schematic representation of the major assays performed.
2.1. Herbal extracts preparation
Leaves of sage (Salvia officinalis) and savoury (Satureja montana)
herbs were milled to power and 1 g was added to 110 mL of boiling
deionized water, which was left to cool by mixing for 5 min. The
solution was then filtered through a No. 1 filter paper (V. Reis,
Portugal), frozen and finally submitted to freeze-drying process.
Freeze-drying process was performed using a vacuum freeze drier (Model
FT33, Armfield, UK), under a vacuum pressure (100 mTorr), with
temperatures of − 46 °C in a freezing chamber and 15 °C in a sample
chamber. These dried extracts were resuspended in water and analyzed by
HPLC (as described below), in order to identify and quantify the
phenolic compounds present in the initial extracts.
2.2. SLN production
The
waxes witepsol H15 (Sasol, Hamburg, Germany) and carnauba yellow no. 1
(Sigma-Aldrich Co., St. Louis, MO, USA) were used as lipid matrices. The
surfactant polysorbate (tween 80) was purchased from Sigma-Aldrich Co.
Solid lipid nanoparticle formulations were produced using 0.5% (w/v) of
lipid matrix, 2% (v/v) of an aqueous solution of polysorbate 80 and the
herbal water extracts at a final concentration of 0.15 mg/mL. Hot melt
ultrasonication was employed as the production method. Hence, the waxes
were warmed to a temperature of 5 °C above the melting point (36 °C for
witepsol and 86 °C for carnauba), then the herbal extracts were added
individually to the melted matrix and submitted to ultrasonication (VCX
130, Sonics & Materials, Newtown, IA, USA) for 1 min at 70% of
intensity. For a complete homogenization of the O/W emulsion, the
aqueous solution of emulsifier was added and mixed for few seconds.
Eight batches resulted from this production: sage and savoury free
extracts, witepsol SLN without extract named WSLN empty, carnauba SLN
without extract named CSLN empty, witepsol SLN loaded with sage extract
named WSLN_Sage, witepsol SLN loaded savoury extract named WSLN_Savoury
and carnauba SLN loaded with savoury named CSLN_Savoury. Each batch was
produced in triplicate and twice. Solutions were left to cool at room
temperature (20 °C). For the assays where dried samples were used, the
SLN emulsions were freeze-dried using a vacuum freeze drier (Model FT33,
Armfield, UK), under a vacuum pressure of 100 mTorr, and temperatures
in the freezing chamber of − 46 °C and in the sample chamber of 15 °C.
