Phytopharmaceuticals. Группа авторов

extracts were filtered and freeze dried until used. Figure 2.7b shows a plot of antiradical activity vs. total polyphenol content measured by Folin–Cicolteu method. Through a calibration curve using different concentrations of gallic acid, the results were expressed as gallic acid equivalents per gram of extract [135]. A positive correlation was observed (R² = 0.828) strongly driven by Lantana camara L. (Verbenaceae Family) extract which showed the highest values for both AA% and gallic acid equivalents, indicating that 82.8% of the antirradical activity observed for the evaluated samples results from natural occurring phenolic compounds contribution.

      While antioxidant capacity and TP relationships have a positive correlation, it may diverge from ideal linearity since Folin–Ciocalteu method is specific for phenolic compounds and antioxidant activity can also be due to a variety of other non-phenolic compounds [136].

      The reducing power of bioactive compounds may be quantified by determining the ferrric reducing (FRAP) or on the capacity to reduce cupric compounds (CUPRAC). FRAP measures the absorbance change at 593 nm produced by Fe (III) in the 2,4,6-tripyridyl-s-thiazine ferric complex TPTZ to the colored Fe (II) derivative [137]. Similarly, CUPRAC uses 2,9-dimetil-4,7-diphenil-1,10-fenantroline, batocuproine and the absorbance change at 490 nm [138]. In the oxygen radical absorbance capacity (ORAC) assay, a probe is measured (β-PE or fluorescein) and its decay by the presence of radicals that are generated by thermic decomposition of azonitriles. Finally, an interesting antioxidant assay is the co-oxidation of β-carotene by lipoxygenase where the oxidation of linoleic acid is monitored spectrophotometrically at 234 nm and the real antioxidant activity of compounds is quantified [128].

      Thin layer chromatography (TLC)-coupled method combine de advantage of separative techniques with chemical activity detection which make them a useful tool in assaying natural extracts. The approach consists on an initial screening by dot blot to select positive extracts. TLCs with different mobile phases are then run on selected samples in order to separate active compounds. Chromatography is revealed by a DPPH assay on the plate surface. TLC coupled autographic assays have the advantages of being simple and several samples could be assayed on the same run with minimal sample handling and high repeatability.

Figure 2.8 shows a DPPH autography on TLC [138]. Samples were aqueous extracts of two species from the Brassicaceae family, Rapistrum rugosum (RR) and Sinapis arvensis (SA). Sulfur containing compounds such as glucosinolates are present in this family. Different types of naturally occurring glucosinolates with different biological activities have been described and many of them have antioxidant capacity [139]. Active compounds appear as yellow spots against purple background. RR showed several active compounds that could be separated along the plate. On the other hand, active compounds on SA remain mainly at the origin while others migrated with low resolution to Rf 0.2.

Two-dimensional TLC can be exploited for qualitative activity analysis in complicated mixtures of several plant extracts at the same time more efficiently and with better resolution than one-dimensional TLC [140]. An innovative technique is based on running high resolution mass spectroscopy (HRMS) in extracting with solvent a autographic separated spot from the TLC (that could be standard low-resolution), allowing the identification of bioactive components in complex systems [141].

Figure 2.8 Right: TLC coupled DPPH autography. Left: TLC plate revealed using UV light (254 nm). Line 1: Rapistrum rugosum extract. Line 2: Sinapis arvensis extract. Mobile phase: toluene-acetone-methylene chloride (40:25:35). Rf: retention factor.

       2.3.2.2 Antiglycant Agents Detection

      The in vitro analysis of antiglycant extracts are based on measuring their inhibitory effect on proteins glycation and related markers in control reactions. The most used models are bovine serum albumin (BSA) and a reducing sugar in a phosphate buffered saline solution (PBS) so the potentially inhibiting extracts are challenged. The systems with and without the extract under analysis are incubated at 37°C or 55°C, or higher temperatures, according to the aim of the study.

      After the isomerization of the Schiff bases, in the advanced phases of the Maillard reaction, very reactive dicarbonyl compounds are formed from proteins residues, called AGEs (advanced glycosilation end products). The main reaction markers for AGEs formation can be crosslinked compounds, fluorescent (such as pentosidine) or nonfluorescent (such as imidazolium dilysine), and not-crosslinked nonfluorescent AGEs, (such as Nε-carboxyethyllysine, CEL and Nε-carboxymethyl-lysine, CML) [142]. Reactive dicarbonyl compounds can also be generated in the lipid oxidation reactions, and some publications stated the correlation between anti-glycant activity and antioxidant activity of natural species [143]. The anti-glycation properties of extracts can be studied by several techniques analyzing different steps of the reaction:

       Fluorescence (excitation/emission l pair 370/440 nm)

       Protein conformation changes detected by polyacrylamide gel electrophoresis using sodium dodecyl sulfate (SDS-PAGE), Coomassie Brilliant Blue R-250 can be used as staining agent to detect proteins [144] and periodic acid Schiff’s staining reveals glycosilated proteins.

       Furosine determination by HPLC [145].

       UV-absorbance and browning at 294/340 nm [146].

       Spectrophotometric analysis of nitro-blue tetrazolium (NBT) reductive assay [147].

       Nε-carboxyethyllysine (CEL) and Nε-carboxymethyl-lysine (CML) (available ELISA Kits).

      In the last years several natural aqueous and/or ethanol extracts with antiglycant capacity have been obtained from different natural sources as Allium sativum, Zingiber officinale, Thymus vulgaris, Petroselinum crispum, Murraya koenigii Spreng, Mentha piperita, Curcuma longa, Allium cepa, Piper nigrum [148]. Different polyphenols, mostly phenylpropanoids and flavonoids, which could be present in high concentrations in tea, cinnamon, rosemary, mate and other herbal plants, are efficient trapping agents of a-dicarbonyl compounds, very active glycating agents which are intermediates of the Maillard reaction [146–148].

2.3.3 Biocompounds Conservation and Controlled Delivery Systems

      Most bioactive compounds from natural sources with health benefits are difficult to incorporate in food, cosmetics or pharmaceutical matrices. Some of the reasons include incompatibility with ingredients and low stability under processes and storage conditions (light, oxygen, temperatures, enzymes, acid or alkali mediums, and humidity), which may lead to the loss of their activity and beneficial effects [149].

Several methods have been developed for encapsulation to preserve bioactive compounds at industrial scale. These methods can be classified into three main groups (Figure 2.9): chemical, physicochemical and physical–mechanical. For the selection of the most adequate encapsulation method, it is necessary to consider the production and cost of the process, the desirable morphology and the coating materials [150]. We are presenting an overall view of the recent studies on encapsulation using two of the more simple and widely employed microencapsulation methods, spray drying and coacervation, and protein systems as bioactives delivery and controlled release media.

Figure 2.9 Microencapsulation techniques [149–155].