Marta
Corzo-Martínez
a,
Celia
Bañares
a,
Alejandro
Díaz
a,
Luis
Vázquez
a,
Guillermo
Reglero
ab and
Carlos F.
Torres
*a
aDept. Production and Characterization of Novel Foods, Institute of Food Science Research (CIAL, CSIC-UAM), Madrid, Spain. E-mail: carlos.torres@uam.es; Fax: +34 910017905; Tel: +34 910017912
bDepartment of Production and Development of Foods for Health, IMDEA-Food Institute, CEI (UAM-CSIC), C/Faraday 7, 28049 Madrid, Spain
First published on 6th January 2020
This work studies the effect of enzymatic glycerolysis on digestibility and bioaccessibility of ratfish liver oil (RLO) rich in alkylglycerols (AKGs), as well as the capability of the glycerolysis product (GP) to act as lipid-based delivery system (LBDS) for a supercritical rosemary extract. For comparison purposes, digestibility and bioaccessibility of two additional lipid systems i.e. original RLO and RLO with addition of GRAS monoolein (MO) as emulsifier agent (RLO + MO), have been evaluated. We have studied the efficiency of the GP and RLO + MO lipid systems as LBDS by combining them with a supercritical rosemary extract (RE), i.e. RE lipid-based formulations. In vitro digestibility and bioaccessibility of un-loaded lipid systems, RE lipid-based formulations and un-carried RE have been determined. The results show a higher digestibility and bioaccessibility of the GP as compared to those of original RLO and RLO + MO. Likewise, a substantial improvement of RE bioaccessibility has been observed when GP is used as lipid carrier of RE. The present work demonstrates that enzymatic glycerolysis is an efficient strategy to obtain highly bioaccessible and potentially bioactive alkylglycerol-based delivery systems, which can be used to increase the bioaccessibility of low water-soluble bioactive compounds.
The potential mechanism of self-emulsifying delivery systems by which they increase bioaccessibility of bioactive compounds and ultimately its bioactivity is as follows. After oral intake of drug or bioactive compound and once in the stomach, lipid system is quickly dispersed in oil drops under gastric lipase and peristalsis effect. Oil dispersion increases drug solubility and dissolution in the aqueous medium, avoiding its precipitation and degradation. Once in the small intestine, pancreatic lipase hydrolyzes triacylglycerol (TAG) into free fatty acids (FFA), di- and monoacylglycerol (DAG and MAG) which along with bile salts and PL from gallbladder form vesicles and micellar structures. These stabilize the bioactive compounds, avoiding their precipitation and interaction with absorption inhibitors and favoring the bioactive transportation until the absorption area.
Oils commonly used to manufacture this kind of formulations are edible plant oils due to their high content in medium chain fatty acids with high dissolution capacity and stability against oxidation.10 Fish oils are not often used in the formulation of lipid carriers due to their high content in PUFAs and, hence, to their lower oxidative stability. However, an interesting and novel alternative to plant oils is the Ratfish Liver Oil (RLO), which offers several advantages with respect to the rest fish oils. On the one hand, due to its higher content in medium chain fatty acids and lower PUFAs content is more stable against oxidation than the rest of fish oils. And, on the other hand, it is exceptionally rich in alkyglycerols (AKG), the ether analogous of acylglycerols.11 Previous studies, some of them carried out in our laboratory, have demonstrated their security and special health-promoting effects in humans, including anticarcinogenic and immunomodulatory activity, hematopoiesis stimulation, increase of sperm motility, and antiviral activity.12–20 Therefore, besides increasing the bioaccessibility of the carried bioactive compound, the use of RLO, rich in AKG, to prepare SEDS has an extra advantage, as it might provide an additional or even synergistic bioactive effect to that of the carried compound, giving rise to highly bioefficient formulations.
AKGs, previously purified from shark liver oil, have been used in previous works as delivery systems for bioactive substances such as butyric acid21 and hydroxytyrosol esters.22 Likewise, Patent ES2294956A123 refers to the procedure for obtaining modified alkoxyglycerols from shark liver oil by fractionation, to obtain a fraction rich in non-esterified AKGs, and subsequent re-esterification with long chain fatty acids, which can be used as lipid carrier of bioactive compounds. However, to the best of our knowledge, studies on the use of self-emulsifying lipid-based delivery systems, rich in AKG, in combination with bioactive compounds for the preparation of highly bioaccessible and bioefficient formulations do not exist.
The use of RLO as lipid carrier presents, however, a limitation. O-Alkyl residue of AKG at sn-1 position of the glycerol backbone affects significantly oil digestibility, which limits its capacity to act as efficient lipid carrier.8,24 This highlights the need of strategies that improves the digestibility, miscibility and emulsifying capacity of this oil. In this respect, enzymatic glycerolysis results of great interest, as it leads to mixtures of minor glycerides and alkylglycerls with better dissolution and self-dispersion capacity than the original oil. Moreover, these mixtures are similar to the natural end products of lipid intestinal digestion and, hence, more biocompatible. In a work previously performed by our group,25 enzymatic glycerolysis, under optimized conditions, shown to be an efficient, cost-effective, environmentally friendly and easily scalable method for the design of potential self-emulsifying systems from ratfish liver oil, with a high content of bioactive alkylglycerols.
In the present work, the in vitro digestibility and bioaccessibility of a lipid system obtained by enzymatic glycerolysis from RLO under optimized conditions have been studied with the aim of evaluating the potential of this glycerolysis product rich in bioactive AKGs to act as efficient lipid-based delivery system of bioactive compounds for obtaining nutritional supplements and functional foods of high bioavailability and bioefficacy.
Food grade monoolein (MO) (99% purity) and cyclopentanone (CPN) used in glycerolysis process, considered as food grade flavoring agent, were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Glycerol was purchased from ICN Biomedicals (Aurora, OH) and the biocatalyst Novozym 435 (Nov435) (immobilized on acrylic resin, ≥5000 U g−1 according to manufacturer) (Candida Antarctica) was kindly supplied by Novozymes A/S (Bagsvaerd, Denmark).
Reagents used for in vitro digestion, including trizma, maleic acid, pancreatin from porcine pancreas, bile salts and cholesterol were purchased from Sigma-Aldrich. Phosphatidyl choline from egg yolk (PC) and Phospholipon 90H (PL) were supplied by Lipoid (Ludwigshafen, Germany). Hydrochloric acid, sodium sulfate anhydrous, sodium chloride, and calcium chloride were from Panreac (Barcelona, Spain).
Regarding reagents used for chromatographic analysis, pure standards of oleic acid (99% purity) and batyl alcohol (99% purity) as well as the menhaden fish oil were obtained from Sigma-Aldrich. Isooctane was obtained from Carlo Erba Reagents (Val de Reuil, France). Hexane, metyl-tertbutyl ether (MTBE) and chloroform were obtained from Lab-Scan (Gliwice, Poland) and formic acid (98% purity) from Panreac (Barcelona, Spain). All these solvents were of HPLC grade.
Rosemary extract (RE): Stabiloton OS was acquired to RAPS GmbH & Co KG (Kulmbach, Germany) with a phenolic diterpenes content of 30%.
- RLO glycerolysis product (GP) with potential self-emulsifying properties. RLO-derived product obtained by an enzymatic glycerolysis process at pilot plant scale, which was previously optimized by our group to result in the maximum diacylglycerol ether (DAGE) and triacylglycerol (TAG) conversion and the monoacylglycerol (MAG) formation in sufficient amount to provide good emulsifying properties to the product mixture (32%, w/w).25 Optimal glycerolysis conditions at pilot plant scale were 40 °C, 48 h, RLO to glycerol molar ratio of 1:1, use of immobilized commercial lipase Nov435 as biocatalyst, enzyme to substrate molar ratio of 1:10 (w:w), and 67% CPN (GRAS solvent).
- Original RLO, which is constituted only by DAGE and TAG.
- RLO with addition of food grade monoolein (MO) as emulsifier agent in a molar ratio RLO:MO of 1:1, which contains DAGE, TAG and MO. This will be called from now on RLO + MO.
On the other hand, a solution trying to simulate biliary secretion was prepared by mixing 200 mg of PC, 250 mg of bile salts, 40 mg of cholesterol (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 1 mL of a 325 mM CaCl2 solution, 3 mL of a 3.25 mM NaCl solution (Panreac Química S.A.U, Barcelona, Spain), and 20 mL of Trizma-maleate buffer 0.1 M pH 7.5. This mixture was homogenized for 2 min at 3500 rpm.
Then, the pre-emulsified sample and the simulated biliary secretion were mixed and homogenized together for 2 more min at 3500 rpm. The whole media was placed in a thermostatically controlled vessel at 37 °C and continuously stirred at 1000 rpm. Simulated intestinal digestion was started by the addition of fresh porcine pancreatin extract, which was prepared as follows: 1.167 g of pancreatin in 7 mL of Trizma-maleate buffer, stirred for 10 min and centrifuged at 1600g at 5 °C for 15 min. 6 mL of aqueous supernatant were added to the reaction medium together with 10 mg of a food grade PLA2 from Streptomyces violaceoruber (103 U mg−1) (Nagase Chemtex Corporation, Fukuchiyama Factory, Kyoto, Japan). After addition of enzymes, reaction was continued for 60 min at 37 °C, taking off aliquots at 0, 5, 10, 30, and 60 min in order to study the evolution of lipid digestion.
In vitro digestion of each sample was performed at least in duplicate.
Organic phases containing separated lipids were collected after each extraction, put together into a vial with anhydrous sodium sulfate for humidity removal and evaporated under a nitrogen stream by using a Stuart Block Heater SBH200D/3 (Staffordshire, U.K.) until constant weight residue. Finally, samples were diluted with chloroform to a final concentration of 20 mg mL−1 before injection in the LC system.
Lipids in the reaction mixtures were identified by comparing their retention times (tR) with those of different standard lipids. Commercial oleic acid and batyl alcohol were used for identification of free fatty acid (FFA) and AKG, respectively. DAGE and TAG were identified by using commercial RLO and menhaden fish oil.27 And finally, products derived from glycerolysis of menhaden fish oil and purified by semi-preparative HPLC were used for identification of MAGE, DAG and MAG. Quantitative analysis was performed by the external standard method, using calibration curves of each standard in the range 0.4–25 mg mL−1. Relative standard deviation (RSD) values were below 10% in all cases.
Quantitative data were expressed as both, conversion of DAGE and TAG during the in vitro digestion (E1), and percentage of each compound of the digestion mixture in weight respect to the total weight of the digestion medium (E2).
100 − [(%final/%initial) × 100] | (E1) |
[Wcomp/Wd.m] × 100 | (E2) |
All measurements were performed by triplicate, data provided being the mean particle size ± SD.
Fig. 1 shows LC-ELSD profiles of RLO original, RLO + MO and GP. Original RLO was mainly comprised of two compounds, diacylglycerol ether (DAGE, peak 1, tR = 6.7 min) and triacylglycerol (TAG, peak 2, tR = 7.7 min), the content in DAGE (80%, w/w) of RLO being much higher than TAG content (20%, w/w). LC-ELSD chromatogram of RLO + MO showed one peak corresponding to MO (peak 5, tR = 18.2 min, ∼28%, w/w) in addition to peaks of DAGE (∼57%, w/w) and TAG (∼15%, w/w). Finally, the glycerolysis product showed a composition more complex than that one of RLO and RLO + MO. The final reaction mixture obtained after optimal glycerolysis conditions (previously indicated in section 2.2.1) was constituted by the reactants DAGE (∼25%, w/w) and TAG (∼1%, w/w), but also by the different products of glycerolysis reaction, namely 2-monoacylglycerol ether (2-MAGE), which coeluted with 1,3-DAG (peak 3, tR = 12.9 min, ∼42%, w/w), 1,2-DAG (peak 4, tR = 14.2 min, ∼2%, w/w) and MAG (peak 5, tR = 18.2 min, ∼32%, w/w). MAGE, DAG and, particularly, MAG have higher polarity and, hence, higher emulsifying capacity than starting DAGE and TAG.
Fig. 1 Chromatographic profile obtained by LC-ELSD of original RLO, RLO + MO and glycerolysis product (GP). 1 = DAGE; 2 = TAG; 3 = 2-MAGE + 1,3-DAG; 4 = 1,2-DAG; 5 = MO/MAG. |
To evaluate the bioaccessibility of studied lipid systems and RE lipid-based formulations, these were subjected to an in vitro gastrointestinal digestion model as explained in the Method section.
As expected, during the gastric phase the lipid profile was not modified with respect to the initial one due to the absence of lipase in the digestion medium (data not shown). However, during the intestinal phase, lipids were hydrolyzed mainly by the action of the pancreatic lipase, so that the digests present a lipid composition different from the initial one.
Fig. 2 depicts the composition of the final digestion products of original RLO (Fig. 2A), RLO + MO (Fig. 2B) and GP (Fig. 2C). For all of them, TAGs were completely hydrolyzed, whereas non-hydrolyzed DAGEs were still detected at the end of the digestion process. This agrees with previous studies (Martin et al., 2011),24 in which a DAGE digestibility lower than that of TAG was observed, suggesting that digestibility of studied lipid systems is determined by their initial content in DAGEs. Thus, as observed in Fig. 2, the highest hydrolysis degree of DAGEs and formation of FFA, DAG, MAGE and MAG were observed for GP, followed by the system RLO + MO. Original RLO, with the highest initial content in DAGEs, was the least digestible of all the studied lipid systems.
Moreover, these results indicate that lipid digestion is favored in the presence of an emulsifier in the medium. Lipids with emulsifying character, as MAG/MO, increase lipid dispersion at the beginning of digestion, which may enhance both the TAG and DAGE hydrolysis and the formation of micellar structures, inside which digestion products can be more easily included.30 Likewise, it is noteworthy the higher content of free AKG (non-esterified) in the final digestion product of GP as compared to that in original RLO and RLO + MO digests. This could be attributed to the higher content in MAGE of GP, which is preferably hydrolyzed before than DAGE to give free AKG.
Regarding GTI digestion of RE formulations, the presence of 4% (w/w) and 9% (w/w) of RE did not alter the digestion of the lipid carrier (GP or RLO + MO), as no notable differences with respect un-loaded lipid systems were observed. However, digestion product of formulation with 16% of RE presented a substantially higher content in DAGE and a lower percentage of FFA, MAGE, DAG and MAG, indicating a lower digestibility of lipid fraction. According to previous works,31–33 this could be due to the inhibition of pancreatic lipase in the presence of a high RE content.
Bioaccessibility of studied lipid systems (RLO, RLO + MO and GP), RE lipid-based formulations and non-carried RE was evaluated by LC-ELSD through the characterization of their MP or bioaccessible fraction. As observed in Fig. 4, MP, mainly constituted by the digestion products (FFA, MAGE, DAG and MAG), is the predominant one for all studied systems. However, according to the results derived from digestibility assays, MP is especially abundant in the digestion product of GP, as ca. 98.5% (w/w) of the lipid fraction is in form of micelles, mixed micelles or vesicles. This indicates that almost all the products released during GTI digestion of GP are bioaccessible. The system RLO + MO and, particularly, the original RLO were substantially less bioaccessible than GP, as showed a lower percentage of lipid fraction in the micellar phase (ca. 75% and 45% (w/w), respectively) but higher in the oil phase (ca. 22% and 41% (w/w), respectively), mainly constituted by non-digested DAGE and MAGE (Fig. 5A and B).
Fig. 5 Lipid composition of the different phases (OP, MP and PP) obtained after centrifugation of the final digestion products of original RLO (A), RLO + MO (B) and GP (C). |
It is particularly of interest the high content in total AKG and non-esterified AKG in the MP of the GP. As shown in Fig. 5C, most of MAGE was in micellar form and, hence, bioaccessible. Likewise, MP of the GP also comprised a content of free AKG (ca. 7.5%, w/w) higher than that of systems RLO + MO (ca. 1%, w/w) and original RLO (ca. 0.7%, w/w). Both MAGE and, particularly, free AKG possess a bioactive potential higher than DAGE in agreement with several studies previously carried out with colon cancer cells.34
Based on digestibility and bioaccessibility results, it was decided to discard the original RLO as a potentially effective RE lipid carrier. That is why in the present work RE was formulated only with the lipid systems GP and RLO + MO. The incorporation of a 4% (w/w) and 9% (w/w) of RE did not affect the bioaccessibility of the lipid fraction. No remarkable differences in the distribution and composition of OP, MP and PP with respect un-loaded lipid systems were observed (data not shown). However, the presence of a 16% (w/w) RE clearly decreased the bioacessibility of the lipid fraction as compared to that of un-loaded lipid systems, as indicated by the more abundant OP and the important loss of MP (Fig. 3B). These results are in concordance with the decrease in the digestibility previously observed in these formulations due to the RE-induced inhibition of pancreatic lipase when RE is at high concentration.
RE bioaccessibility of lipid-based formulations and non-carried RE was evaluated by means of the determination of total polyphenols in the micellar phase. Non-carried RE (4% and 9%, w/w) showed a relatively low intestinal bioaccessibility, as only a ca. 39% of the initial RE content (before digestion) was detected in MP. As commented above, this could be attributed to the degradation or precipitation of RE during the gastric phase.28 However, when 4% and 9% (w/w) of RE was formulated with both GP and RLO + MO, its intestinal bioaccessibility improved notably with respect to that of non-carried RE. Such bioaccessibility improvement was particularly remarkable with GP, formulations GP + 4%/9% RE showing a ca. 91% of the initial RE content in the MP (vs. ca. 84% in the MP of formulation RLO + MO + 4%/9% RE).
Fig. 6 shows several pictures taken during quantification of micelles in the MP of studied lipid systems and RE formulations with the optical microscope. Under the optical microscope, micelles presented spherical and bright shape. At naked view, it can be observed clearly a high concentration of micellar structures in MP of GP and GP + 9% RE (Fig. 6E and F), followed (in decreasing order) by RLO + MO + 9% RE, RLO + MO, original RLO and non-carried RE (Fig. 6A–D). As observed in Table 1, the subsequent count of the micelles confirmed these observations, which correlates with the order from higher to lower digestibility and bioaccessibility of these systems (sections 3.2 and 3.3). Based on these results, we can infer that a direct relationship between the digestibility and bioaccessibility of a system and the micelle concentration in the MP exists.
Fig. 6 Pictures of the MP of all studied systems taken during the count of micelles with the optical microscope. |
Sample | No. of micelles per mL MP |
---|---|
RLO | 61 ± 11 |
RLO + MO | 113 ± 17 |
RLO + MO + 9% RE | 147 ± 10 |
GP | 324 ± 24 |
GP + 9% RE | 376.5 ± 75 |
Non-carried RE | 11 ± 3 |
NTA results also provided data of micelle concentration in the MP of lipid systems and RE formulations, as well as of size micelle distribution. In agreement with the count of micelles with the optical microscope, systems with the highest number of particles per mL of MP were GP and GP + 9% RE with 1.38 × 109 and 1.16 × 109 particles per mL, respectively, vs. 1.12 × 109, 1.08 × 109 and 1.04 × 109 particles per mL of RLO + MO + 9% RE, RLO + MO and original RLO, respectively.
The average size of micelles in all systems was similar, around 200–300 nm. However, as shown in Fig. 7, notable differences in the micelle size distribution of different systems can be observed. Micellas phases of RLO + MO + 9% RE, RLO + MO and original RLO presented a similar size distribution (Fig. 7A), in which it can be distinguished a population of minor particles (ca. 100–250 nm) and two populations of higher size (ca. 250–500 and 500–700 nm), probably due to the micellar aggregation. Such micellar aggregates could be clearly observed in micrographs obtained by HR-TEM (Fig. 8A). Micellar phase of GP and GP + 9% RE (Fig. 7B) showed a size distribution more homogeneous, with a major population of particles of ca. 100–200 nm and a lower concentration of aggregates. In concordance, micrographs obtained by HR-TEM (Fig. 8B) showed individual micelles with spherical shape. The higher concentration of micellar aggregates (>900 nm) in the MP of RLO + MO + 9% RE, RLO + MO and original RLO suggests a lower stability as compared to MP of GP and GP + 9% RE, in which it seems that a more stable emulsion forms after intestinal digestion. However, further studies are needed to confirm this and conclude that GP acts as an efficient self-emulsifying system.
Fig. 7 Particle size distribution obtained by NTA of the MP of RLO, RLO + MO, and RLO + MO + 9% RE (A); and RLO, GP, and GP + 9% RE (B). |
The use of the GP as lipid carrier of rosemary extract efficiently increased stability during GTI digestion, leading to an increase of its intestinal bioaccessibility.
Therefore, the present work has demonstrated that enzymatic glycerolysis is an efficient formulation strategy to obtain highly bioaccessible and potentially bioactive alkylglycerol-based delivery systems, which can be used to increase the bioaccessibility of low water-soluble bioactive compounds. Further studies are needed to evaluate the bioactivity of these new LBDS and resulting formulations when they are combined with bioactive compounds.
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