2.1. Chemicals
Lutein (≥ 96% purity), butylated hydroxyl toluene (BHT), linoleic acid, oleic acid and LC-MS grade solvents were purchased from Sigma-Aldrich (St Louis, MO). Pepsin, bile salts, bovine pancreatin, sodium taurocholate, and monooleoyl glycerol were purchased from Hi-Media (Mumbai, India). HPLC grade solvents methanol, hexane, acetonitrile, dichloromethane (DCM), acetone and sodium sulfate, potassium hydroxide, ammonium acetate, tween 20 and other chemicals were purchased from Sisco Research Laboratory (Mumbai, India).
2.2.1. Sample for lutein
Marigold flowers were purchased from the local agricultural market (Mysore, Karnataka, India), petals were separated and moisture was removed using a hot air oven (45°C). Dried petals were grounded using a mixer grinder and used for lutein extraction as described by Lakshminarayana, Aruna, Sangeetha, Bhaskar, Divakar and Baskaran [18].
2.2. Preparation of lutein nanoemulsion (lutein-NEL)
Oleic acid and linoleic acid (1 % w/v) were prepared in acetone separately, stirred using a magnetic stirrer for 20 min at 600 rpm. Lutein (0.4 mg/ml) was added to linoleic acid under magnetic stirring for 20 min at 600 rpm. Then, to the mixture of linoleic acid-lutein, oleic acid was added at a ratio of 1:1 under magnetic stirring for 30 min at 700 rpm with 0.1% Tween-20 (surfactant), and the mixture was sonicated for 15 min. The solvent was evaporated under nitrogen flux, and the lutein-NEL was redissolved in PBS followed by sonication for 15 min.
2.3. Preparation of blank nanoemulsion (blank-NEL)
The blank-NEL (NEL with no lutein added) was prepared using linoleic acid and oleic acid (1% w/v) was prepared in acetone separately and stirred for 20 min at 600 rpm. Then, at a ratio of 1:1, oleic acid and linoleic acid were mixed under magnetic stirring for 30 min at 700 rpm, and 0.1% Tween-20 was added. Then, the samples were sonicated for 15 min, the solvent was evaporated under nitrogen flux, and the NEL was redissolved in PBS followed by sonication for 15 min and treated as control.
2.4. Particle size distribution of NEL
The hydrodynamic diameter and particle size distribution of the prepared lutein-NEL and control NEL was determined by dynamic light scattering (Zetasizer Nano, Malvern Instruments, UK). Samples were prepared (n = 3) by diluting the lutein-NEL and control NEL separately with ultrapure water (1:10 ratio) and loaded into a polystyrene cuvette (DTS0012®, Malvern, UK). All measurements were performed in triplicate at 25°C with an angle of detection of 173° and a refractive index of 1.33 for the dispersion medium.
2.4.1. Surface charge of nanoemulsion
The surface charge or zeta potential of the diluted nanoemulsion (1/10 v/v in water) was measured (n = 3) using a Zetasizer instrument (Nano-ZS®, Malvern, UK). The dielectric constant of the dispersion medium was 78.5. A capillary cell (DTS1070®, Malvern, UK) was used for the zeta potential analysis.
2.4.2. Transmission electron microscopy (TEM)
TEM was used to examine the size and shape of the nanoparticles. In brief, diluted samples (1/10 in double-distilled water) were deposited on a carbon-coated copper grid and air-dried for 5 min and the excessive samples were removed with filter paper. A droplet of sample stain, sodium phosphotungstate (2 %, w/v), was placed on a dry copper grid for 3–5 min. The samples were observed by Titan Themis transmission electron microscopy (FEI, ThermoScientific™, Waltham, Massachusetts, USA) at 300 kV randomly scanned, and photomicrographs were taken at different magnifications.
2.5. Fourier transform infrared (FTIR) spectroscopy
Infrared spectra of oleic acid, linoleic acid, lutein and lutein nanoemulsion were recorded using a Fourier transform infrared spectrophotometer (Bruker, Tensor II, Germany). Briefly, samples were taken directly, and spectra were analyzed in the range of 4000–400 cm− 1.
2.6. Extraction of lutein from lutein-NEL
Lutein was extracted from lutein-NEL by the procedure described earlier by Toragall, Jayapala and Vallikannan [19]. Briefly, 0.5 ml of lutein-NEL was added to DCM: MeOH (1:2 v/v) vortexed and1.5 ml of hexane was added, vortexed and the hexane layer was collected. The process was repeated with DCM and hexane until the yellow colour disappeared. The hexane layer was pooled, evaporated under nitrogen flux and redissolved in the mobile phase for HPLC analysis.
2.7. Preparation of mixed micelles
Mixed micelles were prepared in phosphate-buffered saline containing mono-oleoyl-glycerol (2.5 mM), sodium taurocholate (12 mM), oleic acid (7.5 mM) and lutein (600 µM). These chemicals were separately dissolved in chloroform and methanol, and solvents were evaporated to dryness using nitrogen. The micelle was resuspended in phosphate-buffered saline (pH 7.2) with vigorous mixing using a vortexer (REMI CM-101 plus, India) and followed by sonication (PCI, Mumbai, India) for 15 min to obtain a clear solution. Lutein content in the lutein-NEL and mixed micelles was quantified using HPLC before it was used for further studies [10].
2.8. Confirmation of lutein core in NEL
The lutein core in the lutein-NEL was examined by phase-contrast microscopy (Olympus, Tokyo). In brief, lutein-NEL were mixed with ‘oil red O’ stain at a working concentration (0.3 %) followed by stirring and sonication. A drop of stained lutein-NEL was placed on a clean glass slide with a coverslip and visualized under phase contrast microscopy [19].
2.9. Solubility of lutein nanoemulsion
To determine the solubility of lutein, lutein-NEL, lutein mixed micelles and free lutein in oleic acid (1% w/v) (0.2 mg) were dispersed in 10 mL of distilled water and incubated in a shaking water bath at 37°C for 24 h. Samples were filtered through Whatman no. 1 filter paper, and lutein was extracted from the filtrate and quantified by HPLC [19].
2.10. Storage stability of lutein NEL
The prepared lutein NEL (n = 3) were immediately transferred into screw-capped glass vials and stored at different storage temperatures at 25°C and 4°C in the dark for 30 days. An aliquotes of the sample were periodically drawn (n = 3) for particle size and lutein content analysis. The percent of lutein retention was at each time point with respect to initial concentration was calculated [20].
2.11. In vitro bioaccessibility of lutein from NEL
In vitro bioaccessibility of lutein from lutein NEL in comparison with free lutein in oleic acid and mixed micelles (control) was measured. The bioaccessibility was examined in simulated gastrointestinal digestion as per method Garrett, Failla and Sarama [21], [22] with slight modifications. Lutein in the free form was dissolved with oleic acid, and mixed micelles and nanoemulsions (600 µM) were taken in screw-tightened glass vials and subjected to in vitro (gastric and intestinal phase) digestion. Gastric digestion: In brief, pepsin (porcine gastric mucosa 88–2500 U/mg protein) was dissolved (3 mL) in a phosphate buffer (3.6 mmol/L CaCl2, 12 mmol/L KCl, 6.4 mmol/L KH2PO4, 1.4 mmol/L MgCl2.6H2O, 49 mmol/L NaCl) was added to the sample containing vial, and the pH was adjusted to 2.0 using 2 M HCl. The vials were filled with nitrogen to prevent the oxidation of lutein, tightly capped and incubated at 37°C in a water bath shaker (Scigenics Orbitek, India) at 120 strokes/min for 1h.
Intestinal phase: After incubation, samples were loaded to room temperature (28°C) and the pH of the digest was raised to 5.0 using 1 M NaHCO3 followed by 6 mL of pancreatin (porcine pancreas 89 U.S.P) prepared in 0.1 M NaHCO3 and 25.38 g/L bile extract (porcine) were added. Furthermore, the pH of the digest was adjusted to 7.4 using 1 N NaOH, followed by incubation for 3 h in a shaking water bath after filling extra space with nitrogen. After the incubation period, the digesta (1 mL) was withdrawn from each sample at different time intervals (0–4 h) and ultracentrifuged at 144,000 x g at 4°C for 60 min (Beckman Optima-100 ultracentrifuge, Indianapolis, US). The aqueous phase containing micelles was used for the extraction and quantification of micellized lutein by HPLC [19]. The percent of transformation, bioaccessibility and bioavailability was calculated as per the equations given below [23]. Values represent the amount of lutein present in the small intestine without chemical degradation or alteration in the structural complexity. Perhaps the in vitro gastrointestinal digestion (GIT) simulated was cannot precisely replicate the complex process occurring in the gastrointestinal tract of in vivo. However, this method can be used for rapid screening of samples to identify important physicochemical interactions for drug/nutraceutical delivery.
$$Transformation \left(\%\right)=\frac{Concentration of lutein in digesta at the end}{Intial concentration of lutein} X 100$$
$$Bioaccessibility \left(\%\right)=\frac{Mass of lutein in micellar}{Mass of lutein in starting emulsion} X 100$$
$$In vitro bioavailability=Bioaccessibility X Tranformation$$
2.12. Lutein bioavailability in rat model
To investigate lutein bioavailability from lutein NEL, the animal experiment was performed after clearance from the CFTRI animal ethics committee (IEAC NO. FT/AHF/AI/108/2019). Weanling Wister rats weighing 50 ± 5 g were housed in polypropylene cages in the institute animal house facility at 28 ± 2°C with a 12 h light:dark cycle and given free access to feed and water. The standard pellet diet (crude protein-18.34%, crude fat-3.28%, crude fiber-5.40%, calcium-1.20%, phosphorus-0.58%, total ash-5.8%, carbohydrate-64% and moisture-7.42%) fed to rats was purchased from M/s Champaka feeds and foods (Bengaluru, India). Rats were acclimatized for one week before treatment started.
A single oral dose of equimolar concentration (600 µM, a physiological dose) of either micellar lutein (control) or lutein NEL and the free lutein-oleic acid mixture (0.2 mL) was gavaged to a group of rats (n = 6) separately. After 8 h of gavage, rats received no sedation before being sacrificed with CO2 anesthetic. The CO2 flow rate was monitored with AIMS CO2 flow meter (AIMS™ SC100) with 25 L/min CO2 for rapid euthanasia. Blood was collected in heparin-coated tubes for the analysis of lutein in the plasma, liver and eyes. To obtain the plasma, the blood was centrifuged at 1000 x g for 15 min at 4°C. The liver and eyes of rats were homogenized with ice-cold isotonic saline (0.9%). The lutein was extracted from plasma (1 mL), liver and eye homogenate (1 mL) and analyzed by HPLC.
2.13. HPLC and LCMS analysis
The purified lutein from the marigold flower petals extracted lutein from lutein NEL, plasma and tissues were analyzed by HPLC (Alliance 2690, Waters, UK). Standard lutein and lutein extracts were injected (10 μL) into an HPLC system equipped with a C-18 column (250*4.6 mm2; SGE company, Mumbai, India) and PDA detector. The mobile phase (acetonitrile/methanol/DCM; 6:2:2 v/v/v) containing 0.1% ammonium acetate was used for the separation of lutein at a flow rate of 1 mL/min by monitoring at 444 nm. LCMS analysis of lutein was carried out as per the method of Toragall, Jayapala and Vallikannan [19] to validate the mass of the lutein extracted with the lutein standard.
2.13. Statistical data analysis
Experiments were carried out in triplicate (NEL preparation and characterization) or n=6/group (animal experiments), and the results were statistically tested for significance (p ≤ 0.05) for the analysis of variance with one-way and two-way ANOVA. All statistical calculations were performed using GraphPad Prism 5.0 software (GraphPad Prism 5 ®, GraphPad Software, San Diego, CA).