2.1. Materials, Solvents and Reagents
Phosphatidylcholine (48% purified from soy lecithin), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin & Ciocalteu’s phenol reagent and quercetin 3-β-glucoside (≥90% pure) were purchased from Sigma-Aldrich, Co (St. Louis, MO, USA). Acetic acid was purchased from Panreac (Barcelona, Spain). Anhydrous sodium carbonate was obtained from VWR® Prolabo®, VWR International. Gallic acid (98%) was acquired from Merck (Darmstadt, Germany) and methanol (99.9%) was bought from Carlo Erba Reagents (Rodano, Italy). The study made use of Milli-Q® water (18.2 MΩ cm) which was attained from a Millipore—Direct Q3 UV System (Molsheim, France). Chromatography solvents are HPLC grade (Merck, Darmstadt, Germany) and the remaining reagents were analytical grade.
The materials for UHPLC-DAD: Merck® gradient grade methanol; acetic acid (glacial) RPE from Carlo Erba; ultrapure water.
Standards UHPLC-DAD: A number of selected phenolic compounds that were used as standards were procured from Sigma-Aldrich® (Madrid, Spain), namely: Protocatechuic acid (CAS 99-50-3); gallic acid (CAS 149-91-7); chlorogenic acid (CAS 329-97-9),;caffeic acid (CAS 331-39-5); epicatechin (CAS 490-46-0); ferulic Acid (CAS 1135-24-6); p-coumaric (CAS 7400-08-0); catechin (CAS 88191-48-4); and fisetin (CAS 345909-34-4); all from Sigma-Aldrich. The purity of the standards was always greater than 95%.
Sticks URIT 10V were purchased from Quilaban LDA (Quilaban LDA, Sintra, Portugal).
Ketamine (Imalgene® 1000) was purchased from Merial (Lisbon, Portugal), and xylazine (Rompun® 2%) was purchased from Bayer (Lisbon, Portugal).
2.2. Equipments
The Vötsch Industrietechnik heating chamber, VC2033 mit TC-Steuerung; Centrifuge 5804 R from Eppendorf (Hamburg, Germany); Rotavapor R-210 from Buchi (Meierseggstrasse, Switzerland; magnetic stirrer (HTS 1003, LMS, Tokyo, Japan); UV-vis spectrophotometer, Hitachi L-2000 (Hitachi High Technology, Tokyo, Japan); Millipore® water system (18.2 Ω cm at 25 °C) was purchased from Millipore—Direct Q3 UV System equipment (Molsheim, France); Delsa Nano C (Coulter, CA, USA); scanning electron microscopy (SEM 5200LV, JEOL, Tokyo, Japan) to evaluate morphology. A HPLC-DAD system (SpectraSystem, Thermo, Darmstadt, Germany) equipped with a binary gradient pump (SpectraSystem P2000, Thermo, Darmstadt, Germany), automatic sampler (SpectraSystem AS1000, Thermo, Darmstadt, Germany), diode array detector (SpectraSystem UV6000LP, Thermo, Darmstadt, Germany), UV controller (SpectraSystem SN4000, Thermo, Darmstadt, Germany) and software Xcalibur™ 2.0.6 (Thermo Fisher Scientific Corporation, Waltham, MA, USA) was used as well as an UHPLC AcquityTM (Waters, Milford MA, USA) equipped with a binary pump, an auto-sampler binary solvent manager, a column thermostatting system and a diode array detector (DAD).
2.3. Methods
2.3.1. Vegetal Extract Preparation
The fruit used in this study, Persimmon (
Diospyros kaki L.), was acquired from a cultivar grown in Portuguese territory (Setúbal region). The extract of persimmon fruit was prepared via the union of a number of methods described [
20,
21] albeit with some adjustments. Approximately 360 g ± 0.1 g of fresh persimmon fruit (FW) was extracted without calyx, using acetone:water (80:20,
v/
v) and it was then mechanically homogenized for 10 min (at room temperature and protected from light). The extracted sample was then centrifuged at 8603×
g for 10 min at room temperature (Eppendorf centrifuge 5804 R (Hamburg, Germany). The supernatant was then collected, and the pellet was extracted three more times using this same procedure. The supernatants were collected together and filtered, the sample was evaporated to almost dryness (35 °C, with slow, adjustable rotation), and finally, the extract was made up of Milli-Q
® water (240 mL) with at least 1.5 g FW/mL to encapsulate. The extract obtained was subsequently subdivided into aliquots and it was stored in falcon tubes at −20 °C, for further analysis.
2.3.2. Quantification of the Total Polyphenolic Content
The procedure to quantify total phenolic content determination was based on a previous work [
22]. An aliquot of the sample with 100 µL was added to 200 µL of Folin-Ciocalteau reagent (diluted in water at 1:10,
v/
v) where after 3 min, 1 mL of sodium carbonate (15%,
w/
v) and 2 mL of water were added. After incubating at room temperature for one hour, the absorbance was measured in the UV-Visible spectrophotometer at 765 nm (Hitachi L-2000, Hitachi High Technology, Tokyo, Japan) against a blank, i.e., a mixture of water and reagents.
A calibration curve of gallic acid was performed in a concentration range from 2 to 500 mg/L. The results were expressed in milligrams of gallic acid equivalents (mg of GAE) per 100 g of fresh fruit and per millilitre of extract. The determinations were made in triplicate and results were expressed as the means ± SD.
2.3.3. Quantification of D. kaki Phenolic Compounds in Extract and Non-Encapsulated PCs by Liquid Chromatography
An Ultra High Performance Liquid Chromatographic (UPLC®, AcquityTM, Waters, Milford, MA, USA) method coupled with a diode array detector (UHPLC-DAD) was developed for simultaneous quantification of the main phenolic compounds of the persimmon extract. The pre-column was an Acquity™ UPLC® BEH C18 (2.1 mm × 5 mm, 1.7 μm particle size). The column was an Acquity™ UPLC® BEH C18 RP 18 (2.1 mm × 50 mm, particle size 1.7 μm). The mobile phase was a gradient of water with 0.1% acetic acid (v/v) (solvent A) and acetonitrile with 0.1% (v/v) acetic acid (solvent B). The mobile phase gradient was: 0 min, 10% of solvent B; 2.5 min 15% of solvent B; 10 min, 30% of solvent B; 10.5 min, 30% of solvent B; 12 min 10% of solvent B.
The temperature of the analytical column was 20 °C and the samples were kept at 5 °C until analysed. The injection volume was 10 μL and the flow of the mobile phase was 0.2 mL/min. In order to correctly identify each compound, specificity was evaluated by comparing the absorption spectra of the chromatographic peaks obtained from the sample with the absorption spectra of analytical standards [
23,
24]. The quantification of the compounds was performed based on the peak area at a characteristic maximum absorption spectrum (λ
max) and the corresponding retention time (R
t) of each phenolic compound. The data was acquired using the software Empower™ version 2.0 (Waters, Milford, MA, USA). The analyses were performed in triplicate.
The chromatographic separation by high-performance liquid chromatography (HPLC) was performed on a reverse phase C18 column (Thermo Scientific) with a particle diameter of 5 μm and a length of 15 cm. The acquisition of chromatograms and UV-Vis spectra was performed using the software Xcalibur™ 2.0.6 (Thermo Fisher Scientific Corporation, Waltham, MA, USA), scanning between 190 and 700 nm, with a range of 1 nm and also at the specific lengths of 280 and 360 nm. The injection volume was 20 µL. The mobile phase consisted of Milli-Q® water–phosphoric acid (99.9: 0.1, v/v) as eluent A, and Milli-Q® water–acetonitrile–phosphoric acid (59.9:40.0:0.1, v/v/v) as eluent B. The mobile phase gradient was: From 0-15 min was from 0% to 20% of solvent B; 10 min with 20% solvent B; 25–70 min, from 20% to 70% solvent B; 70–75 min, with 70% of solvent B; 75–85 min from 70% to 100% solvent B; 85–110 min, with 100% solvent B; 111–120 min 100% of solvent A. The flow rate was 0.7 mL/min. Xcalibur™ software version 2.0.6 (Thermo Fisher Scientific Corporation, Waltham, MA, USA) was used to acquire and process the data. The identification of compounds was done by comparing the retention time (Rt), spectra and spiking samples with pure standard solutions, whenever available, or comparing with data from the literature.
An aliquot of the concentrated extract was diluted (1:7) in Milli-Q water to a concentration equal to the one present in the formulation and was analysed using the method and assay conditions described above. The compounds present in the supernatant of the formulation were identified by comparing and overlapping of the chromatograms obtained for the extract.
2.3.4. Preparation of Standard Stock Solutions and Work Solutions
From each compound, stock solutions at 5 mg/mL were prepared, except for protocatechuic acid with 2 mg/mL. The dilutions (1:100) of those stock solutions were performed with acidified 50% methanol (0.1% acetic acid) and filtered with a 0.2 μm filter. Several standard concentrations were then prepared: Gallic acid (50 μg/mL); chlorogenic acid (125 μg/mL); caffeic acid (50 μg/mL); epicatechin (125 μg/mL); p-coumaric acid (50 μg/mL); ferulic acid (75 μg/mL); (+)-catechin (150 μg/mL); fisetin (50 μg/mL) and protocatechuic acid (50 μg/mL). To this mixture, 6.8 mL of methanol was added and the volume of the volumetric flask was made up to 20 mL with ultrapure water with 0.2% acetic acid. Subsequently, approximately 2 mL of the solution was filtered into vial with 0.2 μm filter and individually injected into the UHPLC-DAD.
The order of elution of phenolic compounds was observed in the chromatogram corresponding to the mix solution. For each phenolic compound, the characteristic maximum absorption spectrum (λ
max) and the retention time (R
t) was determined [
25].
2.3.5. Sample
The persimmon extract solution was prepared with 1.5 mL of mature persimmon extract in a 10 mL volumetric flask and the volume of each flask was completed with acidified 50% methanol (0.1% acetic acid). Approximately, 2 mL of this sample solution was filtered for vials with 0.2 μm filters, for further analysis in UHPLC. The results were expressed in mg/100 g of fresh fruit.
2.3.6. Phytosomes Preparation
Phytosomes were prepared by adding extract to phosphatidylcholine dissolved in 20 mL of ethanol (1:1 or 1:2, molar ratio). This mixture was heated to 25 °C with a rotation of 300 rpm (HTS 1003, LMS, Tokyo, Japan) for 2 h. Thereafter, 40 mL of 2% acetic acid solution was added, and the mixture remained for 24 h in the same conditions previously described by Matias et al. 2015 [
26].
2.3.7. Physical Characterization of Phytosomes
The phytosomes were diluted (1:10) with distilled water and analysed on a Delsa Nano C (Coulter, CA, USA). The mean particle size, polydispersity index (PI) and zeta potential (ζP) were evaluated at room temperature using detection angle of 165° (except for zeta potential at 90°) for samples of the 1:1 and 1:2 formulation after 0, 3 and 6 months of preparation.
2.3.8. Determination of Encapsulation Efficiency (EE)
The efficiency of encapsulation (EE, %) of the extract in the phytosomes was determined by evaluating the fraction of the non-encapsulated extract [
27]. After centrifugation, the supernatant was collected and analysed on HPLC-DAD. Briefly, 2 mL aliquot of the phytosomal formulation was added to 2 mL oil and homogenized. Then, it was centrifuged at 15,000×
g (Eppendorf centrifuge 5804 R (Hamburg, Germany) for 30 min at 4 °C. The lower phase was separated and filtered with a 0.22 μm filter (VWR
TM, Radnor, PA, USA) and injected for HPLC chromatographic analysis. A blank was prepared in the same manner but with 2 mL water and 2 mL oil. The encapsulated extract (%) was calculated by the difference between the total persimmon extract area (corresponding to the extract added to the formulation) and the total chromatogram area of the supernatant (corresponding to non-encapsulated phenolic compounds) [
26,
28,
29,
30]. Applying Equation (1), the encapsulation efficiency (EE, %) of the extract in the phytosomes was determined at 280 and 360 nm. Independent injections into the HPLC column were performed and analysed.
The total phenolic compounds in the supernatant were also calculated by the spectrophotometric (765 nm) quantification of total phenolic compounds by the Folin-Ciocalteu technique. The same calculation was made for the extract sample and the encapsulation efficiency (EE, %) of the extract in the phytosomes was also calculated according to Equation (1). All measures were performed in triplicate.
2.3.9. Stability Test over the Time
The objective of the stability study was to evaluate the stability based on the test of at least three batches of the formulation following international guidelines [
31]. Stability studies should include testing of those attributes of the bioactive compounds that are susceptible to change during storage and are likely to influence quality, safety, and/or efficacy [
31,
32].
Parameters like particle size, PI and zeta potential were accessed over time.
2.3.10. Storage Conditions
In general, a consumer product should be evaluated under storage conditions (with appropriate tolerances) and, if applicable, its sensitivity to moisture or potential for loss of solvent. The storage conditions and duration of the chosen studies should be sufficient to cover the storage, transport, shipping and subsequent use [
31].
In an intermediate stability study, the minimum time period covered by data at submission was 6 months and the storage conditions generally were 30 ± 2 °C/65% RH (relative humidity) ± 5% RH [
32].
The accelerated storage conditions of the study were: 40 ± 2 °C/75% RH ± 5% RH; 25 ± 2 °C / 60% RH ± 5% RH and 4 ± 2 °C, over a period of 6 months. In the accelerated storage condition, a minimum of three points in time, including the start and end time points (for example, 0, 3 and 6 months), of a 6-month period study was recommended [
32].
2.3.11. Closure System and Container
Stability studies were conducted by storing the concentrated polyphenolic extract in a container and closure system, which was the same as the proposed packaging for storage and distribution [
31].
The two formulations (1:1 and 1:2, molar ratio) with 30 mL were each stored for 6 months in glass amber type II colour bottles and sheltered from light at room temperature (~25 °C) and 60% RH, in the refrigerator at 4 °C and 55% RH and at 40 °C in a heating chamber (Vötsch Industrietechnik, VC2033 mit TC-Steuerung, Oldenburg, Germany) with 75% humidity. At the same time, aliquots (7 mL) of the concentrated extract as well as quercetin standard solutions (10 mL) were also placed under the same conditions as the above-mentioned formulations.
2.3.12. Determination of the Antioxidant Activity
The potential for elimination of free radicals by the extract and by the phytosomal formulation (1:1 and 1:2) was determined according to the previous procedure [
33]. In brief, aliquots of 10 μL of each sample were mixed with 990 μL of DPPH solution (0.002% in 70% ethanol). The reaction mixture was incubated at room temperature for 30 min in the dark. The positive control was quercetin at 10 mgmL
−1 in water. As the absorbance control (i.e., at 100%), a sample containing 10 μL water and 990 μL DPPH was prepared. The free radical scavenging potential of the 1:1 and 2:1 phytosomal formulation and free extract samples were expressed as the disappearance of the initial purple colour. The absorbance was measured at 517 nm against a blank sample of 70% ethanol using a UV-Visible Spectrophotometer (Hitachi L-2000 from Hitachi High Technology, Tokyo, Japan). Each sample was tested in triplicate (
n = 3; mean ± SD). The sequestration capacity of DPPH was calculated using the following formula (2):
2.3.13. In Vivo Safety Assessment
Four-month-old female Wistar rats (250–330 g), obtained from Charles River (Barcelona, Spain) were housed in a 12–12 h light-dark cycle with a constant temperature environment of around 22 °C and a relative humidity of 55%. Animals were allowed free access to food and water during acclimatization. All animal experiments were conducted according to the animal welfare organ of the Faculty of Pharmacy, Universidade de Lisboa, approved by the competent national authority Direção-Geral de Alimentação e Veterinária (DGAV) and in accordance with the EU Directive (2010/63/UE) and Portuguese laws (DR 113/2013, 2880/2015 and 260/2016).
Animals were randomly allocated into three experimental groups: The sham group which were dosed with distilled water (n = 3); test group with animals orally dosed with extract-loaded phytosomes (15 mg/kg) (n = 5); and the free extract group (n = 5) where the same dose of the extract was applied. Oral administrations were performed daily, during the 15 days of the protocol, by gastric gavage. During the 15-day experiment, weight and mortality of all animal groups were monitored. The clinical signs of each rat were examined for ataxia, dehydration, dyspnoea, hypothermia, tachypnoea, lack of movement, sustained rapid movement around the cage, seizure, hunched posture and ruffled fur. A scale of 0 to 3 was used by an investigator blinded to treatment groups, where 0 was used for no indication, 1 for minimal indication, 2 for critical indication, and 3 for extreme indication of clinical problem(s).
Glycaemia was measured three times during the 15-day assay, at day 0, at the end of the first week of administration, and at day 15 of the protocol. All measures were done in triplicate in each of the three times of the protocol.
For the analysis of urine parameters, urine of all animal groups was collected on days 0, 7 and 15 and urine density, pH, bilirubin, urobilinogen, blood, glycosuria, proteinuria and ketonic bodies, leucocytes and nitrites were analysed using Sticks URIT 10V (Quilaban LDA, Sintra, Portugal). All measures were done in triplicate in each of the three times of the protocol.
After 15 days, animals were anaesthetised, blood and urine were collected for analysis and then sacrificed.
The evaluation of biochemical and haematological parameters was performed on the blood and serum of all animals. The blood was taken via cardiac puncture, where whole blood was collected into two tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. For haematological analysis, the parameters examined were: Mean corpuscular volumes (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentrations (MCHC); neutrophils (NEU, %); basophils (BAS, %); eosinophils (EO, %); monocytes (MO, %) and lymphocytes (LYM, %); erythrocyte counts (RBC); leukocyte counts (WBC); haematocrit, haemoglobin (Hb); and platelets (Coulter MaxM, Beckman Coulter, High Wycombe, UK). Another part of the blood was collected into tubes without EDTA and was centrifuged after coagulation (2500× g during 10 min). The serum was analysed by an automated clinical chemistry analyser (ADVIA®1200, Synchron CX4, Beckman Coulter, Bucks, UK). Biochemical parameters analysed were cholesterol (CHOL), low density lipoprotein (LDL), high density lipoprotein (HDL), alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), triglycerides (TRIG), urea (UR), total proteins (T-PROT), and creatinine (CR). The results of each tested parameter have been presented for each group of rats as the mean ± SD.
2.3.14. Statistical Analysis
The results were expressed as the means ± standard deviations (SD). An analysis of variance with ANOVA test was applied. For multiple comparison group tests, Tukey’s or Dunnett´s multi-comparison test was applied, using Graph Prisma Version 6.01 (GraphPad Software, San Diego, CA, USA). The differences were considered statistically significant when p ˂ 0.05.