- Email: [email protected]
Novel carriers ensuring enhanced anti-cancer activity of Cornus mas (cornelian cherry) bioactive compounds
Biomedicine & Pharmacotherapy 125 (2020) 109906
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Novel carriers ensuring enhanced anti-cancer activity of Cornus mas (cornelian cherry) bioactive compounds
T
Zarrin Radbeha, Narmela Asefia,*, Hamed Hamishehkarb, Leila Roufegarinejada, Akram Pezeshkic a
Department of Food Science and Technology, Tabriz Branch, Islamic Azad University, Tabriz, Iran Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran b
ARTICLE INFO
ABSTRACT
Keywords: Cornus mas Antioxidant activity Nano-cubosomes Bioactive compounds Colorectal cancer Eudragit® S100
Cornusmas’ bioactive compounds are powerful antioxidants. In this study, we evaluated the antioxidant activity of the encapsulated bioactive compounds of Cornus mas extract (CME) and its release in semi digestive condition via enteric coated nanocarriers (NCs). The two forms of CME, encapsulated into enteric coated nanocarriers (CME-NCs) and free CME, were studied to determine the effect of encapsulation on the stability of antioxidants. Then, their effect on cell cycle, cell viability and apoptosis of cancer cells were studied. The characterization analysis reported the mean particle size and zeta potential value of NCs equal to 22.7 ± 6.58 nm and -16 ± 5 mV. The results showed that CME-NCs could improve IC50 value 1.33 and 1.47 times more than the free CME after 24 and 48 h of incubation. These findings confirmed that CME-NCs could stop the cells proliferation in G1 phase, and caused apoptosis in cancer cell line HT-29.
1. Introduction
(GIT) pass via the liver, and appearing in the systemic circulation. According to many reports, the bioavailability of the parent intact glycoside compounds is less than 1 % [13,28,38]. Based on the recent reports, 10%–25% and 30%–50% of the anthocyanins are degraded in the stomach and the small intestine, respectively [12,15]. Thus a potential solution lies in the nanoparticle-based polyphenol delivery systems that are able to protect them [18], to enhance their absorption across the gastrointestinal tract, improve their bioavailability, and transport them to the target organs [39]. As mentioned above polyphenols could function as a cancer chemopreventive agent [3]. Cancer as a chronic disease proliferates uncontrollably throughout the body, making it a major global health concern [19]. The slow treatment of the disease has urged the scientists to consider new approaches (e.g. nano technology), to tackle the problems associated with carrier delivery. Nano systems can affect of the carrier, while presenting the lowest level of side effects [39]. Despite the significant development of different types of nanoparticles for different purposes, little is known about cell interactions of lipid-based nano-cubosomes. Cubosomes are the formations of bicontinuous cubic liquid crystalline phase using hydrating mixture of lipid (monoolein) and surfactant (poloxamer® 407). They appear as dots square shaped, slightly spherical, with an average diameter of 10−500 nm [41].The dynamic nature of liquid cubic nanostructure membranes enables their aqueous nanochannels to dynamically open or
Given the effective role of bioactive compounds on health, numerous studies have been recently reported on the desirability of utilizing plant sources [42]. Anthocyaninsas a bioactive compound are a group of the phenolic phytochemicals which have a high correlation of antioxidant activity. They have been confirmed to add anti-inflammatory, anti-carcinogenic, anti-mutagenic and chemo preventative effects to food in various in vitro, animal studies and clinical experiments [3,26,43]. Cornelian cherry (Cornus mas L.) is found mainly as a rich source of anthocyanins, in Southeast Asia. The strong antioxidative effect of cornelian cherry is attributed to its bioactive compounds such as phenolic compounds including phenolic acids, flavonoids, tannins and anthocyanins [29,30]. Despite the protective activity of polyphenols, their inefficient delivery systems and poor bioavailability strongly limit their application in medicine and functional food [14].Chokeberries a great source of anthocyanins, was incubated with human saliva lost an approximately 50 % of their anthocyanin, probably due to the binding of anthocyanin to salivary proteins, high oral temperatures and the enzymatic activity of oral micro biota [25]. It is thought that the unchanged anthocyanins are absorbed in the small intestine of humans [34]. In addition to the oral ingestion, the other attribute governing the bioavailability of the anthocyanins is their capability of crossing the epithelial surface of the gastro intestinal tract ⁎
Corresponding author. E-mail address: [email protected] (N. Asefi).
https://doi.org/10.1016/j.biopha.2020.109906 Received 7 October 2019; Received in revised form 7 January 2020; Accepted 12 January 2020 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
close at the cubosomic interface [7]. The cubic liquid-crystalline structure could be determined by means of X-ray diffraction [33,9,32]. In Cubosomes active chemical constituent molecules are anchored through chemical bonds to the polar head of the lipids. Their vesicular structures are similar to liposomes that are used for the carrier potential of hydrophilic, lipophilic and amphiphilic bioactive compounds found in food -particularly fruits- or drugs [4,22,17,5,8]. For control release, cubic phase is more applicable [41,5,6].The enteric coating could protect the biodegradable encapsulating compounds while passing gastro intestinal tract until intesinal. PH-dependent release, along with capability of enclosed active substances, protection especially in low pH, enzyme and moisture of environment have led to Eudragit® S100 as a tempting option to supplement the nanoparticles. Produced via the anionic copolymerization of methacrylic acid and methyl methacrylate, Eudragits® S100 is widely utilized in coating nanoparticles. The sustained intestine delivery of drugs is developed to bypass the stomach and release the loaded drug for long periods into the intestine via coating of Eudragit® polymer [23,40]. To the best of authors’ knowledge, nano encapsulation of Cornus mas’ bioactive compounds using coated nano-liquid crystalline systems with Eudragit® S100, their release and effect on colorectal cancers is yet to be explored. However, the encapsulation of rich source of anthocyanins, Cornus mas extract via the coated nano-cubosomes with Eudragit® S100 could protect these bioactive compounds against the adverse effect of GIT environment conditions and the increase the efficacy in HT-29 cells. The enteric coated nano-cubosomes could be as a novel carrier for the oral consumption. In this regard, the aim of this study was to improve the stability of Cornus mas’ antioxidants activity and investigate the anticancer activity and associated mechanism of CME against human colon carcinoma cells in cell culture. The findings of this study may open new horizons for the use of natural bioactive compounds as a new ingredient for functional food and chemopreventive in colorectal cancer.
min. Having done that, the mixture was centrifuged (Sigma 2-16KL, Germany at 9,000 rpm for 15 min and the supernatant of the mixture was condensed using a rotary vacuum evaporator Hei-VAP Core at 40 °C. Finally, the prepared concentrate was freeze dried Christ Alpha 1–4, Germany) and stored at −20 °C until it was ready to use. 2.2.2. Preparation of nano-capsulation and enteric coating The NCs were prepared with hot homogenization method. Briefly, 10 mg of freeze dried extract was dissolved into 100 all of DMSO. Then, the mixture was added to the melted lipid glycerolmonooleate and homogenized Silent Crusher M, Heidolph, Germany under 10000 rpm speed for 1 min. Following that, the 10 ml of poloxamer® 407, 7.5 mg/ ml solution at the same temperature as melted lipid mixture was added drop by drop to the lipid phase while being homogenized Heidolph Instruments GmbH and Co., Schwabach, Germany at 20000 rpm. The homogenization process takes 20 min. After homogenization, the prepared nanoemulsions were left in a stagnant place to recrystallization of the lipid phase. The NCs coating process with Eudragit® S100 was done using a dip coating method. In order to coating the NCs, Eudragit®S100 1.5 % solution (solving its powder into 1:1 isopropyl alcohol and acetone) was prepared, then the prepared nanoemulsions, were added drop by drop into the coating solution. 2.2.3. Size, Poly dispersity index (PDI), zeta potential (ZP) and morphology of NCs The particle size, PDI and ZP of the NCs were obtained using a dynamic light scattering technique (DLS) particle size analyzer (Zeta Sizer Pro, Malvern Instruments Ltd, UK) at room temperature. All samples for Size and ZP measurements were diluted five times using deionized water. In addition, the morphology of the NCs was studied using a scanning electron microscope (SEM) (MV2300, Vega Tuscan, Czech Republic).
2. Material and methods
2.2.4. Lipid based nanoparticle structure determination The determination of the nano structures generated in the self-assembled lipid based formulation was investigated by X-Ray Diffraction (XRD) PANanalytical (2009, the Netherlands) at the laboratory of Sharif University at 25 °C. The instrument source was a copper anode operating at 40 mA and 40 kV with a wavelength ʎ = 1.54 Å.Goniometer Radius was 240 mm. The experiment was conducted with Start position 0.4550°2 , end position 9.9950°2 , step size 0.01°2 and scan step time 1.76 s. Scattering files were background subtracted and normalized tothe sample transmission then integrated to the one dimensional scattering function I (q), where q is the length of the scattering vector, defined by q = 4 / sin , where is wavelength of 1.54 Å and 2 is the scattering angle.
2.1. Materials 2.1.1. Fruit samples The Cornus mas was obtained from the suburbs of Kaleibar (East Azarbaijan, Iran) on September 2018. All plant materials were verified by the Tabriz Reference Herbarium at the Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 2.1.2. Chemicals RPMI 1640, Fetal Bovine Serum, Pancreatin, Trypsin EDTA 0.25 %, Penicillin-Streptomycin, Dimethyl sulfoxide (DMSO, 99.9 %), Pepsin, 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), Eudragit® S100, poloxamer®407, ethanol 96 % were supplied by Sigma Aldrich Co. (St. Louis, MO). Glyceryl monooleate was purchased from Gattefosse (St-Priest, France). The annexin V/PI apoptosis detection kit was purchased from Exbio Co. (Exbio, Czech Republic). All other chemicals and reagents used in study were of analytical grade.
2.2.5. Encapsulation efficiency (%EE) and loading capacity(%LC) of poly phenol %EE and %LC of CME were measured using the following Equations (Eqs. 1 and 2):
EE % = W0 / Wi × 100
(1)
Where, Wo is the weight of the CME before separation, and Wi is its weight after separation,
2.2. Methods
LC % = WLP / WLNPs × 100
2.2.1. Preparation of Cornus mas extract The extraction process was done using ultra-sonication extraction method (Ultrasonic Bath Sonorex Super, Bandelin, Germany). In order to perform the extraction, the Cornus mas’ fruits were washed with cold water and their seeds were removed manually. Then, the fruit pomace was dried in dark room at 45 °C as standard condition. After that, all the dried fruit pomace was powdered using mill. The prepared powder was mixed with water and ethanol (30:70) mixture and sonicated for 30
(2)
Where WLP is weight of the loaded CME into NCs and WLNPs is solid mass weight of NCs. For measuring the amount of the non-entrapped extract, 2 ml of the prepared CME-NCs was filtered using Amicon® Ultra-Centrifugal filters (MWCO 30 kDa, Millipore, USA) for15 min at 4000 rpm. The amount of the unloaded extract was measured with ultraviolet-visible (UV163VIS) spectrophotometer (Ultrospec 2000, Scinteck, UK) method at 567 nm [11]. 2
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 1. a) Represents size analysis, b) zeta potential analysis and c) Scanning Electron Microscope imaging of Cornus Mas Extract-Nano Carriers.
2.2.6. Bioactive compound release The dialysis bag diffusion method was established to measure the rate of CME release from nano formulation. Briefly, 4 ml of the free CME and CME-NCs were separately diluted with 4 ml PBS pH 7.4 and placed into two different dialysis bags (MWCO 20 kDa) and immersed into50 ml of simulated intestinal and gastric environments containing pancreatin and pepsin enzymes, respectively [37]. The bioactive compound release in vitro simulation system temperature was kept at 37 °C, while the stirring speed was 300 rpm. During the experiment at the different times (0, 5,
10, 15, 20, 30, 40, 50, 60 and 80 h) 1 ml of the samples were withdrawn and replaced with fresh PBS. Finally, the amount of extract released from the dialysis bag was measured with UV–vis spectrophotometry at 567 nm [27]. 2.2.7. Antioxidant and phenolic stability For evaluating the antioxidant and phenolic stability two forms of the free CME and CME-NCs, the DPPH (2,2-diphenyl-1-picryl-hydrazylhydrate) antioxidant activity and the stability of phenolic measurement methods were established. The antioxidant activity of the free CME and CME-NCs at the 1, 15, 30, 45 and60th day of storage was measured by adding 1 ml of the DPPH solution into 1 ml of each sample, the samples were incubated in 37 °C for 30 min. and the absorbance of samples were measured with UV–vis spectrophotometry at 517 nm. The antioxidant activity was measured with fallowing Equation (Eq. 3).
Table 1 Calculated XRD (X-ray diffraction) data for blank cubic phase of glycerol monooleat. peak position
Blank Nanocarrier
q (Å)
d (Å)
hkl
a (nm) Lattice parameter
0.051564 0.136194
120.8 45.8
110 321
17 17
DPPH activity = A Sample / ADPPH × 100
(3)
Where, A Sample is the absorbance of the sample and A DPPH is the absorbance of the 1:1 diluted DPPH. In order to evaluate the phenolic stability, the colorimetric method
Pm3n
3
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 2. RepresentativeLow Angle X-Ray Diffraction and Small Angle X-ray Scattering patterns of blank Nano Carriers self-assembled mixtures at 25 °C. The recorded Braggpeaks are indicative of the formation of periodic liquid crystalline phases. TheLow Angle X-Ray Diffraction pattern of blank Nano Carriers (red) andthe Small AngleX-ray Scattering pattern of blank Nano Carriers (green)reflection of a micellar cubic phase (Pm3n).
with Folin–Ciocalteu reagent at 765 nm wavelength was used. Gallic acid standard curve was plotted in the concentration range of 0.04–0.4 mg/ml.
(Treestar, Inc., San Carlos, CA) [27]. 2.2.11. DAPI staining DAPI (4′,6-diamidino-2-phenylindole) staining is a technique used to evaluate the chromatin fragmentation. HT-29 cells at the density of 2 × 106 cell/well were seeded into 6-well plates, coated with 1 × 1 cm glass lamella and incubated for 48 h. The cultivated cells were treated with blank cubosomes, free CME and CME-NCs for 48 h. The cells were then fixed with 5 % formaldehyde for 3 h and permeabilized with 15 % Triton X-100 in PBS for 15 min. Following that, they were stained with DAPI 0.1 %for 15 min and finally monitored using fluorescence microscopy system (Olympus, Korea) [27].
2.2.8. Cell culture Human colorectal cancer cells (HT-29) were purchased from Pasteur Institute (Tehran, Iran). The cells maintained in RPMI 1640 media contained 10 % FBS, while the temperature of cell culturing incubation was 37 °C with 5 % CO2. The cells were sub-cultured 24–48 h later with an initial concentration of 4 × 104 cells/ml and used in the logarithmic phase in all experiments.
2.2.12. Cell cycle analysis HT-29 cells were seeded at a density of 4 × 106 cells/well in 6-well plates in three groups: blank NCs, free CME, and CME-NCs. After 48 h of treatment, the cells were harvested and fixed with 50 % ethanol, treated with 5 mg/ml RNase A (Pioneer, Daedeok-gu, Daejeon, Korea), stained with 50 mg/ml propidium iodide, and subsequently analyzed using flow cytometry for DNA synthesis and cell cycle status (MACS Quant 10, Miltenyi Biotech GmbH).
2.2.9. In vitro Cellular cytotoxicity assay The MTT assay Method was used to evaluate the cell viability after incubation with the free CME and its CME-NCs. Briefly, 1.5 × 104 three-time passaged HT-29 cells were seeded into each well of 96- well plate. Then, the cells were treated with different concentrations (0.05–6.4 mg/ml) of both free CME and CME-NCs. 24 and 48 h after treatment, the medium of each plate was changed with fresh medium containing 50 μl of MTT 2 mg/ml solution. After 4 h incubation, the incubated media was replaced with 200 μl of DMSO. Then, the absorbance of each well was read at a wavelength of 570 nm using Elisa Reader (Sunrise™, Tecan Group Ltd. Männedorf, Switzerland) Elisa Reader [27].
2.2.13. Statistical analysis All results were expressed as the mean ± standard deviation (SD). The statistical analysis was done using one-way and two-way ANOVA with multiple Dunnett test (Prism, version 8.0, Graph Pad, INC). The value of P less than 0.05 was considered significant.
2.2.10. Annexin V/PI flow cytometry HT-29 cells at the density of 2 × 106 cell/well were seeded into 6well plate and incubated in the medium for 48 h. After 48 h, the upper media was discharged and 2 ml of fresh media containing IC50 concentration of the free CME and CME-NCs was added to each well and incubated for 48 h. Then, the cells were detached with trypsin and washed with ice-cold PBS, and re-suspended in 100 μl binding buffer at ice-cold temperature. After that, the detached cells were stained using an Annexin V/PI apoptosis detection kit (Exbio, Czech Republic) according to the manufacturer's protocol. Finally, the rate of the apoptosis was analyzed using flow cytometry (BD flow cytometry instrument, USA). The obtained data were analyzed using FlowJo software package
3. Results and discussion 3.1. Characterization of Cornus mas extract loaded in coated nanocarriers Several studies have revealed the beneficial prevention effects of natural bioactive components against the colorectal cancer. In this study, we successfully prepared CME-NCs to improve its anticancer activities in vitro. As shown in Fig. 1, the characterization analysis shows the mean particle size (Fig. 1a) and ZP value (Fig. 1b) of micellar 4
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 4. Represents in vitro cellular viability results of Cornus Mas Extract and Cornus Mas Extract-Nano Carriers in HT-29 cell line over 24 and 48 h. The results were calculated as the mean ± standard deviation (n = 3).
cubosome equal to 22.27 ± 6.58 nm and −16 ± 5 mv, respectively. The SEM analysis confirmed the particle size reports (Fig. 1c) and the narrow size distribution of particles reported by the PDI size study. It was shown that a PDI < 0.3 was produced with a narrow size and homogenous particle distribution. This result is supported by salem et al., report [36]. Particles in the size range of sub 100 nm present the best cell penetration [2]. According to Gillet et al. [16], the surface charge of nano particles is a key factor affecting the stability of nano particles. The nano particles with a smaller size had higher ZP values, hence had better stability. Despite the advantages of a negative surface charge, by decreasing the ZP value to a more negative value, the interaction is increased with biological surfaces. EE% and LC% indices of CME in the prepared NC formulation were 95.14 ± 12.31 % and 9.51 ± 1.23 %, respectively. The amount of CME released from the nanoparticles was measured after the complete separation of the non-entrapped CME from the cubosomes. Fig. (1 d) shows that the release of CME from lipid nanocarriers of liquid crystalline was about 14 % after 10 h in an acidic environment (pH 1.2 simulated gastric fluids). Furthermore, at the intestinal
Fig. 3. Showing the storage stability of Cornus Mas Extract and Cornus Mas Extract-Nano Carriers in refrigerator at 4−8 °C. a) DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) antioxidant stability, b) phenolic stability and c) total phenolic contents. The results were calculated as the mean ± standard deviation (n = 3). (*P < 0.05, #P < 0.01).
5
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 5. Illustrates a) cell cycle analysis and b) cell apoptosis induced by Cornus Mas Extract and Cornus Mas Extract-Nano Carriers over 48 h by using Annexin V FITC/ PI(propidium iodide)staining.
simulated environment (pH 7.4, simulated intestinal fluid), CME release was about 35 % over 10 h. Moreover, in the case of nanocarriers after 40 h, CME release at pH 1.2 and pH 7.4 was about 47 % and 71 %, respectively. According to these results, the amount of CME release from nanocarriers of liquid crystalline in the intestinal simulated environment is approximately 24 % is higher than that of stomach. Therefore, surface modification of nanocarriers with Eudragit® S100increase CME release in intestinal environment and decrease it for an acidic environment.
diphenyl-1-picryl-hydrazyl-hydrate) during 60 days of storage. The obtained results showed the time-dependent free radical scavenging ability and the lack of significant scavenging activity of NCs (Fig. 3). According to the results, the antioxidant activity of CME and CME-NCs on the first day was 97.82 and 95.55 %, respectively. However, this amount was altered during the storage period, which was equal to 60 days of storage. The free CME 25 % lost its antioxidant activity, but the CME-NCs lost only 7.99 % of its antioxidant activity. The stability measurements of phenolic compounds show that the encapsulation can protect 30 % of the phenolic aggregation of the free CME. The results obtained from phenolic stability measurements supported those reports for the antioxidant stability. There was a correlation between the content of anthocyanin and the high antioxidant capacity. This result was supported by homoki et al., [21] and Kähkönen et al., [24]. Fig. 3a–c showed a strong antioxidant protective effect of CME-NCs. These results highlight the effect of the carrier, displaying an advantage for the stability of the CME encapsulation into the nano structured lipid system coated with Eudragit® S100. These results agree with other studies that have been conducted by Haris et al., [20], Pool et al. [31], and Rakotoarisoa et al., [32].
3.2. XRD characterization of nano carrier dispersions The XRD (low angle x ray diffraction and small angle x ray scattering) experiment was employed to investigate the internal structure of the obtained liquid crystalline. Table 1 shows the value of scattering vector q corresponding to the scattering peaks, interplanar distance (d), miller indices (hkl) and lattice parameter for blank cubic lattice. The presence of Braggs peaks in Fig. 2 illustrated nanocarriers with a periodic inner organization (cubosomes). Supported by Angelova et al., report [10], the results fit the characteristic peaks of Pm3n crystallographic space group (Q 223) which contain micellar cubosome (micellar cube phase). The determined lattice parameters of the cubic unit cells in the Pm3n cubosome particles are a1& a2 = 17 nm.
3.4. Evaluation of in vitro cell cytotoxicity of CME-NCs The cell proliferation is an important mechanism for the growth, development, and regeneration of eukaryotic organisms. However, it is the primary cause of some disabling diseases such as cancers. In this study, the effects of the free CME and CME-NCs on the cell viability of HT-29 human colon carcinoma cell line were evaluated with MTT cell proliferation assay. After 24 h and 48 h of treatment, a significant reduction of cell viability was observed by the free CME and CME-NCs (Fig. 4). As illustrated in Fig. 4a b, 3.32 mg/ml of the free CME and 2.49 mg/ml of CME-NCs could decrease the cell viability to 50 % during 24 h. The results for IC50 values showed a significant difference (P < 0.01) between the free CME and CME-NCs with 2.36 mg/ml and 1.6 mg/ml after 48 h, respectively. CME-NCs could improve the IC50 value to 1.33 and 1.47 times more than that of the free CME after 24 h
3.3. Antioxidant stability study After the oral ingestion, anthocyanin bioavailability is governed by their ability to cross the epithelial surface of the GIT pass, and appear in systemic circulation. During transition, anthocyanins could be degraded due to several factors [25]. Therefore, they could not show their biological effect including antioxidants activity properly. The encapsulation of CME using Eudragit® coated NCs could preserve them from degradation. In order to determine the amount of polyphenols in the free CME and CME-NCs, the Folin Ciocalteu assay was conducted. To conduct further investigations, the antioxidant activity of the free CME and CME-NCs were measured to scavenge DPPH free radicals (2,26
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 6. Displays fluorescent images of treated and untreated DAPI (4′,6-diamidino-2-phenylindole) stained HT-29 cells, a) control negative, b) treated with Cornus Mas Extract and c) treated with Cornus Mas Extract-Nano Carriers (Red arrows shown healthy cell nuclei and white arrows shown fragmented cell nuclei samples.
and 48 h of incubation, respectively. This may be attributed to better cellular internalization of CME-NCs in comparison with free CME, highlighting the impact of nanoparticles (especially sub 100 nm size) on cellular internalization [2]. Sabzichi et al. reported that programmed cell death or apoptosis in the chemotherapeutic approach was the most important molecular mechanism used in the treatment of anticancer carriers [35]. From the flow cytometry results, after the treatment of HT-29 cells with the free CME and CME NCs, cells were stained with annexin V/PI to evaluate the rate of apoptosis after 48 h. In this study, the non-treated cells were considered as a negative control group. According to the obtained results (Fig. 5), the free CME showed 5.4 % necrosis and 5.5 % apoptosis, while CME-NCs indicated 3.92 % necrosis and 10 % apoptosis. From flow cytometry data, the rate of necrosis on the HT-29 cells treated with the free CME was equal with the apoptosis rate. The encapsulation of CME by liquid crystalline could significantly (P < 0.05) decrease the rate of necrosis. Our results are supported using cell cycle analysis. In this study, a DAPI staining assay was conducted to evaluate the effect of CME on the DNA morphology in the HT-29 cells. The exposure of the cells with IC50 concentration of the free CME and CME-NCs led to the segregation of the cell nuclei into fragments, demonstrating a breakdown in the chromatin that caused DNA condensation. Based on the results, the free CME treated cells showed a low rate of the DNA fragmentation, while CME-NCs increased the rate of DNA fragmentation, compared to the free CME (Fig. 6). The results suggested that the free CME and CME-NCs formulation was able to induce apoptosis, which could be detected by the morphological changes in DNA. According to our results, the encapsulation of the CME significantly decreased the number of the G0/G1 cells from 79.6% to 44.7%. On the other hand, the encapsulation of the CME increased the number of Sub G1 cells from 2.48 % to 21.1 %. Our obtained results suggest that the CME-NCs affected the mitosis phases in the cell duplication. These results were confirmed by literature reports that G0/G1 arrest and apoptosis induced via ginger extract in case of HCT 116and HT 29 colon cancer cell lines [1].
Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments The team of the authors was pleased to acknowledge BehzadMansoori for assistance inflow cytometry analysis. This project was a part of PhD dissertation in the Field of Food Science and Technology, Islamic Azad University, Tabriz Branch. References [1] S. Abdullah, S. Amalina, Z. Abidin, N.A. Murad, S. Makpol, Z.W. Ngah, et al., Ginger extract (Z ingiber officinale) triggers apoptosis and G 0 / G 1 cells arrest in HCT 116 and HT 29 colon cancer cell lines, Afr. J. Biochem. Res. 4 (May) (2010) 134–142. [2] Z.M. Adib, S. Ghanbarzadeh, M. Kouhsoltani, A.Y. Khosroshahi, H. Hamishehkar, The effect of particle size on the deposition of solid lipid nanoparticles in different skin layers: a histological study, Adv. Pharm. Bull. 6 (1) (2016) 31–36, https://doi. org/10.15171/apb.2016.006. [3] B.B. Aggarwal, S. Shishodia, Molecular targets of dietary agents for prevention and therapy of cancer, Biochem. Pharmacol. 71 (2006) 1397–1421, https://doi.org/10. 1016/j.bcp.2006.02.009. [4] S. Andersson, S. Lidin, Structure of the cubosome lipid bilayer aggregate, Zeitschrift Für Kristallographie 210 (1995) 315–318. [5] B. Angelov, A. Angelova, V.M. Garamus, M. Drechsler, R. Willumeit, R. Mutafchieva, et al., Earliest stage of the tetrahedral nanochannel formation in cubosome particles from unilamellar nanovesicles, Langmuir 28 (48) (2012) 16647–16655, https://doi.org/10.1021/la302721n. [6] B. Angelov, A. Angelova, B. Papahadjopoulos-sternberg, S.V. Ho, S. Lesieur, Proteincontaining PEGylated cubosomic particles: freeze-fracture Electron microscopy and synchrotron radiation circular dichroism study, J. Phys. Chem. B 116 (2012) 7676–7686. [7] B. Angelov, A. Angelova, B. Papahadjopoulos-Sternberg, S. Lesieur, J.F. Sadoc, M. Ollivon, P. Couvreur, Detailed structure of diamond-type lipid cubic nanoparticles, J. Am. Chem. Soc. 128 (17) (2006) 5813–5817, https://doi.org/10.1021/ ja060082c. [8] A. Angelova, B. Angelov, Dual and multi-drug delivery nanoparticles towards neuronal survival and synaptic repair, Neural Regen. Res. 12 (June 1) (2017) 886–889, https://doi.org/10.4103/1673-5374.208546. [9] A. Angelova, B. Angelov, B. Papahadjopoulos-Sternberg, M. Ollivon, C. Bourgaux, Proteocubosomes: nanoporous vehicles with tertiary organized fluid interfaces, Langmuir 21 (9) (2005) 4138–4143, https://doi.org/10.1021/la047745t. [10] A. Angelova, V.M. Garamus, B. Angelov, Z. Tian, Y. Li, A. Zou, Advances in structural design of lipid-based nanoparticle carriers for delivery of macromolecular drugs, phytochemicals and anti-tumor agents, Adv. Colloid Interface Sci. 249 (2017) 331–345, https://doi.org/10.1016/j.cis.2017.04.006. [11] S. Bagherpour, A. Alizadeh, M. Mohammadi, Author’ s accepted manuscript, Food Biosci. (2017), https://doi.org/10.1016/j.fbio.2017.07.010. [12] G. Borges, S. Roowi, J. Rouanet, G.G. Duthie, M.E.J. Lean, The Bioavailability of Raspberry Anthocyanins and Ellagitannins in Rats, (2007), pp. 714–725, https:// doi.org/10.1002/mnfr.200700024. [13] G. Cao, H.U. Muccitelli, C. Sánchez-moreno, R.L. Prior, Anthocyanins are absorbed in glycated forms in elderly women : a pharmacokinetic study 1–4, Am. J. Clin. Nutr. 73 (March) (2001) 920–926. [14] R.N. Cavalcanti, D.T. Santos, M.A.A. Meireles, Non-thermal stabilization mechanisms of anthocyanins in model and food systems — an overview, FRIN 44 (2) (2011) 499–509, https://doi.org/10.1016/j.foodres.2010.12.007. [15] J. Correa-betanzo, E. Allen-vercoe, J. Mcdonald, K. Schroeter, M. Corredig, G. Paliyath, Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion, Food Chem. 165 (2014) 522–531, https://doi.org/10.1016/j.foodchem.2014.05.135. [16] A. Gillet, P. Compère, F. Lecomte, P. Hubert, E. Ducat, B. Evrard, G. Piel, Liposome
4. Conclusions We demonstrated that nano-cubosomal delivery system could effectively protect antioxidant activity and increase the anticancer effect of CME. In vitro studies indicated that encapsulating the CME reduced the rate of necrosis, significantly increased the apoptosis and cell cytotoxicity, and significantly increased the G0-G1 phase cells count. In addition, CME–NCs could effectively inhibit colorectal cancer, which could successfully suggest a new ingredient for functional food and chemopreventive. Funding source declaration This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 7
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
[17] [18]
[19] [20]
[21]
[22]
[23] [24]
[25] [26] [27]
[28] [29]
[30]
surface charge influence on skin penetration behaviour, Int. J. Pharm. 411 (1–2) (2011) 223–231, https://doi.org/10.1016/j.ijpharm.2011.03.049. G. Gregoriadis, © 1977 Nature Publishing Group, Nature 265 (1977) 407–411, https://doi.org/10.1007/978-1-4684-4241-0. L.P.B. Guerzoni, V. Nicolas, A. Angelova, In vitro modulation of TrkB receptor signaling upon sequential delivery of Curcumin-DHA loaded carriers towards promoting neuronal survival, Pharm. Res. 34 (2) (2017) 492–505, https://doi.org/10. 1007/s11095-016-2080-4. F.A. Haggar, R.P. Boushey, D. Ph, Colorectal Cancer epidemiology : incidence, mortality, survival, and risk factors, Clin. Colon Rectal Surg. 6 (212) (2009) 191–197, https://doi.org/10.1055/s-0029-1242458. R. Harris, E. Lecumberri, I. Mateos-Aparicio, M. Mengíbar, A. Heras, Chitosan nanoparticles and microspheres for the encapsulation of natural antioxidants extracted from Ilex paraguariensis, Carbohydr. Polym. 84 (2) (2011) 803–806, https://doi.org/10.1016/j.carbpol.2010.07.003. J.R. Homoki, A. Nemes, E. Fazekas, G. Gyémánt, P. Balogh, F. Gál, et al., Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.), Food Chem. 194 (2016) 222–229, https://doi.org/10.1016/j.foodchem.2015.07.130. C.A. Hunter, T.F. Dolan, G.H. Coombs, A.J. Baillie, Vesicular systems (niosomes and liposomes) for delivery of sodium stibogluconate in experimental murine visceral leishmaniasis, Pharm.Pharmacol 40 (1988) 161–165, https://doi.org/10.1111/j. 2042-7158.1988.tb05210. A. Jain, S.K. Jain, M. K. T, Vijay Kumar Thakur (Eds.), Handbook of Polymers for Pharmaceutical Technologies, vol. 3, 2015 chapter 4. M.P. Kähkönen, A.I. Hopia, H.J. Vuorela, J.P. Rauha, K. Pihlaja, T.S. Kujala, M. Heinonen, Antioxidant activity of plant extracts containing phenolic compounds, J. Agric. Food Chem. 47 (10) (1999) 3954–3962, https://doi.org/10.1021/ jf990146l. K. Kamonpatana, M.M. Giusti, C. Chitchumroonchokchai, M. Morenocruz, Susceptibility of anthocyanins to ex vivo degradation in human saliva, Food Chem. 135 (2) (2012) 738–747, https://doi.org/10.1016/j.foodchem.2012.04.110. M.K. Khan, K. Ahmad, S. Hassan, M. Imran, N. Ahmad, Effect of novel technologies on polyphenols during food processing, Innov. Food Sci. Emerg. Technol. 45 (June 2017) (2018) 361–381, https://doi.org/10.1016/j.ifset.2017.12.006. S. Mahmoudi, M. Ghorbani, M. Sabzichi, F. Ramezani, Journal of Drug Delivery Science and Technology targeted hyaluronic acid-based lipid nanoparticle for apigenin delivery to induce Nrf2-dependent apoptosis in lung cancer cells, J. Drug Deliv. Sci. Technol. 49 (August 2018) (2019) 268–276, https://doi.org/10.1016/j. jddst.2018.11.013. T.K. McGhie, G.D. Ainge, L.E. Barnett, J.M. Cooney, J.D. Jensen, Anthocyanin glycosides from berry fruit are absorbed and excreted unmetabolized by both humans and rats, Agri. Food Chem. 51 (2003) 4539–4548. G.E. Pantelidis, M. Vasilakakis, G.A. Manganaris, G. Diamantidis, Food Chemistry Antioxidant Capacity, Phenol, Anthocyanin and Ascorbic Acid Contents in Raspberries, Blackberries, Red Currants, Gooseberries and Cornelian Cherries, February 2018 (2013), https://doi.org/10.1016/j.foodchem.2006.06.021. A.M. Pawlowska, F. Camangi, A. Braca, Quali-quantitative analysis of flavonoids of Cornus mas L. (Cornaceae) fruits, Food Chem. 119 (3) (2010) 1257–1261, https://
doi.org/10.1016/j.foodchem.2009.07.063. [31] H. Pool, D. Quintanar, J.D.D. Figueroa, C. Marinho Mano, J.E.H. Bechara, L.A. Godínez, S. Mendoza, Antioxidant effects of quercetin and catechin encapsulated into PLGA nanoparticles, J. Nanomater. 2012 (2012), https://doi.org/ 10.1155/2012/145380. [32] M. Rakotoarisoa, B. Angelov, S. Espinoza, K. Khakurel, T. Bizien, A. Angelova, Cubic liquid crystalline nanostructures involving catalase and curcumin: BioSAXS study and catalase peroxidatic function after cubosomal nanoparticle treatment of differentiated SH-SY5Y Cells, Molecules 24 (17) (2019) 3058, https://doi.org/10. 3390/molecules24173058. [33] M. Rakotoarisoa, B. Angelov, V.M. Garamus, A. Angelova, Curcumin- and fish oilloaded spongosome and cubosome nanoparticles with neuroprotective potential against H 2 O 2 -Induced oxidative stress in differentiated human SH-SY5Y cells, ACS Omega 4 (2) (2019) 3061–3073, https://doi.org/10.1021/acsomega.8b03101. [34] D.M. Ribnicky, D.E. Roopchand, A. Oren, M. Grace, A. Poulev, M. Ann, et al., Effects of a high fat meal matrix and protein complexation on the bioaccessibility of blueberry anthocyanins using the TNO gastrointestinal model (TIM-1), Food Chem. 142 (2014) 349–357, https://doi.org/10.1016/j.foodchem.2013.07.073. [35] M. Sabzichi, J. Mohammadian, R. Bazzaz, M.B. Pirouzpanah, M. Shaaker, H. Hamishehkar, et al., Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway, Process. Biochem. 60 (February) (2017) 84–91, https://doi.org/10.1016/j.procbio.2017.05.024. [36] D.S. Salem, M.A. Sliem, M. El-sesy, S.A. Shouman, Y. Badr, Journal of Photochemistry & Photobiology, B: biology Improved chemo-photothermal therapy of hepatocellular carcinoma using chitosan-coated gold nanoparticles, J. Photochem. Photobiol. B, Biol. 182 (March) (2018) 92–99, https://doi.org/10. 1016/j.jphotobiol.2018.03.024. [37] T. Sanz, H. Luyten, Release, partitioning and stability of isoflavones from enriched custards during mouth, stomach and intestine in vitro simulations, Food Hydrocollids 20 (2006) 892–900, https://doi.org/10.1016/j.foodhyd.2005.09.003. [38] A. Scalbert, C. Morand, C. Manach, C. Rémésy, Absorption and metabolism of polyphenols in the gut and impact on health, Biomed. Pharmacother. 56 (2002) 276–282. [39] T. Squillaro, A. Cimini, G. Peluso, A. Giordano, M.A.B. Melone, Nano-delivery systems for encapsulation of dietary polyphenols : an experimental approach for neurodegenerative diseases and brain tumors, Biochem. Pharmacol. 154 (March) (2018) 303–317, https://doi.org/10.1016/j.bcp.2018.05.016. [40] M.B. Subudhi, A. Jain, A. Jain, P. Hurkat, S. Shilpi, A. Gulbake, S.K. Jain, Eudragit S100 coated Citrus pectin nanoparticles for Colon targeting of 5-Fluorouracil, Materials (2015) 832–849, https://doi.org/10.3390/ma8030832. [41] M. Thadanki, P.S. Kumari, K.S. Prabha, Overview of cubosomes : a nano particle, ResearchGate 1 (3) (2011) 535–541. [42] K.U. Yilmaz, S. Ercisli, Y. Zengin, M. Sengul, E.Y. Kafkas, Preliminary characterisation of cornelian cherry (Cornus mas L.) genotypes for their physico-chemical properties, Food Chem. 114 (2) (2009) 408–412, https://doi.org/10.1016/j. foodchem.2008.09.055. [43] L. Zhao, F. Temelli, L. Chen, Encapsulation of anthocyanin in liposomes using supercritical carbon dioxide : effects of anthocyanin and sterol concentrations, J. Funct. Foods 34 (2017) 159–167, https://doi.org/10.1016/j.jff.2017.04.021.
8
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Novel carriers ensuring enhanced anti-cancer activity of Cornus mas (cornelian cherry) bioactive compounds
T
Zarrin Radbeha, Narmela Asefia,*, Hamed Hamishehkarb, Leila Roufegarinejada, Akram Pezeshkic a
Department of Food Science and Technology, Tabriz Branch, Islamic Azad University, Tabriz, Iran Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran b
ARTICLE INFO
ABSTRACT
Keywords: Cornus mas Antioxidant activity Nano-cubosomes Bioactive compounds Colorectal cancer Eudragit® S100
Cornusmas’ bioactive compounds are powerful antioxidants. In this study, we evaluated the antioxidant activity of the encapsulated bioactive compounds of Cornus mas extract (CME) and its release in semi digestive condition via enteric coated nanocarriers (NCs). The two forms of CME, encapsulated into enteric coated nanocarriers (CME-NCs) and free CME, were studied to determine the effect of encapsulation on the stability of antioxidants. Then, their effect on cell cycle, cell viability and apoptosis of cancer cells were studied. The characterization analysis reported the mean particle size and zeta potential value of NCs equal to 22.7 ± 6.58 nm and -16 ± 5 mV. The results showed that CME-NCs could improve IC50 value 1.33 and 1.47 times more than the free CME after 24 and 48 h of incubation. These findings confirmed that CME-NCs could stop the cells proliferation in G1 phase, and caused apoptosis in cancer cell line HT-29.
1. Introduction
(GIT) pass via the liver, and appearing in the systemic circulation. According to many reports, the bioavailability of the parent intact glycoside compounds is less than 1 % [13,28,38]. Based on the recent reports, 10%–25% and 30%–50% of the anthocyanins are degraded in the stomach and the small intestine, respectively [12,15]. Thus a potential solution lies in the nanoparticle-based polyphenol delivery systems that are able to protect them [18], to enhance their absorption across the gastrointestinal tract, improve their bioavailability, and transport them to the target organs [39]. As mentioned above polyphenols could function as a cancer chemopreventive agent [3]. Cancer as a chronic disease proliferates uncontrollably throughout the body, making it a major global health concern [19]. The slow treatment of the disease has urged the scientists to consider new approaches (e.g. nano technology), to tackle the problems associated with carrier delivery. Nano systems can affect of the carrier, while presenting the lowest level of side effects [39]. Despite the significant development of different types of nanoparticles for different purposes, little is known about cell interactions of lipid-based nano-cubosomes. Cubosomes are the formations of bicontinuous cubic liquid crystalline phase using hydrating mixture of lipid (monoolein) and surfactant (poloxamer® 407). They appear as dots square shaped, slightly spherical, with an average diameter of 10−500 nm [41].The dynamic nature of liquid cubic nanostructure membranes enables their aqueous nanochannels to dynamically open or
Given the effective role of bioactive compounds on health, numerous studies have been recently reported on the desirability of utilizing plant sources [42]. Anthocyaninsas a bioactive compound are a group of the phenolic phytochemicals which have a high correlation of antioxidant activity. They have been confirmed to add anti-inflammatory, anti-carcinogenic, anti-mutagenic and chemo preventative effects to food in various in vitro, animal studies and clinical experiments [3,26,43]. Cornelian cherry (Cornus mas L.) is found mainly as a rich source of anthocyanins, in Southeast Asia. The strong antioxidative effect of cornelian cherry is attributed to its bioactive compounds such as phenolic compounds including phenolic acids, flavonoids, tannins and anthocyanins [29,30]. Despite the protective activity of polyphenols, their inefficient delivery systems and poor bioavailability strongly limit their application in medicine and functional food [14].Chokeberries a great source of anthocyanins, was incubated with human saliva lost an approximately 50 % of their anthocyanin, probably due to the binding of anthocyanin to salivary proteins, high oral temperatures and the enzymatic activity of oral micro biota [25]. It is thought that the unchanged anthocyanins are absorbed in the small intestine of humans [34]. In addition to the oral ingestion, the other attribute governing the bioavailability of the anthocyanins is their capability of crossing the epithelial surface of the gastro intestinal tract ⁎
Corresponding author. E-mail address: [email protected] (N. Asefi).
https://doi.org/10.1016/j.biopha.2020.109906 Received 7 October 2019; Received in revised form 7 January 2020; Accepted 12 January 2020 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
close at the cubosomic interface [7]. The cubic liquid-crystalline structure could be determined by means of X-ray diffraction [33,9,32]. In Cubosomes active chemical constituent molecules are anchored through chemical bonds to the polar head of the lipids. Their vesicular structures are similar to liposomes that are used for the carrier potential of hydrophilic, lipophilic and amphiphilic bioactive compounds found in food -particularly fruits- or drugs [4,22,17,5,8]. For control release, cubic phase is more applicable [41,5,6].The enteric coating could protect the biodegradable encapsulating compounds while passing gastro intestinal tract until intesinal. PH-dependent release, along with capability of enclosed active substances, protection especially in low pH, enzyme and moisture of environment have led to Eudragit® S100 as a tempting option to supplement the nanoparticles. Produced via the anionic copolymerization of methacrylic acid and methyl methacrylate, Eudragits® S100 is widely utilized in coating nanoparticles. The sustained intestine delivery of drugs is developed to bypass the stomach and release the loaded drug for long periods into the intestine via coating of Eudragit® polymer [23,40]. To the best of authors’ knowledge, nano encapsulation of Cornus mas’ bioactive compounds using coated nano-liquid crystalline systems with Eudragit® S100, their release and effect on colorectal cancers is yet to be explored. However, the encapsulation of rich source of anthocyanins, Cornus mas extract via the coated nano-cubosomes with Eudragit® S100 could protect these bioactive compounds against the adverse effect of GIT environment conditions and the increase the efficacy in HT-29 cells. The enteric coated nano-cubosomes could be as a novel carrier for the oral consumption. In this regard, the aim of this study was to improve the stability of Cornus mas’ antioxidants activity and investigate the anticancer activity and associated mechanism of CME against human colon carcinoma cells in cell culture. The findings of this study may open new horizons for the use of natural bioactive compounds as a new ingredient for functional food and chemopreventive in colorectal cancer.
min. Having done that, the mixture was centrifuged (Sigma 2-16KL, Germany at 9,000 rpm for 15 min and the supernatant of the mixture was condensed using a rotary vacuum evaporator Hei-VAP Core at 40 °C. Finally, the prepared concentrate was freeze dried Christ Alpha 1–4, Germany) and stored at −20 °C until it was ready to use. 2.2.2. Preparation of nano-capsulation and enteric coating The NCs were prepared with hot homogenization method. Briefly, 10 mg of freeze dried extract was dissolved into 100 all of DMSO. Then, the mixture was added to the melted lipid glycerolmonooleate and homogenized Silent Crusher M, Heidolph, Germany under 10000 rpm speed for 1 min. Following that, the 10 ml of poloxamer® 407, 7.5 mg/ ml solution at the same temperature as melted lipid mixture was added drop by drop to the lipid phase while being homogenized Heidolph Instruments GmbH and Co., Schwabach, Germany at 20000 rpm. The homogenization process takes 20 min. After homogenization, the prepared nanoemulsions were left in a stagnant place to recrystallization of the lipid phase. The NCs coating process with Eudragit® S100 was done using a dip coating method. In order to coating the NCs, Eudragit®S100 1.5 % solution (solving its powder into 1:1 isopropyl alcohol and acetone) was prepared, then the prepared nanoemulsions, were added drop by drop into the coating solution. 2.2.3. Size, Poly dispersity index (PDI), zeta potential (ZP) and morphology of NCs The particle size, PDI and ZP of the NCs were obtained using a dynamic light scattering technique (DLS) particle size analyzer (Zeta Sizer Pro, Malvern Instruments Ltd, UK) at room temperature. All samples for Size and ZP measurements were diluted five times using deionized water. In addition, the morphology of the NCs was studied using a scanning electron microscope (SEM) (MV2300, Vega Tuscan, Czech Republic).
2. Material and methods
2.2.4. Lipid based nanoparticle structure determination The determination of the nano structures generated in the self-assembled lipid based formulation was investigated by X-Ray Diffraction (XRD) PANanalytical (2009, the Netherlands) at the laboratory of Sharif University at 25 °C. The instrument source was a copper anode operating at 40 mA and 40 kV with a wavelength ʎ = 1.54 Å.Goniometer Radius was 240 mm. The experiment was conducted with Start position 0.4550°2 , end position 9.9950°2 , step size 0.01°2 and scan step time 1.76 s. Scattering files were background subtracted and normalized tothe sample transmission then integrated to the one dimensional scattering function I (q), where q is the length of the scattering vector, defined by q = 4 / sin , where is wavelength of 1.54 Å and 2 is the scattering angle.
2.1. Materials 2.1.1. Fruit samples The Cornus mas was obtained from the suburbs of Kaleibar (East Azarbaijan, Iran) on September 2018. All plant materials were verified by the Tabriz Reference Herbarium at the Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 2.1.2. Chemicals RPMI 1640, Fetal Bovine Serum, Pancreatin, Trypsin EDTA 0.25 %, Penicillin-Streptomycin, Dimethyl sulfoxide (DMSO, 99.9 %), Pepsin, 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), Eudragit® S100, poloxamer®407, ethanol 96 % were supplied by Sigma Aldrich Co. (St. Louis, MO). Glyceryl monooleate was purchased from Gattefosse (St-Priest, France). The annexin V/PI apoptosis detection kit was purchased from Exbio Co. (Exbio, Czech Republic). All other chemicals and reagents used in study were of analytical grade.
2.2.5. Encapsulation efficiency (%EE) and loading capacity(%LC) of poly phenol %EE and %LC of CME were measured using the following Equations (Eqs. 1 and 2):
EE % = W0 / Wi × 100
(1)
Where, Wo is the weight of the CME before separation, and Wi is its weight after separation,
2.2. Methods
LC % = WLP / WLNPs × 100
2.2.1. Preparation of Cornus mas extract The extraction process was done using ultra-sonication extraction method (Ultrasonic Bath Sonorex Super, Bandelin, Germany). In order to perform the extraction, the Cornus mas’ fruits were washed with cold water and their seeds were removed manually. Then, the fruit pomace was dried in dark room at 45 °C as standard condition. After that, all the dried fruit pomace was powdered using mill. The prepared powder was mixed with water and ethanol (30:70) mixture and sonicated for 30
(2)
Where WLP is weight of the loaded CME into NCs and WLNPs is solid mass weight of NCs. For measuring the amount of the non-entrapped extract, 2 ml of the prepared CME-NCs was filtered using Amicon® Ultra-Centrifugal filters (MWCO 30 kDa, Millipore, USA) for15 min at 4000 rpm. The amount of the unloaded extract was measured with ultraviolet-visible (UV163VIS) spectrophotometer (Ultrospec 2000, Scinteck, UK) method at 567 nm [11]. 2
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 1. a) Represents size analysis, b) zeta potential analysis and c) Scanning Electron Microscope imaging of Cornus Mas Extract-Nano Carriers.
2.2.6. Bioactive compound release The dialysis bag diffusion method was established to measure the rate of CME release from nano formulation. Briefly, 4 ml of the free CME and CME-NCs were separately diluted with 4 ml PBS pH 7.4 and placed into two different dialysis bags (MWCO 20 kDa) and immersed into50 ml of simulated intestinal and gastric environments containing pancreatin and pepsin enzymes, respectively [37]. The bioactive compound release in vitro simulation system temperature was kept at 37 °C, while the stirring speed was 300 rpm. During the experiment at the different times (0, 5,
10, 15, 20, 30, 40, 50, 60 and 80 h) 1 ml of the samples were withdrawn and replaced with fresh PBS. Finally, the amount of extract released from the dialysis bag was measured with UV–vis spectrophotometry at 567 nm [27]. 2.2.7. Antioxidant and phenolic stability For evaluating the antioxidant and phenolic stability two forms of the free CME and CME-NCs, the DPPH (2,2-diphenyl-1-picryl-hydrazylhydrate) antioxidant activity and the stability of phenolic measurement methods were established. The antioxidant activity of the free CME and CME-NCs at the 1, 15, 30, 45 and60th day of storage was measured by adding 1 ml of the DPPH solution into 1 ml of each sample, the samples were incubated in 37 °C for 30 min. and the absorbance of samples were measured with UV–vis spectrophotometry at 517 nm. The antioxidant activity was measured with fallowing Equation (Eq. 3).
Table 1 Calculated XRD (X-ray diffraction) data for blank cubic phase of glycerol monooleat. peak position
Blank Nanocarrier
q (Å)
d (Å)
hkl
a (nm) Lattice parameter
0.051564 0.136194
120.8 45.8
110 321
17 17
DPPH activity = A Sample / ADPPH × 100
(3)
Where, A Sample is the absorbance of the sample and A DPPH is the absorbance of the 1:1 diluted DPPH. In order to evaluate the phenolic stability, the colorimetric method
Pm3n
3
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 2. RepresentativeLow Angle X-Ray Diffraction and Small Angle X-ray Scattering patterns of blank Nano Carriers self-assembled mixtures at 25 °C. The recorded Braggpeaks are indicative of the formation of periodic liquid crystalline phases. TheLow Angle X-Ray Diffraction pattern of blank Nano Carriers (red) andthe Small AngleX-ray Scattering pattern of blank Nano Carriers (green)reflection of a micellar cubic phase (Pm3n).
with Folin–Ciocalteu reagent at 765 nm wavelength was used. Gallic acid standard curve was plotted in the concentration range of 0.04–0.4 mg/ml.
(Treestar, Inc., San Carlos, CA) [27]. 2.2.11. DAPI staining DAPI (4′,6-diamidino-2-phenylindole) staining is a technique used to evaluate the chromatin fragmentation. HT-29 cells at the density of 2 × 106 cell/well were seeded into 6-well plates, coated with 1 × 1 cm glass lamella and incubated for 48 h. The cultivated cells were treated with blank cubosomes, free CME and CME-NCs for 48 h. The cells were then fixed with 5 % formaldehyde for 3 h and permeabilized with 15 % Triton X-100 in PBS for 15 min. Following that, they were stained with DAPI 0.1 %for 15 min and finally monitored using fluorescence microscopy system (Olympus, Korea) [27].
2.2.8. Cell culture Human colorectal cancer cells (HT-29) were purchased from Pasteur Institute (Tehran, Iran). The cells maintained in RPMI 1640 media contained 10 % FBS, while the temperature of cell culturing incubation was 37 °C with 5 % CO2. The cells were sub-cultured 24–48 h later with an initial concentration of 4 × 104 cells/ml and used in the logarithmic phase in all experiments.
2.2.12. Cell cycle analysis HT-29 cells were seeded at a density of 4 × 106 cells/well in 6-well plates in three groups: blank NCs, free CME, and CME-NCs. After 48 h of treatment, the cells were harvested and fixed with 50 % ethanol, treated with 5 mg/ml RNase A (Pioneer, Daedeok-gu, Daejeon, Korea), stained with 50 mg/ml propidium iodide, and subsequently analyzed using flow cytometry for DNA synthesis and cell cycle status (MACS Quant 10, Miltenyi Biotech GmbH).
2.2.9. In vitro Cellular cytotoxicity assay The MTT assay Method was used to evaluate the cell viability after incubation with the free CME and its CME-NCs. Briefly, 1.5 × 104 three-time passaged HT-29 cells were seeded into each well of 96- well plate. Then, the cells were treated with different concentrations (0.05–6.4 mg/ml) of both free CME and CME-NCs. 24 and 48 h after treatment, the medium of each plate was changed with fresh medium containing 50 μl of MTT 2 mg/ml solution. After 4 h incubation, the incubated media was replaced with 200 μl of DMSO. Then, the absorbance of each well was read at a wavelength of 570 nm using Elisa Reader (Sunrise™, Tecan Group Ltd. Männedorf, Switzerland) Elisa Reader [27].
2.2.13. Statistical analysis All results were expressed as the mean ± standard deviation (SD). The statistical analysis was done using one-way and two-way ANOVA with multiple Dunnett test (Prism, version 8.0, Graph Pad, INC). The value of P less than 0.05 was considered significant.
2.2.10. Annexin V/PI flow cytometry HT-29 cells at the density of 2 × 106 cell/well were seeded into 6well plate and incubated in the medium for 48 h. After 48 h, the upper media was discharged and 2 ml of fresh media containing IC50 concentration of the free CME and CME-NCs was added to each well and incubated for 48 h. Then, the cells were detached with trypsin and washed with ice-cold PBS, and re-suspended in 100 μl binding buffer at ice-cold temperature. After that, the detached cells were stained using an Annexin V/PI apoptosis detection kit (Exbio, Czech Republic) according to the manufacturer's protocol. Finally, the rate of the apoptosis was analyzed using flow cytometry (BD flow cytometry instrument, USA). The obtained data were analyzed using FlowJo software package
3. Results and discussion 3.1. Characterization of Cornus mas extract loaded in coated nanocarriers Several studies have revealed the beneficial prevention effects of natural bioactive components against the colorectal cancer. In this study, we successfully prepared CME-NCs to improve its anticancer activities in vitro. As shown in Fig. 1, the characterization analysis shows the mean particle size (Fig. 1a) and ZP value (Fig. 1b) of micellar 4
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 4. Represents in vitro cellular viability results of Cornus Mas Extract and Cornus Mas Extract-Nano Carriers in HT-29 cell line over 24 and 48 h. The results were calculated as the mean ± standard deviation (n = 3).
cubosome equal to 22.27 ± 6.58 nm and −16 ± 5 mv, respectively. The SEM analysis confirmed the particle size reports (Fig. 1c) and the narrow size distribution of particles reported by the PDI size study. It was shown that a PDI < 0.3 was produced with a narrow size and homogenous particle distribution. This result is supported by salem et al., report [36]. Particles in the size range of sub 100 nm present the best cell penetration [2]. According to Gillet et al. [16], the surface charge of nano particles is a key factor affecting the stability of nano particles. The nano particles with a smaller size had higher ZP values, hence had better stability. Despite the advantages of a negative surface charge, by decreasing the ZP value to a more negative value, the interaction is increased with biological surfaces. EE% and LC% indices of CME in the prepared NC formulation were 95.14 ± 12.31 % and 9.51 ± 1.23 %, respectively. The amount of CME released from the nanoparticles was measured after the complete separation of the non-entrapped CME from the cubosomes. Fig. (1 d) shows that the release of CME from lipid nanocarriers of liquid crystalline was about 14 % after 10 h in an acidic environment (pH 1.2 simulated gastric fluids). Furthermore, at the intestinal
Fig. 3. Showing the storage stability of Cornus Mas Extract and Cornus Mas Extract-Nano Carriers in refrigerator at 4−8 °C. a) DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) antioxidant stability, b) phenolic stability and c) total phenolic contents. The results were calculated as the mean ± standard deviation (n = 3). (*P < 0.05, #P < 0.01).
5
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 5. Illustrates a) cell cycle analysis and b) cell apoptosis induced by Cornus Mas Extract and Cornus Mas Extract-Nano Carriers over 48 h by using Annexin V FITC/ PI(propidium iodide)staining.
simulated environment (pH 7.4, simulated intestinal fluid), CME release was about 35 % over 10 h. Moreover, in the case of nanocarriers after 40 h, CME release at pH 1.2 and pH 7.4 was about 47 % and 71 %, respectively. According to these results, the amount of CME release from nanocarriers of liquid crystalline in the intestinal simulated environment is approximately 24 % is higher than that of stomach. Therefore, surface modification of nanocarriers with Eudragit® S100increase CME release in intestinal environment and decrease it for an acidic environment.
diphenyl-1-picryl-hydrazyl-hydrate) during 60 days of storage. The obtained results showed the time-dependent free radical scavenging ability and the lack of significant scavenging activity of NCs (Fig. 3). According to the results, the antioxidant activity of CME and CME-NCs on the first day was 97.82 and 95.55 %, respectively. However, this amount was altered during the storage period, which was equal to 60 days of storage. The free CME 25 % lost its antioxidant activity, but the CME-NCs lost only 7.99 % of its antioxidant activity. The stability measurements of phenolic compounds show that the encapsulation can protect 30 % of the phenolic aggregation of the free CME. The results obtained from phenolic stability measurements supported those reports for the antioxidant stability. There was a correlation between the content of anthocyanin and the high antioxidant capacity. This result was supported by homoki et al., [21] and Kähkönen et al., [24]. Fig. 3a–c showed a strong antioxidant protective effect of CME-NCs. These results highlight the effect of the carrier, displaying an advantage for the stability of the CME encapsulation into the nano structured lipid system coated with Eudragit® S100. These results agree with other studies that have been conducted by Haris et al., [20], Pool et al. [31], and Rakotoarisoa et al., [32].
3.2. XRD characterization of nano carrier dispersions The XRD (low angle x ray diffraction and small angle x ray scattering) experiment was employed to investigate the internal structure of the obtained liquid crystalline. Table 1 shows the value of scattering vector q corresponding to the scattering peaks, interplanar distance (d), miller indices (hkl) and lattice parameter for blank cubic lattice. The presence of Braggs peaks in Fig. 2 illustrated nanocarriers with a periodic inner organization (cubosomes). Supported by Angelova et al., report [10], the results fit the characteristic peaks of Pm3n crystallographic space group (Q 223) which contain micellar cubosome (micellar cube phase). The determined lattice parameters of the cubic unit cells in the Pm3n cubosome particles are a1& a2 = 17 nm.
3.4. Evaluation of in vitro cell cytotoxicity of CME-NCs The cell proliferation is an important mechanism for the growth, development, and regeneration of eukaryotic organisms. However, it is the primary cause of some disabling diseases such as cancers. In this study, the effects of the free CME and CME-NCs on the cell viability of HT-29 human colon carcinoma cell line were evaluated with MTT cell proliferation assay. After 24 h and 48 h of treatment, a significant reduction of cell viability was observed by the free CME and CME-NCs (Fig. 4). As illustrated in Fig. 4a b, 3.32 mg/ml of the free CME and 2.49 mg/ml of CME-NCs could decrease the cell viability to 50 % during 24 h. The results for IC50 values showed a significant difference (P < 0.01) between the free CME and CME-NCs with 2.36 mg/ml and 1.6 mg/ml after 48 h, respectively. CME-NCs could improve the IC50 value to 1.33 and 1.47 times more than that of the free CME after 24 h
3.3. Antioxidant stability study After the oral ingestion, anthocyanin bioavailability is governed by their ability to cross the epithelial surface of the GIT pass, and appear in systemic circulation. During transition, anthocyanins could be degraded due to several factors [25]. Therefore, they could not show their biological effect including antioxidants activity properly. The encapsulation of CME using Eudragit® coated NCs could preserve them from degradation. In order to determine the amount of polyphenols in the free CME and CME-NCs, the Folin Ciocalteu assay was conducted. To conduct further investigations, the antioxidant activity of the free CME and CME-NCs were measured to scavenge DPPH free radicals (2,26
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
Fig. 6. Displays fluorescent images of treated and untreated DAPI (4′,6-diamidino-2-phenylindole) stained HT-29 cells, a) control negative, b) treated with Cornus Mas Extract and c) treated with Cornus Mas Extract-Nano Carriers (Red arrows shown healthy cell nuclei and white arrows shown fragmented cell nuclei samples.
and 48 h of incubation, respectively. This may be attributed to better cellular internalization of CME-NCs in comparison with free CME, highlighting the impact of nanoparticles (especially sub 100 nm size) on cellular internalization [2]. Sabzichi et al. reported that programmed cell death or apoptosis in the chemotherapeutic approach was the most important molecular mechanism used in the treatment of anticancer carriers [35]. From the flow cytometry results, after the treatment of HT-29 cells with the free CME and CME NCs, cells were stained with annexin V/PI to evaluate the rate of apoptosis after 48 h. In this study, the non-treated cells were considered as a negative control group. According to the obtained results (Fig. 5), the free CME showed 5.4 % necrosis and 5.5 % apoptosis, while CME-NCs indicated 3.92 % necrosis and 10 % apoptosis. From flow cytometry data, the rate of necrosis on the HT-29 cells treated with the free CME was equal with the apoptosis rate. The encapsulation of CME by liquid crystalline could significantly (P < 0.05) decrease the rate of necrosis. Our results are supported using cell cycle analysis. In this study, a DAPI staining assay was conducted to evaluate the effect of CME on the DNA morphology in the HT-29 cells. The exposure of the cells with IC50 concentration of the free CME and CME-NCs led to the segregation of the cell nuclei into fragments, demonstrating a breakdown in the chromatin that caused DNA condensation. Based on the results, the free CME treated cells showed a low rate of the DNA fragmentation, while CME-NCs increased the rate of DNA fragmentation, compared to the free CME (Fig. 6). The results suggested that the free CME and CME-NCs formulation was able to induce apoptosis, which could be detected by the morphological changes in DNA. According to our results, the encapsulation of the CME significantly decreased the number of the G0/G1 cells from 79.6% to 44.7%. On the other hand, the encapsulation of the CME increased the number of Sub G1 cells from 2.48 % to 21.1 %. Our obtained results suggest that the CME-NCs affected the mitosis phases in the cell duplication. These results were confirmed by literature reports that G0/G1 arrest and apoptosis induced via ginger extract in case of HCT 116and HT 29 colon cancer cell lines [1].
Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments The team of the authors was pleased to acknowledge BehzadMansoori for assistance inflow cytometry analysis. This project was a part of PhD dissertation in the Field of Food Science and Technology, Islamic Azad University, Tabriz Branch. References [1] S. Abdullah, S. Amalina, Z. Abidin, N.A. Murad, S. Makpol, Z.W. Ngah, et al., Ginger extract (Z ingiber officinale) triggers apoptosis and G 0 / G 1 cells arrest in HCT 116 and HT 29 colon cancer cell lines, Afr. J. Biochem. Res. 4 (May) (2010) 134–142. [2] Z.M. Adib, S. Ghanbarzadeh, M. Kouhsoltani, A.Y. Khosroshahi, H. Hamishehkar, The effect of particle size on the deposition of solid lipid nanoparticles in different skin layers: a histological study, Adv. Pharm. Bull. 6 (1) (2016) 31–36, https://doi. org/10.15171/apb.2016.006. [3] B.B. Aggarwal, S. Shishodia, Molecular targets of dietary agents for prevention and therapy of cancer, Biochem. Pharmacol. 71 (2006) 1397–1421, https://doi.org/10. 1016/j.bcp.2006.02.009. [4] S. Andersson, S. Lidin, Structure of the cubosome lipid bilayer aggregate, Zeitschrift Für Kristallographie 210 (1995) 315–318. [5] B. Angelov, A. Angelova, V.M. Garamus, M. Drechsler, R. Willumeit, R. Mutafchieva, et al., Earliest stage of the tetrahedral nanochannel formation in cubosome particles from unilamellar nanovesicles, Langmuir 28 (48) (2012) 16647–16655, https://doi.org/10.1021/la302721n. [6] B. Angelov, A. Angelova, B. Papahadjopoulos-sternberg, S.V. Ho, S. Lesieur, Proteincontaining PEGylated cubosomic particles: freeze-fracture Electron microscopy and synchrotron radiation circular dichroism study, J. Phys. Chem. B 116 (2012) 7676–7686. [7] B. Angelov, A. Angelova, B. Papahadjopoulos-Sternberg, S. Lesieur, J.F. Sadoc, M. Ollivon, P. Couvreur, Detailed structure of diamond-type lipid cubic nanoparticles, J. Am. Chem. Soc. 128 (17) (2006) 5813–5817, https://doi.org/10.1021/ ja060082c. [8] A. Angelova, B. Angelov, Dual and multi-drug delivery nanoparticles towards neuronal survival and synaptic repair, Neural Regen. Res. 12 (June 1) (2017) 886–889, https://doi.org/10.4103/1673-5374.208546. [9] A. Angelova, B. Angelov, B. Papahadjopoulos-Sternberg, M. Ollivon, C. Bourgaux, Proteocubosomes: nanoporous vehicles with tertiary organized fluid interfaces, Langmuir 21 (9) (2005) 4138–4143, https://doi.org/10.1021/la047745t. [10] A. Angelova, V.M. Garamus, B. Angelov, Z. Tian, Y. Li, A. Zou, Advances in structural design of lipid-based nanoparticle carriers for delivery of macromolecular drugs, phytochemicals and anti-tumor agents, Adv. Colloid Interface Sci. 249 (2017) 331–345, https://doi.org/10.1016/j.cis.2017.04.006. [11] S. Bagherpour, A. Alizadeh, M. Mohammadi, Author’ s accepted manuscript, Food Biosci. (2017), https://doi.org/10.1016/j.fbio.2017.07.010. [12] G. Borges, S. Roowi, J. Rouanet, G.G. Duthie, M.E.J. Lean, The Bioavailability of Raspberry Anthocyanins and Ellagitannins in Rats, (2007), pp. 714–725, https:// doi.org/10.1002/mnfr.200700024. [13] G. Cao, H.U. Muccitelli, C. Sánchez-moreno, R.L. Prior, Anthocyanins are absorbed in glycated forms in elderly women : a pharmacokinetic study 1–4, Am. J. Clin. Nutr. 73 (March) (2001) 920–926. [14] R.N. Cavalcanti, D.T. Santos, M.A.A. Meireles, Non-thermal stabilization mechanisms of anthocyanins in model and food systems — an overview, FRIN 44 (2) (2011) 499–509, https://doi.org/10.1016/j.foodres.2010.12.007. [15] J. Correa-betanzo, E. Allen-vercoe, J. Mcdonald, K. Schroeter, M. Corredig, G. Paliyath, Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion, Food Chem. 165 (2014) 522–531, https://doi.org/10.1016/j.foodchem.2014.05.135. [16] A. Gillet, P. Compère, F. Lecomte, P. Hubert, E. Ducat, B. Evrard, G. Piel, Liposome
4. Conclusions We demonstrated that nano-cubosomal delivery system could effectively protect antioxidant activity and increase the anticancer effect of CME. In vitro studies indicated that encapsulating the CME reduced the rate of necrosis, significantly increased the apoptosis and cell cytotoxicity, and significantly increased the G0-G1 phase cells count. In addition, CME–NCs could effectively inhibit colorectal cancer, which could successfully suggest a new ingredient for functional food and chemopreventive. Funding source declaration This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 7
Biomedicine & Pharmacotherapy 125 (2020) 109906
Z. Radbeh, et al.
[17] [18]
[19] [20]
[21]
[22]
[23] [24]
[25] [26] [27]
[28] [29]
[30]
surface charge influence on skin penetration behaviour, Int. J. Pharm. 411 (1–2) (2011) 223–231, https://doi.org/10.1016/j.ijpharm.2011.03.049. G. Gregoriadis, © 1977 Nature Publishing Group, Nature 265 (1977) 407–411, https://doi.org/10.1007/978-1-4684-4241-0. L.P.B. Guerzoni, V. Nicolas, A. Angelova, In vitro modulation of TrkB receptor signaling upon sequential delivery of Curcumin-DHA loaded carriers towards promoting neuronal survival, Pharm. Res. 34 (2) (2017) 492–505, https://doi.org/10. 1007/s11095-016-2080-4. F.A. Haggar, R.P. Boushey, D. Ph, Colorectal Cancer epidemiology : incidence, mortality, survival, and risk factors, Clin. Colon Rectal Surg. 6 (212) (2009) 191–197, https://doi.org/10.1055/s-0029-1242458. R. Harris, E. Lecumberri, I. Mateos-Aparicio, M. Mengíbar, A. Heras, Chitosan nanoparticles and microspheres for the encapsulation of natural antioxidants extracted from Ilex paraguariensis, Carbohydr. Polym. 84 (2) (2011) 803–806, https://doi.org/10.1016/j.carbpol.2010.07.003. J.R. Homoki, A. Nemes, E. Fazekas, G. Gyémánt, P. Balogh, F. Gál, et al., Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.), Food Chem. 194 (2016) 222–229, https://doi.org/10.1016/j.foodchem.2015.07.130. C.A. Hunter, T.F. Dolan, G.H. Coombs, A.J. Baillie, Vesicular systems (niosomes and liposomes) for delivery of sodium stibogluconate in experimental murine visceral leishmaniasis, Pharm.Pharmacol 40 (1988) 161–165, https://doi.org/10.1111/j. 2042-7158.1988.tb05210. A. Jain, S.K. Jain, M. K. T, Vijay Kumar Thakur (Eds.), Handbook of Polymers for Pharmaceutical Technologies, vol. 3, 2015 chapter 4. M.P. Kähkönen, A.I. Hopia, H.J. Vuorela, J.P. Rauha, K. Pihlaja, T.S. Kujala, M. Heinonen, Antioxidant activity of plant extracts containing phenolic compounds, J. Agric. Food Chem. 47 (10) (1999) 3954–3962, https://doi.org/10.1021/ jf990146l. K. Kamonpatana, M.M. Giusti, C. Chitchumroonchokchai, M. Morenocruz, Susceptibility of anthocyanins to ex vivo degradation in human saliva, Food Chem. 135 (2) (2012) 738–747, https://doi.org/10.1016/j.foodchem.2012.04.110. M.K. Khan, K. Ahmad, S. Hassan, M. Imran, N. Ahmad, Effect of novel technologies on polyphenols during food processing, Innov. Food Sci. Emerg. Technol. 45 (June 2017) (2018) 361–381, https://doi.org/10.1016/j.ifset.2017.12.006. S. Mahmoudi, M. Ghorbani, M. Sabzichi, F. Ramezani, Journal of Drug Delivery Science and Technology targeted hyaluronic acid-based lipid nanoparticle for apigenin delivery to induce Nrf2-dependent apoptosis in lung cancer cells, J. Drug Deliv. Sci. Technol. 49 (August 2018) (2019) 268–276, https://doi.org/10.1016/j. jddst.2018.11.013. T.K. McGhie, G.D. Ainge, L.E. Barnett, J.M. Cooney, J.D. Jensen, Anthocyanin glycosides from berry fruit are absorbed and excreted unmetabolized by both humans and rats, Agri. Food Chem. 51 (2003) 4539–4548. G.E. Pantelidis, M. Vasilakakis, G.A. Manganaris, G. Diamantidis, Food Chemistry Antioxidant Capacity, Phenol, Anthocyanin and Ascorbic Acid Contents in Raspberries, Blackberries, Red Currants, Gooseberries and Cornelian Cherries, February 2018 (2013), https://doi.org/10.1016/j.foodchem.2006.06.021. A.M. Pawlowska, F. Camangi, A. Braca, Quali-quantitative analysis of flavonoids of Cornus mas L. (Cornaceae) fruits, Food Chem. 119 (3) (2010) 1257–1261, https://
doi.org/10.1016/j.foodchem.2009.07.063. [31] H. Pool, D. Quintanar, J.D.D. Figueroa, C. Marinho Mano, J.E.H. Bechara, L.A. Godínez, S. Mendoza, Antioxidant effects of quercetin and catechin encapsulated into PLGA nanoparticles, J. Nanomater. 2012 (2012), https://doi.org/ 10.1155/2012/145380. [32] M. Rakotoarisoa, B. Angelov, S. Espinoza, K. Khakurel, T. Bizien, A. Angelova, Cubic liquid crystalline nanostructures involving catalase and curcumin: BioSAXS study and catalase peroxidatic function after cubosomal nanoparticle treatment of differentiated SH-SY5Y Cells, Molecules 24 (17) (2019) 3058, https://doi.org/10. 3390/molecules24173058. [33] M. Rakotoarisoa, B. Angelov, V.M. Garamus, A. Angelova, Curcumin- and fish oilloaded spongosome and cubosome nanoparticles with neuroprotective potential against H 2 O 2 -Induced oxidative stress in differentiated human SH-SY5Y cells, ACS Omega 4 (2) (2019) 3061–3073, https://doi.org/10.1021/acsomega.8b03101. [34] D.M. Ribnicky, D.E. Roopchand, A. Oren, M. Grace, A. Poulev, M. Ann, et al., Effects of a high fat meal matrix and protein complexation on the bioaccessibility of blueberry anthocyanins using the TNO gastrointestinal model (TIM-1), Food Chem. 142 (2014) 349–357, https://doi.org/10.1016/j.foodchem.2013.07.073. [35] M. Sabzichi, J. Mohammadian, R. Bazzaz, M.B. Pirouzpanah, M. Shaaker, H. Hamishehkar, et al., Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway, Process. Biochem. 60 (February) (2017) 84–91, https://doi.org/10.1016/j.procbio.2017.05.024. [36] D.S. Salem, M.A. Sliem, M. El-sesy, S.A. Shouman, Y. Badr, Journal of Photochemistry & Photobiology, B: biology Improved chemo-photothermal therapy of hepatocellular carcinoma using chitosan-coated gold nanoparticles, J. Photochem. Photobiol. B, Biol. 182 (March) (2018) 92–99, https://doi.org/10. 1016/j.jphotobiol.2018.03.024. [37] T. Sanz, H. Luyten, Release, partitioning and stability of isoflavones from enriched custards during mouth, stomach and intestine in vitro simulations, Food Hydrocollids 20 (2006) 892–900, https://doi.org/10.1016/j.foodhyd.2005.09.003. [38] A. Scalbert, C. Morand, C. Manach, C. Rémésy, Absorption and metabolism of polyphenols in the gut and impact on health, Biomed. Pharmacother. 56 (2002) 276–282. [39] T. Squillaro, A. Cimini, G. Peluso, A. Giordano, M.A.B. Melone, Nano-delivery systems for encapsulation of dietary polyphenols : an experimental approach for neurodegenerative diseases and brain tumors, Biochem. Pharmacol. 154 (March) (2018) 303–317, https://doi.org/10.1016/j.bcp.2018.05.016. [40] M.B. Subudhi, A. Jain, A. Jain, P. Hurkat, S. Shilpi, A. Gulbake, S.K. Jain, Eudragit S100 coated Citrus pectin nanoparticles for Colon targeting of 5-Fluorouracil, Materials (2015) 832–849, https://doi.org/10.3390/ma8030832. [41] M. Thadanki, P.S. Kumari, K.S. Prabha, Overview of cubosomes : a nano particle, ResearchGate 1 (3) (2011) 535–541. [42] K.U. Yilmaz, S. Ercisli, Y. Zengin, M. Sengul, E.Y. Kafkas, Preliminary characterisation of cornelian cherry (Cornus mas L.) genotypes for their physico-chemical properties, Food Chem. 114 (2) (2009) 408–412, https://doi.org/10.1016/j. foodchem.2008.09.055. [43] L. Zhao, F. Temelli, L. Chen, Encapsulation of anthocyanin in liposomes using supercritical carbon dioxide : effects of anthocyanin and sterol concentrations, J. Funct. Foods 34 (2017) 159–167, https://doi.org/10.1016/j.jff.2017.04.021.
8