Date seed extract-loaded oil-in-water nanoemulsion: Development, characterization, and antioxidant activity as a delivery model for rheumatoid arthritis

Abstract

INTRODUCTION Rheumatoid arthritis (RA) is a most common inflammatory disorder. It is portrayed at the cell level by changes in the natural and adaptive immunity, which creates a constant synovial joint provocative reaction that may influence other organs.[1] Studies in support of RA have given the proof that RA is a dynamic insusceptible cell-intervened illness, which is started and advanced by variant actuation of T cells with related B-cell hyperactivity. The deregulation of invulnerable cell action causes incessant synovial joint inflammation that is considered fundamentally by T-cell-interceded actuation of synovial fibroblasts.[2] This drives the dynamic obliteration of cartilage and subchondral bone bringing about joint disaster.[3] Free radicals are involved in constant incendiary illnesses including RA and some other conditions. Free radicals assume a significant job in the seriousness of rheumatoid joint pain and patients, for the most part, endure with excessive oxidative stress. Antioxidants either engineered or natural are intense foragers of free radicals and effectively affect human well-being and illness avoidance.[4] It has been reported that antioxidants have plausible role for uplifting the inflammatory condition in RA patients.[5] Several literature reported that synthetic antioxidants (butylated hydroxyanisole, butylated hydroxytoluene) may be the cause of cancer development. So, natural antioxidants are extremely desirable.[6] Phoenix dactylifera (date palm) belongs to the family Palmaceae and genus Phoenix, cultivated globally, especially in Middle East and North Africa for its palatable organic nature. A date is a high-vitality nourishment having high value of carbohydrates (70%–80%) and low fat content, and is a great wellspring of phenolic compounds, vitamins, carotenoids, zinc, iron, calcium, magnesium, phosphorous, and potassium. Date seeds are generally considered as a waste item and either disposed of or utilized in as animal feed. Nevertheless, date seeds have been appeared to have highly extractable valuable components.[7] The preventive effects of natural antioxidants in leafy foods are associated with four significant groups of compounds such as polyphenols, alkaloids, vitamins, and carotenoids. Edible portion of date and date seed has antioxidant activity in vitro because of the existence of phenolic compounds.[8] Defensive effect of phenolic compounds as antioxidants is related to lipoperoxidative impairment, which relies on the hydrogen donating limit of hydroxyl bunch in every molecule.[9] The nanotechnology-based drug delivery system such as nanomicelles, nanoemulsions, nanosuspension, nanoparticles, and some other nanoformulations has displayed a promising value in enhancing the delivery of hydrophobic and hydrophilic molecules since past decades.[10] Nanoemulsion provides very low interfacial tension with substantial oil-in-water interfacial regions, high dynamic strength, and optical transparency approximating to microemulsion.[1112] Nanoemulsions offer a higher solubilization limit than a straightforward micelle arrangement and their thermodynamic steadiness offers favorable circumstances over flimsy scatterings, for example, emulsion and suspension.[13] Nanoemulsions may also be employed as an alternate for lipidic nanocarriers (liposomes and vesicles) to enhance bioavailability of many active biomolecules when compared with the traditional formulations.[14] Thus, to improve the antioxidant activity of date seed, a robust and stable nanoemulsion system containing date seed extract was developed and optimized. The developed nanoemulsions were further evaluated for mean droplet size and its surface morphology, viscosity, conductivity, refractive index (RI), in vitro drug release, and in vitro antioxidant activity. MATERIALS AND METHODS Materials and chemicals Date seeds were purchased from local in a full ripe condition and authenticated by Dr. Y. T. Kamal, Assistant Professor, Department of Pharmacognosy and Phytochemistry, College of Pharmacy, Sattam Bin Abdul Aziz University, KSA (accession no. PSA/PHAR/COG/15/04). Sefsol 218 and Kolliphor RH40 were acquired as a bequest from Nikko Chemicals (Tokyo, Japan) and BASF (Mumbai, India), respectively. PEG 400 was procured from S.D. Fine-Chem (India). All chemicals used were of analytical grade like Isopropyl myristate (IPM), Olive oil, Methanol (HPLC grade) were purchased from Merck (Mumbai). Methodology Methanolic extract of date seeds Date seeds (200g) were extracted with 500 mL of methanol using Soxhlet apparatus for 24 h at 50°C–60°C. After extraction, the solvent was evaporated to dryness using a rotary vacuum evaporator (HAHN SHIN, HS-2005 V-N) at 40°C under an inert atmosphere to obtain a dark brown-colored residue. This obtained material was weighed and percentage yield was calculated.[15] Nanoformulation development and its characterization To achieve stable and robust nanoemulsion, a through screening of various components was done. For this, solubility studies of methanolic extract of date seed were performed with different oils, surfactants, and cosurfactants. Afterward, construction of pseudoternary phase diagram was carried out followed by testing of their thermodynamic stability of developed nanoemulsions. Screening of oil, surfactants, and cosurfactants by solubility studies An important criterion for a stable nanoemulsion is to find out the solubility of a drug in selected oils. In this sequence, solubility of date seed extract was analyzed in different oils (Sefsol 218, Capryol 90, oleic acid, and isopropyl myristate [IPM]). An excess quantity of date seed extract was poured in stoppered vials containing 2 mL of each oil and blended utilizing a vortex.[13] To get equilibrium, these vials were further kept at 25°C ± 1.0°C in an isothermal shaker (IKA KS 400i, Germany) for 72 h. Afterward, these vials were centrifuged (REMICM are -8 Plus and CM-12 Plus, India) at 10,000rpm for 15 min. The supernatant obtained was filtered over a 0.45-µm membrane filter and the quantity of date seed extract was further estimated in various oils by a UV spectrophotometer (UV-1800, Schimadzu) at a wavelength of 280nm. A qualitative solubility test was performed for selecting a surfactant and cosurfactant. Final selection was done based on the miscibility of extract loaded oil with surfactants. Preparation of nanoemulsion Nanoemulsions were formulated by aqueous titration method followed by construction of pseudoternary phase diagrams to determine the concentration range of oil, Smix (surfactant–cosurfactant mixture), and distilled water.[13] Different Smix ratios were prepared by mixing surfactants and cosurfactants in different quantities (1:0, 1:1, 1:2, 2:1), one or the other in increasing concentration with respect to each other. For every phase diagram, oil and a definite Smix ratio were blended properly in distinct volume ratios from 1:9 to 8:1 in individual glass vials. Then the blend (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1:2, 1:3, 1:3.5, 1:5, 1:6, 1:7, and 1:8) was titrated slowly with aqueous phase. Each titration was visually examined for transparency and flowability. The physical condition of the nanoemulsion was set apart on the phase diagrams with three pivots demonstrating an aqueous phage, oil, and Smix. For each stage graph, nanoemulsion territory was drawn and the more extensive district showed the improved self-nanoemulsifying proficiency. From every phase diagram, constructed, various formulations were selected from nanoemulsion region altering the proportion of oil (10-30%v/v) at minimum concentration of Smix. Furthermore, the selected nanoemulsions were studied for stability and dispersibility. Thermodynamic stress stability studies Designated nanoemulsions were analyzed for the stress stability tests (heating–cooling cycle, centrifugation, and freeze–thaw cycle): Heating–cooling cycle: Formulations were exposed to 45°C and 0°C for 48 h for each temperature round and examined for any type of precipitation or phase separation. Centrifugation study: Selected nanoemulsions were further centrifuged at 5000rpm (REMI) for 30 min and diagnosed for any possible non-homogeneity of formulations. Freeze–thaw cycle: In this study, nanoemulsions were maintained at the temperatures −20°C and +20°C for three cycles and each cycle for 24 h and these formulations were observed for homogeneity.[16] Dispersibility tests Every selected formulation (1 mL) was gently added to a medium of 0.1N HCl (500 mL) in an USP Type II dissolution apparatus (75rpm) at 37°C ± 0.5°C, in order to evaluate its proficiency of self-emulsification.[17] All formulations were observed visually as per the grading system given in Table 1.Table 1: Dispersibility testFormulations that agreed with the stability and dispersibility criteria were selected for preparation of date seed extract-loaded nanoemulsion followed by further characterization. Preparation of drug-loaded nanoemulsion by aqueous titration method The drug-loaded nanoemulsion (DLN) was fabricated by dissolving 50 mg (single dose)[8] of methanolic date seed extract in oil (5%, 10%, 15%, 20% vol/vol). Respective Smix ratio was mixed with oil using vortex followed by gently adding aqueous phase to achieve nanoemulsion. Physicochemical characterization and evaluation of nanoformulation Visual inspection Visual inspection was performed to distinguish the drug-loaded nanoemulsion and macroemulsion. Droplet size measurement The average droplet size of nanoemulsions was established by dynamic light scattering method with the help of zetasizer ZS 90 (Malvern Instruments, Worcestershire, UK). Scattering of light was done at the room temperature at a 90° angle. Polydispersity index (PDI) of the formulations was also recorded.[16] Viscosity measurement Viscosity of the developed nanoemulsions was also determined by a Brookfield DV III ultra V6.0 RV cone and plate rheometer (Brookfield Engineering Laboratories, Middleboro, Massachusetts) with spindle #CPE40 at 25°C ± 0.5°C. Electro conductivity studies To find the type of developed nanoemulsions, conductivity (σ) studies were performed using a CDM 230 conductometer (Radiometer, Copenhagen, Denmark) with the cell constant value of 0.11/cm and the frequency of 94 Hz at 25°C ± 1°C. Refractive index and percent transmittance RI of the developed nanoemulsions was measured by placing a single drop of nanoemulsion on the slide and analyzed using an Abbe refractometer (Bausch and Lomb Optical Company, Rochester, New York) at 25°C. In order to get % transmittance, the sample was examined using a UV spectrophotometer (Shimadzu, Japan) at 280nm with respect to distilled water.[18] Surface morphology Surface morphology of the developed formulation was explored by transmission electron microscopy (TEM; TOPCON 002B, Topcon).[13] One drop of the selected formulation was taken and attenuated with distilled water (1:100); after filtering with a syringe filter (0.22 μm), the sample was placed on the carbon grid with 2% phosphotungstic acid and allowed to stay for 30s. This dried grid was placed on a slide and covered up with a cover slip. The prepared slide was analyzed using the microscope. Drug content There is a possibility of precipitation of active drug due to the presence of surfactant and cosurfactant in the formulation by the centrifugation method. Hence, the amount of drug in the developed formulations was calculated by using UV spectroscopy. The selected formulations were diluted with methanol and the absorbance was recorded at 280nm. Drug content was expressed as a percentage of date seed extract determined in the formulation to the theoretical quantity of the drug added. In vitro drug release profile The optimized nanoformulations were further investigated for the drug release profile by the dialysis bag method. Dialysis membrane was preactivated in a solvent system of phosphate buffer (pH 7.4) and methanol (3:1) for 1 h. One milliliter of date seed extract-loaded nanoemulsion and suspension was placed into a separate preactivated dialysis bag (MW: 12,000–14,000Da). Both the ends of dialysis bag were tied properly and suspended in a baker filled with solvent system (20 mL). The whole set up was affirmed at 37°C ± 1°C with continuous stirring at 75rpm for 24 h. At predetermined time intervals (0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h), drug samples (1 mL) were withdrawn and filtered over a 0.22-μm membrane filter. Withdrawn quantity of medium was replaced with an equal volume of fresh medium to achieve the sink condition. The analysis was done in triplicate. The withdrawn samples were analyzed by a UV spectrophotometer (UV-1800, Shimadzu) at 280nm for the drug content. The release profile of the nanoemulsions were compared with the drug suspension.[19] Antioxidant activity The radical scavenging potential of methanolic extract of date seed in contrary to 1,1-diphenyl-2-picrylhydrazyl (DPPH) was determined by analytical techniques UV spectrophotometry, at 517nm. An aliquot (20, 40, 60, 80, 100, and 120 μg/mL) for methanolic extract of date seed was dissolved in different test tubes comprising 5 mL of methanol and 0.5 mL of 1mM DPPH. α-Tocopherol (vitamin E) was used as the standard with the identical concentrations as that of test samples. A placebo solution with equal volume of methanol and DPPH was made, and this solution mixture was incubated at room temperature for half an hour.[20] The antioxidant activity was estimated using the following equation: where Ab denotes absorbance of blank and As denotes absorbance of sample. All experiments were done in triplicate. The calculated IC50 values are stated as mean ± SD. RESULT AND DISCUSSION Extraction of components from date seed Methanol was used as a solvent for extraction of components using Soxhlet apparatus for 24 h. After 24 h, the solvent was evaporated under reduced pressure to obtain chocolate-colored solid mass and the percent yield was calculated to be 12% wt/wt [Figure 1].Figure 1: Methanolic extract of crownSolubility studies Solubility examinations were carried out to identify an appropriate oil phase for the effective formulation of date seed extract-loaded nanoemulsion to attain the drug in the solubilized form with optimal drug loading. In the selected oils, the solubility of the date seed extract was observed maximum in Sefsol 218 (70 mg/mL) followed by IPM (32.48 mg/mL), triacetin (18.5 mg/mL), and oleic acid (20 mg/mL) [Figure 2]. Therefore, Sefsol 218 was chosen as oil phase for the formulation development. It has been reported that nanoemulsion comprising Sefsol 218 as oily phase provides a broader and stable nanoemulsion region when combined with a surfactant, Kolliphor RH40, and cosurfactant, PEG400.[21] Therefore, after performing a miscibility test of Sefsol 218-solubilized extract with Kolliphor RH40 and PEG400, it was observed that the selected surfactant and cosurfactant were completely miscible to each other.Figure 2: Quantitative solubility studies of date seed extract in different oilsTernary phase diagram study Ternary diagram is utilized to ascertain the presence of different zones of nanoemulsion. It graphically delineates the proportions of the three factors as positions in a symmetrical triangle. Typically, three distinct types of phages are observed during construction of a ternary phage diagram that is nanoemulsion state (transparent bluish nature), coarse emulsion (unstable, milky white) and liquid crystal (translucent gel like state). However, in this study, our was in the nanoemulsion region and have been which showed the of nanoemulsion [Figure Ternary phase diagrams of date seed extract nanoemulsions, 2:1), and this study, four different were observed for the nanoemulsion formulation Sefsol 218 as oil phase and RH40 as surfactant, and cosurfactant was used in this It was from the diagram that low quantity of oil be solubilized with a higher concentration of It a range for nanoemulsion of a cosurfactant in the nanoemulsion An is used for the surface the surfactant to the interfacial and the liquid which are established when the surfactant is This be to the that interfacial tension and liquid interfacial are in a by the of single surfactant, generally the of a cosurfactant. The ternary of which was of Sefsol 218 RH40 and PEG 400 with an Smix ratio of 1:1, the development of a region of nanoemulsion. nanoemulsions with light bluish with the maximum solubilization of oil were in this The of maximum nanoemulsions in this region may be to interfacial tension oil and water because maximum of oil were solubilized with surfactant in this Furthermore, surfactant and cosurfactant in optimized amount causes oil phase The of PEG 400 as cosurfactant the of and creates the interfacial to up various to nanoemulsion over a of from the ternary of phase diagram showed higher nanoemulsion in the higher surfactant this was when compared with Hence, quantity of water was to in this the nanoemulsion was to to the surfactant This be because of quantity of RH40 in the diagram from formulation the development of nanoemulsions in the It be due to an amount of PEG 400 which causes a reduced interfacial tension when compared with other Thermodynamic stability tests All the developed nanoemulsions were evaluated for thermodynamic formulations were selected for preparation of drug-loaded nanoemulsion followed by further characterization. test are reported in Table 2: Thermodynamic stability test of different formulation selected from phase of drug-loaded nanoemulsion Formulations that the stability tests and minimum amount of surfactant were selected for drug it is that a of drug is in one mL of oil, drug was loaded in various oil concentrations which stability tests formulation selected from phase diagram and thermodynamic stability study test at a of of characterization of nanoemulsions Visual were for the developed and nanoemulsions were any [Figure of a nanoemulsion and prepared by aqueous titration size measurement and morphology Droplet size and size were analyzed as in 5 and It was observed that the formulations were in the size range with a low The formulation the droplet size ± with ± The value the of nanoemulsions. The that drug-loaded nanoemulsions were in with the of droplet size as by a Therefore, the were of the that selection of oil, surfactant, and cosurfactant with definite concentrations are the factors for size with a stable nanoemulsion as in Droplet size of optimized potential of optimized droplet percent transmittance, refractive viscosity, conductivity, and drug electron microscopy of optimized content Drug content (date seed in the optimized nanoemulsions was evaluated which was in the range of The drug content up to different formulations In vitro drug release studies of optimized formulations and and suspension of date seed extract were and that release of drug from optimized formulations is significant than release of drug from suspension as in from the nanoemulsions were higher as compared to that from drug suspension due to its which offers a high surface for solubility as as dissolution drug release showed that the formulations and and drug suspension ± ± ± ± and ± of at 24 h. It is due to the droplet size and of and which limit the drug the of release formulation was selected for antioxidant In vitro release profile of date seed extract from different optimized nanoemulsion formulations and and drug suspension in phosphate activity Antioxidant potential of date seed extract was determined by the DPPH in which the of a stable free radical (DPPH) was Antioxidant activity was based on the that when mixing the DPPH solution as an with an it hydrogen which to the reduced form The of the reaction mixture from to and its absorbance was measured at a wavelength of 517nm. The standard was taken to the antioxidant activity of methanolic of date The IC50 for DPPH scavenging action was determined graphically [Figure Methanolic extract of date seed and nanoemulsion formulation at the concentration of 120 showed and activity to It was observed that antioxidant activity of date seed extract was to the concentration of A IC50 value higher antioxidant activity. The antioxidant activity of date seed is to the presence of phenolic compounds and The in the were by and it was that were in antioxidant which are for and scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by the methanolic extract of date seed and compared with standard drug Each value mean ± IC50 values of methanolic extract of date seed, nanoemulsion and were ± ± and ± In this nanoemulsion containing date seed extract was developed by utilizing Sefsol 218 as oil RH40 as surfactant, and PEG 400 as cosurfactant and further for in vitro The developed nanoemulsions were in size with a high drug content range and were also The in vitro release study showed drug release when compared with drug from antioxidant activity that optimized nanoemulsion has more antioxidant potential when compared with drug the of our it be that the developed methanolic extract of date seed nanoemulsion was to have more antioxidant potential and high drug release when compared with The be a for the of However, is a for further studies in of to it support and of There are of

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