Abstract
Chrysin is a natural flavonoid with a wide range of bioactivities. Only a few investigations have assessed the analgesic activity of chrysin. The lipophilicity of chrysin reduces its aqueous solubility and bioavailability. Hence, self-nanoemulsifying drug delivery systems (SNEDDS) were designed to overcome this problem. Kollisolv GTA, Tween 80, and Transcutol HP were selected as oil, surfactant, and cosurfactant, respectively. SNEDDS A, B, and C were prepared, loaded with chrysin (0.1%w/w), and extensively evaluated. The optimized formula (B) encompasses 25% Kollisolv GTA, 18.75% Tween 80, and 56.25% Transcutol HP was further assessed. TEM, in vitro release, and biocompatibility towards the normal oral epithelial cell line (OEC) were estimated. Brain targeting and acetic acid-induced writhing in a mouse model were studied. After testing several adsorbents, powdered SNEDDS B was formulated and evaluated. The surfactant/cosurfactant (S/CoS) ratio of 1:3 w/w was appropriate for the preparation of SNEDDS. Formula B exhibited instant self-emulsification, spherical nanoscaled droplets of 155.4 ± 32.02 nm, and a zeta potential of − 12.5 ± 3.40 mV. The in vitro release proved the superiority of formula B over chrysin suspension (56.16 ± 10.23 and 9.26 ± 1.67%, respectively). The biocompatibility of formula B towards OEC was duplicated (5.69 ± 0.03 µg/mL). The nociceptive pain was mitigated by formula B more efficiently than chrysin suspension as the writhing numbers reduced from 8.33 ± 0.96 to 0 after 60 min of oral administration. Aerosil R972 was selected as an adsorbent, and its chemical compatibility was confirmed. In conclusion, our findings prove the therapeutic efficacy of chrysin self-nanoemulsion as a potential targeting platform to combat pain.
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Introduction
Chrysin, 5,7-dihydroxyflavone (Fig. 1), is a natural flavonoid that is present in bee propolis, honey, and various plants [1]. Chrysin is categorized by the U.S. Food and Drug Administration (FDA) as a “generally recognized as safe” (GRAS) nutraceutical [2]. It has a broad spectrum of biological activities. The antioxidant [3], anti-inflammatory [4], antinociceptive, antidiabetic [5], anticancer [6], antidepressant [7], antiepileptic, anticonvulsant [8], and neuroprotective [9,10,11] activities of chrysin have been reported. Few studies only investigated the antinociceptive and analgesic effects of chrysin [4, 12,13,14].
Chemical structure of chrysin [120]
Pain is a common sign of illnesses and the body’s defense mechanism against stimulants. When nociceptors (pain receptors) are excited by stimulants at free nerve terminals, pain develops [15]. If acute pain is not properly managed, it can turn into chronic pain and lead to serious health problems [16, 17]. Pain is commonly classified as either nociceptive or neuropathic according to pathophysiology. Peripheral pain, though, is not always nociceptive. Neuropathic pain may arise from abnormal signal processing in either the central or peripheral nervous systems [18].
Because of its ease of use, low cost, non-invasiveness, and self-administration, the oral route is one of the most popular administration routes. However, factors such as poor aqueous solubility, limited permeability, and the pre-systemic first-pass metabolism negatively affect the oral delivery of drugs [19]. Chrysin is classified according to the biopharmaceutical classification system (BCS) as class II [20]. Accordingly, low water solubility of chrysin severely reduces its oral bioavailability which is the major constraint in its use [2]. The incorporation of chrysin within appropriate delivery systems has therefore been proposed as a solution to such an obstacle.
Innovative drug delivery systems such as polymeric nanoparticles [21, 22], micelles [20], phytosomes [23] nanoemulsions [4, 24,25,26], solid lipid nanoparticles [27], and nanostructured lipid carriers [6, 28] have been studied, and their promotion of chrysin bioactivities was reported [28]. Upon dilution, self-nanoemulsifying drug delivery systems (SNEDDS) produce self-nanoemulsions (NEs) as nanosized transparent systems. It is reported that SNEDDS could enhance the solubility of lipophilic drugs and subsequently their oral bioavailability [19]. Such an improvement in bioavailability could enhance the delivery of lipophilic drugs to the brain [29]. Conclusively, SNEDDS would help escape hepatic metabolism as well as increase the deliverability and bioavailability of lipophilic drugs [2, 30]. To the best of our knowledge, the antinociceptive activity of chrysin-loaded SNEDDS has not been studied before.
From such a perspective, the present study aimed to design, prepare, evaluate, and optimize SNEDDS as an attempt to enhance the antinociceptive activity of chrysin and boost its ameliorative effect against pain. To fulfill our aim, acetic acid-induced writhing in mice was applied as a pain model to investigate the efficiency of the chrysin-loaded SNEDDS by counting the number of writhing.
Materials and Methods
Materials
Chrysin was obtained from Alfa Aesar, Thermo Fisher Scientific Co (Germany). Maisine® CC, Peceol™, Labrafac lipophile WL 1349™, Labrasol® ALF, Labrafil® M 1944 CS, Labrafil M 2125 CS™, Plurol Oleique CC 497®, Capryol PGMC®, Lauroglycol FCC™, Lauroglycol 90™, Capryol 90®, and Transcutol HP® were a gift from Gattefosse (Lyon, France). Methanol HPLC grade was purchased from Fisher Chemical (Loughborough, UK). Kollicream IPM and Kollislov GTA were obtained from BASF Pharma (Germany). Tween 20, Tween 80, and Tween 40 were purchased from El-Nasr Pharmaceutical Chemicals Co. (Egypt). Aerosil 200 and Aerosil R972 were developed by Evonik Industries AG (Germany). Chitin was supplied by Sigma Chemical Company (St, Louis, MO, UK). Starch and sodium fluorescein were obtained from Adwic Co. (Egypt). Normal human oral epithelial cell line (OEC) was brought from Nawah Scientific Inc. (Mokatam, Cairo, Egypt)
Solubility Study
Solubility of chrysin was measured in different oils: Maisine CC, Peceol TM, Labrofac 1349, Kollicream IPM, and Kollislov GTA; surfactants: Tween 20, Tween 80, Tween 40, Labrasol ALF, Labrofil 1944, and Labrofil M2125; and cosurfactants: Plurol Oleqiue, Capryol PGMC, Lauroglycol FCC, Lauroglycol 90, Capryol 90, and Transcutol HP. In brief, chrysin was added in excess to a 5-mL solvent, vortexed (Model VM-300, Gemmy Industrial Corp., Taiwan), and shaken in a thermostatically controlled agitating water bath (Grant Instrument, Cambridge Ltd., UK) at 37 ± 0.5°C for 24 h. The suspensions obtained were centrifuged for 20 min at 10,000 rpm (Benchtop Centrifuge, Sigma Laborzentrifugen GmbH, Germany) in order to separate the undissolved chrysin. Then, the supernatant was filtered (Millipore filter 0.45 µm, EMD Millipore, Billerica, MA, USA), diluted with methanol, and assayed spectrophotometrically at 267 nm (UV/visible double beam spectrophotometer, Labomed Inc., USA). Every measurement was made against the corresponding chrysin-free solvent, which was prediluted in an identical way to act as a blank avoiding any interference. The study was conducted in triplicate, and the mean ± standard deviation was used to describe the findings.
Pseudo-ternary Phase Diagrams Using Water Titration Method
The pseudo-ternary phase diagrams were constructed using Kollisolv GTA, Tween 80, and Transcutol HP as oil, surfactant, and cosurfactant, respectively. The diagrams consisted of three-sided triangles representing the oil phase, the aqueous phase, and the mixture of surfactant and cosurfactant (S/CoS mix). The S/CoS mixtures of Tween 80 and Transcutol HP were prepared at different ratios (1:1, 1:2, 2:1, 3:1, 1:3, 5:1, and 1:5 w/w, respectively) by vigorous vortex in separate tubes for 30 s. Kollisolv GTA and each S/CoS mixture at ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 w/w, respectively, were weighed and vortexed. After that, distilled water was added in daily increments of 10 to 90% to the prepared oil and S/CoS mixtures, but without chrysin incorporation, then vortexed for 5 min, and kept at ambient temperature to equilibrate for 24 h. Following equilibrium, the mixture’s transparency and clarity were visually observed. Switching points from clear to turbid were represented as phase transitions from nanoemulsion to ordinary emulsion, respectively. Using the obtained pseudo-ternary phase diagrams, the ratio and the concentration ranges that yielded the largest area of nanoemulsion (NE area) were selected and used in the formulation of SNEDDS which would include chrysin.
Preparation of Chrysin-Loaded SNEDDS
Three percentages of oils: 10, 25, and 40 % w/w of Kollisolv GTA, were selected for the preparation of three chrysin-loaded SNEDDS designated as A, B, and C, respectively. Table I shows the composition of the prepared chrysin-loaded SNEDDS. First, chrysin (0.1 g % w/w) was dissolved in Kollisolv GTA for 1 h under magnetic stirring (Magnetic stirrers, HPS-20D, Taisite lab Scientific Inc., USA). Then, Transcutol HP was added to the oily mixture and magnetically stirred for 1 h at 1100 rpm followed by Tween 80 addition. Finally, the entire system was stirred for a further 1 h.
Characterization of Chrysin-Loaded SNEDDS
Percent Transmittance (%T)
The chrysin-loaded SNEDDS were diluted with distilled water at a ratio of 1:100 v/v, and the percent transmittance (%T) was measured spectrophotometrically at 650 nm utilizing distilled water as a blank [31].
Robustness to Dilution
By diluting the chrysin-loaded SNEDDS by 100 and 1000 v/v times with distilled water, simulated gastric fluid (SGF) pH 1.2, and phosphate buffer pH 7.4 (PB 7.4), robustness to dilution was determined. The diluted SNEDDS were kept for 24 h and any indication of phase separation, precipitation, or turbidity was observed. The preparations without phase separation or precipitation were regarded as robust to dilution [32].
Self-Emulsification Time
The chrysin-loaded SNEDDS were added to distilled water, SGF, and PB 7.4 at a ratio of 1:500 v/v, respectively under magnetic stirring of 100 rpm at 37 ± 0.5°C. Just after addition, the time needed for nanoemulsification was recorded [33]. The produced nanoemulsions formed were stored for 24 h and their appearance, optical clarity, precipitation, and phase separation were visually observed [31].
Viscosity Determination
The viscosity of the prepared SNEDDS was measured without dilution using VR 3000 MYR, model V1-L, Visco-tech Hispania SL., Tarragona, Spain. The viscometer was operated at 100 rpm using spindle L2 [34].
Droplet Size, Polydispersity Index (PDI), and Zeta Potential
Each formula was diluted with distilled water (1:100 v/v) and sonicated Sonix IV, SS 101H 230, ETL Testing Laboratories Inc., USA for 10 min. Then, dynamic light scattering (DLS) and laser Doppler micro-electrophoresis techniques were applied using a Zetasizer Nano ZS system (Malvern Instruments Ltd, Malvern, UK) for droplet size, PDI, and zeta potential measurement, respectively [35].
Thermodynamic Stability
Centrifugation at 3500 rpm for 30 min was applied to the prepared SNEDDS. Then, three cycles of a heating-cooling cycle: heating (45 ± 0.5°C) and cooling (4 ± 0.2°C) for 48 h were applied to those systems that exhibited no sign of phase separation after centrifugation. Those that withstand the heating-cooling cycles without phase separation underwent three freeze-thaw cycles operated between − 21°C and 25 ± 0.5°C for 48 h [36,37,38].
Cloud Point Measurement
The cloud point is the temperature above which the clear nanoemulsion turned cloudy upon increasing temperature gradually. The prepared SNEDDS were diluted by 250 folds with distilled water, placed in a water bath, and heated gradually. The temperature at which sudden cloudiness was visually perceived and noted as the cloud point [36].
Formula B was chosen as the optimal one depending on the obtained results, and hence, it was subjected to further investigation.
Evaluation of the Optimal Formula B
Transmission Electron Microscope (TEM)
The morphology of the optimal formula B was examined by TEM (JEOL JEM-2100; JEOL Ltd., Tokyo, Japan) using a digital micrograph and soft imaging viewer software. Formula B was diluted tenfold with water to form a nanoemulsion. One drop of the nanoemulsion was applied on the surface of a carbon-coated copper grid and left for one minute to allow droplets’ adherence; then, the excess was wiped up. One drop of 2% uranyl acetate solution was applied for two minutes to support inspection. Then, microphotographs were acquired at a proper magnification [39, 40].
Fourier Transform-Infrared Spectroscopy (FT-IR)
Samples of chrysin, Transcutol HP, Tween 80, Kollisolv GTA, and formula B were scanned over a range of 500–4000 cm−1 using FT-IR-spectrophotometer (Bruker ALPHA II PLATINUM ATR, Germany) to obtain their FT-IR spectra.
In Vitro Release Study
The in vitro release of chrysin was studied using the dialysis bag technique. Three release media, namely, SGF, simulated intestinal fluid (SIF; phosphate buffer pH 6.8), and PB 7.4 containing 1% v/v Tween 80 were applied [41]. Dialysis cellulose tubing Spectra/Por™ semipermeable cellulose membrane (MWCO of 12,000–14,000 Da, Spectrum Medical Industries Inc., Los Angeles 90054, USA) was equilibrated in the release media for 24 h before the experiment. The dialysis bag was sealed from one end and the weight of optimal formula equivalent to 2 mg chrysin was transferred inside the bag. Then, the bag was sealed and placed in 100 mL of the release medium kept at 37 ± 0.5°C and 100 rpm for 48 h using a thermostatically controlled shaking incubator (GFL Gesellschaft für Labortechnik, Burgwedel, Germany)[28]. Aliquots of 2.5 mL each were withdrawn at predetermined time intervals (0.5, 1, 2, 4, 6, 8, 10, 24, and 48 h). The withdrawn samples were replenished with a fresh medium equilibrated at the same condition. Chrysin concentrations were assayed spectrophotometrically at 267 nm against the applied release medium as blank. Another experiment was also carried out as mentioned before using chrysin suspension (0.1% w/w) to study the release profiles of the pure drug. The suspension was prepared using 1% sodium carboxy methyl cellulose (1% w/w Na CMC) [24].
Kinetic Analysis of Release Data
The mechanism by which chrysin was released from SNEDDS formula B and pure chrysin suspension in different release media could be determined using kinetic models. The kinetic models including zero-order, first-order [42], Higuchi’s [43], and Korsmeyer-Peppas [44] models were applied. Based on the highest coefficient of determination (r2), the kinetic model related to the best kinetic release profile was selected.
Biocompatibility of the Optimized Formula B with Normal Oral Epithelial Cell Line
The biocompatibility studies of chrysin and SNEDDS formula B were investigated on normal human oral epithelial cell line (OEC) using methyl thiazolyl tetrazolium (MTT) assay. After cell confluency in 96-well plates, the cells were treated with formula B at amounts equivalent to 1.95, 3.9, 7.8, 15.6, and 31.25 μg chrysin/mL and incubated for 48 h. The untreated OEC served as the control group. The plain SNEDDS was considered and treated similarly. For interest, the safety of pure chrysin on OEC was elicited. The concentrations of pure chrysin were the same as that in formula B. Following the incubation period, 20 μL MTT solution at a concentration of 5 mg/mL in PBS was added to the treated cells in each well and mixed. At 37°C, each plate was incubated for a further four hours. Then the formed formazan crystal was dissolved with 100 μL of absolute DMSO. A multi-well plate reader was used to measure the absorbance of formazan solutions at 570 nm (BMGLABTECH® FLUO star Omega, Germany)[45]. The percent cell viability was calculated as follows [46]:
In Vivo Evaluation of the Optimal Formula B
Brain Targeting
Six male Swiss Albino mice weighing 25–30 g were used. Mansoura University Research Ethical Committee consented (Ethical Approval Code: 2023-219) to the animal use protocol following “Principles of Laboratory Animal Care” (NIH publication No. 85–23, revised 1985). Sodium fluoresceine (NaFl) aqueous solution (1% w/v) and chrysin/NaFl-loaded formula B were prepared. NaFl (10 mg) was directly dissolved in 1 gm of distilled water to prepare NaFl solution (10 mg/g). In the case of chrysin/NaFl-loaded formula B, both NaFl at a concentration of 10 mg/g and chrysin (1 mg/g) were incorporated as previously described under “Preparation of Chrysin-Loaded SNEDDS”. Randomly, the animals were divided into two groups (three animals/ group):
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Group I: NaFl-treated group
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Group II: Chrysin/NaFl-treated group
The animals of Groups I and II were injected intravenously into the tail veins with NaFl (40 mg/kg) [47] and chrysin/NaFl-loaded formula B (40 mg NaFl and 4 mg chrysin/kg), respectively [2, 21]. Fifteen minutes later, the animals were killed by cervical dislocation and the whole brain was excised carefully. Each brain was weighed, and then, it was homogenized in 4 mL of borate buffer (pH 10) for 40 sec (DAIHAN Scientific HG-15 D Homogenizer, Korea). The brain homogenate was centrifuged for 10 min at 4000 rpm. To precipitate the proteins, 1 mL of the supernatant was mixed with 4 mL of ethanol. After that, the mixture was centrifuged at 4500 rpm for 20 min. Subsequently, the supernatant was filtered (0.45 μm) and assayed spectrofluorometrically for NaFl (Agilent Cary Eclipse fluorescence spectrophotometer, USA) at an excitation wavelength of 500 nm and emission wavelength at 518 nm [47]. The amount of NaFl was expressed as an amount of ng/mg brain. The calibration curve for NaFl was constructed already between the concentration of NaFl versus the intensity of fluorescence at the above-mentioned excitation and emission wavelengths.
Pharmacodynamic Study
Acetic acid-inducing pain and writhing were used as a model for assessing the analgesic effect of the optimal SNEDDS formula B [48]. Forty-two Swiss Albino mice weighing 25–30 g were used. The Research Ethical Committee at Mansoura University passed the animal use protocol (Ethical Approval Code: 2023-219) under “Principles of Laboratory Animal Care” (NIH publication No. 85–23, revised 1985). The animals were allocated at random to three groups specified as groups I, II, and III.
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Group I: (18 mice) chrysin-treated group receiving chrysin suspension (0.1 %w/w) in 1% w/w Na CMC.
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Group II: (18 mice) optimal formula B-treated group.
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Group III: (6 mice) control group receiving normal saline equivalent to the volume of other formulations.
Animals of groups I and II were given chrysin oral doses equivalent to 10 mg/kg by an oral gavage [49]. Each group was divided into three subgroups (6 mice/subgroup). At 20, 60, and 90 min post oral administration, each subgroup received 0.3% acetic acid solution at a dose of 10 mg/kg by intraperitoneal injection (i.p.). Five minutes after acetic acid injection, the number of writhing was counted for 10 min. A writhing was indicated as the animal’s abdomen stretching with concurrent stretching of at least one of its hind limbs [50].
Preparation of Formula B Powder Using Adsorption Technique
One gram of formula B was transferred to glass mortar. Different adsorbents, namely, Aerosil 200, Aerosil R972, chitin, starch, Aerosil R972: chitin (1:1), Aerosil R972: starch (1:1), and chitin: starch (1:1) were added each alone gradually and mixed well with a pestle until getting free-flowing powder [51, 52]. The final powder was weighed and sieved through sieve number 40 to break any lumps. Then, the powder was stored over anhydrous calcium chloride in a desiccator until further evaluation [40]
Determination of Flowability
The flowability of the produced powders was determined by measuring the angle of repose (θ) [53]. Based on values of the angle of repose, Aerosil R972 was selected as the proper adsorbent and hence was subjected to further evaluation.
Evaluation of the Powdered SNEDDS
Compressibility Index and Hausner’s Ratio
Based on the predetermined bulk and tapped densities of the powdered SNEDDS, the compressibility index (Carr’s index), and Hausner’s ratio were calculated using the equations proposed by the USP [54].
Drug Content
The powdered SNEDDS (100 mg) was added to methanol: water (1:1 v/v) mixture, vortexed for 5 min, sonicated for 20 min for proper extraction of chrysin, and then filtrated. The filtrate was suitability diluted and chrysin content was determined spectrophotometrically at 267 nm against plain powdered SNEDDS as a blank.
FT-IR
The FT-IR spectra of chrysin, Aerosil R972, and the powdered SNEDDS were recorded as mentioned under “Fourier Transform-Infrared Spectroscopy (FT-IR).”
Differential Scanning Calorimetry (DSC)
DSC thermograms of chrysin, Aerosil R972, and the powdered SNEDDS were attained using a differential scanning calorimeter (LABSYS evo SETARAM, France) [55] over a temperature range of 50–400°C.
X-Ray Diffraction (XRD)
XRD diffractograms of chrysin, Aerosil R972, and the powdered SNEDDS were analyzed (Bruker D8 DISCOVER X-ray diffractometer, Germany) [56]. Samples were operated with Ni-filtered Cu Kα radiation (λ = 0.15416 nm), under a voltage of 45 kV and current of 40 mA. Over a diffraction angle (2θ) ranging from 5 to 100°C, the rate of scanning at 2 min−1 was established.
Statistical Analysis
Results were presented as mean ± standard deviation (SD) and mean ± standard error of the mean (SEM) for in vitro and in vivo data, respectively. Utilizing GraphPad Prism V 8.3 (GraphPad Software Inc., CA, USA), statistical comparisons were made by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Also, a Student t-test was applied whenever possible. Statistics were deemed significant if p < 0.05.
Results and Discussion
Solubility Study
The solubility of chrysin in the various oils, surfactants, and cosurfactants was screened (Supplementary Table I). Among the studied ingredients, it was found that the significantly highest solubility of chrysin was achieved by Kollisolv GTA (2.52 ± 0.74 mg/mL), Tween 80 (19.95 ± 2.99 mg/mL), and Transcutol HP (19.07 ± 1.47 mg/mL) which specified as oil, surfactant, and cosurfactant, respectively (p < 0.05). Hence, SNEDDS were prepared using the above ingredients.
Tween 80 is a hydrophilic non-ionic surfactant that is less impacted by pH and ionic strength variations than other surfactants [57]. Moreover, it is safe and can enhance drug permeability and absorption [58]. The high HLB of Tween 80 [15], which promotes faster lipophilic drug solubilization and spontaneous emulsification ability, can be also used to explain the highest solubility of chrysin in Tween 80 [59]. It has been reported that drug delivery to neurons could be improved by Tween 80 through receptor-mediated endocytosis incorporating the low-density lipoprotein receptors [60]. Transcutol HP (diethylene glycol monoethyl ether), an amphiphilic cosurfactant, could increase the solubility of chrysin which might improve its loading capacity [61, 62].
Pseudo-ternary Phase Diagrams
For investigation of the emulsification ability, various ratios of S/CoS (1:1, 1:2, 2:1, 3:1, 1:3, 5:1, and 1:5 w/w, respectively) were studied. Then, the areas of the nanoemulsion (NE) regions were compared where the larger the area the greater the self-nanoemulsification efficiency [63]. It was observed that as the S/CoS ratio decreased the NE area of the phase diagram increased (Supplementary Figure 1). Intuitively, the ratio of S/CoS of 1:3 and 1:5 exhibited the largest NE area. This finding could be attributed to the fact that Transcutol HP decreases the surface tension between the two phases in the nanoemulsion to stabilize NE droplets [64]. Thus, the interfacial layer would be flexible with increasing stability of the system. Although both ratios 1:3 and 1:5 have the same NE areas, ratio 1:3 was selected for the preparation of chrysin-loaded SNEDDS, because it contains a lower amount of Transcutol HP [62]. Figure 2 manifests the pseudo-ternary phase diagram at an S/CoS ratio of 1:3, respectively. The chrysin-loaded SNEDDS were prepared at the selected S/CoS ratio of 1:3 using three oil ratios: 10, 25, and 40% w/w.
Pseudo-ternary phase diagram of Kollisolv GTA, water, and Tween 80: Transcutol HP at an S/CoS ratio of 1:3, respectively
Characterization of Chrysin-Loaded SNEDDS
Table II summarizes the percent transmittance, self-emulsification time, viscosity, particle size, PDI, zeta potential, and cloud point of chrysin-loaded SNEDDS.
Percent Transmittance (%T)
The %T values of chrysin-loaded SNEDDS were found to be not less than 99.25 ±0.12% which proved the clarity of the formulations with no precipitation or separation.
Robustness to Dilutions
Upon dilution of the prepared formulations by 1:100 and 1:1000 using distilled water, SGF, and PB 7.4, clear nanoemulsions were produced without turbidity. Moreover, no separation or precipitation was observed even after 24-h storage. Such a result indicated the robustness of the prepared SNEDDS to dilutions with different media and potential stability and suitability for oral administration.
Self-Emulsification Time
After oral administration, SNEDDS should disperse completely and rapidly upon dilution in the gastrointestinal tract (GIT) to form in situ clear nanoemulsion [33]. As shown in Table II, the dilution of SNEDDS by 1:500 with distilled water, and SGF resulted in the formation of clear nanoemulsions immediately, except for formula C in SGF which required about 62.00 ± 2.83 sec for the disappearance of oily spots completely and formation of a clear nanoemulsion. This could be related to the high percentage of the oil phase in formula C that increased the interfacial tension between oil and aqueous phase causing longer time for dispersion. Accordingly, formulations A and B with a lower amount of oil percentage were preferred over formula C [65, 66].
Viscosity
Table II illustrates the viscosity of the prepared SNEDDS with values ranging from 22.33 ± 1.53 to 18.00 ±0.00 mPa.s. It was found that the viscosity decreases gradually when the oil proportion in the formulation increases, in other words when the S/COS percent decreases. At the same time, the viscosity values recorded by the SNEDDS formulations were low enough to produce an acceptable self-emulsification time [36, 67]. Viscosity can affect the stability of SNEDDS. The higher the viscosity of a preparation, the more stable it is, because there is less tendency for the droplets to collide with each other [68].
Droplet Size, Polydispersity Index (PDI), and Zeta Potential
The droplet size, PDI, and zeta potential of the prepared SNEDDS are displayed in Table II. It could be observed that the droplet size of A, B, and C did not exceed 155.4 nm (149.5 ± 5.43, 155.4 ± 32.02, and 151.3 ± 10.17 nm, respectively). Also, the prepared systems exhibited PDI of 0.34 ± 0.07, 0.38 ± 0.09, and 0.40 ± 0.07 of A, B, and C, respectively. It is widely recognized that the small droplet size of SNEDDS increases the drug’s surface area which improves the bioavailability of drugs [69]. The narrow size distribution of chrysin-loaded nanodroplets and the consistency of the formulations were evidenced by PDI values less than 0.5, which is consistent with numerous investigations [25, 33, 37, 70]. As surface charge is crucial to colloidal stability, the zeta potential of each formulation was also assessed. Table II illustrates that the tested formulations had negative charges ranging from − 9.87 ± 5.34 to − 12.67 ± 2.68 mV. Other researchers have detected similar results [63, 71,72,73]. This might be attributed to the free fatty acids and glycols in the Tween 80, Transcutol HP, and Kollisolv GTA as well as the ionization of the chrysin hydroxyl groups [33, 69, 74]. Besides, nonionic surfactants produce a negatively charged interface at neutral pH due to the differential adsorption of the hydroxyl ion (OH‾) and hydrated oxonium ion (H3O+) from the surrounding aqueous environment [33]. Hence, a stable dispersion of nanoemulsion droplets could be maintained, and coalescence reduced owing to the electrostatic repulsion between negatively charged particles [75]. Also, the nonionic surfactants can stabilize the system sterically by coating the surface of droplets, in addition to electrostatic stabilization [76].
Thermodynamic Stability
Through in situ solubilization, SNEDDS develops nanoemulsions that are stable against phase separation, cracking, creaming, and precipitation [33]. The resilience of SNEDDS formulations against the previously mentioned instabilities during temperature fluctuations and centrifugal stress is determined by the thermodynamic examination. During several thermodynamic phases (centrifugation, heat-cool cycles, and freeze-thaw cycles), the prepared chrysin-loaded SNEDDS showed no evidence of precipitation or phase separation, indicating the thermodynamic stability of the systems [36].
Cloud Points
Since the cloud point is a sign that a stable nanoemulsion has been formed, it is a crucial parameter of SEDDS. An irreversible phase separation of the system would happen if the surrounding temperature increased above the cloud point. Consequently, the components would be dehydrated which in turn negatively impacts drug absorption [69]. Hence, the cloud points of SNEDDS ought to be above the body temperature to secure the in vivo formation of stable nanoemulsion upon oral administration. Table II documents the cloud points of the prepared SNEDDS. It was observed that the turbidity of all formulations appeared at temperatures not less than 86.33 ± 0.58°C indicating the suitability of the prepared SNEDDS for oral administration.
From the above experiments, the data proved that SNEDDS A and B are better than C, and the surfactant concentration must be as low as possible because it may cause toxicity and irritation of GIT after oral SNEDDS administration [39]. Hence, the SNEDDS B was recommended as the optimal formula for further investigations because of its lower percentage of the S/CoS mixture (Table I).
Evaluation of the Optimal Formula (B)
Transmission Electron Microscopy (TEM)
The morphology of the SNEDDS droplets following dilution was identified using TEM. Figure 3 shows the morphology of the formed nanoemulsion droplets. Spherical nanoscaled droplets were observed with a dark center surrounded by a bright S/CoS frame (core-shell structure). It could be concluded that such a frame might prevent the coalescence of the droplets and enhance their stability [39]. It should be kept in mind that the droplet diameter that appeared under TEM represents the size of droplets in the dry state. Instead, the hydrodynamic diameter of the droplets with the surrounding solvated layer (surface-hydrated droplets) dispersed in a properly diluted sample was measured using the DLS technique. Also, the droplet mobility in a dispersant (water) should be taken into consideration in the case of the DLS-based analysis [77, 78]. The TEM-visualization of a lower droplet size than that seen by Zetasizer may be explained by the aforementioned facts. Similar results have been documented in scholarly works [74, 79].
TEM photomicrograph of the optimized formula B
Fourier Transform-Infrared Spectroscopy (FT-IR)
FT-IR was performed to evaluate the molecular states of chrysin, Tween 80 (surfactant), Transcutol HP (co-surfactant), Kollisolv GTA (oil), and the optimized SNEDDS (formula B). Figure 4 displays the FT-IR spectra of the above ingredients. The chrysin spectrum (a) showed peaks at 2706 and 2633 cm−1 which were attributed to the C–H stretching band of CH, as well as a peak at 1651 cm−1 that originated from carbonyl group stretching. The peaks at 1609, 1575, and 1449 cm−1 are assigned to C=C stretching. In addition, a peak at 1357 cm−1 was attributed to the C–O stretching [80, 81]. The spectrum of Kollisolv GTA (b) showed two distinct absorption peaks at 1742 and 1219 cm−1 [82]. The spectrum of Transcutol HP (c) exhibited peaks at 3433 and 2869 cm−1 of OH group and C-H stretching, respectively. In addition, the bands at 1110 and 1067 cm−1 are due to the C-O stretching [83]. For Tween 80 (d), peaks at 3472 and 1735 cm−1 originated from the hydroxyl group and C=O ester group, respectively. Peaks at 2922 and 2862 cm−1 are attributed to the (-CH2) band. Also, the band at 1104 cm−1 is due to asymmetric C–O stretching vibrations [84, 85]. The spectrum of formula B (e) illustrates the sum of the components' peaks without extra ones that indicate the absence of the chemical interaction between the components, but the peaks slightly shift with diminished intensities due to physical interaction and dilution effect [86].
FT-IR spectra of chrysin (a), Kollisolv GTA (b), Transutol HP (c), Tween 80 (d), and SNEDDS formula B (e)
In Vitro Release Study
One of the most crucial physicochemical traits of colloidal drug delivery systems is the in vitro release profile, which is essential for anticipating in vivo performance and relating it to the system microstructure. Figure 5a–c manifests the in vitro release profiles of chrysin from its suspension and formula B at pH 1.2, 6.8, and 7.4 to represent the gastric, intestinal, and systemic circulation environments, respectively. Regardless of pH, chrysin suspension did not release more than 10%; 7.72 ± 2.84, 2.94 ± 1.92, and 9.26 ± 1.67% in SGF, SIF, and the physiological pH, respectively. Additionally, chrysin suspension showed a lag time of about 8 h at pH 6.8 (Fig. 5b). However, the percentage of chrysin released from SNEDDS built up to a comparable pattern without any lag time during the first ten hours, after which distinct release patterns were observed. The percentage released of chrysin from formula B reached 41.74 ± 5.29, 29.72 ± 4.65, and 56.16 ± 10.28 % in SGF, SIF, and the physiological pH (PB 7.4), respectively, by the end of the experiment. As illustrated in Fig. 5, the release of chrysin from SNEDDS B was pH-dependent, so a minimum release could be perceived at pH 6.8. As the pH of the medium went further from 6.8 to 1.2, or 7.4, the percentage released increased (Fig. 5a, c, respectively). It could be attributed to the weakly acidic nature of chrysin (pKa = 6.72) [87]. In SGF, with a much lower pH than pKa, more chrysin molecules would be protonated (positively ionized), solubility increased, and eventually, the release improved. By increasing pH to 6.8, the protonation of chrysin molecules would be dropped to about 50%, causing the least solubility and release. On the contrary, neutral chrysin molecules might be deprotonated (negatively ionized) at PB 7.4 with a rise in solubility and an enrichment of the rate and extent of the released chrysin. It could be concluded that the chrysin release from SNEDDS formula B was markedly higher than the corresponding one from the suspension in different media. Qu and his colleagues reported similar findings [88]. The above results proved the superiority of formula B as a nanocarrier of chrysin with a high surface area-to-volume ratio, which in turn secured higher solubility of chrysin associated with faster rates and enhanced extent of release. Such a pH-dependent release behavior of chrysin has been reported in the literature [74, 89, 90].
In vitro release profiles of chrysin suspension and SNEDDS formula B at pH 1.2 (a), 6.8 (b), and 7.4 (c).
Kinetic Analysis of Release Data
The in vitro release data was analyzed using a variety of mathematical methods. The kinetic theory for the spherical geometry model eventually resulted in selecting a particular model based on the highest r2. The release kinetic analysis is displayed in Table III. It was found that the release of chrysin from optimum formula B was well described by first-rate kinetics at pH 1.2 and 7.4. Similar results have been reported in the literature [88]. On the other hand, the optimal formula B best fitted the Higuchi model at pH 6.8, which indicated that drug release from formula B was diffusion-controlled in SIF. Naseri and co-workers documented comparable findings [79]. Besides, the kinetic study showed that the release of chrysin suspension happened in accordance with Higuchi diffusion irrespective of the medium’s pH. The Korsmeyer-Peppas equation was used for further analysis of the release data, which revealed that n values ranged from 0.4776 to 0.6358. Thus, chrysin release occurred via a non-Fickian diffusion (Anomalous transport) that is a combination of chrysin diffusion and dissolution [91]. Such results are consistent with the obtained in vitro release profiles and have proven that the pH of the surrounding environment influenced not only the rate and extent of release but also its kinetics.
Biocompatibility of the Optimized Formula B with Normal Oral Epithelial Cell Line
Figure 6a documents the biocompatibility of chrysin, the optimized formula B, and its corresponding plain one expressed as percent cell viability of the normal oral epithelial cell line (OEC). It was reported that chrysin was safe for normal cells at specified concentrations while it could experience certain intrinsic cell toxicity [92, 93]. In this study, the highest safety of chrysin (Fig. 6a) alone over the studied concentration range of 1.95–31.25 µg/mL was detected with percentage cell viability not lower than 75.53 ± 3.88%. However, OEC exhibited a marked sensitivity towards the plain formula upon 48 h incubation, encompassing Kollisolv GTA, Tween 80, and Transcutol HP (IC50 = 2.63 ± 0.08 µg/mL). Such a result might be attributed to the high internalization of the plain formula into the cells. Moreover, Tween 80 can inhibit the P-glycoprotein pump and hence allows the flowing of the plain formula intracellularly. By incorporation of chrysin in Formula B, the biocompatibility towards OEC was duplicated with an IC50 value of 5.69 ± 0.03 µg/mL. This means that chrysin might increase the safety of SNEDDS formula B on normal oral epithelial cells. The forthcoming brain targeting experiment would open new vitals for SNEDDS as an appropriate choice for the administration of chrysin.
Biocompatibility of chrysin, SNEDDS formula B, and its corresponding plain one towards normal oral epithelial cell line (OEC) for 48 h incubation (a) and number of writhing of chrysin suspension-treated group (Group I), formula B-treated group (Group II), and control group (Group III) (b)
In Vivo Evaluation of the Optimal Formula B
Brain Targeting
This experiment was conducted to ensure that the chrysin-loaded SNEDDS formula B can escape opsonization and pass the blood-brain barrier (BBB). Since fluorescein can penetrate the blood-brain barrier, it was used to monitor brain targeting [94, 95]. In this experiment, the linearity of the calibration curve constructed between NaFl concentration versus fluorescence intensity was expressed using the following equation:
where Y is the measured fluorescence intensity and X is the concentration of NaFl (ng/mL).
In group I (NaFl-treated group), the amount delivered to the brain was 2.337 ± 0.26 ng/mg. However, the amount of NaFl delivered was 2.345 ± 1.085 ng/mg in group II (chrysin/NaFl-SNEDDS-treated group). Surprisingly, both groups are statistically insignificant (P > 0.05). Such a result indicates that SNEDDS could deliver chrysin to the brain without any negative interference and might help in combating pain. By intravenous injection of SNEDDS, the oil droplets encapsulating the drug would be self-emulsified so that the drug could be protected from enzymatic and chemical destruction and distributed throughout the body until reaching the biophase of BBB [96].
Pharmacodynamic Study
Acetic acid-induced writhing was applied as a pain model in mice to evaluate the antinociceptive activity and analgesic effect of chrysin suspension and SNEDDS formula B. Table IV and Fig. 6b demonstrate the number of writhing produced after acetic acid i.p. injection in the chrysin suspension-treated group, formula B-treated group, and control group. It was observed that the number of writhing of the control group (24.5 ± 1.93) was significantly (P < 0.0001) reduced by the oral administration of chrysin, whether as chrysin-loaded SNEDDS, formula B (group II), or as chrysin suspension (group I). Successfully, group II significantly (P < 0.001) ameliorated the pain more efficiently than drug suspension group I (Table IV). Compared to chrysin suspension, the writhing number was reduced by a tenth after 20 min of the oral administration of formula B. Auspiciously, the antinociceptive effect reached its maximum one hour after the oral administration of chrysin-loaded SNEDDS formula B. Contrarywise, the antinociceptive effect of chrysin suspension was practically constant without a significant change throughout the studied intervals (P > 0.05). Conclusively, these results disclosed the efficacy of SNEDDS as a nanocarrier to enhance its antinociceptive and analgesic activities of chrysin.
It is worth mentioning that the liberation of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 8 (IL-8) by resident peritoneal macrophages and mast cells is the mechanism of acute inflammation that is induced by i.p. injection of acetic acid [97]. Chrysin can inhibit the major pro-inflammatory cytokines (TNF-α, IL-1β, and IL-8) by suppressing macrophage phenotype M1 involved in the cascade of acute inflammation [98]. Simultaneously, chrysin can induce macrophage phenotype M2 phenotype, producing the anti-inflammatory cytokines IL-10 and transforming growth factor β (TGF-β) in peritoneal macrophages of mice [99]. These findings demonstrated that chrysin acts as a prophylactic anti-inflammatory agent to lessen the severity and duration of inflammatory cell infiltration [100]. Further, it has been stated that chrysin has an anti-oxidative activity by lowering the activity of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels, which may have a protective impact on injuries [101].
Many studies have shown that SNEDDS can enhance oral absorption by forming large-surfaced droplets of in situ clear nanoemulsion in the GIT, maintaining drug solubilization, facilitating the enzymatic digestion of the lipidic formulation to create amphiphilic molecules, and enhancing lymphatic transport [62, 102]. Hence, SNEDDS can avoid the pre-systemic first-pass metabolism [103]. In addition, the SNEDDS-forming lipid Kollisolv GTA, surfactant Tween 80 [104, 105], and cosurfactant Transcutol HP [62] have P-glycoprotein (P-gp) inhibitory effects, which considerably reduce the P-gp efflux mechanism and enhance intestinal permeation after oral administration [106, 107]. Thankfully, the usage of these functional excipients was advantageous since they might offer an efficient P-gp modulation without the added expense or security risk of using an additional modulator.
Considering the aforementioned concepts, loading chrysin into SNEDDS proved beneficial since it provided an effective pairing between the antinociceptive activity of chrysin and a functional nanocarrier to enhance the drug’s pharmacological efficacy and modulate its issues.
Evaluation of the Powdered SNEDDS
Determination of Flowability
The angle of repose of different adsorbents ranged from 35.70 ± 0.34 to 58.10 ± 0.24° (See Supplementary Table 2). Aerosil R972 was chosen as an appropriate solid carrier with an angle of repose of 35.70 ± 0.34°. Hence, Aerosil R972-powdered SNEDDS could be classified as a good flowable powder [54]. Such a result could be attributed to the hydrophobic nature of Aerosil R972 (silicon dioxide treated with dimethyl chlorosilane) permitting efficient adsorption of the oily component of SNEDDS in comparison to the other adsorbent. Both hydrophobicity and surface area of the solid carrier have been reported as key elements in the development of effective solid SNEDDS [108]. In addition, the oil phase has an intrinsic granulating effect [109]. Conclusively, the powdered SNEDDS was further prepared using Aerosil R972.
Compressibility Index and Hausner’s Ratio
Based on the values of the tapped and bulk densities, which were 0.152 ± 0.003 and 0.119 ± 0.004 g/mL, respectively, the powdered SNEDDS showed a Hausner’s ratio of 1.27 ± 0.062 and a Carr’s index of 21.46 ± 3.8%. While the bulk density is inclusive of the interparticle spaces and pores, the tapped density, which provides the true density of the solid material, is exclusive of these voids and gaps. The small bulk density of the powdered SNEDDS suggested that there were substantial spaces or voids between the closely spaced particles. Nonetheless, Hausner’s ratio and Carr’s index are used to express the lower difference between the bulk and tapped densities, which was suggestive of the particle flow [109, 110]. According to USP guidelines, the powdered SNEDDS could be categorized as having passable flow characters [54].
Drug Content
Chrysin content in the powdered SNEDDS was 95.78 ± 2.92% which was found to be within the USP compendial limits [67]. The reduction in drug content after addition to the powder might be due to processing and handling.
FT-IR
Figure 7a shows FT-IR spectra of chrysin, Aerosil R972, and powdered SNEDDS. The spectrum of chrysin (i) was discussed under “Fourier Transform-Infrared Spectroscopy (FT-IR).” The spectrum of Aerosil R972 (ii) exhibited a peak at 1076 cm−1 corresponding to the presence of Si–O–Si (siloxane groups), and a stretching band appeared at 807 cm−1 [111, 112]. The spectrum of powdered SNEDDS formula B (iii) showed the prominent bands of Aerosil R972 while chrysin bands appeared with diminished intensity. Furthermore, no major shifts or new bands were observed. Such a result stated the physical adsorption of chrysin-loaded SNEDDS formula B by Aerosil R972 (dimethyl chlorosilane-treated SiO2). Interestingly, the chemical interaction between Aerosil R972 and adsorbate molecules has been reported to be retarded by the steric hindrance effect of the silanol groups coupled to dichloro-dimethyl silane moieties [113]. Therefore, the FTIR data confirmed that all of the components were compatible with chrysin. Other researchers observed similar findings [109, 114].
FT-IR spectra (a), DSC thermograms (b), and XRD diffractograms (c) of chrysin (i), Aerosil R972 (ii), and powdered SNEDDS formula B (iii)
Differential Scanning Calorimetry (DSC)
Figure 7b illustrates DSC thermograms of chrysin, Aerosil R972, and the powdered SNEDDS. It could be observed that chrysin has a sharp endothermic peak at 289.9°C (i), which corresponds to its melting point, indicating the crystalline nature of chrysin [27]. All over the tested temperature range, no peaks could be detected in the thermogram of Aerosil R972 (ii), which could be attributed to its amorphousness [114]. Regarding the powdered SNEDDS thermogram (iii), the sharp chrysin peak disappeared, signifying the complete solubilization of chrysin in SNEDDS and successive incorporation of the molecularly dispersed form in the powdered SNEDDS [115]. Our findings concurred with those of other research studies [116,117,118,119].
X-Ray Diffraction (XRD)
Figure 7c demonstrates XRD diffractograms of chrysin, Aerosil R972, and the powdered SNEDDS. Characteristic sharp peaks of chrysin appeared at 7.4, 14.9, 17.8, and 27.7° of 2θ confirming the crystalline nature of chrysin (i) [23]. The amorphous nature of Aerosil R972 with no peak could be proved in its XRD diffractogram (ii). The absence of the characteristic peaks of chrysin in the powdered chrysin-loaded SNEDDS (iii) proved the complete solubilization of chrysin in SNEDDS and efficient incorporation in the powder, which agreed with DSC results [30]. Analogous results were noted by other scholars [109, 114, 118].
Conclusion
Natural flavonoids like chrysin have a variety of biological actions. The analgesic efficacy of chrysin has only been evaluated in a handful of studies. Chrysin’s lipophilicity causes low aqueous solubility, which leads to low bioactivity. Self-nanoemulsifying drug delivery systems (SNEDDS) were therefore designed to address this issue. The properties of the developed systems were intensively investigated. Our results revealed the therapeutic efficacy of chrysin self-nanoemulsion as a possible platform for pain management. It might be speculated that this nanoemulsion could be ratified therapeutically.
Data Availability
The datasets generated and/or analyzed during the current study are available upon a reasonable request.
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The authors would like to sincerely thank Gattefosse, for their kind donation of different lipids and oils.
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Samar Mohamed Elhoseny: methodology, analysis, investigation, software, data visualization, figures, and writing—original draft. Noha Mohamed Saleh: conceptualization, supervision, and writing—review and editing. Mahasen Mohamed Meshali: conceptualization, supervision, and writing—review and editing.
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Elhoseny, S.M., Saleh, N.M. & Meshali, M.M. Self-Nanoemulsion Intrigues the Gold Phytopharmaceutical Chrysin: In Vitro Assessment and Intrinsic Analgesic Effect. AAPS PharmSciTech 25, 54 (2024). https://doi.org/10.1208/s12249-024-02767-0
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DOI: https://doi.org/10.1208/s12249-024-02767-0