Curcumin was procured as a gift sample from Arjuna Naturals, India. Virgin coconut oil was purchased from Tamil Nadu Test House Pvt. Ltd, Tamil Nadu. Capmul MCM and Captex 300 EP/NF were generous gift samples received from Abitec Corporation, India. Donepezil hydrochloride was purchased from Alkem Laboratories Ltd., India. Kolliphor ELP and RH40 were gifted by BASF India. Ellman’s reagent (DTNB) was purchased from Loba Chemie Pvt. Ltd, Mumbai, India. Tween 80, Tween 20, polyethylene glycol 400 (PEG 400) and propylene glycol were purchased from Loba Chemie, India. Acetonitrile, methanol, ethanol and glacial acetic acid were purchased from Merck, India. All other chemicals were analytical grade.
Determination of saturation solubility
Saturation solubility of curcumin was determined by adding curcumin in surplus amount to the selected oils [Virgin coconut oil (VCO), Capmul MCM (CPM), Captex 300 EP/NF (CPT)], surfactants, and co-surfactants. Samples were taken in Eppendorf tubes. This mixture was vortexed and sonicated for 15 min, respectively. The samples were kept in a water shaker bath (Classic Scientific, India) at 37 °C for 48 h for attaining equilibrium and facilitating the solubilization of curcumin. The samples were centrifuged at 15,000 rpm for 15 min to remove the undissolved curcumin. The supernatant was taken and subsequent dilutions were done. The curcumin content in samples was quantified using UV spectrophotometer (Jasco V-630, Easton USA) at 421 nm as detection wavelength.
Determination of emulsification potential of surfactant
Emulsification study was performed for the selection of suitable surfactant and co-surfactant. The mixtures of oil and each surfactant in the ratio of (1:1) was taken in Eppendorf tubes, and samples were vortexed for 15 min followed by sonication for 10 min. The samples were further allowed to stabilize overnight and observed on next day for phase separation. The samples were further diluted 1000 times in distilled water and percentage transmittance was recorded using UV visible spectrophotometer (Jasco V-630, Easton USA) at 650 nm against distilled water as reference.
Construction of pseudo-ternary phase diagram
Pseudo-ternary phase diagrams were plotted to determine the microemulsion region corresponding to the specific composition of the mixture. Tween 80 was used as a surfactant and PEG 400 was used as a co-surfactant. The mixture of VCO and CPM in the ratio of 1:1 w/w was used as an oil phase. The mixture of surfactant and co-surfactant in three different ratios (1:1, 2:1, 3:1 w/w) was used as Smix. The oil phase and Smix phase was blended in a different ratio ranging from (1:9 to 9:1 v/v). Distilled water was added as aliquots. Change in appearance from clear to turbid was considered as the endpoint. The readings were recorded, and pseudo-ternary phase diagrams were generated using TriDraw software (version 2.6).
Effect of oil and Smix concentration on globule size
The effect of oil and Smix concentration in microemulsion was assessed by changing one factor at a time and keeping other factor constant. The effect of Smix concentration was assessed by keeping oil concentration constant at 10% and varying the Smix concentration from 60 to 70%. Similarly, the effect of oil concentration was determined by changing oil concentration from 5 to 25% with Smix concentration constant at 70%. Globule size measurement was determined by using a nanoparticle analyzer (Horiba SZ-100 V2 series).
Preparation of curcumin-loaded microemulsion
The Cur-ME was prepared by simple mixing of the components. Briefly, oil (20%) was mixed with Smix (60%) using a vortex mixer. Curcumin was added and dissolved at a concentration of 10 mg/mL considering the final volume of the formulation. Water (20%) was finally added to the previous mixture and mixed for 15 min followed by sonication for 15 min. The final formulation was kept overnight for stabilization.
In vitro characterization
The microemulsion was observed for appearance like color, drug precipitation and phase separation.
Determination of globule size, polydispersity index, and zeta potential
Globule size, polydispersity index (PDI), and zeta potential of microemulsion were determined by the dynamic light scattering method. The microemulsion was diluted suitably with distilled water, and abovementioned parameters were determined by using particle size analyzer Horiba nanopartica Scientific (SZ-100 V2 series), India. The study was performed in triplicates (n = 3).
Determination of pH, dilution potential, and centrifugation
The pH of the microemulsion formulation was assessed by using a digital pH meter (Toshniwal Process Instruments Pvt. Ltd, India). The microemulsion was diluted 100 times with distilled water, vortexed for 5 min and kept for 48 h to determine the dilution potential. After 48 h, the samples were observed for the precipitation of drug and phase separation. The microemulsion was centrifuged at 6000 rpm for 15 min for centrifugation study and observed for phase separation or drug precipitation. All the measurements were done in triplicate (n = 3).
Drug content by HPLC
The drug content of microemulsion was determined by using the reported RP-HPLC method . The final formulation was suitably diluted with methanol. The samples were further centrifuged at 10,000 rpm for 15 min, and the supernatant was injected into HPLC (Jasco UV-1575, PU1580). The samples were quantified at 421 nm as detection wavelength.
In vitro drug release
In vitro release of curcumin from formulations was determined by using the dialysis bag method . The dialysis bags were activated in phosphate buffer pH 7.4 before 24 h. Curcumin solution (Cur-Sol) and Cur-ME (equivalent to 5 mg curcumin) were transferred to respective dialysis bags in which the other end was tied tightly using a thread. These bags were transferred to dissolution vessels containing 200 mL media maintained at 37 °C at 50 rpm. In vitro drug release study was performed using dissolution apparatus (Electrolab USP II) and was quantified using UV Visible spectrophotometer (Jasco V-630, Easton USA).
In vivo Plasma Pharmacokinetics in Wistar rats
The protocol was approved by Institutional Animal Ethical Committee (IAEC) of Poona college of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Pune, MH, India (IAEC/PCP/PCL29/2020–2021) and in vivo plasma pharmacokinetic study was performed. The animals were fasted overnight before the experiment. The animals were divided into two groups (n = 18); Cur-Sol and Cur-ME. The Cur-Sol and Cur-ME were administered orally at a dose of 150 mg/kg to the respective groups. The blood samples were collected from the animals at 0.083, 0.25, 0.5, 1, 2, 3, 6, 9, and 12 h, respectively. The blood samples were centrifuged at 3500 rpm for separating the plasma. Plasma was stored at −80 °C until analysis. Plasma was mixed with methanol for precipitating the proteins. Samples were further centrifuged at 10,000 rpm for 15 min, and the supernatant was collected and injected into RP-HPLC (Jasco UV-1575, PU1580). The pharmacokinetic parameters were assessed by using PK Solver, MS-Excel add-in Program .
In vivo brain pharmacokinetics in adult zebrafish
The in vivo brain pharmacokinetic study was performed as per the reported method . The protocol for the study was approved (IAEC/PCP/PCL28/2020–2021) by Institutional Animal Ethical Committee (IAEC) of Poona college of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Pune, MH, India, with CPCSEA (1703/PO/Re/S/01). Adult zebra fish (Danio rerio) (2.5 ± 0.5 cm length and 250 ± 50 mg weight) were fasted overnight before the experiment. The zebrafish were divided into two groups as Cur-Sol and Cur-ME with 25 fish each. The fishes were partially anesthetized using cold anesthesia during dose administration. The Cur-ME and Cur-Sol were orally administered at a dose of 25 mg/kg using a micropipette by holding them in upright position. The fishes were sacrificed using cold anesthesia at each time points (5, 15, 30, 60, and 120 min). The skull roof was opened by surgical procedure and the brain was removed and immediately transferred into the Eppendorf tube containing methanol. The extraction of drug from the brain was carried out by the homogenization process. Further, the samples were centrifuged at 10,000 rpm for 15 min. The supernatant was collected and injected into RP-HPLC (Jasco UV-1575, PU1580). The pharmacokinetic parameters were assessed by using PK Solver, MS-Excel add-in Program.
In vivo pharmacodynamics in TMT induced neurodegeneration in rats
The protocol was approved by Institutional Animal Ethical Committee (IAEC) of Poona college of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Pune, MH, India (IAEC/PCP/PCL29/2020–2021). Male wistar rats weighing 200–230 g were used for the study. The animals were divided into five groups (n = 8) as follows: group 1—control, group 2—neurodegeneration control, group 3—standard (donepezil 1 mg/kg), group 4—Cur-Sol and group 5—Cur-ME. Morris water maze is a maze of 150 cm diameter filled with water at 25 ºC till 40 cm. A hidden platform was placed 2 cm below the water level. The animals were trained in the Morris Water Maze for seven days to reach the platform from four different cues. The time taken to reach the platform (escape latency) and distance traveled (path length) were measured. On day 8, all group of animals except control group were injected with of trimethyltin chloride (TMT) at the dose of 7 mg/kg, intraperitoneally (i.p.) for induction of neurodegeneration . Respective treatment was administered from 15th day to 28th day. The Morris water maze test was performed on 0th, 7th, 14th, 21st, and 28th days of the study to evaluate spatial memory. The animals were sacrificed on 28th day and brains isolated for evaluating biochemical parameters and histopathology.
The brain homogenization was done as per the reported method  in Tris–HCl buffer (pH = 7.4). The brain glutathione (GSH)  and malondialdehyde (MDA)  were determined as per the reported studies.
The sample of the brain was isolated and preserved in a collection tube containing neutral buffered formalin. The sample was stained with Hematoxylin and Eosin (H&E) for histopathological study. The changes occurred in the rat brain was compared with the vehicle control group at 10 × magnification.
The HPLC method used in this study was according to earlier reported methods . The Pearless C18 Chromatopak, India, column was used. The mobile phase composition was 2% acetic acid pH 3 and acetonitrile at a ratio of (40:60). The pump was operated at 1.0 mL/min flow rate and the elution was monitored at 425 nm wavelength using a UV detector (Jasco UV 1575). The retention time of curcumin was observed at 9 min. The raw data were assessed by Bowin HPLC software (Jasco Easton. USA).
All the experimental data was expressed as mean ± SD. The statistical analyses were carried out with help of GraphPad Prism demo v8 using one-way ANOVA followed by Dunnet’s ‘t’ test and two-way ANOVA followed by Bonferroni’s multiple comparison test.