Formulation and Development of Curcumin–Piperine-Loaded S-SNEDDS for the Treatment of Alzheimer’s Disease

Curcumin (CUR) and piperine (PIP) are very well-known phytochemicals that claimed to have many health benefits and have been widely used in foods and traditional medicines. This study investigated the therapeutic efficacy of these compounds to treat Alzheimer’s disease (AD). However, poor oral bioavailability and permeability of curcumin are a major challenge for formulation scientists. In this research study, the researcher tried to enhance the bioavailability and permeability of curcumin by a nanotechnological approach. In this research study, we developed a CUR–PIP-loaded SNEDDS in various oils. Optimised formulation NF3 was subjected to evaluate its therapeutic effectiveness on AD animal model in comparison with untreated AD model and treated group (by market formulation donepezil). On the basis of characterisation results, it is confirmed that NF3 formulation is the best formulation. The optimised formulation shows a significant dose-dependent manner therapeutic effect on AD-induced model. Novel formulation CUR–PIP solid-SNEDDS was successfully developed and optimised. It is expected that the developed S-SNEDDS can be a potential, safe and effective carrier for the oral delivery of curcumin to the brain. To date, this article is the only study of CUR–PIP-loaded S-SNEDDS for the treatment of AD.


Introduction
The treatment of Alzheimer's disease (AD) is one of the most unsuccessful examples of medical science [1]. Currently, around the world, 55 million people are suffering from dementia, making it the seventh leading cause of death among all diseases [2,3]. Neurological disease contributes to 6.3% of the global burden of disease, and due to population increase, this number is estimated to increase in upcoming years [4]. It is reported that death cases due to AD have increased by more than 145% whereas death cases due to HIV, stroke, and heart diseases have decreased [5].
Two major changes are associated with AD: one is inside the neuron and other is outside the neuron. Inside the neuron, an abnormal form of a protein gets accumulated called tau tangles, which blocks the transportation of nutrients and other essential molecules necessary for neuron survival and proper function. Outside the neuron, accumulation of protein fragments takes place called β-amyloid (plaques), which causes the death of nerve cells or damages them. The death of neurons is called neurodegeneration which interrupts the nerve cells' communication at synapses [6].
The blood-brain barrier (BBB) reduced the intracellular gap and prevents the brain to take most of the pharmaceuticals from circulation [7,8]. However, nanoparticles with size less than 200 nm enter through endocytosis [9] and lipophilic molecules with a positive charge (+ zeta potential) [10] increase the chances to easily pass through the BBB [9]. Thus, for the treatment of neurodegenerative diseases, there is a need to develop a nanoformulation.
Turmeric is the rhizome of Curcuma longa, the primary chemical constituents of turmeric are curcuminoids, and the most important and abundant curcuminoid is curcumin [11]. Due to its safety and non-toxicity, curcumin has been approved by the FDA (Food and Drug Administration) as a natural food additive [12]. Scientists' interest increased in curcumin due to its unique molecular structure. It has multitargeted action including its anti-inflammatory, antioxidant and neuroprotective action [13], disaggregate tau filaments [14] and target amyloid aggregation (plaques) which is one of the major reasons for AD [15]. Various studies indicate that among those aged between 70 and 79 years, there is 4.4 times lower incidence of Alzheimer's disease in India than in the USA. Researchers investigated that those people who consume curry performed better in cognitive function tests than those who rarely or never consume curry [16].
However, this is a challenge for formulation scientists as curcumin has very low oral bioavailability and is not easily absorbed into the body and tends to be broken down quickly which can be resolved by new technological approaches [15,24]. Piperine is an alkaloid present in black pepper (Piper nigrum) and long pepper (Piper longum) [11]. It has been reported that piperine enhances the bioavailability of various drugs [25] and was observed that curcumin co-administration with piperine increases the bioavailability of curcumin by 154% in rats and 2000% in humans [11,26,27]. The combined CUR-PIP SNEDDS improved curcumin solubility, stability as well as bioavailability [28].
Currently, to increase the solubility and oral absorption of lipophilic drugs, lipid-based techniques to formulate SNEDDS are utilised [24]. SNEDDS is a composite blend of oil, surfactant, and co-surfactant. SNEDDS maintains the drug in a dissolved state, inside small globules of oil, with a hydrophilic head group [23]. When these SNEDDS are introduced into an aqueous phase on mild agitation, they form ultrafine droplets of oil-in-water (o/w) nanoemulsions [29,30]. L-SNEDDS (liquid) may be filled in soft gelatin capsules while S-SNEDDS (solid) may be filled in hard gelatin capsules. After oral administration, they reach the gastrointestinal tract (GIT). Gastrointestinal motility of the stomach provides the shaking to spontaneously form nanoemulsion with GI fluid [24]. These droplets increase the surface area of the drug and hence increase absorption [30]. The process of formulation is represented in Fig. 1.

Preformulation Study or Selection of SNEDDS Ingredients
Solubility Studies Curcumin is a hydrophobic drug, and our objective is to develop stable oil-in-water nanoemulsions. Therefore, the amount of curcumin is consolidated in oil phase. However, for maximum drug loading, low soluble drugs require more amounts of oil. To produce a stable nanoemulsion, the formulation will need more quantity of surfactant and co-surfactant, which may make SNEDDS preparation toxic and increase the cost of the formulation [31,32] as well as increase the concentration of oil and lower the stability of emulsions [33].
To select the perfect SNEDDS components, high-solubilising-capacity (for drug) ingredients should be selected. The solubility of curcumin was found in numerous oils of sunflower, virgin coconut, black seed, olive, sesame, castor and eucalyptus; surfactants (Labrasol, Cremophor RH 40, Tween 60, Tween 20, Span 80, Span 60, Span 40 and Span 20) and cosurfactants (ethanol, PEG 800, PEG 400 and Transcutol P) were prepared by the shake-flask method [34]. UV spectroscopy is used to determine the solubility of curcumin (mg/mL).
To determine the solubility of curcumin, 3-ml small vials containing different oils or surfactants or co-surfactants were taken and an equal quantity of curcumin (excess amount in which some quantity remains undissolved) was added to each vial. The vials were perfectly closed and continuously stirred on a mechanical stirrer for 48 h at a temperature of 37 ± 5 °C. After equilibrium has been achieved, mixtures were centrifuged at 10,000 rpm for 20 min. The undissolved drug was separated, filtered and appropriately diluted with methanol. The concentration of drugs in the solution was assayed by a UV spectrophotometer [31,32,35,36].
Appearance and Homogeneity On behalf of solubility results studies, briefly, olive oil, castor oil, black seed oil and virgin coconut oil were selected as oil; Cremophor RH-40 and Labrasol were selected as surfactants; and Transcutol P selected as a co-surfactant. Composite ratio of selected surfactant, co-surfactant and oils (w/w/w) was taken according to Table 1 and prepared by vortex mixing.
For appearance and homogeneity, 1 ml SNEDDS (blank) was titrated with 100 ml of deionised water at 37 °C and mixed continuously on a magnetic stirrer (at 100 rpm) until equilibrium was reached (or for 1 min). After equilibrium, the mixture was visually observed and reported [37].

Preparation of L-SNEDDS
After identification of the self-nanoemulsifying composition ratio, L-SNEDDS formulations were prepared according to optimised ratio (w/w/w) shown in Table 1 in different oils by a previously reported method [38]. Quantities of components were accurately weighed in a glass vial and mixed with a magnetic stirrer at 100 rpm for 30 min to get a clear homogenous mixture. Curcumin and piperine were kept constant for all formulations. Then, 100 mg curcumin (20% of L-SNEDDS) and 20 mg piperine (5% of L-SNEDDS) were mixed in little-by-little quantity with constant stirring at 60 °C. The resultant mixture of formulations was homogenated by vortex mixing until a clear, monophasic solution was achieved. Formulated L-SNEDDS were placed in a tightly packed glass vial at 25 °C for further development and characterisations.

Solidification of L-SNEDDS
CUR-PIP S-SNEDDS was prepared by a previously reported adsorption method [39]. Optimised L-SNEDDS preparation was transformed to S-SNEDDS by the adsorption method. The benefit of the adsorption technique is to have more Fig. 1 Formulation of S-SNEDDS and its mechanism of action for AD surface area with good physical adsorption, high dissolution efficiency, and good uniformity and reproducibility [40]. In a glass mortar, CUR-PIP-loaded L-SNEDDS was added dropwise in 1:1 w/w proportion with adsorbent neusilin-US2. After every addition, the blend was mixed properly until a uniform solid powder was obtained. The resultant damp mass was collected from the apparatus and processed through the mesh size 250 µm (mesh no. 60). Prepared S-SNEDDS (powder form) was filled directly into the hard gelatin capsules and sample quantity of S-SNEDDS was placed at 25 °C in a desiccator for further characterisations and in vivo study.

Phase Separation and Stability Study of Emulsions
For phase separation, each of the optimised L-SNEDDS formulations (50 µl) was mixed with 5 ml of double-distilled water in a vial and cyclo mixed for 60 s at room temperature.
Then, each mixture was stored and observed at intervals of 2, 4, 6, 8, 12 and 24 h [41]. Stable L-SNEDDS emulsions were further evaluated to find the effects of dilution and diluted mediums (in pH 1.2 and 6.8) for the stability of emulsions to be formed. For this, 1 ml of L-SNEDDS formulation was diluted with 1000 ml distilled water, 0.1 N HCl and phosphate buffer solution which provide a similar in vivo gastric condition [39]. Each formulation response was determined after a 3-h stabilisation period [42].

Efficiency of Self-Emulsification (Dispersibility Test)
Emulsification time is an important point to describe the SNEDDS stability and gastric fluid characteristics. Self-emulsification properties of preparations were obtained by performing visual assessments [43]. Prepared SNEDDS must be dispersed spontaneously in an aqueous medium on mild shaking. The emulsification time of the drug-loaded L-SNEDDS formulations was evaluated according to USP (United State Pharmacopeia) by dissolution apparatus II [44]. For sample preparation, 1 ml of L-SNEDDS was diluted  [45,46]. According to the capability to form an emulsion in a particular time interval and appearance of nanoemulsions, SNEDDS emulsions are classified into a number of grades: A, B, C, D and E [46].
Grade A: They are quickly forming nanoemulsions (in 1 min) and have a clear or transparent appearance [43]. Grade B: Grade B are also rapidly forming nanoemulsions (in 1 min), but slightly less clear, having turbid transparency. Grade C: They are fine milky, hazy emulsions formed in less than 2 min. Grade D: Grade D are dull and greyish-white emulsions taking longer emulsification time (> 2 min). Grade E: Grade E exhibit slow self-emulsification capability with large oil droplets existing on the surface of the formulation. Grade E takes more than 2 min to form an emulsion.
When grades A and B SNEDDS are dispersed with GIT fluid, they will remain as nanoemulsions. However, grade C to grade E formulations could fail [47].

Drug Loading Efficiency
The quantity of curcumin and piperine present in preparation was determined by UV spectrophotometry. CUR-PIP-loaded SNEDDS (50 mg) was diluted with 100 ml methanol. To remove undissolved drugs, formulations were gently stirred at 37 °C and centrifuged at 2000 rpm for 30 min [29]. After suitable dilutions, samples were prepared in triplicate (n = 3). The resultant solution was analysed by a UV spectrophotometer (UV-1800; Shimadzu Japan) [48]. The drug loading efficiency of SNEDDS was determined by using the formula [49]

Thermodynamic Stability Studies
To evaluate the effect of various stress conditions of nanoemulsions, thermodynamic stability studies were conducted [50].
Heating-cooling cycle In this thermodynamic stress, preparations were stored at temperatures + 4 and + 45 °C separately for 48 h.

Freeze-thaw cycle
In the freeze-thaw cycle, formulations were stored at − 21 and + 25 °C for more than 48 h at each temperature and visually observed.

Determination of Droplet Size, PDI and Zeta Potential
Average droplet size and polydispersity index (PDI) of the nanoemulsion of L-SNEDDS were measured using a Zetasizer Nano (Nano ZS; Malvern Instruments Ltd, UK). Thereafter, 1-ml formulation was diluted with 1000 ml double-distilled water and loaded in the cuvette, and samples were observed at 25 °C [45]. For zeta potential (surface charge), L-SNEDDS was diluted in 100 ml distilled water by gentle mixing [51].

In Vitro Dissolution Study
In this study, drug-loaded S-SNEDDS and pure drugs (curcumin and piperine) were subjected to an in vitro dissolution test by using a USP II dissolution test apparatus (paddle type; Optics Technology, Delhi, India). It is reported that the drug takes about 2 h to pass the stomach (strong acidic pH) and then move to the intestine where pH suddenly increases [52]. As pH changes from stomach to intestine, we performed an in vitro study in different mediums. The starting 2-h formulation was dissolved in 0.1 N HCl medium at pH 1.2 (stomach) and then moved to phosphate buffer at pH 6.8 (intestine). Temperature of bath apparatus was sustained at 36.5 to 37.5 °C and paddle speed was maintained at 100 rpm. At regular intervals of time (10, 30, 60, 90, 120, 150, 180, 210 and 240 min), 5-ml samples were collected and 5 ml of fresh one was added to balance the medium volume. Withdrawn samples were filtered, diluted and then analysed for curcumin and piperine by using a UV spectrophotometer (UV-1800; Shimadzu, Japan) [28].

FTIR
Fourier-transform infrared spectroscopy (FTIR) is considered to be the best tool to examine any possible chemical interaction of drugs and excipients used in the formulation [47,53]. FTIR spectra of pure curcumin, piperine and S-SNEDDS formulations were scanned. The samples were scanned over the scanning range 4000-650 cm −1 by FTIR spectrophotometer Model RZX (Perkin Elmer UK) [39].

PXRD Study
Powder X-ray diffraction (PXRD) patterns of pure curcumin, pure piperine and all the S-SNEDDS formulations were recorded by using an X-ray diffractometer (X'Pert; Malvern Panalytical Ltd, Eindhoven, Netherlands). An appropriate amount of sample powder was taken and equipped with an intended wavelength K-Alpha1 (1.54060 Å) operating a generator power of 45 kV and current of 40 mA. The samples were filled in the sample holder and patterns were recorded over angular range intervals of 2 to 60° (2θ) at a scanning speed of 67 s per step [39].

Animal Studies
Thirty adult Swiss albino male mice weighing 18-22 g were taken from and housed in suitable temperature (21-25 °C) and humidity 50-70% with standard 12-h light and dark cycle. The mice were allowed free access to plenty of water and food [54]. All experiments were performed as per the guidelines of CPCSEA (

AD Model Preparation
Aluminium chloride (AlCl 3 ) was dissolved in distilled water, and D-galactose (D-gal) powder with extra purity was dissolved in 0.9% normal saline. AD model preparations were freshly prepared at least two times per week and stored at 4 °C. AlCl 3 was given intragastrically in a dose of 20 mg/kg/ day and D-gal intraperitoneally (i.p.) in a dose of 120 mg/ kg/day continuously for 40 days to induce AD in mice [54]. Animals of the vehicle control group were also injected intraperitoneally and administeredintragastrically the same amount,of normal saline [55].

Treatment Preparations
NF3 S-SNEDDS formulation was optimised as best formulation and selected for animal study. CUR-PIP nanoemulsion treatment was given to two groups of animals: for group 3, 25 mg curcumin + 5 mg piperine/kg/day; for group 4, 50 mg curcumin + 10 mg piperine/kg/day [56][57][58]. Donepezil hydrochloride (DNP) 5 mg tablet was grinded to make fine powder and dissolved in distilled water and then administered orally to mice at a dose of 1.0 mg/kg/day [54]. All treatments administered to mice were dissolved in normal saline 2.5 ml/kg volume through oral gavage for 21 days [59,60].

Grouping of Animals
Mice were divided into five groups, having six mice in each group (n = 6) [58,61]. The total duration of the study was 12 weeks. The dose of curcumin 25 and 50 mg/kg was decided on the bases of previous studies [56][57][58]62].

Group 1 (vehicle control):
Mice in this group acted as vehicle control and received the same amount of water and food as in all other groups. The vehicle control group received normal saline solution 2.5 ml/kg [63]. An equal volume was used to dissolve the drug [59].

Behavioural Experiments
After the end of treatments, from the next day behavioural or learning tests were started. There were three behavioral tasks/tests performed: novel object recognition task, Y-maze spontaneous alternation and Morris water maze. Between each test, animals got a few days of resting time to minimise the effects of the prior test [64]. Mice were acclimatised to an experimental room to familiarise themselves for at least 1 h before conducting experiments.

NOR Task
It is a normal behaviour of mice to spend more time with a novel object in comparison with a familiar object. This tendency is applied in analysing the learning and memory capability of mice [65]. Mice that have object interaction for less than 7 s in any trial was removed from the study [64]. The testing apparatus (arena) is a custom-built dark quadrangular wooden area (40 × 40 × 40 cm) [66]. In all sessions, light in the testing area was dimmed to 30 ± 5 lx to reduce the anxiety of animals.
Habituation phase Mice were familiarised individually for 10 min with the arena, 24 h before the training stage. The first 5 min of the habituation phase was used to assess the locomotor activity of the mice.
Training phase Two identical objects (pen holders with the same colour and size) were placed in the left and right corners of the arena. As expected, no significant difference was observed; both objects were explored equally. After 10 min, mice were returned to their cages [65,67].
Test phase After 1-h interval from training, one familiar object (pen holder) and one novel object (tin can) were placed in another back corner. The test duration was 5 min [68, 69]. After each trial, the arena was cleaned with 70% isopropyl alcohol to avoid odour cues. Xnote stopwatch was used as a timer for each object (familiar and novel) separately. Timer was started with any contact with nose, mouth or jmrodriguez e755claw; accidental contacts were not included [65,70].

Spontaneous Alternation Task (Y-Maze)
In behavioural science, spontaneous alternation is a test for learning and spatial working memory [71]. It was performed by using Y-maze (like a Y-shaped letter) with equally measured arms. Each arm of this Y-maze will be 35 cm long, 5 cm wide and 10 cm high, and each tip of the arm wall was coloured with black and white pattern. To make the mice comfortable, light of testing area was adjusted from 25 to 35 lx [64]. Elevated Y-maze was placed horizontally; mice were individually left at the end of the arm and allowed to move all over the maze freely.
In the spontaneous alteration protocol, mice do not need to be familiarised formerly with the maze. In the form of reward, food is placed in both arms A and B, and all the doors remain open. When the mice were placed at the base of the Y (arm C), mice choose either arm A or B; if mice choose arm A to explore, they get the food reward. Next time, mice were placed in the same arm (C), all the doors were still open, and mice can choose to go either in arm A or B. If mice choose arm A (already explored), then they do not get the food reward. However, if mice choose arm B, then then they get the food as reward and this represents that mice have alteration behaviour. This test is for spatial learning because a mouse needs to learn that once-if it goes down to one arm and consumes the food reward, then it needs to go down the other arm for the second food reward. This tendency can be increased by making them hungry and then giving them food as reward. The number of times mice choose the correct arm to explore would be higher in healthy mice than mice that have learning or memory defects. The mice that have less than three arm entries in starting 1 min were excluded from the study [64].
Arms of the maze were named as A, B and C. Continuous entry into three different arms (i.e. C → B → A, C → A → B…) was considered a correct entry. Re-entry into the same arm (i.e. C → A → C, B → A → B…) will be considered as entry error. The number of entries and alterations was recorded manually for each and every mouse over a period of 8 min. After each trial, arms of the maze were cleaned with 70% isopropyl alcohol to ensure that odour and excreta of previous animal had been removed. Spontaneous alternation percentage was calculated by a previously reported formula [54].

Morris Water Maze
Morris' test was performed in a round pool 122 cm in diameter and 62.5 cm in height. The pool was filled with water up to the height of 40 cm. Water was made hazy by adding milk. The pool temperature was maintained at 24 ± 2 °C by adding warm water. The pool surface was divided into four equal quadrants (N, W, E and S) by using a software. Mice were dropped down gently into the water by hand at a sidewall of the pool. The mice were first dropped from the back side (otherwise the animal gets stressed if dropped from the head side). The navigating trial was carried out for 5 days. In the first 2 days of training, mice were trained to track down the visible platform (a pole with 10 cm diameter) which teaches the animal that there is a platform, which is the way to come out from the water. Then, for further training, the platform was hidden (dipped 2 cm below the water) for the next 3 days while the pole's position remains unchanged during whole learning test. For each trial, mice were put down through the same location (opposite quadrants of the pole) in the water and 60 s was allocated to find the pole. At the end of each trial, animals were allowed to stay on the pole for 20 s to recognise the location of the pole. Mice that have difficulty reaching the pole within 60 s were guided in the direction of the pole and allowed to stay there for 20 s [62]. Mice were trained three times a day with a break of approximately 30 min in each trial. When the training was over then after 24 h, spatial probe test was started. On the test day, the pole was hidden, and mice were allowed to find the platform for 60 s. The latent time taken to find the platform by each group was recorded.

Solubility Study
The maximum solubility of curcumin was found in virgin coconut oil, castor oil, black seed oil and olive oil; in surfactants, Cremophor RH40 and Labrasol; and in % Alterations = Number of alternations (Number of entries − 2) × 100 co-surfactants, Transcutol P (results shown in Fig. 2). Similar results were observed in previous research studies [72][73][74]. By using solubility studies, suitable bio-active oils, surfactants and co-surfactants that possess a fine solubilising capability of curcumin were selected (result represented in Fig. 2) which is further used for formulation development. A small ratio of Labrasol (surfactant) is also mixed with formulation components, as Labrasol increases the membrane lipid fluidity and permeability [75].

Appearance and Homogeneity
In formulation development, produced samples that had a clear or slightly bluish appearance are indicated as nanoemulsions. Immediate coalescence of droplets with drug precipitation and phase separation of the emulsion on storage for 48 h at room temperature indicate the formation of poor or unstable emulsion [39]. All the blank liquid SNEDDS preparations expressed good self-emulsification capability within 60 s. The optimised formulation has best transparency appearance, less emulsification time and better homogeneity as compared to other preparations of the same oil.
The designed anhydrous SNEDDS (drug-free) preparations showed mutual miscibility upon aqueous dispersion. Some particular SNEDDS preparations such as 2-3, 6-8 and 27 preparations showed excellent homogeneity and self-emulsification time. Even after CUR and PIP loading, preparation 3 (further named NF3), preparation 8 (further named NF8) and preparation 27 (further named NF27) showed a good transparent appearance. The result is given in Table 1.

Phase Separation and Stability Study of Emulsions
All optimised SNEDDS formulations were found stable (neither precipitation nor phase separation of the drug) at intervals of 2, 4, 6, 8, 12, and 24 h when optimised L-SNEDDS were subjected to increased dilutions in distilled water, 0.1 N HCl and phosphate buffer solution. All formulations show stable nanoemulsion (results shown in Table 2).

Efficiency of Self-Emulsification (Dispersibility Test)
Formulations NF3, NF8 and NF27 have a self-emulsification time less than 1 min with clear transparency, while formulation NF23 has slightly turbid transparency. According to SNEDDS capability to form a nanoemulsion in different media, the results are given in Table 3 (Nanoemulsion formulation graded according to their appearance mention in Efficiency of Self-Emulsification).

Drug Loading Efficiency
All SNEDDS formulations have curcumin loading efficiency between 86.81 and 98.71% and piperine loading efficiency between 90.52 and 99.12%. It was observed that formulation NF3 has the highest drug (CUR-PIP) loading efficiency (results shown in Table 4).

Thermodynamic Stability Studies
In SNEDDS nanoemulsion, both the drug curcumin and piperine are present in solubilised form, and emulsion stability decreased with phase separation and drug precipitation.
Thermodynamical stability study results have shown that all SNEDDS emulsions can withstand a wide range of temperature changes (− 20 and + 45 °C). Not one SNEDDS formulation showed any sign of drug precipitation and phase separation (results represented in Table 5).

Determination of Droplet Size, PDI and Zeta Potential
Results indicate that all the SNEDDS formulations' average droplet size was in the range 22.01 to 309.5 nm. It was previously observed that the concentration of given curcumin in the brain is highest for the smallest size of the particle [66], so the prepared SNEDDS formulation will be an effective formulation to deliver the drugs to the brain.
In the present study formulations, PDI value was observed between 0.172 and 0.298, indicating that all SNEDDS formulations had narrow size distribution and high homogeneity. Generally, the PDI value less than 0.3 indicates that SNEDDS formulations have high uniformity in particle sizes [67], while a high value of PDI indicates that formulations have high variation in particle sizes [51].
The surface charge (zeta potential) of the formulation's droplets affects the stability [67]. All SNEDDS formulations have a surface charge from + 1.87 to − 5.81 mV. It was observed that in the presence of surfactant and co-surfactants, negative zeta potential (− mv) of the emulsion may be obtained [67], and it is also reported that the droplets that have positive zeta potential (+ mv) have more interaction with GIT mucus (as the intestinal cells have negative charges). It is expected that the SNEDDS NF3 would enhance the GI absorption of CUR-PIP in an in vivo study [69]. All results are represented in Table 6.

In Vitro Dissolution Studies
The CUR-PIP-loaded S-SNEDDS formulations were evaluated through an in vitro release study, and data indicates that all four preparations showed curcumin and piperine release that was increased in comparison to their suspensions.

Powder X-Ray Diffraction Studies
Pure curcumin (Fig. 6A) and pure piperine (Fig. 6B) show highly crystalline diffraction while S-SNEDDS formulations NF3, NF8, NF23 and NF27 (Fig. 6C-F) showed the absence of peaks at diffraction angles, representing an amorphous pattern. XRPD patterns of all optimised S-SNEDDS formulations and pure drugs are adjusted in Fig. 6.

Novel Object Recognition Task
The behaviour of mice in all groups varied significantly. Mice treated with D-gal + AlCl 3 (AD model group) showed poor ability to recognise novel object as compared to control and treatment (per se) groups. Significant improvement was observed in learning and memory in groups treated with CUR-PIP-loaded S-SNEDDS (dose I, dose II) and DNP, as mice of these groups interact more time with the novel object over the familiar object (discrimination ratio and percentage preference for a novel object are given in Fig. 7A and B, respectively). Dunnett's multiple comparisons test indicates the differences between control and treatment (per se) groups.

Spontaneous Alternation Task (Y-Maze)
As shown in Fig. 8, the percentage of spontaneous alternation rate of mice in AD group was decreased significantly in comparison to the vehicle control group (****p < 0.0001).
In comparison, the percentage of spontaneous alternation rate of treatment group (dose I, dose II and DNP) was increased significantly in comparison to AD model group.  CUR-PIP-loaded S-SNEDDS dose I and dose II percentage alternation increased significantly in a dose-dependent manner (**p < 0.01 and ****p < 0.0001, respectively).

Morris Water Maze
On the test day (last day of the experimental schedule), a significant decrease in escape latency time was observed in the treatment groups as compared with AD model group. Differences were examined by one-way ANOVA (mixed) and significant differences were found as AD group versus CUR-PIP S-SNEDDS treated groups dose I **p < 0.01, dose II ****p < 0.0001 and DNP treated group ****p < 0.0001 (results are shown in Fig. 9).

Statistical Analysis
All results for experiment NOR test, spontaneous alternation task and MWM tests are expressed as mean ± SEM and statistically analysed by GraphPad Prism 9.3.1 (GraphPad Software, USA). For comparing the data among different groups, ordinary one-way ANOVA (analysis of variance) followed by Dunnett's multiple comparisons test was used. The differences between groups were considered significant if associated *p-value is < 0.05.

Conclusion
In the present study, CUR-PIP-loaded S-SNEDDS formulation was successfully developed with black seed oil, castor oil, olive oil and virgin coconut oil as oil phase; Labrasol and Cremophor RH40 as a surfactant; and Transcutol P as cosurfactant. On the basis of homogeneity, self-emulsification time and appearance, four formulations, NF3, NF8, NF23 and NF27, were selected and loaded with CUR-PIP. Formulated L-SNEDDS were further converted into solid form by using Neusilin US2. Neusilin US2 helps to increase the drug dissolution and stability of S-SNEDDS. From the above characterisation studies, it is concluded that the formulation NF3 is an ideal formulation out of four S-SNEDDS, as it has the smallest nano-sized droplets, positive zeta potential, PDI value less than 0.3 and shows better drug release; when compared to pure curcumin and piperine, it has a four to five fold percentage release with a fast rate.
Selected NF3 formulations were subjected to pharmacodynamic studies against AD model. In an in vivo study, treated group (dose I, dose II and DNP) show significantly increased learning and memory enhancement as compared with the disease control group. It has been found that optimised CUR-PIPloaded S-SNEDDS formulation has a significant effect on AD. Therefore, laboratory studies suggested that CUR-PIP-loaded S-SNEDDS could be a promising dosage form for the treatment of AD. Graph showing the escape latency of AD induced group (D-gal + AlCl 3 ) versus the control group and treatment groups (dose I, dose II and DNP). All data are expressed as the mean ± SEM (n = 6). **p < 0.01, ****p < 0.0001 vs. AD group

Future Prospects
Curcumin has various effects on AD but less effective due to its bioavailability. However, when curcumin is administered with piperine, the bioavailability of curcumin increases. This research article is the first one which reported an in vivo study that observed the synergistic effect of curcumin and piperine for the treatment of AD and reported that nanoformulation has a significant effect on AD. However, clinical studies are required to identify the therapeutic effect of prepared formulation.