Bilosomes as a Potential Carrier to Enhance Cognitive Effects of Bacopa monnieri Extract on Oral Administration

The Indian system of medicine, Ayurveda employs Bacopa monnieri extract (BME) for memory enhancement. This study attempts to prepare and test a more potent formulation by incorporating BME in nanovesicles. BME-loaded liposomes and bilo-somes (bile salt-stabilized liposomes) were formulated using soy phosphatidylcholine. Liposomes and bilosomes had homogeneous size distribution


Introduction
A cognitive decline challenges the aging brain.In the context of a steadily growing geriatric population, it is essential to address this emerging public health problem with indigenous solutions. 1It has been reported that 50 to 60% of those above 65 years of age suffer from neurodegen-reports, 55.2 million people are affected by dementia globally, and 60% are in low-and middle-income countries.Dementia cases may rise to 78 million by 2030 and 139 million by the end of 2050.Dementia associated with various neurological conditions remains the seventh leading cause of death worldwide.The World Health Organization (WHO) has called for urgent action in dementia management and risk reduction. 4ootropic formulations are expected to address the above concerns.Nootropics are cognition enhancers, but a cognition enhancer is not necessarily a nootropic. 5A cognition enhancer improves memory and attention, whereas nootropics enhance mental abilities.Both learning and memory are variations in synaptic neural connectivity and psychological processes.In recent times, there has been a tremendous demand for herbal products to treat chronic ailments in developing countries and the West. 6acopa monnieri (BM), family Scrophulariaceae, is known as Brahmi in India and has been used since 500AD.In Ayurveda BM is known as "Medha Rasayana" to treat anxiety, impaired memory, epilepsy, and cognitive processes such as understanding, memory, and recall. 7Bacopa monnieri extract (BME) scavenges free radicals and protects against deoxyribonucleic acid (DNA) damage in a dose-dependent manner.BME is an antioxidant (half as potent as vitamin E) and exhibits anti-stress, immunomodulatory, anti-aging, cognition facilitating, and anti-inflammatory effects.The memory-enhancing property of BM is credited to its antilipid peroxidation property and has been prescribed for longterm therapy at low doses. 8The pharmacological activities of BM have been extensively studied and have been linked to the saponin constituents known as "bacosides."Bacosides are a complex mixture of structurally related compounds such as jujubogenin or pseudojujubogenin glycosides. 7Alkaloids brahmine and herpestine have been isolated from Brahmi.Tetra cyclic triterpenoid saponin, Bacoside A, and B (crystalline mixture of several saponins) are the main bioactive constituents of the plant.Bacoside A is the most abundant of these.Other saponins include bacoside A1, A3, bacoside saponins A, B, C, D, E, and F. The plant constituents also include d-mannitol, hersaponin, betulic acid, and flavonoids. 9he phytoconstituents often fail to offer therapeutic benefits owing to their poor bioavailability caused by hepatic first-pass metabolism or incomplete absorption.The technological advancements in drug delivery can overcome these challenges enabling the phytonutrients to achieve therapeutic concentrations at the site of action effortlessly.A novel vesicular drug delivery system aims to deliver the active ingredient to the site of action at a rate determined by the body's need during treatment. 10Nanomedicine is likely to offer better therapeutic success than conventional drug delivery approaches. 11Several studies on tumors and central nervous system (CNS) disorders such as Alzheimer's disease (AD) use nano-vesicular formulations. 12The therapeutic index, solubility, stability, and rapid degradation of drug molecules have been improved using vesicular drug delivery systems. 13Liposomes entrap the compounds, avoid fast removal or degradation from the systemic circulation, and facilitate their penetration through the blood-brain barrier (BBB) and distribution in the brain tissue, making liposomes an efficient drug delivery system for the brain. 12,14Liposomal delivery generally requires lower doses because of improved bioavailability and tissue distribution.Liposomes contain multiple components, the most important being phospholipids and cholesterol.Soy phosphatidylcholine (SPC) is the most widely used phospholipid in liposomal preparations. 10iposomes mimic cell membranes by exhibiting excellent entrapment ability, safety, and biocompatibility.Although parenteral delivery of liposomes has been successful, oral delivery is thwarted by various factors such as gastrointestinal (GI) instability.Liposome stability and permeability can be significantly improved for oral administration by modifying the composition of the lipid bilayers or by adding polymers or ligands for targeting. 15Liposomes administered orally would be destroyed partially by gastric acid, leading to the release of the drug payload.Surviving liposomes would subsequently move from the stomach into the small intestine, where intestinal enzymes break down liposomes.Liposomes that survive this stage penetrate the mucus layers and contact intestinal epithelia.Even at this point, liposomes can still be destroyed.Only liposomes that survive the digestive process would be absorbed by M cells as integral vesicles to the lymphatic system. 16 variety of methods are employed to improve liposomal stability.Adding bile salts enhances lipid biomembranes' flexibility, allowing them to survive the adverse effects of bile acids in the GI tract.Imaging studies have shown that bilosomes that survive the GI environment can be absorbed as entire vesicles. 17Bilosomes are vesicular carriers used to protect proteins and peptides from degradation in the gastrointestinal tract (GIT) to a certain extent.Vesicles are developed by incorporating bile salts into the lipid bilayers, making them resistant to the disruption in GIT.As a result, the vesicle protects the entrapped agents from the harsh GI environment. 18In this novel drug delivery system, carriers such as deoxycholic acid are introduced into the membrane of liposomes. 19Bile salts improve the penetration of lipophilic drugs across biological membranes, thereby enhancing oral bioavailability. 20Here we attempt to enhance the nootropic activity of BM through bilosomal delivery, facilitating value addition to the traditional knowledge system in Ayurveda.

Materials
The alcoholic extract of BME was a gift sample from Elixir Extracts Pvt. Ltd., Kochi, Kerala.Soya phosphatidylcholine (SPC or soy lecithin) was procured from Hi-Media Laboratories, Mumbai.Cholesterol was purchased from Merck, Mumbai.Sodium deoxycholate, pepsin (from porcine gastric mucosa), pancreatin (from the porcine pancreas) was purchased from Sigma-Aldrich.Chloroform was purchased from Loba Chemie, Mumbai.All chemicals used were of analytical or pharmaceutical grade.
Froth Test for Saponins in the Bacopa monnieri Extract BME (50 mg) was mixed with 5 mL of distilled water in a stoppered test tube, shaken well for $5 minutes, and allowed to stand for 30 minutes.The honeycomb-like froth formation indicated the presence of saponins. 21

Fourier Transform Infrared Spectroscopy Studies
The Fourier transform infrared spectroscopy (FT-IR) spectra of BME and extract-loaded nanoformulations were recorded in a wavelength range of 4000 to 500 cm -1 on a M-15, Bruker Alpha Fourier transforms infrared spectrophotometer, USA, employed with an ATR crystal.Transmittance peaks were identified and compared with the literature reports.

Method 1: Solvent Evaporation Technique
To prepare liposomes (LS11, LS12, and LS13), equal volumes of BME, SPC, and various amounts of cholesterol were dissolved in 10 mL of ethanol in a beaker.►Table 1 shows the ratio of materials used in the formulation.The mixture was added to 80 mL of distilled water under stirring at 15000 rpm on a magnetic stirrer (on a magnetic stirrer) maintained at 80°C.Stirring was continued for 15 minutes, maintaining the temperature to evaporate the ethanol.The mixture was allowed to cool and was sonicated for 10 minutes (50% amplitude) using a probe sonicator (Sonics, USA). 22thod 2: Thin-film Hydration Method (LS2) BME-loaded liposomes (LS21, LS22, and LS23) were prepared following a thin-film hydration process in a rotary flash evaporator.BME, SPC, and cholesterol were dissolved in 10 mL of chloroform in a round bottom flask; the quantities are represented in ►Table 1.A rotary evaporator was used to evaporate the organic solvent, maintained at 65°C under reduced pressure for 30 minutes.The resulting thin, dry film was left overnight for complete evaporation of organic solvents and then hydrated using a phosphate buffer (pH 7.4) to obtain a crude dispersion of BME-loaded liposomes.Then crude dispersion was further sonicated for 10 minutes (50% amplitude) using a probe sonicator.23

Formulation of Bilosomes by Thin-film Hydration Method
BME-loaded bilosomes (BS1, BS2, and BS3) were prepared using a rotary flash evaporator by a thin-film hydration process, slightly modifying the previous method.As given in ►Table 1, BME and SPC were dissolved in 10 mL of chloroform in a round bottom flask.The organic solution was evaporated at 65°C under reduced pressure for 30 minutes until a thin, dry film was formed in a rotary flash evaporator.It was left overnight to achieve complete evaporation of the organic solvent.Crude dispersion of BMEloaded bilosome was achieved by hydrating the thin, dry film with 10 mL of distilled water containing bile salt (sodium deoxycholate).The dispersion was then sonicated for 10 minutes using a probe sonicator. 24

Vesicle Size and Zeta Potential Analysis
The average vesicle size and size distribution (polydispersity index, PDI) of formulated liposomes and bilosomes were analyzed by dynamic light scattering (DLS) using Nano ZS, Malvern Instruments, UK.The surface charge of vesicles was measured in terms of zeta potential (Nano ZS, Malvern Instruments, UK).Only those formulations with desirable size distribution were considered for further characterization.

High-Resolution Optical Microscopy
On account of the acceptable size range, formulations LS23 and BS3 were taken for further studies.The vesicles formed were examined for their morphology under a high-power optical microscope (BIOVIS particle analyzer, India).A drop of the liposome/bilosome sample was sufficiently diluted with distilled water, smeared on a glass slide, protected with a coverslip, and inspected under microscopic magnification (100Â objective), and the images were recorded.

Transmission Electron Microscopy of Formulations
The morphology and vesicular size of BME-loaded liposomes LS23 and bilosomes BS3 were further analyzed by highresolution transmission electron microscopy (HR TEM) using JEM-2100 Electron Microscope, Jeol, Japan.After suitable dilution, a drop of the formulation was adsorbed on a copper grid, negatively stained with 1% phosphotungstic acid, and air-dried at room temperature for 10 minutes.The morphological appearance of the selected vesicles was recorded by TEM. 23tract Entrapment Efficiency of Vesicles Flocculation was induced for LS23 and BS3 by adding potassium chloride solution (1 mL of 3M KCl) before centrifugation.The unentrapped BME in the formulations was removed by ultracentrifugation (Beckman, USA) at 40,000 rpm for 1 hour at 4°C.The concentration of unentrapped extract in the supernatant was analyzed by UV-Vis spectrophotometry at 321 nm (Spectroquant Prove 600, Merk, Germany). 25,26he percentage entrapment efficiency was determined using the following equation

In Vitro Drug Release Study
Drug release from nanovesicles was studied using the dialysis method.Dialysis kits (Pur-A-Lyzer Maxi Dialysis Kit, Sigma-Aldrich, India) of 12 to 14 kDa molecular weight cut-off were used for this method.Dialysis kits were presoaked in distilled water for 10 minutes and later loaded with 3 mL of the formulations.Loaded dialysis kits were fixed on the floating rack and placed in a beaker containing 200 mL of phosphate-buffered saline (PBS; pH 7.4) as a release medium, maintained at 37 AE 0.5°C with continuous magnetic stirring at 50 rpm.Samples of 5 mL were withdrawn at different intervals of 0.5, 1, 2, 3, 4, 6, 7, 9, 24, 48, 69, 72 h, and replaced with the medium.The concentration was determined by UV-visible spectrophotometry by measuring the absorbance after suitable dilution. 27 Vitro Gastrointestinal Stability of Nanovesicles Simulated gastric fluid (SGF) was prepared by dissolving 2 g of NaCl and 7 mL of concentrated HCl in 1 L of deionized water, then adjusting the pH to 1.2.To mimic gastric digestion, pepsin (0.032 mg/mL) was added to the above solution.Simulated intestinal fluid (SIF) was prepared by dissolving 6.8 g of dipotassium phosphate, 190 mL of 0.1 M sodium hydroxide, 3.32 g of sodium chloride, 8.76 g of calcium chloride , and 0.1 g of sodium deoxycholate (bile salt); in water.The solution was diluted to 1 L, and the pH was adjusted to 7.4.Pancreatin was added to the above solution at a 0.032 mg/mL concentration for intestinal digestion.The liposomes and bilosomes were added to SGF and SIF in separate standard flasks, mixed well, and incubated in the water bath for 30 minutes at 37 AE 0.5°C.Samples were withdrawn after 1 minute and 30 minutes.Vesicle size, PDI, and zeta-potential of liposomes and bilosomes were determined during GI digestion. 28

Selection of Dose
To assess the nootropic activity of BME, the dose was fixed at 200 mg/kg, (1/10th of the previously reported maximum dose in acute toxicity studies, i.e., 2000 mg/kg).Formulations equivalent to the above amount of drug were used for evaluation. 29eatment Schedule and Evaluation of Nootropic Activity The animals in Group I and II were administered orally once daily with vehicle (0.6% w/v sodium carboxymethyl cellulose [Na CMC], 10 mL/kg) for 7 days.Group III was administered with BME (200 mg/kg) orally once daily for 7 days.For seven days, groups IV and V were administered with the formulation LS23 and BS3 containing BME equivalent to 200 mg/kg.On the seventh day, after half an hour of BME administration, each group except Group II received scopolamine (0.5 mg/kg) intraperitoneally. 30fter 45 minutes of scopolamine administration on day 7, the nootropic activity was evaluated using the Y maze.To evaluate short-term memory, the percentage of trials in which the mice entered all three arms on consecutive choices alternatively (without returning to the previous arm) was recorded as an alternation. 31

Statistical Analysis
The data for nootropic effect of BME, LS23 and BS3 formulations are expressed as mean AE standard error of the mean (SEM).The data obtained were subjected to a one-way analysis of variance (ANOVA) followed by Dunnett's test using GraphPad Prism software.p-Values less than 0.05 are considered statistically significant (indicated by ÃÃ ).

Identification of Bacopa monnieri Extract
The procured BME was brownish-green with a characteristic odor and powdery appearance.In the froth test, a characteristic honey-comb-like froth was formed, indicating the presence of saponin glycosides.Steroid saponin glycoside, such as bacoside A 3 in the extract, helps with memory enhancement.

Formulation of Liposomes and Bilosomes
BME-loaded liposomes and bilosomes were prepared as described in the methodology section, The BME was dissolved in ethanol to solubilize lipophilic active constituents, improving the entrapment.►Table 1 depicts the formulation chart of liposomes and bilosomes, showing the preparation method and composition.Solvent evaporation yielded larger vesicles; therefore, the thin-film hydration method prepared the final formulations of liposomes and bilosomes.The difference between the formulation of liposomes and bilosomes was the incorporation of bile salt in the latter during their hydration process.Adding cholesterol modulates membrane permeability and elasticity, reducing vesicle size. 32

Characterization of Liposomes and Bilosomes
Vesicle Size, Zeta-potential, Morphology We used Malvern Zetasizer, which employs the dynamic light scattering technique, to determine the vesicle size of the formulations.Results are shown in ►Table 2. Although liposomes prepared by solvent evaporation method are known for their high entrapment efficiency and ease of preparation, they yielded large vesicles even after sonication.Vesicle size and entrapment efficiency are directly related because increasing the vesicle size increase the volume of extract encapsulated.Additionally, increasing the amount of soy lecithin enhances the medium's lipophilicity, increasing the entrapment efficiency and vesicle size.Due to the large vesicle size, liposomes prepared by solvent evaporation were excluded from advanced characterization.
The amount of cholesterol affects the vesicle size and drug release from liposomes. 33Cholesterol increases membrane hydrophobicity, favoring the inclusion of hydrophobic molecules. 34Furthermore, the increased vesicle size for SPC liposomes could be attributed to the packing parameter and phase transition temperature.Saturated phospholipids with shorter carbohydrate chains have lower packing parameters, favoring smaller vesicles' formation.On the contrary, phospholipids with monounsaturated carbohydrate chains have the highest packing parameter and lower phase transition temperature, helping the formation of larger liposomes. 35odium deoxycholate or bile salts impart a negative charge to the vesicles, increasing the zeta potential.Smaller vesicles are observed in bilosomes with better zeta potential, possibly due to bile salt's presence.Also, the vesicle size is known to increase due to the repulsion between the bilayers in multilamellar bilosomes, which was not observed here. 24 detailed comparison of vesicle size is given in ►Table 2.
The polydispersity index (PDI) is an essential measure of dispersion characteristics related to size uniformity.PDI values of more than 0.4 imply high heterogeneity, and those 0.2 or less indicate homogeneity.PDI of LS23 was found to be 0.5 AE 0.15, and BS3 was found to be 0.386 AE 0.01, showing moderate homogeneity in size distribution. 36he zeta potential values for BME-loaded liposomes LS23 and bilosomes BS3 were satisfactory, with values of À16.8 and À15.1 mV, respectively, implying moderate stability of the formulations due to electrostatic repulsion.The incorporation of bile salts and cholesterol showed increased zeta potential.Formulations with lower zeta potential and high polydispersity were excluded from further evaluation.
►Fig. 1 and ►Fig. 2 show the high-resolution optical microscopy and TEM images of BME-loaded liposomes LS23 and BME-loaded bilosomes BS3.These studies confirmed that both systems have a well-defined spherical shape and proper structure.Vesicle size analysis proved that both formulations were good candidates for clinical applications, well within the optimum size requirements for good biological activity.

Encapsulation Efficiency
Evaluating extract loading efficiency helps estimate the amount of BME entrapped in the vesicles.The encapsulation efficiency was determined to be 45% for LS23, and 85%for BS3.The phospholipid composition of the liposomal membrane influences the drug loading efficiency and drug release kinetics.The membrane's hydrophobicity, thickness, and compactness determine the drug loading efficiency and leakage of molecules entrapped in liposomes. 37As far as BS3 is concerned, the encapsulated fraction of extract in-creased proportionately with an increase in SPC concentration.It may be concluded that SPC concentration has a twoway effect on entrapment efficiency. 38ncapsulation of extract was lower in LS23 without bile salt in the bilayers.Nevertheless, the entrapment efficiency of BS3 increased with increased SPC in the presence of bile salt.Reports state that the surface-active properties enable sodium deoxycholate to perturb the acyl chains of the lipid matrix and incorporate them into the surface of the membrane bilayer perpendicularly.Thus, sodium deoxycholate increases membrane flexibility, thereby improving the solubility of a highly lipophilic drug in the membrane.Thus, incorporating bile slats might facilitate BME penetration into the lipid bilayer.
Entrapment efficiency increased significantly with bile salts and high lipid content, implying that lipid and bile salts are the main factors influencing entrapment.The change in the vesicle size also affects the entrapment efficiency as a function of the sodium deoxycholate-to-PC molar ratio.Sodium deoxycholate in the exterior aqueous medium can decrease the surface tension of the vesicles, possibly resulting in a reduction in vesicle size. 39Increased bile salt concentrations would result in the development of micelles in the dispersion medium, improving drug solubility in the medium and lowering percentage entrapment.Furthermore, high concentrations of bile salts have a fluidizing effect on lipid bilayers, resulting in the loss of the encapsulated medication.Raising the molar   ratio of SPC would increase the lipophilicity of the systems, increasing the amount of entrapped drugs. 24 Vitro Drug Release Studies ►Fig. 4 shows the in vitro drug release pattern of BMEloaded liposomes LS23 and bilosomes BS3.A biphasic release profile was observed in nanovesicular formulations, i.e., a rapid initial burst release followed by sustained release.The burst release of BME might be due to the detachment of surface-loaded extract and unentrapped one.In contrast, the sustained release period could be due to BME release from the vesicular bilayer into the release medium over time. 40

In Vitro Release Kinetics
The release rates are shown in ►Table 3. The optimized liposomal and bilosomal suspensions (LS23 and BS3) followed first-order release kinetics with a significant regression coefficient.The drug release process was analyzed using the exponential model Korsmeyer-Peppas and Higuchi.The mechanism of drug release followed the Higuchi model with a higher regression coefficient (R 2 ) than the Korsmeyer-Peppas model.The Higuchi model showed drug release by diffusion through the bilayers. 41 Vitro GI Stability of Liposomes and Bilosomes Change in Vesicle Size and PDI of Nanovesicles during Gastric Digestion ►Fig.5 A shows the stability of BME-loaded liposomes LS23 and bilosomes BS3 in the gastric fluid.In the absence of pepsin, the vesicle size of BME-loaded liposomes was 285 nm with a PDI of 0.5.After 1 minute of digestion with SGF, the vesicle size increased to 397 nm, further increasing to 595 nm after 30 min.PDI decreased to 0.40 after 1 minute of digestion and reduced to 0.24 after 30 minutes.In the absence of pepsin, the vesicle size of BME-loaded bilosomes was 84 nm with a PDI of 0.5.After 1 minute digestion with SGF, vesicle size increased to 1421 nm, which dropped to 210 nm after 30 min.PDI was 0.23 after 1 minute of digestion and did not decrease much after 30 minute.According to the findings, liposome's increased size during digestion suggests vesicle aggregation or fusion.The significant drop in pH during the gastric phase, from neutral (pH 7.4 liposomal formulation) to severely acidic (pH 1.5); changes in pH alter the strength and extent of colloidal interaction between particles, permitting liposome coalescence.
Additionally, an osmotic effect due to the pH gradient across the phospholipid membrane (inside-outside the vesicles) could promote destabilization and fusion. 28Studies to stabilize liposomes with bile salts and other surfactants reported an initial increase in the size followed by a decrease in the present study. 42The reduction in the vesicle size after 30 minutes of digestion may correspond to the detachment of   loose aggregates formed initially, and this phenomenon rules out the possibility of a fusion of vesicles.

Change in Vesicle Size and PDI of Nanovesicles during Intestinal Digestion
►Fig.5B illustrates the stability of BME-loaded liposomes LS23 and bilosomes BS3 in simulated intestinal fluid.In the absence of pancreatin, the vesicle size of BME-loaded liposomes was 285 nm with a PDI of 0.5.After 1 minute of digestion with SIF, the vesicular size decreased to 282 nm and 276 nm after 30 min.PDI decreased to 0.37 after 1 minute and further reduced to 0.33 after 30 minute.In the absence of pancreatin, the vesicle size of BME-loaded bilosomes was 84 nm with a PDI of 0.38.After 1 minute of digestion with SIF, the vesicular size increased to 814.7 nm, then decreased to 651 nm after 30 minute.PDI was 0.43 after 1 minute of digestion and 0.74 after 30 minutes.Previous studies showed that the pancreatic enzyme in SIF could modify the organized structure of assembled lipids or damage the liposomal membrane severely, indicating that the liposomes exhibited lower stability in SIF than in simulated gastric fluid. 43The kinetic stability of the liposomal bilayer can be influenced by its composition and surface properties.Cholesterol, a principal constituent in many biological membranes, can increase bilayer thickness and membrane ordering and even reduce membrane permeability.Incorporating cholesterol has been assumed to provide physicochemical stabilization to liposomes. 44

In Vivo Cognitive Enhancement Activity Studies of Nanovesicles
BMs memory-enhancing and cognitive effects are believed to be mediated by bacoside A (bacoside A3, bacopacide II, bacopasaponin C, and bacopaside X). 45 A recent report shows that BME inhibits selected human cytochrome P450 (CYP) drug metabolizing enzymes, in addition to altering the expression of rat hepatic and intestinal CYP drug metabolizing enzymes and intestinal P-glycoprotein. 46Thus, it is conceivable that the bacoside constituents in BME may be metabolized in vivo to active forms before exerting their pharmacological effects.►Fig.6A and 6B depicts the nootropic effect of BME-loaded liposomes and bilosomes on Swiss albino mice, compared with a formulation.Bilosomal delivery of BME significantly improved behavioral characteristics in dementia-induced mice than the extract, possibly because of the better absorption from GIT and better permeability via the BBB.►Table 4 provides the number of Y maze alternations reflecting the nootropic effect of BME and nanovesicles (LS23 and BS3).The bilosomes treated group showed more alternations in the Y-maze apparatus than the BME and liposomal vesicles.Better nootropic performance might be attributed to higher entrapment efficiency and, smaller bilosomes enhanced GIT penetration.The protective effects of sodium deoxycholate on vesicle integrity might have improved BME's oral absorption and bioavailability. 47Including negatively charged lipids changes the vesicle surface charge, affecting biodistribution, and clearance. 48opolamine, a muscarinic antagonist, phosphorylates tau proteins leading to the formation of β-amyloid in the brain, resulting in memory impairment. 49Studies have shown that ethanolic extract of BM could prevent β-amyloid-induced toxicity in cultured neurons by inhibiting acetylcholinesterase enzyme and lipid peroxidation. 50In transgenic mice bearing human PS-1 and AßPP mutations, long-term treatment of BME reduces β-amyloid in different brain regions, possibly the reason behind cognitive enhancement. 51In the animal model of dementia, Brahmi Ghrita (an ayurvedic preparation containing BM in ghee base) has shown a positive effect, possibly by reversing the depletion of acetylcholine, decreasing the choline acetyltransferase activity, and reducing muscarinic, cholinergic receptor binding in the frontal cortex and hippocampus, but also by alleviating cholinergic degeneration, lowering norepinephrine, and increasing 5-hydroxytryptamine levels in the hippocampus, hypothalamus, and cerebral cortex.ÃÃÃ p < 0.001 compared with that of Group I. ##p < 0.01 compared with that of Group II (repeated measure ANOVA followed by Tukey's post hoc test).

Conclusions
Due to suboptimal bioavailability, many phytoconstituents fail to validate ethnopharmacological claims in full measure.One cannot employ higher doses to overcome obstacles such as incomplete absorption, first-pass effect, etc.In this context, the present study has demonstrated the possibility of enhancing the pharmacological effects of BME by loading them in bilosomes.Various phytoconstituents with promising therapeutic or nutritional benefits can be formulated as vesicles.Highquality bioavailability evidence would entail using more advanced and direct techniques such as radiolabeling.

Funding/Acknowledgments
The authors are grateful to the Management of Nitte (Deemed to be a University) for the laboratory facilities and support for conducting this study.We acknowledge the kind help offered by Dr. Unnikrishnan M K, Professor, NGSM Institute of Pharmaceutical Sciences, for the language corrections while writing the manuscript.
Nootropic activity was evaluated with the help of a Y maze spontaneous alternation test using Male Swiss albino mice.The animals were housed and maintained in large, spacious polyacrylic cages at ambient room temperature with a 12hour light/12-hour dark cycle.The animals were fed with water and a standard rat pellet diet ad libitum.The study was approved by the Institutional Animal Ethics Committee (IAEC) of NGSM Institute of Pharmaceutical Sciences with approval number NGSMIPS/IAEC/JUNE-2020/191.Male Swiss albino mice (22 to 30 g) were randomly divided into five groups of five animals each.Group I: Scopolamine (0.5 mg/kg) Group II: Normal control (saline) Group III: Scopolamine þ BME (200 mg/Kg) Group IV: Scopolamine þ Liposome (LS23) Group V: Scopolamine þ Bilosome (BS3) formulation.Therefore, infrared spectroscopy can be a tool to select chemically compatible excipients.The compatibility between soy lecithin, cholesterol, sodium deoxycholate, and BME was established using an FTIR spectrophotometer.Absorption peaks are shown in ►Fig.3A.FTIR spectra obtained with the alcoholic BME were concordant with the literature available.The prominent peaks associated with BME, namely O-H bonded (3650-3600), C-H stretch (3000-2850), C ¼ O (Ketone) (1725-1705), C ¼ C (Alkene) (3319-3383), CH 3 -R(Alkyl) (2944-2882) and C-O bond (furanosyl, pyranosyl) (1075-1018) were observed.The constituent peaks were present in both formulations (►Fig.3B and 3C), thereby ruling out the possibility of any chemical incompatibility between the extract and the lipids used.

Fig. 4
Fig. 4 In vitro drug release pattern of BM-loaded liposomes LS23 and BME loaded bilosomes BS3 (values are average of three trials).

Fig. 6
Fig. 6 Number of alternations of test and control groups.All values of mean AE SEM (n ¼ 5).Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001 compare with that of group I. ##p < 0.01 compare with that of Group II (repeated measure ANOVA followed by Tukey's post hoc test).

Table 1
Formulation chart of liposome and bilosome formulations showing the method of preparation and their composition

Table 2
Data for estimation of vesicle size, polydispersity index (PDI), zeta potential and extract Loading of vesicles