FIELD OF THE INVENTION
The present invention relates to a herbal formulation, particularly in form of herbal nanoemulsion exhibiting enhancement in learning and memory, lipid peroxidation, total antioxidant status and gene expression in Alzheimer’s disease (AD). More particularly, the present invention relates to a herbal nanoemulsion comprising flax seed oil (FSO) and beetroot extract (BTE). The present invention also relates to a process of preparation of herbal nanoemulsion for the effective management and reversal of AD and associated conditions.

BACKGROUND OF THE INVENTION
Alzheimer’s disease (AD) is a neurological ailment that affects most individuals on a progressive scale all over the globe. AD is distinguished by a deterioration in the neuronal cells of the various regions of the brain including the hippocampus, amygdala and cerebral cortex. This degeneration leads to a decrease in the production and release of the neurotransmitter acetylcholine (Ach), as well as a deposition of amyloid beta (Aß), which ultimately results in dementia. More than 55 million people around the world have dementia. Between 60-80 % of all dementia cases are AD. Researchers further predict the number of people living with AD will increase to almost 153 million by 2050. In the majority of instances, approximately 69% of the patients hail from Asian nations including India and China. According to the findings of recent research, AD places a significant burden, both financially and socially, on the families and carers of senior AD patients who are impacted by the disease. This is mostly the result of delayed diagnosis and inadequate treatment. Only 20-50% of people who have AD are diagnosed and get the appropriate primary care for their condition. In most cases, delaying treatment is caused by a lack of access to professional therapy, care and coordinated support services.
Although most developed countries have access to conventional AD treatment options, the therapeutic index is still inadequate in reversing the state of AD. In the case of generic drugs, they are ineffective and unaffordable for most people living in impoverished nations. Chemically manufactured medications may be toxic and lead to unpleasant side effects; in contrast. Pharmaceutical drugs are both prohibitively costly and difficult to get.
The rising prevalence, linked impairment, and frightening health quality with a raised death rate in both emerging and established nations sparked significant interest in therapeutic management research with a suitable compound for controlling AD in terms of the drug’s efficacy, accessibility and safety. According to recent research, the incidence of AD is less likely to be identified in patients who consume a therapeutic diet.
Worldwide, people rely on flax seed oil (Linum usitatissimum) and beetroot (Beta vulgaris) extract (BTE) for their nutritional needs. Flax seed oil (FSO) is rich in several nutrients, including the important fatty acids omega-6 (n-6) and omega -3 (n-3) polyunsaturated fatty acids (PUFAs) found in plants including linoleic acid (LA) and alpha-linolenic acid (ALA), as well as lignans, proteins and vitamin E. A few different hypotheses attempt to explain how omega-3 fats may slow or stop the deterioration of nerve cells related to AD. Due to the fact that the body converts n-3 fatty acids into eicosanoids that have neuroinflammatory properties, n-3 fatty acids are thought to have anti-neuroinflammatory benefits. It has been proposed that n-3 PUFAs, as a ligand of cannabinoid receptors, enhance memory and learning by enhancing neurotransmitter release, synapse formation and neurogenesis in the brain.
In a similar vein, beetroot (Beta vulgaris L.) has been used for centuries due to its widespread availability and well-documented health benefits. It is a vegetable that has a strong antioxidant potential, which may be explained by the concentrations of betalains in the vegetable. The name betalains refer to a group of pigments that may be either red (betacyanin) or yellow (betaxanthins). Indeed, beetroot was formerly widely employed as a food dye. Betalain is a naturally occurring antioxidant that shows intriguing promise in the treatment of oxidative stress-related diseases such as neuroinflammation, metabolic syndrome, and cardiovascular diseases. These conditions are all linked to oxidative stress. Betalain has recently shown potential as an inhibitor of specific chemical events in the brain that are implicated in the development of AD according to recent studies.
US8017147B2 discloses a synergistic mixture (which may be utilized as a food or a drink or a supplement or a drug or a cosmetic or a hygienic product) that is formulated and is capable of improving a person's well-being, lowering the risks of cardiovascular and/or Alzheimer's diseases and/or lowering blood sugar using natural and synthetic ingredients. Numerous ratios may be formulated for aroma, colour, flavor, flow (viscosity), taste and uniformity. Moreover, ingredients for sugar substitutes, natural preservatives, nano-dispersion, nano-emulsion, nano-encapsulation of ingredients and apparatus for personalized nutrition are also described. It provides that Sprouted Flaxseed has many health benefits. Sprouting doubles, the antioxidant (ORAC) value of flaxseed. Nutrients such as enzymes, amino acids, and vitamins are substantially increased and become more bioavailable, allowing for better absorption. The “anti-nutrients” such as phytic acid, enzyme inhibitors and insoluble fibers are decreased. It provides various mixtures showing synergism and also mentions beta vulgaris as one of the usable plant ingredients.
Sidra Rehman et. Al. (2022) “The Insight of In Silico and In Vitro evaluation of Beta vulgaris phytochemicals against Alzheimer’s disease targeting acetylcholinesterase” explores the potential bioactive phytochemicals of B. vulgaris as an alternative therapeutic agent against AD by conducting in vitro and in silico studies. The phytochemical compounds were analyzed for their binding affinities towards the acetylcholinesterase (AChE) enzyme. Virtual screening identified three hit compounds including betanin, myricetin and folic acid with least binding score compared to the reference drug, donepezil (-17 kcal/mol). Further, in vitro studies for the anti-acetylcholinesterase activity of betanin and glycine betaine were performed. Dose-response analysis showed 1.271 µM and 1.203 µM 50% inhibitory concentration (IC50) values for betanin and glycine betaine compounds respectively. The findings indicate that phytoconstituents of B. vulgaris can be implicated as an alternative therapeutic drug candidate for cognitive disorders like Alzheimer’s disease.
Doha Mohamed (2021) “Protective effect of dietary supplements against streptozotocin-induced Alzheimer’s disease in mice” prepared two dietary supplements (DS) rich in betalains, anthocyanins, and omega-3 fatty acids and evaluated the protective effect of DS against AD. Two dietary supplements (DS I and DS II) were prepared; the first one was a mixture of anthocyanin-rich extract of purple carrot and flaxseed oil (DS I), while the second was a mixture of betalains-rich extract of beetroot and flaxseed oil (DS II). AD was induced in mice by intracerebroventricular (ICV) injection of streptozotocin (STZ) (3 mg/kg). Biochemical changes in brain tissue and plasma were determined. The Behavioural of mice was evaluated through Y–maze test, the Morris water maze, and the novel object recognition test. Changes in brain tissues were assessed through histopathological examination. In vitro, the antioxidant activities of DS I and DS II were evaluated. Also, the contents of total phenolics, anthocyanins, betalains, and fatty acids profiles were assessed. Results showed that both DS investigated in the present study showed significant improvement (P < 0.05) in acetylcholinesterase, antioxidant enzymes, tumour necrosis factor-a (TNF-a) and malondialdehyde (MDA)in brain tissue and butyrylcholinesterase in plasma in association with amelioration in the behavioural tests and histopathological changes of the brain tissue. Thus, both DS showed protective effects against STZ-induced AD in mice due to the presence of anthocyanins, betalains, and omega-3 fatty acids.
Megan Craig (2022) “Bioactive-Loaded Nanotechnology Applications for Drug Discovery” teaches that Plant-based medicines have some drawbacks, such as low lipid solubility, instability, and the need for a well-validated method for ingredient extraction and purification. Furthermore, manufacturers must overcome these restrictions and ensure that the product is stable enough for patients to consume safely. Newer improved drug delivery systems (DDS) for plant-based therapies have been designed to eliminate these constraints. Liposomes, phytosomes, ethosomes, transferosomes, nanostructured lipid carriers (NLCs), cubosomes, solid lipid nanoparticles (SLNs), exosomes, microspheres, nanoparticles, and nanoemulsions are all examples of these. It further teaches that Linseed Oil nanoemulsion obtained from Linum usitatissimum seed provides improved stability and physicochemical properties for topical applications, suitable for atopic dermatitis evaluated through in vitro and in silico studies. The free drug suffers drawbacks of Poorer stability and penetration through the skin membrane.
Despite having tremendous potential, there is no stable formulation of flax seeds and beetroot for the management of AD and associated conditions.

OBJECT OF THE INVENTION
In order to obviate the drawbacks in the existing state of the art, the main object of the present invention is to provide an herbal composition of Linum usitatissimum (Flax) and Beta vulgaris (beet) for the effective management and reversal of AD and associated conditions.
It is another object of the present invention to provide an herbal nanoemulsion of flax seed extracts and beetroot extracts for the effective management and reversal of AD and associated conditions.
It is a further object of the present invention to provide an herbal nanoemulsion comprising bioactive fractions obtained from flax seeds and beetroot extracts for the effective management and reversal of AD and associated conditions.
It is a further object of the present invention to provide an herbal nanoemulsion comprising bioactive fractions prepared from flax seeds and beetroot extracts showing reversed streptozotocin effects and reducing the beta-amyloid plaques in the AD brain.
It is yet another object of the present invention to provide a safe, non-toxic, herbal-based nanoemulsion for the treatment of AD and associated conditions.
It is yet another object of the present invention to provide the process of preparation of an herbal nanoemulsion comprising bioactive fractions prepared from flax seeds and beetroot extracts for the management and treatment of AD and associated conditions.

SUMMARY OF THE INVENTION
Accordingly, the present invention provides an herbal nanoemulsion for the management of AD and associated conditions, said nanoemulsion is an oil-in-water (O/W) nanoemulsion comprising flax seed extract, beetroot extract, surfactant and water.
More particularly, the present invention provides an herbal nanoemulsion for the management of AD and associated conditions, said nanoemulsion is an oil-in-water (O/W) nanoemulsion comprising bioactive fractions obtained from flax seed extracts, beetroot extracts, surfactant and water.
More particularly, the present invention provides an herbal nanoemulsion for the management of AD and associated conditions, said nanoemulsion is an oil-in-water (O/W) nanoemulsion comprising flax seed oil extracts (FSO), beetroot extracts (BTE), surfactant and water.
In yet another embodiment the present invention provides a process for the preparation of nanoemulsion comprising bioactive fractions of seed extracts of Linum usitatissimum and root extracts of Beta vulgaris.
In another embodiment, the present invention provides a process for the preparation of nanoemulsion comprising flax seed oil extracts (FSO) and beet root extracts (BTE).

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts the binding interactions of palmitic acid and (n-3)–a-Linolenic acid (ALA) compounds with CB1, CB2 and FAAH enzyme.
Fig. 2 depicts the binding interactions of betanidin and betanin compounds with CB1, CB2 and FAAH enzymes.
Fig. 3 depicts the % Cumulative Drug Release (CDR) of nanoemulsion (BFSON-6, FSOF and BTEF).
Fig. 4 depicts the effect of FSOF (flax seed oil formulation), BTEF (beetroot extract formulation), donepezil and BFSON on mean transfer latency in MWM task in STZ-induced AD in mice.
Fig. 5 depicts the effect of FSOF (flax seed oil formulation), BTEF (beetroot extract formulation), donepezil and BFSON on olfactory discrimination task in STZ-induced AD in mice.
Fig. 6 depicts the effect of FSOF (flax seed oil formulation), BTEF (beetroot extract formulation), donepezil and BFSON-6 on transfer latency task in STZ-induced AD in mice.
Fig. 7 depicts the effects of BFSON-6 on STZ-induced oxidative stress in mice’s hippocampus
Fig. 8 depicts the neuroprotective effect of BFSON-6 formulation in STZ-induced AD in mice.
Fig. 9 depicts Histopathological analysis of mice’s hippocampus tissue: A) Normal control mice exhibiting the normal histological structure of pyramidal neurons. B) Mice’s hippocampus of STZ-injected mice (3?mg/kg) showing disorganization and areas of cell loss of small pyramidal cells (arrow). C) Mice treated with donepezil (2?mg/kg) showing the apparent normal histological structure of neurons. D) Mice treated with FSOF showing necrosis of sporadic pyramidal neurons (arrow). E) BTEF-treated mice showing necrosis of some pyramidal neurons (arrow). F) BFSON-treated mice showing necrosis of some pyramidal neurons (arrow).

DETAILED DESCRIPTION OF THE INVENTION WITH ENABLING EMBODIMENTS AND NON-LIMITING EXAMPLES
The present invention provides an herbal nanoemulsion for use in the therapeutic management of Alzheimer’s disease (AD) and associated conditions. The herbal nanoemulsion comprises an organic solvent fraction of flax seeds (Linum usitatissimum) and aqueous alcoholic extracts of beetroot (Beta vulgaris).
The organic solvent is selected from hexane, methanol, acetone, petroleum ether, ethanol and dichloromethane.
The aqueous alcoholic is selected from hydromethanolic solvent, acidic water, and acidified alcoholic solution.

In an embodiment, the flax seed extract is prepared by subcritical hexane oil extraction to obtain a yellow colour oil extract of flax seeds (FSO). The beetroot extract is prepared by hydromethanolic extraction to obtain a red colour beet root extract in powder form (BTE).
The FSO and BTE have been qualitatively and quantitatively analyzed by Gas chromatography–mass spectrometry (GC-MS) and Liquid chromatography–mass spectrometry (LC-MS) respectively. In FSO and BTE, a total of six compounds and ten compounds have been chemically characterized respectively. Palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and behenic acid have been the main constituents in FSO. Similarly, 2,17-bidecarboxybetanin, 2,17-decarboxy-neobetanin, 2-decarboxybetanin, 2-decarboxy-neobetanin, 15-decarboxy-betanin, 17-decarboxybetanidin, 17-decarboxy-betanin, 17-decarboxy-neobetanin, betanidin and betanin have been the main constituents in BTE.
Binding interactions of identified compounds from FSO and BTE extracts via LC-MS, molecular docking analyses were performed on cannabinoid receptors CB1 and CB2 and FAAH using the Discovery studio 2.0 LibDock program. The compounds identified from the FSO and BTE exhibited significant binding interactions with FAAH, CB1 and CB2. As shown in Fig. 1, FSO’s compounds palmitic acid, stearic acid, (n-9)–Oleic acid, (n-6)–Linoleic acid, (n-6)–g-Linolenic acid and (n-3)–a-Linolenic acid (ALA) showed excellent binding energy with CB1; 90.12, 36.53, 68.38, 89.19, 91.56 and 91.56 Kcal/mol, CB2; 78.23, 99.94, 83.79, 90.65, 78.23 and 78.23 Kcal/mol, FAAH; 100.23, 95.23, 86.36, 72.15, 100.23 and 100.23 Kcal/mol respectively. Similarly, as shown in Fig. 2, BTE’s compounds viz 2,17-bidecarboxybetanin, 2,17-decarboxy-neobetanin, 2-decarboxybetanin, 2-decarboxy-neobetanin, 15-decarboxy-betanin, 17-decarboxybetanidin, 17-decarboxy-betanin, 17-decarboxy-neobetanin, betanidin and betanin showed binding energies with CB1; 86.12, 92.31, 88.90,87.19, 83.87, 83.10, 78.90, 73.12, 89.17, 76.15 and 88.32 Kcal/mol, CB2; 86.17, 89.09, 89.43, 91. 90, 85.67, 89.12, 89.17, 88.13, 82.16 and 88.12 Kcal/mol, FAAH; 95.12, 98.15, 97.21, 10.32, 90.78, 83.87, 80.10, 10.43, 85.09, 90.12 Kcal/mol. Moreover, these compounds showed various binding interactions including hydrogen bonds, hydrophobic bonds, vander-walls and pi-pi bond interaction with the different amino acids of the active binding sites of the CB1, CB2 and FAAH respectively. Leu341, Thr344, Ser401, Tyr397, Arg150, Tyr209, Leu213, Arg238, Asp1158, Ser241, Gly216 and Ser193 found within the substrate recognizing active sites of CB1, CB2 and FAAH receptors were involved in binding with identified compounds.
In another embodiment, the nanoemulsion is prepared with the FSO, BTE, water and a surfactant. The surfactant is selected from sorbitan fatty acid esters, (Spans), polysorbates (Tweens), and poloxamer (Pluronics) (Table 1). In a preferred embodiment, the surfactant is tween 80.
BFSON has been prepared using cold homogenization and probe sonication process. The quantity of beetroot extract remained constant in the formulation and the formulation has been optimized as mentioned in Table 1.

Table 1 Optimization of BFSON
Run Order Quantity Time for probe sonication (min)
BTE (g) FSO (ml) Tween 80 (ml) Water (ml)
BFSON-1 2 6 6 88 10
BFSON-2 2 6 6 88 20
BFSON-3 2 12 6 82 10
BFSON-4 2 12 6 82 20
BFSON-5 2 6 12 82 10
BFSON-6 2 6 12 82 20
BFSON-7 2 3 6 91 10
BFSON-8 2 3 6 91 20

In another embodiment, the present invention provides a herbal nanoemulsion for the management of AD and associated conditions, said nanoemulsion comprising bioactive fractions obtained from flax seed oil extracts, beetroot powder extracts, water and tween 80.
The ratio between beetroot extract and flax seed oil in the polyherbal nanoemulsion is in the range of 100:3 to 100:12.
In an embodiment, the ratio between beetroot extract and flax seed oil in the polyherbal nanoemulsion 100:6.
In yet another embodiment, the present invention provides a herbal nanoemulsion for the treatment of AD and associated conditions, said nanoemulsion comprises bioactive fractions obtained from flax seed extract, beetroot extract, tween 80 and water.
In yet another embodiment, the amount of the tween 80 in the nanoemulsion ranges from 6 to 12 ml. In a preferred embodiment, the amount of the tween 80 in the nanoemulsion is 12.
In another embodiment, the present invention provides a process for preparing the herbal nanoemulsion comprising bioactive fractions of flax seeds, beetroot, surfactant and water, said process comprising:
(a) preparing beetroot extract in an aqueous alcoholic solvent,
(b) preparing flax seed extract in an organic solvent,
(c) adding surfactant and the beet root extract of step (a) in a continuous phase, wherein said continuous phase is preferably water,
(d) emulsifying for 15 min at 1000 rpm,
(e) homogenizing by adding flax seed extract of step (b) drop-wise,
(f) conducting probe sonication for 10 min to 20 min to obtain the nanoemulsion.

In an embodiment, the beetroot extract is hydromethanolic beetroot extract (BTE).
In an embodiment, the organic solvent is hexane and the flax seed extract is flax seed oil (FSO).
In an embodiment, the continuous phase is water.

The nanoemulsion has been characterized for the determination of globule size (GS), polydispersity index (PDI) and zeta potential (ZP) in 8 run orders by known methods. The GS, ZP and PDI are shown in Table 2.

Table 2 Characterization (Zeta size, Zeta potential and PDI) of herbal nanoemulsion
Run Order Quantity of BTE Quantity of FSO Quantity of tween 80 Time for probe sonication (min) Zeta size (Experimental) Zeta potential PDI
BFSON-1 100 12 6 10 187.65 -14.5 0.226
BFSON-2 100 3 6 20 200.71 -21.8 0.326
BFSON-3 100 3 6 10 144.75 -25.1 0.215
BFSON-4 100 3 6 20 155.85 -27.3 0.189
BFSON-5 100 6 12 10 109.99 -23.7 0.145
BFSON-6 100 6 12 20 104.11 -29.6 0.108
BFSON-7 100 12 6 10 185.31 -6.6 0.196
BFSON-8 100 3 6 20 186.85 -16.9 0.221

Accordingly, the nanoemulsion of 1:2 ratio of FSO and surfactant displayed the smallest GS (100.2±0.36) and produced more uniform stable droplets at 20 mins sonication time.
To determine the bioactive fraction (also referred to herein as drug) and entrapment efficiency (EE), the nanoemulsion has been centrifuged to create a clear supernatant and the supernatant was analysed using a UV/Vis spectrophotometer, with HPLC water serving as a standard.
The following formula has been used to calculate the % EE

The total bioactive fraction of the nanoemulsion particle is in the range of 1.10±0.78mg to 2.54±0.05 and the entrapment efficiency ranges from 93.12±0.18 to 98.64±1.21. The pH of the nanoemulsion is 6.9±0.13.
The acute toxicity study reported that the nanoemulsion is safe up to 2000 mg/kg dose.
Stability tests of nanoemulsion
The herbal nanoemulsion is stable for at least three months when stored at two different temperatures, i.e., 4°C±2 and 27°C±2. The developed nanoformulation (BFSON) offered a sustained release of bioactives/drug with a CDR of 96.69% for a period of 48h. However, the conventional formulations of flax seed oil (FSOF) and beetroot extract (BTEF) demonstrated a comparatively faster rate of bioactives/drug release i.e., 95.87% at 4h and 95.55% at 8h respectively. Comparative release behaviour of BFSON and the conventional formulations FSOF and BTEF has been provided in Fig. 3.
Stability studies of nanoemulsion have been conducted on storage at refrigerated temperature (4?C±2) and room temperature (27?C±2). In order to determine whether or not phase separation happened in the nanoemulsion, the nanoemulsion samples have been placed in a 20 ml measuring cylinder and kept at 4?C±2 and 27?C±2 for a period of 21 days (arancibia2016). In the case of O/W nanoemulsion, the density of the continuous phase is greater than that of the dispersed phase. As a consequence of this, creaming takes place as a result of phase separation (Bernardi2011, Romes2021). The values of the creaming index (CI) have been determined in accordance with the phase separation in order to ascertain the nanoemulsion’s degree of stability. Using the following formula, CI was calculated (arancibia2016).

Hs is the volume of the cream layer (mL) and He is the volume of the emulsion (mL).
Similarly, the ZP, GS and PDI values of the nanoemulsion of run orders 5 and 6 have been measured at both temperatures 4?C±2 and 27?C±2 after 30, 60 and 90th days respectively (Table 2, Table 3).

Table 3 Effect of temperature on the characterization of nanoemulsion at 27?C±2
Parameters BFSON-5 (days) BFSON-6 (days)
30 60 90 30 60 90
PS 101.32±0.11 115.90±0.23 120.90±0.99 100.47±0.41 103.36±0.74 113.00±0.12
PDI 0.258±0.10 0.250±0.14 0.269±0.45 0.175±0.00 0.184±0.11 0.191±0.15
ZP -31.14±0.29 -32.14±0.00 30.35±0.08 -29.12±0.17 -28.41±0.15 -30.12±0.96

Table 4 Effect of temperature on the characterization of nanoemulsion at 4?C
Parameters BFSON-5 (days) BFSON-6 (days)
30 60 90 30 60 90
PS 112.21±0.00 120.14±0.41 130.26±0.52 105.41±0.42 111.19±0.79 119.23±0.99
PDI 0.2 74±0.11 0.279±0.47 0.284±0.15 0.189±0.19 0.192±0.46 0.205±0.57
ZP -30.74±0.00 -34.00±0.28 -35.84±0.76 -30.11±0.29 -31.49±0.00 -32.67±0.76

Pharmacokinetic Studies:
The kinetics of the drug’s release from the nanoemulsion is characterized using five distinct models of the kinetics of drug release. These models are used in the process of determining the kinetics and mechanisms involved in the release of the drug from the nanoemulsion. The value of the correlation coefficient (r) in each of the several models is used to decide which model provides the best fit to the release data. The model with the highest r-value is the most convincing candidate for explaining the observed scatter in the release statistics. It was necessary to use a variety of kinetic models, such as the zero-order model, the first-order model, the Hixson-Crowell model, the Higuchi model, and the Korsmeyer-Peppas plot in order to get a comprehensive understanding of the kinetics of the drug release. Korsmeyer-Peppas release kinetics (0.888), which indicates that the drug release from the system follows Super case II transport, was used in all of the aforementioned models for the optimal nanoemulsion (Fig. 3).
In-vivo anti-Alzheimer activity
Swiss albino mice of either sex (25-35 gms) were randomly divided into five different groups comprised of six animals in each. Streptozotocin (STZ, 3 mg/kg, intracerebroventricular (I.C.V.) route, single dose) was administered for induction of AD. Nanoemulsion and BTE formulation were administered after the fifteenth day of administration of STZ by oral route for consecutively ten days and behavioural, biochemical, gene expression, histopathological parameters were evaluated using different assays. The nanoemulsion prevented memory impairments induced by STZ in three memory tasks, i.e. Morris water maze test (WMM), crop gap test (CGT) and olfactory recognition test (ORT), which are commonly used to validate behavioural impairment in STZ-treated rats (Fig. 4-6).
Morris water maze test
Animals' spatial memory and learning were examined in the Morris water maze (Morris, 1984). The Morris water labyrinth is made up of a sizable pool with a circumference of 120 cm, a height of 50 cm, and a depth of 30 cm with water temperature settings (25±1?C), all of which were set up in a dimly lit room. A circular platform with a diameter of 8 cm and that is submerged in the water to a height of 1 cm was placed within the pool so that the mice may climb it and get out of the water so they can continue swimming. To acclimate the mice to the Morris water maze, trials were given for 10–14 consecutive days. Every trial began from the same place. The mice had 120 seconds to locate the secret platform, after which they would have 30 seconds to rest and investigate it.
The paths taken by each mouse were recorded and examined using a video camera (ANY-maze video tracking system). Path efficiency, average swimming speed (m/s), total distance travelled, and escape latency (time needed to reach the platform as a goal) were among the starling collection of parameters that were observed (an index of the efficiency of the path taken by the animal to get from the first position to the last position, i.e. the shortest distance from the first position to the platform divided by the swimming distance, with a value of 1 indicating perfect efficiency). The treatment schedules were followed according to Table 5.
Table 5: Experimental design (n=6) according to treatment schedule
Groups Groups
Group I Control
Group II STZ (3 mg/kg, I.C.V.)
Group III Donepezil (2 mg/kg, oral)
Group IV FSOF (0.5 ml, oral)
Group V FSOF (1 ml, oral)
Group VI BTEF (0.5 ml, oral)
Group VII BTEF (1 ml, oral)
Group VIII BFSON-6 (0.5 ml, oral)
Group IX BFSON-6 (1 ml, oral)

Repeated experiments and a two-way ANOVA revealed a statistically significant overall impact of treatment, as well as an interaction between time and treatment. The efficiency of formulations of each of BTEF, FSOF and BFSON on STZ-induced AD in mice was evaluated. After the STZ administration mice showed forgetfulness characteristics and increased escape latency time of 241.89±2.36 sec to reach the platform as compared to the control mice group escape latency time of 16.12±1.25 sec. After the treatment of 10 days with BTEF, FSOF and BFSON, overall performance to find the platform in the water tank was increased and escape latencies were decreased significantly when compared to the control mice group. In FSOF (1ml), BTEF (20 mg/kg) and BFSONs (1 ml) treated groups escape latencies were 21.65±2.84, 31.76±0.71, 8.96±2.96 sec (Figure 4). When these results were compared with the marketed drug Donepezil administered group, results were found significant 16.89±2.52 sec as shown in Figure 4.
Olfactory recognition test
Animals were dehydrated for 12 hours prior to training and then given ad libitum water for 30 minutes during the test. The olfactory apparatus consisted of a rectangular box (30 X 30 X 55 cm) containing an empty odour container. Inducing agents, standard drugs, and test compounds were administered to the animals. The mice were first introduced to the cage for 2 minutes and 20 minutes before testing, and the containers were placed in two corners of the box. The odour was then presented by placing the odour stimulus, such as peppermint oil, mint oil, chocolate solution, and so on, in a container and placing it in a cage corner. The odor's habituation was determined over three trials of two minutes each, with a 20-minute intertribal interval. The following measures were taken: latency to investigate the odour (s), total duration (s), and the number of sniffs per trial. The time it took the animal to place its nose within 1 cm of the container was the latency to investigate the odour. Each sniff lasted as long as the nose was within 1 cm of the container. The total duration of sniffing for each animal during the trial was then calculated. The number of sniffs per trial was determined by the number of times the animal's nose came within 1 cm of the container. Throughout the first three trials, the same odour was used, and on the fourth trial, a new odour was presented. The treatment plan was based on the radial arm maze assay.
It is well established that both human and animal sniffing memories are formed and developed in the hippocampus region of the brain. Since this task combines an exploration behaviour component with a memory retention component such that an animal must have thoroughly investigated the familiar olfaction during the pretest phase in order to differentiate it from the new olfaction later during the test phase. In the sample phase testing, which employed two identical flavour waters, all animal groups showed any substantial variation in flavouring discrimination. During the test phase, FSOF, BTEF and BFSON-treated groups spent substantially more time examining the new flavour than the familiar one (p?