The novel test formulation designed was constituted with pyridoxine hydrochloride (vitamin B₆), calcitriol, zinc chloride, magnesium (II) gluconate, and β-carotene (retinol, Provit A), which were purchased from TCI, Japan. Copper chloride, cyanocobalamin (vitamin B₁₂), calcium chloride, vitamin E (alpha-tocopherol), cholecalciferol (vitamin D₃), iron (II) sulfate, and sodium carboxymethyl cellulose (Na-CMC) were procured from Sigma-Aldrich, USA. Ascorbic acid (vitamin C) and sodium selenate were obtained from Alfa Aesar, India. Cannabidiol isolate and Panax ginseng extract were obtained from Panacea Phytoextracts, India and Standard Hemp Company, USA, respectively. Imipramine Hydrochloride was purchased from Sigma, USA. For the estimation of brain sleep biomarker such as melatonin, BDNF (Brain-derived neurotrophic factor), PGD2, Leptin, Orexin-A, and acetylcholine, specific ELISA kits were used such as for detection, which were procured from CUSABIO and My Bio Source, USA respectively.

Randomly breed male Sprague Dawley (SD) rats with body weight ranges from 200 to 300 gm were used in this study. The animals were purchased from M/s. Vivo Bio Tech, Hyderabad, India. Animals were randomly divided into nine groups based on their body weights consist of 6 animals of each group. They were kept individually in sterilized polypropylene cages with stainless steel top grill having provision for holding pellet feed and drinking water bottle fitted with stainless steel sipper tube. The animals were maintained as per standard protocol throughout the experiment.

The test formulation consisted each constituents was divided into two parts. One part of each ingredients of the test formulation did not receive any sort of treatment and was labeled as the untreated or control sample, while the second part of each ingredients of the test formulation was treated with the Trivedi Effect^® - Energy of Consciousness Healing Treatment (Biofield Energy Treatment) by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi under standard laboratory conditions for ~3 minutes. The novel test formulation was consisted of zinc chloride, iron (II) sulfate, copper chloride, vitamin B₆, vitamin B₁₂, vitamin D₃, sodium selenate, calcium chloride, ascorbic acid, vitamin E, beta carotene, Panax ginseng extract, cannabidiol, and magnesium (II) gluconate. Besides, three group of animals also received Biofield Energy Healing Treatment (known as the Trivedi Effect^®) by Mr. Mahendra Kumar Trivedi under similar laboratory conditions for ~3 minutes in the research laboratory of Dabur Research Foundation, New Delhi, India. The energy transmission was done without touching the samples or animals. After that, the Biofield Energy Treated samples was kept in the similar sealed condition and used as per the study plan. In the same manner, the control test formulation group was subjected to sham healer under the same laboratory conditions for comparison purposes. The sham healer did not have any knowledge about the Biofield Energy Treatment. The Biofield Energy Treated animals were also taken back to experimental room for further proceedings.

Seven days after acclimatization, animals were randomized and grouped based on the body weight. The test formulation was prepared freshly prior to dosing and administered to the animals using an oral intubation needle attached to an appropriately graduated disposable syringe. The dose volume was 10 mL/kg in morning and evening based on body weight. The experimental groups were divided as G1 as normal control; G2 as disease control (UCS: Unpredictable Chronic Stress with 0.5% CMC); G3 as reference item (UCS along with imipramine hydrochloride, 30 mg/kg); G4 includes UCS along with untreated test formulation; G5 as UCS along with Biofield Energy Treated test formulation); G6 group includes UCS along with Biofield Energy Treatment per se to animals from day -15; G7 as UCS along with Biofield Energy Treated test formulation from day -15; G8 group includes UCS along with Biofield Energy Treatment per se plus Biofield Energy Treated test formulation from day -15), and G9 group denoted UCS along with Biofield Energy Treatment per se animals plus untreated test formulation. G1 and G2 animals were treated orally with 0.5% w/v CMC-Na in distilled water for 8 weeks (From day 1 to 56). Group G3 animal was treated orally with reference item, imipramine hydrochloride at a dose of 30 mg/kg body weight for 8 weeks. The freshly prepared suspensions of untreated test formulation and Biofield Energy Treated Proprietary Product was administered orally to the G4 and G5 group animals, respectively, at a dose of 1257.80 mg/kg body weight in the morning and 2012.75 mg/kg body weight in the evening, respectively for 8 weeks. G6 group was not to be dosed with the test formulation. In addition; G7 and G8 group were dosed similar to the G4 and G5 dosing regimen, but from the day of Biofield Energy Treatment (i.e. from day-15 to day 56). G9 group, Biofield Energy Treated per se animal was treated with untreated test formulation for 8 weeks. Body weight and clinical signs were taken daily throughout the experimental period. All the animals except G1 group received stress induced procedures such as stress procedures like sound stress, tilted cages and crowd stress, cold and warm water swim stress, food and water deprivation, stress due to change in the light and dark cycle were undergo seven different types of unpredictable stress procedures after scheduled dosing daily at specified interval to the end of the experiment for 8 weeks after the initiation of stress, which vary every week interval i.e. shuffling of stress type. At the end of (8 week) experimental period, all the animals were individually subjected gross necropsy to collect brain tissue for the experimental purpose.

The brain homogenate from all the animals groups after experimental period was subjected for the estimation of sleep biomarker such as melatonin, BDNF, PGD2, Leptin, Orexin-A, and acetylcholine was performed. The entire assay was estimation using ELISA method as per manufacturer s recommended standard procedure. This was a quantitative method and the principle was based on the binding of protein and their specific antibody.

The data were represented as mean ± standard error of mean (SEM) and subjected to statistical analysis using Sigma-Plot statistical software (Version 11.0). For multiple comparison One-way analysis of variance (ANOVA) followed by post-hoc analysis by Dunnett s test and for between two groups comparison Student s t-test was performed. The p≤0.05 was considered as statistically significant.

Melatonin, a ubiquitous natural neurotransmitter secreted in the brain by the pineal gland with most diverse biological actions such as energy metabolism, circadian rhythm, and the immune system 42. It imparts significant protective action on neurons, especially against wide variety of oxidative stress by removing reactive oxygen, which suggest its role against many targets for the development of many neurological diseases 4344. Melatonin in CNS also reacts with various reactive intermediates in order to protect against tissue damage 45. Brain melatonin level was estimated in all the experimental test groups and the results are presented in Figure 1. The data showed the melatonin level in the unpredictable chronic stress (G2) group was 75.31 ± 4.1 pg/mL, which was significantly decreased by 30.9% as compared with the normal control (G1, 109.04 ± 3.4 pg/mL). Imipramine treatment (G3) increased brain melatonin level (80.37 ± 4.4 pg/mL) by 6.7% as compared to the G2. The untreated test formulation to the untreated rats (G4) showed an increased brain melatonin level (78.98 ± 0.5 pg/mL) by 4.9% as compared to the G2. The Biofield Energy Treated test formulation to the untreated rats (G5) showed a significant increased brain melatonin level (88.58 ± 4.2 pg/mL) by 17.6% (p≤0.01) and 12.2% as compared to the G2 and G4 groups, respectively. Biofield Energy Treatment per se to the rats (G6) increased the brain melatonin level (84.45 ± 2.0 pg/mL) by 12.1% and 6.9% as compared to the G2 and G4 groups, respectively. 15-days pre-treatment of Biofield Energy Treated test formulation (G7) increased the brain melatonin level (87.39 ± 4.1 pg/mL) by 16.0% and 10.7% as compared to the G2 and G4 groups, respectively. 15-days pre-treatment of Biofield Energy Treated test formulation to the Biofield Energy Treated rats (G8) showed decreased brain melatonin level (72.82 ± 0.8 pg/mL) as compared to the G2 and G4. The untreated test formulation to the Biofield Energy Treated rats (G9) decreased brain melatonin level (73.85 ± 2.3 pg/mL) as compared to the G2 and G4.

BDNF, a neurotrophin which is important for growth, survival, and neuron maintenance as an important mechanism involved in emotional and cognitive function. BDNF was found to be very protective to neuron in stress situations, which is the leading cause of death globally with total case of more than 300 millions. BDNF role has been widely correlated in depression, aging, and posttraumatic stress disorder 4647. Brain BDNF level was measured in all the experimental groups and is graphically presented in the Figure 2. Brain BDNF level (7.15 ± 0.30 pg/mL) in the G2 was significantly decreased by 8.3% as compared to the normal control (G1, 7.79 ± 0.27 pg/mL). Imipramine (G3) showed significantly increased brain BDNF level (7.52 ± 0.20 pg/mL) by 5.2% as compared to the G2. G4 (7.02 ± 0.56 pg/mL) and G6 (6.75 ± 0.40 pg/mL) groups showed a decreased brain BDNF level as compared to the G2. G7 (7.38 ± 0.38 pg/mL) group showed an increased brain BDNF level by 3.3% and 5.2% as compared to the G2 and G4 groups, respectively. G8 (5.64 ± 0.38 pg/mL) and G9 (6.28 ± 0.31 pg/mL) groups showed a decreased brain BDNF level as compared to the G2.

Prostaglandin D2 (PGD2), a prostaglandin (PG), which is most abundant PGs involved in various physiological processes in the mammalian brain. It is mostly produced in organs, such as the brain, bone marrow, thymus, spleen, oviduct, uterus, prostate, ovary, testis, and epididymis. PGD2 is involved in major depressive disorder as one of the main physiological and pharmacological role in the central nervous system. Various reports suggest that the level of PGD2 was decreased in stressed conditions and depressive disorder physiology. Thus, the level of PGD2 can be maintained by using some alternative way of treatment would be the best mode of treatment in depressive disorders 48. All the test groups were tested for the presence of brain PGD2 level, which was measured and was graphically presented in the Figure 3. Brain PGD2 level (0.80 ± 0.01 ng/mL) in the G2 was significantly decreased by 11.6% as compared to the normal control (G1, 0.91 ± 0.02 ng/mL). Imipramine (G3) showed significantly increased brain PGD2 level (0.85 ± 0.02 ng/mL) by 6.3% as compared to the G2. G4 (0.85 ± 0.01 ng/mL) group showed an increased brain PGD2 level by 6.2% as compared to the G2. G6 (0.90 ± 0.01 ng/mL), G7 (0.95 ± 0.02 ng/mL), G8 (0.99 ± 0.03 ng/mL), and G9 (1.05 ± 0.01 ng/mL) groups showed a significant increased brain PGD2 level by 6.1%, 11.2%, 16.6%, and 23.1%, respectively, as compared with the G4, untreated test formulation group. Prostaglandins D2 (PGD2) level was significantly (p≤0.001) increased by 18.1%, 23.7%, and 30.7% in the G7, G8 (15 days pre-treatment of Biofield Energy Treated Test formulation to the Biofield Energy Treatment per se rats), and G9 (untreated test formulation to the Biofield Energy Treatment per se to the rats) groups, respectively as compared with the G2.

Leptin, is a hormone mainly released in adipose tissue from fat cells, it provide signal to the brain especially in the hypothalamus region. Its role in weight loss and its relation with appetite and high intake of food has been widely studies 49. However, it has been found to have significant role in extra-hypothalamic effects, which protects the brain/CNS from various form of neurodegenerative disorders like Alzheimer’s disease or the development of the mood disorders. Thus, regulation of leptin level in CNS is the core area of research now a days for various neurodegenerative disorders due to the action of leptin on neuron structure and function, as well as on glial cells 505152. The level of brain leptin level was estimated among all the experimental groups and is graphically presented in the Figure 4. Brain leptin level (7.24 ± 0.30 ng/mL) in G2 group was significantly increased by 42.3% as compared to the normal control (G1, 5.09 ± 0.36 ng/mL). Imipramine (G3) showed significantly decreased the brain leptin level (6.96 ± 0.44 ng/mL) by 3.9% as compared to the G2. G4 (6.18 ± 0.28 ng/mL) group showed an increased brain leptin level by 6.2% as compared to the G2. G5 (5.01 ± 0.42 ng/mL), G6 (4.31 ± 0.24 ng/mL), G7 (4.69 ± 0.31 ng/mL), and G8 (4.83 ± 0.39 ng/mL) groups showed a significant decreased brain leptin level by 18.9%, 30.2%, 24%, and 21.8% respectively, as compared with the G4, untreated test formulation group. However, brain leptin was also significantly decreased by 30.8%, 40.5%, 35.2%, 33.3%, and 15.6% in the G5, G6, G7, G8, and G9 groups, respectively as compared with the G2 group.

Orexin (also known as hypocretin) is a kind of peptide released in neurons of the peripherical, lateral, and posterior hypothalamus, it regulates sleep/wakefulness cycle and feeding behaviour. Besides, brain leptin also helps in coordination of emotion, energy homeostasis, reward system, and arousal. Thus, it has been found to have its important in various CNS disorder due to its huge biological role in sleep cycle or awake 5354. The level of brain orexin-A level was estimated among all the experimental groups and is graphically presented in the Figure 5. Brain orexin-A level (205.76 ± 22.3 ng/mL) in G2 was significantly increased by 71.9% as compared to the normal control (G1, 119.73 ± 11.6 ng/mL). Imipramine (G3) showed significantly decreased brain orexin-A level (169.48 ± 19.7 ng/mL) by 17.6% as compared to the G2. G4 (162.96 ± 24.4 ng/mL) group showed an increased brain orexin-A level by 20.8% as compared to the G2. G5 (129.34 ± 21.7 ng/mL), G6 (138.59 ± 8.5 ng/mL), G7 (122.5 ± 23.4 ng/mL), G8 (122.50 ± 23.4 ng/mL), and G9 (147.66 ± 19.6 ng/mL) groups showed a significant decreased brain orexin-A level by 20.6%, 15%, 24%, 29.8%, and 9.4%, respectively as compared with the G4, untreated test formulation group. However, brain orexin-A was also significantly decreased by 37.1% (p≤0.05), 32.6%, 40.5% (p≤0.05), 44.4% (p≤0.05), and 28.2% in the G5, G6, G7, G8, and G9 groups, respectively as compared with the G2 group.

The neurotransmitter acetylcholine, is a chemical messenger that actions on skeletal mUCSles to contract. It is released by nerve cells in the brain, and found in between the nerve synapses, or gaps, between nerve cells. It plays an important role in signalling for mUCSle movement, learning and memory formation, sensation of pain, endocrine system regulation, and rapid eye movement (REM) sleep cycles. Degeneration of the acetylcholine pathway associated with the pathologies of Alzheimer's disease 55. The level of brain Ach level was estimated among all the experimental groups and is graphically presented in the Figure 6. Brain Ach level (456.29 ± 11.1 pg/mL) in G2 was significantly increased by 29.4% as compared to the normal control (G1, 352.70 ± 11.1 pg/mL). Imipramine (G3) showed significantly decreased brain Ach level (424.28 ± 32.8 pg/mL) by 7% as compared to the G2. G4 (441.86 ± 15.8 pg/mL) group showed a decreased brain Ach level by 3.2% as compared to the G2. G5 (262.53 ± 11.2 pg/mL), G6 (231.80 ± 7.5 pg/mL), G7 (273.34 ± 23.2 pg/mL), G8 (237.80 ± 10.5 pg/mL), and G9 (251.06 ± 12.4 pg/mL) groups showed a significant decreased brain Ach level by 40.6%, 47.5%, 38.1%, 46.2%, and 43.2%, respectively as compared with the G4, untreated test formulation group. However, brain Ach was also significantly (p≤0.001) decreased by 42.5%, 49.2%, 40.1%, 47.9%, and 45% in the G5, G6, G7, G8, and G9 groups, respectively as compared with the G2 group.

In this research plan, four groups were considered as preventive maintenance groups. These groups were G6 (Biofield Energy Treatment per se to animals at -15 days), G7 (Biofield Energy Treated test formulation from day -15), G8 (Biofield Energy Treatment per se to animals along with Biofield Treated test formulation from day -15), and G9 (Biofield Treatment per se at -15 days to animals with untreated test formulation). The results showed the significant slowdown of the disease progression, stress disease related all other symptoms/complications and also reduced the chances of disease sUCSeptibility in these groups. Specifically, group G6 (preventive Biofield Energy Treatment group per se at -15 days) showed the best results as a prophylactic/preventive treatment group compared to the other groups. Based on the overall data, it suggests that the Biofield Energy Healing Therapy was found to be most effective and benefited in order to prevent and protect from the occurrence of any type of diseases in rat model. It indicated that this therapy can act as a preventive maintenance therapy to prevent the occurrence of the disease, slow down the disease progression and disease related complications of the existing aliments that will ultimately improve the overall health and quality of life in human.