Calcitriol, pyridoxine hydrochloride (vitamin B₆), magnesium (II) gluconate, β-carotene (retinol, provit A), and zinc chloride were obtained from TCI, Japan. Cyanocobalamin (vitamin B₁₂), copper chloride, vitamin E (alpha-tocopherol), cholecalciferol (vitamin D₃), calcium chloride, iron (II) sulfate, and sodium carboxymethyl cellulose (Na-CMC) were purchased from Sigma-Aldrich, USA. Sodium selenate and ascorbic acid (vitamin C) were obtained from Alfa Aesar, India. Panax ginseng extract and cannabidiol isolate were procured from Panacea Phytoextracts, India and Standard Hemp Company, USA, respectively. For the estimation of brain biomarkers, specific ELISA kits were used such as for detection of 25-Hydroxy Vit D₃, TNF-α, and IL-6 kit were procured from CUSABIO, USA while acetylcholine (ACh) level was estimated using MyBioSource, USA.

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 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 isolate and magnesium (II) gluconate. Each ingredient of the novel test formulation was divided into two parts. The test formulation was divided into two parts, one part of the test compound was not received any sort of treatment and were defined as the untreated or control sample. The second part 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 laboratory conditions for ~3 minutes. Besides, three group of animals also received Biofield Energy Healing Treatment (known as the Trivedi Effect^®) by Mr. Trivedi under similar laboratory conditions for ~3 minutes. The blessing/treatment was given to the test items remotely without touching in the laboratory of Dabur Research Foundation, near New Delhi, India. After that, the Biofield Energy Treated samples was kept in the similar sealed condition and used as per the study plan. Similarly, the control test formulation was subjected to sham healer for ~3 minutes, under the same laboratory conditions. 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. Dosing for groups G7 and G8 were initiated on day -15 and continued till end of the experiment. However, G1 to G6 and G9 groups were dosed from day 1 till the end of experiment. All the animals except G1 group received vitamin D₃ deficient diet (VDD) daily to the end of the experiment. Three weeks after the initiation of induction of VDD, all the groups were dose with the respective formulations.

About 100 mg of the brain and lung tissue was rinsed with 1X PBS, homogenized in 1 mL of 1X PBS and stored overnight at -20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000g, at 2 to 8°C. The supernatant was removed and assayed immediately. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

Brain homogenate was subjected for the estimation of acetylcholine, 25 (OH) D₃, and IL-6; while lung homogenate was subjected for the estimation of TNF-α level. All the brain biomarkers estimation was performed using ELISA method as per manufacturer s recommended standard procedure.

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.

Vitamin D deficiency is a worldwide problem as it associates with an increased mortality for all causes. In the global population, more than 70% is vitamin D deficient and it is a key factor for maintenance of calcium and phosphate metabolism and bone homeostasis 4445. 25 OH vitamin D₃ level in the brain of rats fed with vitamin D₃ deficient diet (G2) was found to be 603.24 ± 90.82 µg/L, which was significantly (p≤0.05) decreased (847.53 ± 35.65 µg/L) by 28.82% as compared to the normal control (G1) group. However, positive control, calcitriol (G3) treatment showed significantly (p≤0.001) increased level of 25 OH vitamin D₃ level (1136.41 ± 64.79 µg/L) by 88.39% as compared to the G2 group. Similarly, the untreated test formulation given to the untreated rats (G4) showed increased the brain 25 OH vitamin D₃ level (713.01 ± 90.64 µg/L) by 18.20% as compared to the G2. However, G5 group (Biofield Energy Treated test formulation to the untreated rats) showed an increased brain 25 OH vitamin D₃ level (724.66 ± 82.11 µg/L) by 20.13% as compared to G2, while 1.63% increased level as compared to the untreated test formulation group (G4). Biofield Energy Treatment per se to the animals (G6) showed increased the brain 25 OH vitamin D₃ level (748.73 ± 78.38 µg/L) by 24.12% and 5.01% as compared to the G2 and G4 group, respectively. 15 days pre-treatment of Biofield Energy Treated test formulation (G7) showed significantly increased brain 25 OH vitamin D₃ level (879.90 ± 90.69 µg/L) by 45.86% and 23.41% in the G2 and G4 groups, respectively. 15 days pre-treatment of Biofield Energy Treated test formulation to the Biofield Energy Treated rats (G8) group showed significantly increased the brain 25 OH vitamin D₃ level (692.49 ± 37.07 µg/L) by 14.79% as compared to the G2. Untreated test formulation to the Biofield Energy Treated animals (G9) showed significantly increased the brain 25 OH vitamin D₃ level (783.97 ± 41.00 µg/L) by 29.96% and 9.95% as compared to the G2 and G4 groups, respectively. Thus, the test formulation can be used against cardiovascular diseases, type 2 diabetes mellitus, autoimmune disorders, cancer, mental disorders, and infectious diseases in vitamin D deficient states. Figure 1.

Acetylcholine (ACh) is an organic chemical, which plays a vital role in the brain and body of many types of animals, including humans, as a neurotransmitter, a chemical message released by nerve cells to send signals to other cells (neurons, muscle cells, and gland cells). In the brain, acetylcholine acts as a neurotransmitter and as a neuromodulator 46. The effect of the test formulation on the level of brain ACh was determined and the data are presented in Figure 2. Brain ACh level in vitamin D₃ Deficient diet (G2) group was 126.84 ± 10.26 pg/mL, which showed slight decreased value as compared to the normal control (G1, 127.06 ± 14.18 pg/mL) group. Calcitriol treatment (G3), showed an increased ACh level (139.23 ± 8.47 pg/mL) by 9.77% as compared to the G2. G4 group animals showed decreased brain ACh level (89.41 ± 6.32 pg/mL) by 29.51% as compared to G2. However, G5 and G6 groups showed decreased level of ACh by 15.8% and 4.2%, respectively as compared with the G4. However, 15 days pre-treatment of Biofield Energy Treated test formulation (G7) group showed an increased the brain ACh level (144.25 ± 45.58 pg/mL) by 61.33% as compared with the G4 group. In addition, G8 and G9 groups were reported with reduced level of ACh by 16.29% and 20.23% respectively as compared with the G4 group.

IL-6 is a soluble mediator with a pleiotropic effect on inflammation, immune response, and hematopoiesis 47. The present study estimated the level of IL-6 in brain homogenate in various experimental groups (Figure 3). IL-6 level with vitamin D₃ deficient diet (G2) was found to be 1.31 ± 0.19 pg/mL, which showed significant (p≤0.05) increased value by 91.4% as compared to the normal control (G1, 0.68 ± 0.14 pg/mL). Calcitriol, positive control (G3) showed an increased the brain IL-6 level (1.48 ± 0.15 pg/mL) by 13.0% as compared to the G2. However, G4 group showed an increased the brain IL-6 level (1.65 ± 0.26 pg/mL) by 25.68% as compared to G2. In addition, G5 (0.93 ± 0.21 pg/mL), G6 (1.14 ± 0.10 pg/mL), G7 (1.30 ± 0.17 pg/mL), G8 (0.89 ± 0.14 pg/mL), and G9 (0.65 ± 0.11 pg/mL) groups showed significant decreased values of IL-6 by 43.44% (p≤0.01), 30.93%, 21.42%, 45.99% (p≤0.01), and 60.85% (p≤0.01), respectively as compared with the untreated test formulation group (G4). However, similar decreased IL-6 pattern were reported in all the experimental test groups as compared with the G2 group. Overall, our results revealed significant decreased values of IL-6 level in brain in all the experimental test groups as compared to G4.

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine and has a major role in airway inflammation and airway remodeling in asthma 48. It plays a central role in inflammation, immune modulation, and lymphocyte activation in various immune-mediated disorders. TNF-α level in the lung of rats fed with vitamin D₃ Deficient diet (G2) was 30.92 ± 1.40 pg/mL, which was significantly (p≤0.01) increased by 44.4% as compared to normal control (G1, 21.41 ± 2.56 pg/mL). Calcitriol, positive control (G3) showed increased lung TNF-α level (46.02 ± 8.60 pg/mL) by 48.85% as compared to the G2. Untreated test formulation to the animals (G4), increased TNF-α level (52.63 ± 2.99 pg/mL) by 70.2% as compared to the G2. However, other experimental test groups such as G5, G6, G7, G8, and G9 showed significantly reduced level of pro-inflammatory cytokine TNF-α level by 24.86%, 32.55%, 30.12%, 42.69%, and 4.7% respectively, as compared with the untreated test formulation group (G4). Figure 4.

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 a significant slowdown of disease progression and all other disease-related symptoms/complications and also reduced the chances of disease susceptibility in these groups. Based on the overall data, it suggests that the Biofield Therapy was found to be most effective and beneficial to prevent and protect from the occurrence of any type of disease in the rat model. The data indicated that this therapy could act as a preventive maintenance therapy to prevent the occurrence of disease, slowdown the disease progression, when disease-related complications are present which will ultimately improve the overall health and quality of life.