Pyridoxine hydrochloride (vitamin B₆), zinc chloride, magnesium (II) gluconate, and β-carotene (retinol, provit A) were purchased from TCI, Japan. Cyanocobalamin (vitamin B₁₂), calcium chloride, vitamin E (Alpha-Tocopherol), cholecalciferol (vitamin D₃), iron (II) sulfate, and carboxymethyl cellulose sodium were procured from Sigma-Aldrich, USA. Ascorbic acid (vitamin C) and sodium selenate were obtained from Alfa Aesar, India. Panax ginseng extract and Cannabidiol Isolate were obtained from Panacea Phytoextracts, India and Standard Hemp Company, USA, respectively. Dexamethasone was obtained from Clear synth, India. For the estimation of brain antioxidant and inflammatory biomarker panel, such as myeloperoxidase (MPO), superoxide dismutase (SOD), lipid peroxidation (LPO), tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein-2 (MIP-2) were procured from CUSABIO, USA using specific ELISA kits.

Randomly breed maleSprague 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 10-12 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.

Each ingredient of the novel test formulation was divided into two parts. One part of the test compound did not receive 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. Mahendra Kumar Trivedi under similar laboratory conditions for ~3 minutes. The Blessing (prayer)/Treatment was given to the test items/animals (present in the laboratory of Dabur Research Foundation, near New Delhi, India), remotely from USA for about 3 minutes via online web-conferencing platform. 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 for ~3 minutes treatment, under the same laboratory conditions. The sham healer did not has any knowledge about the Biofield Energy Treatment. The Biofield Energy Treated animals were also taken back to experimental room for further proceedings

Myeloperoxidase (MPO), was estimated in the presence of the test formulation and the data is graphically presented in Figure 1. The data suggested that the disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) + 0.5% CMC) group (G2) showed value of MPO as 6.23 ± 0.33 ng/mL, which was increased by 90.27% as compared with the normal control (G1, 3.28 ± 0.08 ng/mL). However, positive control (Dexamethasone) treatment (G3) showed the level of MPO in brain i.e. 5.37 ± 0.49 ng/mL, which was decreased by 13.9% as compared to the G2 group. The level of MPO in brain tissues was decreased by 8.89% and 2.49% in the G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15 and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus untreated test formulation from day -15) groups, respectively as compared to the disease control (G2) group. On the other hand, the level of MPOwas significantly (p≤0.001) reduced by 19.43%, 34.91%, 25.43%, 25.29% and 30.33% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group. Myeloperoxidase (MPO) is an inflammatory enzyme and therapeutic target for attenuating oxidative damage and neuro-inflammation in ischemic stroke in brain tissues. It is highly expressed in different inflammatory cells, like neutrophils, activated microglia, monocytes/macrophage, astrocytes, neurons, etc. 27. Polymorphism of MPO enzyme may associated with the severity of brain damage 28. Overall, in this experiment the Biofield Energy Treated test formulation reduced the level of MPO in the brain tissues, which could be helpful for the management of oxidative stress and inflammatory conditions.

The effect of the test formulation and Biofield Energy Treatment per se was assessed by estimating the level of brain superoxide dismutase (SOD), and the results are graphically presented in the Figure 2. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) + 0.5% CMC) group (G2) showed value of SOD as 1.66 ± 0.09 U/mL, which was decreased by 9.12% as compared to the normal control group i.e., 1.83 ± 0.14 U/mL. However, positive control (Dexamethasone) treatment (G3) showed the level of SOD in brain i.e. 1.55 ± 0.08 U/mL. The level of SOD was increased by 8.77%, 45.02%, 16.59%, 6.11%, and 35.99% in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15), G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E.coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the untreated test formulation group (G4). SOD are a group of metalloenzymes with diverse therapeutic activities in various physiological and pathological conditions such as inflammatory diseases, ischemia, aging, rheumatoid arthritis, neurodegenerative diseases, and cancer 29. The enzyme can serve as an anti-inflammatory agent and can also prevent precancerous cell changes 30. Overexpression of extracellular SOD can protects brain injury induced by chronic hypoxia 31. Therefore, in this experiment the Biofield Energy Treated test formulation significantly increased the level of brain SOD, which could be beneficial inflammation and oxidative damage.

The level of lipid peroxidation (LPO) end product in terms of malondialdehyde (MDA) was detected in all the experimental groups and the data are presented in Figure 3. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) and positive control (Dexamethasone) treatment (G3) groups showed value of MDA as 8.33 ± 0.64 µM and 12.26 ± 0.70 µM, respectively. The level of MDA was decreased by 4.35% in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation) group as compared to the untreated test formulation group (G4) group.

The effect of the test formulation and Biofield Energy Treatment per se on the level of brain interleukin-6, and the results are graphically presented in the Figure 5. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of IL-6 as 7.87 ± 0.79 pg/mL, which was increased by 43.95% as compared with the normal control (G1, 5.47 ± 0.28 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed the level of IL-6 i.e., 7.24 ± 0.25 pg/mL. The level of IL-6 was significantly decreased by 21.86% and 16.73% in the G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15) and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Further, the expression of IL-6 was significantly decreased by 7.42%, 37.51% (p≤0.001), 20.28% (p≤0.001), 21.55% (p≤0.001), and 33.4% (p≤0.001) in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the untreated test formulation group (G4). The level of IL-6 is upregulated in various infection in CNS or injury or in a number of CNS diseases due to neuro-inflammation. IL-6 levels in CSF were also significantly higher than plasma levels in patients with traumatic brain injury 35. Further, recent study revealed that IL-6 polymorphism associated with severe traumatic brain injury 36. Overall, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of IL-6, which could be suppressed the neuroinflammatory conditions in the CNS and simultaneously reduce the risks of inflammatory diseases specially in the brain.

Expression of brain macrophage inflammatory protein-2 (MIP-2) after administration of Biofield Treated test formulation and Biofield Blessing directly to the rats was detected in all the experimental groups and the data is presented in Figure 6. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of MIP-2 as 4591.13 ± 401.95 pg/mL, which was increased by 199.83% as compared with the normal control (G1, 1531.27 ± 67.27 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed decreased brain MIP-2 level by 29.69% i.e., 3228.11 ± 346.83 pg/mL as compared to the G2 group. The level of MIP-2 was decreased by 4.75%, 46.13%, 14.14%, 17.34%, and 24.26% in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli + Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se + Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Similarly, MIP-2 level was significantly decreased by 8%, 47.97%, 17.08%, 20.16% and 26.84% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group. MIP-2 is one of the chemokine is produced in response to any types of infection or injury. For example, in liver injury the activated Kupffer cells are known as the major source of MIP-2 and causes liver inflammation 37. In brain injury or inflammation, astrocytes is a prime source of chemokine (MIP-2), that plays an important function for induction of inflammation in the CNS 38. As per literature report, one of the possible reason for brain inflammation is the polymorphonuclear neutrophils (PMNs) that mediates the brain inflammation followed by hypoxia/reoxygenation (H/R). They also studied the MIP-2 mRNA expression and found that H/R upregulated MIP-2 gene expression 39. Taken together, our data suggest that the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of MIP-2 in brain tissues, which could prevent the brain neuro-inflammation.

Experiment includes four preventive maintenance groups (G6, G7, G8 and G9). The findings showed the significant slowdown of inflammation-related symptoms and also reduced the chances of disease susceptibility. All-inclusive, it indicate that the Trivedi Effect^® was found to be most effective and benefited to protect different kinds of diseases and also improve the overall health and quality of life.