Pyridoxine hydrochloride (vitamin B₆), atorvastatin, zinc chloride, magnesium (II) gluconate, and β-carotene (retinol, provit A) were purchased from TCI, Japan. Copper chloride, cyanocobalamin (vitamin B₁₂), calcium chloride, vitamin E (Alpha-Tocopherol), cholecalciferol (vitamin D₃), iron (II) sulfate, captopril, L-NAME, 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. Standard normal chow diet and high fat diet were purchased from Altromin, USA and Research Diets, USA.

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. HYLASCO Biotechnology (India) Pvt. Ltd., India. Animals were randomly divided into nine groups based on their body weights consist of 15 animals of each group (at the time of induction period) and 10 animals of each group (at the time of treatment period). 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 novel test formulation was consisted of zinc chloride, iron (II) sulfate, copper chloride, vitamin B₆, vitamin B₁₂, vitamin D₃, vitamin B₉, sodium selenate, calcium chloride, ascorbic acid, beta carotene, Panax ginseng extract, cannabidiol and magnesium (II) gluconate. 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/Blessing (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 Biofield Energy Healing Treatment/ Blessing (prayer) was done remotely, for about 3 minutes via online web-conferencing platform. After that, the Biofield Energy Treated/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 have any knowledge about the Biofield Energy Treatment/Healing. The Biofield Energy Treated/Blessed animals were also taken back to experimental room for further proceedings.

Animals were subjected for the measurement of systolic and diastolic blood pressure and heart rate in conscious rats through the tail-cuff method. Each animal was introduced into a restrainer and kept in a quiet and warm environment for 10 minutes. The rat tails were cleaned/dilated using a cotton soaked in hot water. A rubber cuff (proximally) and a photoelectric sensor of pulsations (more distally) were placed around the tail. The sensor was connected to an amplifier and pulsations were recorded on a Non-Invasive Blood Pressure Amplifier, BioPac Inc.

Hematological parameters such as total leukocyte count (TLC), and differential leukocyte counts (DLC), were analyzed using Hematology analyzer (Abbott Model-CD-3700) in blood samples.

Glucose, total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), very low-density lipoprotein (VLDL), alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase (SGOT), and serum glutamate-pyruvate transaminase (SGPT) were analyzed using serum by Biochemistry Analyzer, Spectralab A– plus, Italy.

The test formulation was tested for important hepatic and cardiac biomarkers such as albumin (g/dl), alkaline phosphatase (U/L), bilirubin total (mg/dL), SGOT (U/L), SGPT (U/L), total protein (mg/dL), globulin (mg/dL), A/G ratio, and creatine kinase-MB (U/L) were analyzed using serum by Biochemistry Analyzer, Spectralab A– plus, Italy.

All the experimental animals were daily analyzed for their change in body weight, feed intake, and organ weight parameters, which was calculated by weighing the daily feed supply and the left-over amount that evaluate the average daily feed intake. The average intake of feed was recorded in every three days interval throughout the experimental period. After terminal bleeding, the animals were sacrificed and the following organs such as liver, lung, kidney, brain, heart, eye, pancreas, spleen, thymus, adrenal gland, intestine, intestine and reproductive organs, i.e., testis, prostate, epididymis and vas deferens were collected. These organs were trimmed off any adherent tissue and fat, as appropriate and weighed. The organ to body weight ratio percentage was identified by comparing the weight of each organ with the final body weight of individual rat 31. All the data were reported through the study treatment regimen.

Relative organ weight was calculated as per Equation 1.

All the animals in different test groups were analyzed for various clinical sign and symptoms in accordance with in-house protocol. Abnormal behaviour in animals was recorded with the time of onset and disappearance.

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.

Blood pressure and heart rate were measured after treatment with the test formulation and the data were graphically presented in the Figure 1. The data suggested that the disease control (L-NAME + high fat diet (HFD) + 0.5% CMC) group (G2) showed systolic blood pressure (SBP) as 179.68 ± 2.49 mm of Hg, which was increased by 63.32% as compared with the normal control (G1, 110 ± 2.89 mm of Hg). However, positive control (captopril + atorvastatin) treatment (G3) showed the level of SBP i.e. 130.8 ± 1.19 mm of Hg, which was significantly (p≤0.001) decreased by 27.20% as compared to the G2 group. The level of SBP was significantly (p≤0.001) decreased by 12.60%, 13.39%, 14.65%, 17.74%, 14.36%, and 14.69% in the G4 (L-NAME + HFD along with untreated test formulation), G5 (L-NAME + HFD + the Biofield Energy Treated test formulation), G6 (L-NAME + HFD + Biofield Energy Treatment per se to animals from day -15), G7 (L-NAME + HFD + the Biofield Energy Treated test formulation from day -15), G8 (L-NAME + HFD + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (L-NAME + HFD along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control (G2) group (Figure 1A). Moreover, the level of diastolic blood pressure (DBP)was significantly (p≤0.001) reduced by 19.73%, 25.95%, 24.41%, 30.79%, 24.67%, and 25.23% in the G4, G5, G6, G7, G8, and G9 groups, respectively as compared to the G2 group. On the other hand, DBP was reduced by 7.75%, 5.83%, 13.79%, 6.16%, and 6.86% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 1A). Besides, heart rate (HR) was significantly reduced by 7.01% (p≤0.05), 6.02%, 4.86%, 6.58% (p≤0.05), 8.06% (p≤0.05), and 6.99% (p≤0.05) in the G4, G5, G6, G7, G8, and G9 groups, respectively as compared to the disease control (G2) group (Figure 1B).

G2 as disease control (L-NAME + high fat diet (HFD) + 0.5% CMC); G3 as reference item (L-NAME + HFD + Captopril + Atorvastatin); G4 includes L-NAME + HFD along with untreated test formulation; G5 as L-NAME + HFD along with the Biofield Energy Treated test formulation; G6 group includes L-NAME + HFD along with Biofield Energy Treatment per se to animals from day -15; G7 as L-NAME + HFD along with the Biofield Energy Treated test formulation from day -15; G8 group includes L-NAME + HFD along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted L-NAME + HFD along with Biofield Energy Treatment per se animals plus the untreated test formulation. Values are presented as mean ± SEM (n=7 to 9). *p≤0.05 and ***p≤0.001 vs. Disease control (G2).

The experimental results of the Biofield Energy Healing Treatment perse and Biofield Energy Treated test formulation on important haematology profile in different groups are summarized in Table 2. The study results suggest that Biofield Energy Treated test formulation showed an improved animal hematology profile compared with the untreated test formulation group. The hematology parameters such as total leucocyte count (TLC) count was increased by 12.8%, 2.34%, 17.45%, and 7.38% in the G5 (L-NAME + HFD + the Biofield Energy Treated test formulation), G7 (L-NAME + HFD + the Biofield Energy Treated test formulation from day -15), G8 (L-NAME + HFD + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (L-NAME + HFD along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively as compared with the G4 group. The level of neutrophil was increased by 11.70%, 60.11%, and 16.49% in the G5, G8, and G9 groups, respectively as compared with the G4 group. The levels of lymphocytes were increased by 11.49%, 6.42%, and 3.89% in the G5, G7, and G9 groups, respectively as compared with the G4 group. The level of monocyte was increased by 16.36%, 30.91%, and 12.73% in the G5, G8, and G9 groups, respectively, as compared with the G4 group. Similarly, level of eosinophil was reduced by 25%, 11.11%, 20%, and 15% in the G4, G6, G7, and G9 groups, respectively as compared with the G2 group.

Lipid biomarker analysis was performed after treatment with the Biofield Energy Treated and untreated test formulations are summarized in the Table 3. The analyzed glucose and lipid biomarkers were tested such as total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The experimental data showed that the level of total cholesterol was significantly decreased by 24.75% (p≤0.05), 22.64% (p≤0.05), 15.78%, 25.56% (p≤0.05), 22.56%, and 34.27% in the G4 (L-NAME + HFD along with untreated test formulation), G5 (L-NAME + HFD + the Biofield Energy Treated test formulation), G6 (L-NAME + HFD + Biofield Energy Treatment per se to animals from day -15), G7 (L-NAME + HFD + the Biofield Energy Treated test formulation from day -15), G8 (L-NAME + HFD + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (L-NAME + HFD along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control (G2) group. Additionally, the level of total cholesterol was reduced by 12.65% in the G9 group as compared to the untreated test formulation group (G4). However, the level of triglyceride was reduced by 70.66%, 80.80%, 83.36%, 86.87%, 79.05%, and 81.60% in the G4, G5, G6, G7, G8, and G9 group, respectively as compared with the G2 group. Moreover, the level of triglyceride was significantly (p≤0.001) reduced by 34.55%, 43.29%, 55.23%, 28.57%, and 37.28% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the G4 group. Similarly, LDL was reduced by 11.33%, 8.87%, 5.54%, and 20.32% in the G4, G5, G8, and G9 group, respectively as compared with the G2 group, while decreased by 10.14% in the G9 group as compared to the G4 group. VLDL level was also significantly (p≤0.001) reduced by 70.64%, 80.81%, 83.61%, 86.82%, 79.19%, and 81.63% in the G4, G5, G6, G7, G8, and G9 group, respectively as compared with the G2 group. The level of VLDL was decrease by 34.65%, 43.15%, 55.12%, 29.13%, and 37.44% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the G4 group. Besides, level of good cholesterol HDL was increased by 12.15% in the G6 group as compared to the G4 group.

The effects of the test formulation on hepatic and cardiac biomarkers are shown in Table 4. The level of obesity and coronary heart disease biomarker like atherogenic index (AI) was significantly (p≤0.001)decreased in the experiment by 64.92%, 78.36%, 83.21%, 84.68%, 74.06%, and 72.98% in the G4, G5, G6, G7, G8, and G9 groups respectively, as compared with the G2 group. Moreover, the level of AI was reduced by 38.31%, 52.11%, 56.32%, 26.05%, and 22.99% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the G4 group. Similarly, the level of uric acid (UA) was significantly (p≤0.001)decreased in the experiment by 48.49%, 57.51%, 52.36%, 45.49%, 43.78%, and 40.77% in the G4, G5, G6, G7, G8, and G9 groups respectively, as compared with the G2 group. The level of SGPT was significantly (p≤0.001)decreased by 42.35%, 45.96%, 48.01%, 37.19%, 37.69%, and 42.93% in the G4, G5, G6, G7, G8, and G9 groups respectively, as compared with the G2 group. However, CK-MB level was significantly reduced by 7.97%, 10.19%, 21.97% (p≤0.01), 10.47%, 4.94%, and 16.94% in the G4, G5, G6, G7, G8, and G9 groups, respectively as compared with the G2.

Animal weight parameters, feed intake, and relative organ weight parameters were studied, and it was found that change in animal weight, daily feed intake, and organ weight were found to be normal and no significant changes were observed throughout the experimental period. Organ to body weight ratio is the valuable index for any experimental test procedure with respect to the documentation of swelling, atrophy, or hypertrophy after exposure of test samples. The results of animal tested organ weight parameters with respect to body weight are summarized in the Table 5. Thus, the relative organ weight parameters as summarized did not found with any significant change in the organ weight throughout the experiment suggested that the test formulation was found to be safe for the treatment. Overall, the animal weight data, relative organ weight, and feed intake data suggested no significant changes were reported as compared with untreated and disease control group (G2), it suggests that Biofield Energy Treated test formulation and Biofield Energy Treatment per se were found safe in all the tested animals.