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. For the estimation of serum antioxidative biomarker panel, specific ELISA kits were used for detection of antioxidants in serum (SOD, GPx, LPO and MPO, Oxidized LDL,) and catalase in heart and kidney tissues, were procured from CUSABIO, 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.

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/Blessing and were defined as the untreated 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 were also Blessed (known as the Trivedi Effect^®) by Mr. Mahendra Kumar Trivedi under similar laboratory conditions for ~3 minutes. The Biofield Energy Healer was located in the USA, however the test formulation were located in the research laboratory of Dabur Research Foundation, New Delhi, India. The energy transmission was done remotely, for about 3 minutes via online web-conferencing platform. After that, the Biofield Energy Treated/Blessed sample 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/Blessing. The Biofield Energy Treated/Blessed 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 dispo sable 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 (vehicle, 0.5% w/v CMC-Na); G2 as disease control (L-NAME + 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 include 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. The normal control animals group (G1) was receive normal drinking water and a normal diet throughout the experimental period. The animals in groups G2-G9 were received L-NAME (20 mg/kg, i.p.) and a high fat diet (HFD) throughout the experimental period. At the end of the experimental period (8 weeks treatment), the animals were sacrifice and blood was collected and separate serum subjected for antioxidants in serum (SOD, GPx, LPO and MPO, Oxidized LDL,) and catalase in heart and kidney tissues estimation.

At the end of the treatment of 8^th week of the experimental period, all the animals were individually subjected for blood collection using retro-orbital route and the blood was collected in the plain vial in all the animals of different experimental groups. The serum from all the groups was stored at -20°C for further estimation. Alternatively, aliquot all the samples and store samples at -20°C or -80°C. After organ collection animals were humanely sacrificed to collect heart and kidney portion that was homogenized and subjected for the analysis of catalase using suitable ELISA method. Avoid repeated freeze-thaw cycles, which may alter the level of antioxidant level in tissues during final calculations.

The serum from all the groups was subjected for the estimation of level of antioxidants such as SOD, GPx, LPO and MPO, Oxidized LDL, along with Catalase in heart and kidney tissue homogenate. All the antioxidative biomarker panel 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 antigen and antibody in sandwich manner assay.

Serum superoxide dismutase (SOD) was estimated after post treatment with the test formulation, and the data are shown in Figure 1. The SOD data suggested that the disease control (L-NAME + high fat diet (HFD) + 0.5% CMC) group (G2) showed value of SOD as 0.98 ± 0.15 U/mL, which was decreased by 62.44% as compared with the normal control (G1, 2.60 ± 0.12 U/mL). Moreover, the positive control (captopril + atorvastatin) treatment (G3) showed the level of serum SOD i.e. 2.64 ± 0.14 U/mL. The level of SOD was significantly increased by 215.98%, 198.46%, 208.73%, 191.73%, 211.75%, and 198.82% in the G4 (L-NAME + HFD + 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 + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Overall, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly increased the level of antioxidant enzyme SOD in sample, which might be helpful for the management of cardiovascular disorders induced due to oxidative stress. Excessive production and inadequate removal of ROS, are responsible in the pathogenesis of various cardiovascular diseases, including atherosclerosis, hypertension, etc. ²⁷. Another study reported that SOD is the first-line of defense antioxidant enzyme against deleterious effects of oxy-radicals in all the living cells. It breaks down the most dangerous free radical superoxide anion to molecular oxygen and hydrogen peroxide and prevents subsequent formation of hydroxyl radicals and plays an important role in the cellular antioxidant mechanism ²⁸. Thus, Biofield Energy Treatment would be the best alternative treatment approach to treat stress induced cardiovascular dysfunctions using improved anti-oxidation action.

Figure 1. The effect of the test formulation on the level of serum superoxide dismutase (SOD) in Sprague Dawley rats. G1 as normal control (vehicle, 0.5% w/v CMC-Na); 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=10).

The effect of the Biofield Energy Treated test formulation and Biofield Energy Treatment per se on the level of serum glutathione peroxidase (GPx), is shown in Figure 2. The disease control (L-NAME + high fat diet (HFD) + 0.5% CMC) group (G2) showed the value of GPxas 4.08 ± 0.44 nmol/min/mL, which was decreased by 37.78% as compared with the normal control (G1, 6.55 ± 0.43 nmol/min/mL) group. However, positive control (captopril + atorvastatin) treatment group (G3) showed an increase the level of GPx in serum by 103.03% i.e. 8.28 ± 0.83 nmol\min/mL as compared to the G2 group. The level of GPx was significantly increased by 54.86%, 61.94%, 118.49%, 82.96%, 141.89%, and 262.02% in the G4 (L-NAME + HFD + 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 + 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 GPx was elevated by 41.05%, 18.11%, 56.16%, and 133.71% in the G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group. Glutathione peroxidase-1 (Gpx1) plays a crucial role in cellular defense by converting hydrogen peroxide (H₂O₂) and organic hydroperoxides to non-reactive products. Lack of GPx1 that accelerates a cardiac-specific hypertrophy and dysfunction in angiotensin II that leads to hypertension ²⁹. Overall, in this study the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly increased the antioxidant function by increasing the level of GPx, which could be beneficial in the cardiovascular patients.

Figure 2. The effect of the test formulation on the level of Serum glutathione peroxidase (GPx) in Sprague Dawley rats

The level of serum lipid peroxidase (LPO) was measured in all the experimental groups and the data are shown in Figure 3. The disease control (L-NAME + high fat diet, HFD + 0.5% CMC) group (G2) group showed the value of LPO as 14.57 ± 0.93 µM, which was decreased by 23.60% as compared with the normal control (G1, 11.79 ± 0.33 µM) group. While, the positive control (captopril + atorvastatin) treatment group (G3) decreased the level of LPO by 17.43% i.e. 12.03 ± 0.64 pg/mL as compared to the G2 group. The level of LPO was decreased by 17.98%, 14.21%, 30.98%, 38.66%, and 32.67% in the G4 (L-NAME + HFD + untreated 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 + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Moreover, the level of LPO was reduced by 15.85%, 25.22%, and 17.91% in the G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group. Lipid peroxidation is the process in which the membrane bound enzymes, proteins, and receptors are inactivated through loss of cell membrane integrity ³⁰. Lipid per-oxidation can affects lipoproteins, cellular membranes, and other molecules containing the lipids in accordance with the oxidative stress. The process of LPO was initiated due to several reasons like acute and chronic stress, cellular damage, altered metabolism, etc. It leads to various diseases like atherosclerosis, kidney damage, Parkinson's disease, preeclampsia etc. ³¹. Overall, here the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of lipid peroxidation end product, malondialdehyde (MDA) level, which could be beneficial in the cardiovascular symptoms.

Figure 3. The effect of the test formulation on the level of serum Lipoperoxidase (LPO) in Sprague Dawley rats.

The effect of the test formulation and Biofield Energy Treatment per se was estimated by measuring the level of serum myloperoxidase (MPO), and the results are shown in the Figure 4. The disease control (L-NAME + high fat diet, HFD + 0.5% CMC) group (G2) showed value of MPO as 0.43 ± 0.03 ng/mL, which was increased by 78.02% as compared with the normal control (G1, 0.24 ± 0.02 ng/mL) group. Further, the positive control (captopril + atorvastatin) treatment (G3) showed decreased serum MPO level by 25.68% i.e., 0.32 ± 0.03 ng/mL as compared to the G2 group. The level of MPO was decreased by 15.03%, 28.46%, 10.87%, 12.41%, and 13.35% in the G4 (L-NAME + HFD + 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 + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Similarly, MPO level was decreased by 16.86% in the G5 group as compared to the untreated test formulation (G4) group. The MPO is an indicator for leukocyte infiltration, which is commonly found in inflamed tissue such as chronic processes like neurodegenerative diseases and atherosclerosis ³². Therefore, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of MPO, which could be beneficial in the cardiovascular disease conditions.

Figure 4. The effect of the test formulation on the level of serum Myloperoxidase (MPO) in Sprague Dawley rats.

The effect of the test formulation and Biofield Energy Treatment per se was estimated using the level of Oxidized Low Density Lipoprotein (LDL); the results are shown in Figure 5. The level of oxidized LDL in the disease control (L-NAME + high fat diet, HFD + 0.5% CMC) group (G2) was 28.70 ± 3.12 pg/mL, which was increased by 414.71% as compared with the normal control (G1, 5.58 ± 1.25 pg/mL). Further, the positive control (captopril + atorvastatin) treatment (G3) showed decreased serum oxidized LDL level by 75.38%, 7.07 ± 1.03 pg/mL as compared with the G2. The level of oxidized LDL was decreased by 42.15%, 65.38%, 65.11%, 71.53%, 79.26%, and 66.57% in the G4 (L-NAME + HFD + 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 + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Besides, the level of oxidized LDL was decreased by 40.15%, 39.67%, 50.78%, 64.13%, and 42.21% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group. Various studies have demonstrated an atherogenic role of oxidized-LDL in the progression of atherosclerotic cardiovascular disease (ASCVD) ^{33, 34, 35}. Overall, here the Biofield Energy Treated test formulation and Biofield Energy Treatment per se has significantly reduced the level of oxidized LDL in serum sample, which could be suppressed the free-radical levels and simultaneously reduce the risks of cardiovascular diseases by antioxidative potentials.

Figure 5. The effect of the test formulation on the level of serum oxidized low density lipoprotein (LDL) level in Sprague Dawley rats.

Catalase enzyme activity in heart tissue was estimated in the presence of Biofield Treated test formulation and Biofield Energy treatment per se to the animals, and graphically shown in the Figure 6. The level of CAT enzyme in the disease control (L-NAME + high fat diet, HFD + 0.5% CMC) group (G2) was 194.53 ± 13.62 nmol/min/mL, which was decreased by 18.70% as compared with the normal control (G1, 239.26 ± 27.42 nmol/min/mL). Further, the positive control (captopril + atorvastatin) treatment (G3) increased the level of CAT by 82.81% as 355.62 ± 31.84 nmol/min/mL as compared with the G2. The level of CAT enzyme was increased by 86.18%, 68.20%, 63.69%, 126.03%, 124.54%, and 112.23% in the G4 (L-NAME + HFD + 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 + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Besides, the level of CAT was increased by 21.40%, 20.61%, and 14% in the 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 + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the untreated test formulation (G4) group. One research work in mice explored that the overexpression of cardiac-specific CAT, that detoxifies the excess H₂O₂, and thus protect from oxidative stress and delayed cardiac aging ³⁶. Another study demonstrated that the antioxidant enzymes as potential targets in cardioprotection and treatment of various cardiovascular diseases ³⁷. Overall, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of oxidative free-radical in heart tissues, which could be suppressed the oxidative stress conditions and simultaneously reduce the risks of cardiovascular diseases.

Figure 6. The effect of the test formulation on the level of catalase activity in heart tissues in Sprague Dawley rats.

Catalase (CAT), is a powerful antioxidant enzyme that scavenge free-radical to the body. Besides, it has a powerful antioxidant support, anti-aging, and anti-degenerative effects, enhanced overall life-span, fat reduction, and prevention of DNA damage due to various stress factors ³⁸. CAT assay in kidney homogenate was estimated in the presence of Biofield Treated test formulation and Biofield Energy treatment per se to the animals, which was measured in all the experimental groups and was graphically presented in the Figure 7. The level of CAT enzyme in the disease control (L-NAME + high fat diet, HFD + 0.5% CMC) group (G2) was 352.69 ± 17.11 nmol/min/mL, which was increased by 20.06% as compared with the normal control (G1, 441.18 ± 24.14 nmol/min/mL). Further, the positive control (captopril + atorvastatin) treatment (G3) showed the level of CAT as 299.83 ± 14.59 nmol/min/mL as compared with the G2. The level of CAT enzyme was increased by 8.33% and 9.18% in the G6 (L-NAME + HFD + Biofield Energy Treatment per se to animals from day -15) and G7 (L-NAME + HFD + the Biofield Energy Treated test formulation from day -15) groups, respectively, as compared to the disease control group (G2). Besides, the level of CAT was increased by 22.48%, 23.43%, and 10.95% in the 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), and G9 (L-NAME + HFD + Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the untreated test formulation (G4) group (Figure 7). Therefore, it is assumed that the Trivedi Effect^®-Biofield Energy Treatment based test formulation and Biofield Energy Healing Treatment per se showed good antioxidative property.

Figure 7. The effect of the test formulation on the level of catalase activity in kidney tissues in Sprague Dawley rats

Experiment includes four preventive maintenance groups (G6, G7, G8 and G9). The findings showed the significant slowdown of cardiovascular-related symptoms and also reduced the chances of disease susceptibility. Based on the overall data, it suggests that the Biofield Therapy was found to be most effective and benefited to protect from the manifestation of the existing aliments that will ultimately improve the overall health and quality of life in human.