Evaluation of Cardiac Performance after Treatment with the Biofield Energy Treated Proprietary Test Formulation on L-NAME and High Fat Diet-Induced Cardiovascular Disorders in Sprague Dawley Rats

Study was aimed to evaluate effect of Biofield Treated Proprietary Formulation and Biofield Treatment per se on cardiac performance on NG-nitro-Larginine methyl ester hydrochloride (L-NAME) and high fat diet (HFD)-induced cardiovascular disorders in Sprague Dawley rats. Nine groups were assigned, in which four were preventive maintenance groups. The constituents of test formulation were divided into two parts; one section was defined as the untreated test formulation, while the other portion of the test formulation and three groups of animals received Biofield Energy Healing Treatment remotely for about 3 minutes by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi. Systolic blood pressure (SBP) was significantly (p≤0.001) decreased by 13.39%, 14.65%, 17.74%, 14.36%, and 14.69% in the G5, G6, G7, G8, and G9 groups, respectively as compared to disease control (G2) group. Diastolic blood pressure (DBP) was significantly (p≤0.001) reduced by 25.95%, 24.41%, 30.79%, 24.67%, and 25.23% in G5, G6, G7, G8, and G9 groups, respectively than G2. Heart rate (HR) was significantly (p≤0.05) reduced by 6.58%, 8.06%, and 6.99% in G7, G8, and G9 groups, respectively than G2. Total leucocyte count (TLC) count was increased by 12.8% and 17.45% in G5 and G8 groups, respectively than G2 group. Neutrophils and lymphocytes were increased by 60.11% (G8) and 11.49% (G5), respectively than G2. Eosinophils were reduced by 11.11%, 20%, and 15% in G6, G7, and G9 groups, respectively than G2. Total cholesterol was significantly decreased by 22.64% (p≤0.05), 15.78%, 25.56% (p≤0.05), 22.56%, and Journal of Hypertension and Cardiology


Introduction
High blood pressure is one of the most important risk factors for cardiovascular diseases (CVDs), which is the leading cause of mortality. About 54% of strokes and 47% of coronary heart diseases has been occurs in the worldwide due to high blood pressure [1]. There are various normal physiological parameters, which denote the healthy status of an individual, if present within the normal range. However, the presence of some agents/ compounds indicates the physiological uncomforting of the individual that might results due to an operation of different energy consuming mechanisms within the body in the process of maintaining the homeostasis and thereby involves numerous biomarkers [2,3]. Moreover, the changes in the body due to oxidative stress involves various physiological and endocrine alterations, along with disturbances in the biochemical and metabolic systems [4,5]. Furthermore, such alterations consequently affect the important system of the body such as cardiovascular, renal, and CNS, followed by the hepato-biliary and pancreatic systems [6,7]. Similarly, imbalance in the metabolic parameters such as, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), cholesterol, glucose, triglycerides (TG), etc. There are many risk factors associated with CVDs such as abnormal blood lipid and sugar levels, obesity, smoking, and high blood pressure. Cholesterol plays the detrimental roles in the pathogenesis of atherosclerosis and CVDs [8]. Besides, the physiological parameters involved in the health and function of various vital organs like liver enzymes (SGOT, SGPT), kidney markers (uric acid, albumin); and in the heart like atherogenic index (AI), creatine kinase-myocardial band (CKMB) etc. have been enumerated [9,10]. Thus, in order to study the change in vital function of heart in presence of L-NAME and High Fat Diet (HFD)-induced cardiovascular disorders in Sprague Dawley rats, a novel test formulation was designed with the combination of vital minerals (selenium, zinc, iron, calcium, copper, and magnesium), essential vitamins (cyanocobalamin, ascorbic acid, pyridoxine HCl, vitamin B9, and cholecalciferol), and nutraceuticals (β-carotene, Ginseng, cannabidiol isolate (CBD)). All the minerals and vitamins incorporate in this test formulation have significant physiological roles [11][12][13]. Besides, cannabidiol itself has wide range of pharmacological profile and has been reported to role in different disorders [14,15], while ginseng extract is regarded as one of the best immune boosters for overall immunity [16]. The

Experimental Procedure
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 animals were divided into nine (9) different groups are shown in Table 1.
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 HFD throughout the experimental period. At the end of the experimental period (8 weeks treatment), recorded systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR). After that, all the animals were individually subjected for blood collection using retro-orbital route to the experimental purpose such as hematology and biochemistry. The organs were isolated and recorded as wet weight.

Measurement of Blood Pressure and Heart Rate
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.

Assessment of Hematology Parameters
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.

Assessment of Hepatic and Cardiac Biomarkers
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.

Determination of Body Weight, Feed Intake, and Organ weight parameters
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.

Clinical Sign and Symptoms
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.

Statistical Analysis
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.

Measurement of Blood Pressure and Heart Rate
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  The effect of the test formulation on A. blood pressure (systolic and diastolic) and B. heart rate 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=7 to 9). *p≤0.05 and ***p≤0.001 vs. Disease control (G2).  11.11%, 20%, and 15% in the G4, G6, G7, and G9 groups, respectively as compared with the G2 group.

Measurement of Glucose and Lipid Biomarkers
Lipid biomarker analysis was performed after treatment with the Biofield Energy Treated and untreated test formulations are summarized in the

Evaluation of Hepatic and Cardiac Biomarkers
The effects of the test formulation on hepatic and cardiac biomarkers are shown in