N-acetyl cysteine (NAC), 2′,7′-Dichlorofluorescin diacetate (DCFDA), 3-(4,5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT), and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO). Antibiotics solution (penicillin-streptomycin) was purchased from HiMedia, India. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco, India. Creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) kits were obtained from Biovision, USA. Annexin-V kit was purchase from Guava Technologies, USA. The positive control, trimetazidine (TMZ) was procured from Zliesher Nobel, USA. All the other chemicals used in this experiment were analytical grade procured from India.

The test item (DMEM) was used in this experiment and one portion was considered as the untreated group, where no Biofield Treatment was provided. Further, the untreated group was treated with “sham” healer for comparison purpose. The sham healer did not have any knowledge about the Biofield Energy Healing Treatment. The other portion of the test item was received one-time Biofield Energy Treatment and referred as the BT-I and was also given two-times Biofield Energy Treatment and defined as the BT-II. Both the test items (BT-I and BT-II) were received Biofield Energy Healing Treatment (known as The Trivedi Effect^®) under laboratory conditions for ~3 minutes through Mahendra Kumar Trivedi’s unique Biofield Energy Transmission process. Biofield Energy Healer was located in the USA, however the test items were located in the research laboratory of Dabur Research Foundation, New Delhi, India. Biofield Energy Healer in this experiment did not visit the laboratory, nor had any contact with the test samples. After that, the Biofield Energy Treated and untreated test items were kept in similar sealed conditions and used for the study as per the study plan.

The cell viability was performed by MTT assay in H9c2 cells (ATCC^® CRL-1446™). The cells were counted and plated in a 96-well plate at the density corresponding to 10 X 10³ cells/well/180 µL in DMEM + 10% FBS. The cells in the above plate(s) were incubated for 24 hours in a CO₂ incubator at 37°C, 5% CO₂, and 95% humidity. Following incubation, the medium was removed and the following treatments were given. In the test item group, 200 µL of the test item was added to wells. Besides, in the positive control group, added 180 µL of SFM with 20 µL of positive controls were added from the respective 10X stock solutions. After incubation for 48 hours, the effect of test item on cell viability was assessed by MTT assay. 20 µL of 5 mg/mL of MTT was added to all the wells and incubated at 37°C for 3 hours. The supernatant was aspirated and 150 µL of DMSO was added to all wells to dissolve formazan crystals. The optical density (OD) of each well was read at 540 nm using Biotek Reader.

Effect of the test items on viability of H9c2 cells was determined using Equation (1):

% Cell viability =(100-% Cytotoxicity)……………..(1)

Where, % Cytotoxicity = {(O.D. of untreated cells – O.D. of cells treated with test item)/ OD of untreated cells}*100

The concentrations resulting in ≥70% cell viability were taken as safe/non-cytotoxic for cytokine estimation.

Cells were trypsinized and a single cell suspension of H9c2 was prepared. Cells were counted on an hemocytometer and seeded at a density of 5 X 10³ cells/well/180 µL in DMEM + 10% FBS in a 96-well plate. Cells were incubated in a CO₂ incubator for 24 hours at 37°C, 5% CO₂,and 95% humidity. After 24 hours, the medium was removed and the following treatments were given. In the test item group, 180 µL of the test item was added to wells. In the positive control group, 160 µL of SFM and 20 µL of positive control from the respective 10X stock solution was added to wells. After 24 hours of treatment, cells were treated with t-BHP at a final concentration of 250 μM (20 µL from the respective 10X stock) for 4 hours. After 4 hours, the protective effect of the test item on cell viability was assessed by MTT assay. The protective effect of the test item on survival of H9c2 cells against t-BHP induced damage was determined using Equation (2)

((A-B)/(C-B))*100……………(2)

Where, A = O.D. of test item/positive control + t-BHP treated cells

B= O.D. of cells (t-BHP alone)

C = O.D. of untreated cells

Cells were trypsinized and a single cell suspension of H9c2 was prepared. Cells were counted (using hemocytometer) and seeded (at a density of 0.12 X 10⁶ cells/well/500 µL) in DMEM + 10 % FBS in 48-well plates. Cells were incubated in a CO₂ incubator for 24 hours at 37°C, 5 % CO₂ and 95% humidity. After 24 hours, medium was removed and following treatments were given. Test items (BT-I and BT-II) groups (450 µL of Biofield Treated DMEM), positive controls (trimetazidine and N-acetyl cysteine) groups (400 µL of SFM), and (untreated DMEM) group (500 µL of SFM) were added to the respective wells and incubate for 24 hours. After that, cells were treated with 300 µM of t-BHP (50 µL from the respective 10X stock) for 2.5 hours. Supernatants were collected from each well and stored at -20°C till analyzed. Estimation of LDH in culture supernatants was done using Lactate Dehydrogenase Activity Colorimetric Assay Kit as per manufacturer’s instructions. LDH activity (nMoles/min/mL) was determined and the protective effect of test item was calculated using Equation (3):

((A-B)/(A-C))*100……………(3)

Where, A = LDH activity in cells (t-BHP alone)

B= LDH activity in test items/positive controls + t-BHP induced cells

C = LDH activity in untreated cells

Cells were trypsinized and a single cell suspension of H9c2 was prepared. Cells were counted on a hemocytometer. Cells were seeded at a density of 0.12 X 10⁶ cells/well/500 µL in DMEM + 10 % FBS in 48-well plates. Cells were incubated in a CO₂ incubator for 24 hours at 37°C, 5 % CO₂ and 95 % humidity. After 24 hours, medium was removed and treatments were given.

Test items (BT-I and BT-II) groups (450 µL of Biofield Treated DMEM), positive control (N-acetyl cysteine) group (400 µL of SFM), t-BHP per se group (450 µL of SFM), and negative control (untreated) group (500 µL of SFM) were added to the respective wells and incubate for 24 hours. After incubation for 24 hours, cells were treated with 300 µM of t-BHP (50 µL from the respective 10X stock) for 2.5 hours. Supernatants were collected from each well and stored at -20°C till analyzed. Estimation of CK-MB in culture supernatants was done using Creatine Kinase Activity Colorimetric Assay Kit as per manufacturer’s instructions. CK-MB activity (nMoles/min/mL) was determined and protective effect of test item on CK-MB activity was calculated using Equation (4):

((A-B)/(A-C))*100……………(4)

Where, A = CK-MB activity in cells (t-BHP alone)

B= CK-MB activity in test items/positive controls + t-BHP treated cells

C = CK-MB activity in untreated cells

Cells were trypsinized and a single cell suspension of H9c2 was prepared. Then, the cells were counted with the help of a hemocytometer and seeded (at a density of 20 X 10³ cells/well/180 µL in DMEM + 10 % FBS) in 96-well plates. Cells were incubated in a CO₂ incubator for 24 hours at 37 °C, 5 % CO₂ and 95 % humidity. Then, medium was removed and treatments were given. About 180 µL of the test item (TI), 160 µL of SFM, 180 µL of SFM, and 200 µL of SFM was added to wells of test items, positive controls, t-BHP per se, and untreated DMEM groups, respectively and incubate for 24 hours. After incubation for 24 hours, cells were stained with DCFDA and washed the wells once with Hank's Balanced Salt Solution (HBSS) + 2% FBS solution and 180 µL of SFM was added to each well. Protective effect of TI on ROS activity was calculated using Equation (5):

((A-B)/(A-C))*100……………(5)

Where, A = Mean FU in Control cells (t-BHP alone)

B= Mean FU in TI/positive control + t-BHP treated cells

C = Mean FU in untreated cells

Cells were trypsinized and a single cell suspension of H9c2 was prepared. Cells were counted using hemocytometer and seeded at a density of 0.25 million/well/1 mL in DMEM + 10% FBS in 96-well plates. Further, the cells were incubated in a CO₂ incubator for 24 hours at 37°C, 5 % CO₂ and 95% humidity. After 24 hours, medium was removed and the following treatments were given. The TI group received 900 µL of Tis (BT-I and BT-II), positive control (N-acetyl cysteine) group received 800 µL of SFM, t-BHP group received 900 µL of SFM, and the untreated DMEM group provided 1 mL of SFM to the corresponding wells and incubate for 24 hours. After that, cells were treated with 300 µM of t-BHP (100 µL from the respective 10X stock) for 2.5 hours. Then, the cells were stained with Annexin reagent for apoptotic population as follows: cells were gently harvested by trypsinisation into prelabeled centrifuge tubes followed by pelleted and resuspended in 200 µL of SFM. At 100 µL of cell suspension was stained with 100 µL of Annexin reagent for 30 minutes in a dark condition at room temperature. Cells were acquired at flow cytometer (Guava technologies). The protective effect of the TI was calculated using Equation (6):

((A-B)/(A-C))*100……………(6)

Where, A = % Apoptotic population in t-BHP

B = % Apoptotic population in test items/positive control + t-BHP treated cells

C = % Apoptotic population in untreated cells

All the values were represented as Mean ± SEM (standard error of mean) of three independent experiments. The statistical analysis was performed using SigmaPlot statistical software (v11.0). For two groups comparison student’s t-test was used. For multiple group comparison, one-way analysis of variance (ANOVA) was used followed by post-hoc analysis by Dunnett’s test. Statistically significant values were set at the level of p≤0.05.

Evaluation of cell viability after treatment with the positive controls and the test items in H9c2 cells is shown in Figure 1 The cardioprotective activity of Biofield already been published by Branton A, 2019 ³⁵. The positive controls, trimetazidine (TMZ) showed more than 88% at the concentrations between 0.1 to 100 µg/mL and N-acetyl cysteine (NAC) showed greater than 74% cell viability upto 500 µg/mL. Besides, the Biofield Energy Treated test items, BT-I (one-time Biofield Energy Treated DMEM) and BT-II (two-times Biofield Energy Treated DMEM) showed 89.03% and 98.49% cell viability, respectively. Overall, the Biofield Energy Treated test items found as a safe and non-toxic profile of the test substances and further used in this experiment for the assessment of various cardiac parameters.

Figure 1. Effect of the test items and positive controls on cell viability in H9c2 cells after 24 hours of treatment. UNT: Untreated; TMZ: Trimetazidine; NAC: N-acetyl cysteine; BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM

For the assessment of cardioprotective activity of the test compounds, tert-butyl hydroperoxide (t-BHP) is a well-recognized oxidative stress inducer in the in vitro cell-based assays ^{36, 37}. The cytoprotective activity of the Biofield Energy Treated test items on the restoration of cell viability in H9c2 cells was determined against t-BHP induced cell damage and the result is shown in Figure 2. Trimetazidine (TMZ) resulted, restoration of cell viability by 54.1%, 41.3%, 14.33%, and 4.16% at 0.1, 1, 10, and 50 µg/mL, respectively compared to the t-BHP induced group. Besides, the test group’s like the one-time Biofield Energy Treated DMEM (BT-I) showed 16.01% and two-times Biofield Energy Treated DMEM exhibited 4.43% restoration of cell viability with respect to the t-BHP induced group. The cellular antioxidant capacity can reduced due to excess production of free radicles that leads to inflammation ³⁸. This excess levels of free radicles can affect the normal functions of cell membrane, and ultimately altered the genetic materials and cause various age-related disorders such as diabetes, cardiovascular, autoimmune diseases, and cancer ^{39, 40, 41}. The results suggest that Biofield Treatment has significantly protects t-BHP induced cardiotoxicity, which could be due to The Trivedi Effect^®. Therefore, Biofield Energy Healing Treatment could be used for the management of cardiovascular disorders.

Figure 2. Assessment of cytoprotective effect of the test items in H9c2 cells against tert-butyl hydroperoxide (t-BHP) induced damage. TMZ: Trimetazidine; BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM

The distribution of lactate dehydrogenase (LDH) is mainly abundant in the heart and skeletal muscle, is a tetrameric enzyme, and is mainly responsible for anaerobic respiration of cells ^{42, 43, 44}. The effect of test items on the level of lactate dehydrogenase (LDH) is presented in Figure 3. The level of LDH activity was significantly increased by 892.05% in the tert-butyl hydroperoxide (t-BHP) induced group as compared to the untreated DMEM group (3.65 ± 0.5 nMoL/min/mL). The positive control, trimetazidine (TMZ) exhibited 8.04%, 18.64%, and 96.13% inhibition of lactate dehydrogenase (LDH) compared to the untreated DMEM group. Besides, One-time Biofield Energy Treated DMEM group (BT-I) and two-times Biofield Energy Treated DMEM group (BT-II) group showed 50.37% and 49.35%, respectively as compared to the untreated DMEM group.

Figure 3. The effect of the test items on lactate dehydrogenase (LDH) against tert-butyl hydroperoxide (t-BHP) induced damage. TMZ: Trimetazidine; BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM.

The impact of the Biofield Energy Treated test items on cardiac marker, creatine kinase-myocardial band (CK-MB) is shown in Figure 4. The level of CK-MB was significantly increased by 4.96% in the tert-butyl hydroperoxide (t-BHP) induced group as compared to the untreated DMEM group (1.14 ± 0.16 nMol/min/mL). The positive control, N-acetyl cysteine (NAC) showed 4.86%, 24% (p≤0.001), and 90.57% (p≤0.001) significant inhibition of CK-MB enzyme activity in a concentration-dependent manner at 25, 50, and 100 µM, respectively compared to t-BHP induced group. Further, the Biofield Treated test items group, BT-I (one-time Biofield Energy Treated DMEM) and BT-II (two-times Biofield Energy Treated DMEM) showed significant (p≤0.001) inhibition of CK-MB enzyme level by 49.48% and 59.79%, respectively as compared to the t-BHP induced group. This reduction of tissue-specific cardiac biomarker (CK-MB) is very essential for the diagnosis of cardiac functions apart from of troponin T (cTnI) and myoglobin (Myo) ^{45, 46}. As CK-MB is a sensitive and specific indicator for the diagnosis of an acute myocardial infarction (AMI) ⁴⁷. Overall, the Biofield Treated test items (BT-I and BT-II) has significantly inhibited the levels of cardiac tissue-specific enzyme CKMB, which was induced by t-BHP.

Figure 4. The effect of the test items (24 hours of pretreatment) on Creatine Kinase-Myocardial Band (CK-MB) activity against tert-butyl hydroperoxide (t-BHP) induced damage after 4 hours of treatment. NAC: N-acetyl cysteine; BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM. ***p≤0.001 vs. t-BHP at 300 µM.

Reactive oxygen species (ROS) play a vital role in the development of cardiovascular disorders specifically atherosclerosis ⁴⁸. Due to misbalance of ROS production and antioxidant defense capacity results in oxidative stress ^{49, 50}. Although ROS is normally required in cell signaling pathways, while excessive production of ROS leads to cell damage ⁵¹. The number of fluorogenic substrates that serve as hydrogen donors have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products ⁵². In this assay, the effect of the Biofield Treated test items on the ROS induced by t-BHP in terms of number of fluorescence unit (FU) is shown in Figure 5. The positive control, trimetazidine (TMZ) showed 5.46% reduction of FU at 25 µM as compared to the untreated DMEM group. Further, one-time Biofield Energy Treated DMEM (BT-I) showed 6.64% reduction of mean FU compared to the untreated group. Results found that the BT-I have significantly protect cardiomyocytes from oxidative stress. This inhibition of ROS could be due to the Biofield Energy Treatment through the change in protons and neutrons in the nucleus caused by weak interactions.

Figure 5. The effect of the test items on reactive oxygen species in terms of fluorescence unit (FU) in H9c2 cells after 24 hours of treatment. FU: Fluorescence unit; TMZ: Trimetazidine; BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM.

Cardiovascular disorders is the leading cause of morbidity and mortality in the developed world. Apoptosis, a process of programme cell death that plays a vital role in various pathologic conditions related to cardiovascular system ⁵³. Inhibition of apoptotic pathway is one of the potential treatment approach for the management of cardiovascular disorders ^{54, 55}. The apoptotic process ensures that damaged, aged, or excess cells are deleted in a regulated manner that is not harmful to the host ⁵⁶. Thus, increased level of percent apoptotic cells is directly link with the overall health. The effect of the test items on the level of percent apoptotic cells is shown in Figure 1. The positive control, N-acetyl cysteine (NAC) was significantly increased the level of percent apoptotic cells by 94.08%, 77.63%, and 57.89% at 100, 200, and 300 µM, respectively compared to the untreated DMEM group. Further, Biofield Treated test items BT-I (one-time Biofield Energy Treated DMEM) and BT-II (two-times Biofield Energy Treated DMEM) showed significantly increased the percent of apoptotic cells by 63.16% and 97.37% (p≤0.05), respectively compared to untreated DMEM. Overall, results suggested that Biofield Energy can increase the level of percent apoptotic cells, which could be able to remove damaged, unwanted, aged, and excess cells from the body Figure 6.

Figure 6. Effect of the test items on percent apoptotic cells in H9c2 cells after 24 hours of treatment. NAC: N-acetyl cysteine; BT-I: One-time Biofield Energy Treated DMEM; BT-II: Two-times Biofield Energy Treated DMEM. *p≤0.05 vs. untreated DMEM.