Pyridoxine hydrochloride (vitamin B₆), calcitriol, 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, and sodium carboxymethylcellulose (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. Imipramine Hydrochloride was purchased from Sigma, USA. H9C2 (rat cardiomyocytes), C2C12 (mouse myoblast cells), HaCaT (human keratinocytes), and SH-SY5Y (human neuroblastoma cells) cell lines were procured from NCCS, Pune. Positive control, calcitriol purchased from TCI Chemicals, However, cell line medium such as DMEM, DMSO, FBS, EDTA, and MTT were procured from Genexlife, Protaq Biomedical, Genexlife, Parshuram and Parshuram traders, respectively.

H9C2, C2C12, HaCaT, and SH-SY5Y cell lines were used as test system in the present study. The cell lines were maintained in DMEM growth medium for routine culture supplemented with 10% FBS. Growth conditions were maintained as 37°C, 5%CO₂, and 95% humidity and subcultured by trypsinisation followed by splitting the cell suspension into fresh flasks and supplementing with fresh cell growth medium. Three days before the start of the experiment (i.e., day-3), the growth medium of near-confluent cells was replaced with fresh phenol-free DMEM, supplemented with 10% charcoal-dextran stripped FBS (CD-FBS) and 1% penicillin-streptomycin 31.

The experimental groups consisted of cells in baseline control, vehicle control group (0.05% DMSO with Biofield Energy Treated/Blessed and untreated DMEM), positive control group (Calcitriol) and four different experimental test groups. The experimental groups included the combination of the Biofield Energy Treated/Blessed and untreated test formulation/Medium (DMEM). It consisted of four major treatment groups on specified cells with Untreated (UT)-DMEM + UT-Test item (UT-TI), UT- DMEM + Biofield Energy Treated/Blessed test item (BT-TI), BT- DMEM + UT-TI, and BT- DMEM + BT-TI.

The novel test formulation was consisted of zinc chloride, iron (II) sulfate, copper chloride, vitamin B₆, vitamin B₁₂, vitamin D₃, sodium selenate, calcium chloride, ascorbic acid, vitamin E, beta carotene, Panax ginseng extract, cannabidiol isolate and magnesium (II) gluconate. Each ingredient of the test formulation was divided into two parts, one part of the test compound was not received 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/Blessing Treatment (Biofield Energy Treatment) by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi under laboratory conditions for ~3 minutes in the research laboratory, Dabur Research Foundation, New Delhi, India without touching the samples. After that, the Biofield Energy Treated/Blessed 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, under the same laboratory conditions. The sham healer did not have any knowledge about the Biofield Energy Treatment/Blessing. The Biofield Energy Treated/Blessed test medium was also taken back to experimental room for further culture methods.

The single cell suspension of H9C2, C2C12, HaCaT, and SH-SY5Y cells were prepared in DMEM with 10% FBS. The cells were counted on a hemocytometer, while the cells were seeded with specific cell density ((5000) cells in H9C2, while 10,000 C2C12, HaCaT, and SH-SY5Y cells/well/180 µL in DMEM + 10% FBS in 96-well plates). The cells were incubated in a CO₂ incubator for 48 hours. After 24 hours, medium was removed, and following treatments were given in medium along with the 10% FBS in various experimental groups. After incubation for 24 hours, the effect of the test formulation on cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. About 20 µL of 5 mg/mL of MTT was added to all the wells and incubated at 37°C for 3 hours. The cells were centrifuged to obtain the pellet. The supernatant was removed and 150 µL of DMSO was added to all wells to dissolve formazan crystals. Further, all the wells were reported using optical density (OD) values at 540 nm using Synergy HT microplate reader. The effect of the test formulation on viability of cells was determined using equation 1.

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

Where; % Cytotoxicity = {(O.D. of Control cells - O.D. of cells treated with test formulation)/ OD of Control cells} *100

The single cell suspension of H9C2, C2C12, HaCaT, and SH-SY5Y cells were prepared in DMEM and 10% FBS using a hemocytometer. The cells were seeded density of 30,000 cells/well/0.5 mL in 48-well plates and incubated in a CO₂ incubator for 24 hours and 95% humidity. The cells were centrifuged to obtain the pellet. Supernatants were removed and cells were resuspended in DMEM with 10% FBS. After treatment in all the experimental test groups, the cells were I ncubated in a 5% CO₂ incubator for 48 hours, while H9C2 cells were incubated for 72 hours. After 24 hours of incubation, culture supernatants were collected from each well and stored at -20°C until analysis. The cells were fixed using 1% glutaraldehyde for 20 minutes, RT and processed for elasticity measurement by AFM at IISC, Bangalore. Young modulus was calculated from the force/ distance indentation curves generated during AFM analysis using XEI data processing and analysis software.

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.

The cytotoxic effect of the test formulation was evaluated on H9C2, C2C12, HaCaT, and SH-SY5Y cells using MTT assay. The results were compared with respect to defined positive control such as calcitriol. The cells were treated with the test formulation for 48 hours. The effect on viability of cells was determined after 48 hours of treatment by MTT assay (Figure 1). The cells were treated with the test formulation and in various experimental test groups. Calcitriol (PC) demonstrated 164.8%, 110.5%, 77.7%, and 96.4% cell viability at concentration 500 nM in the H9C2, C2C12, HaCaT, and SH-SY5Y cell lines, respectively. In addition, the test formulation resulted more than 73.11% cell viability at the concentration range of 0.01 µg/mL to 10 µg/mL. The results of percentage cell viability range in all the tested cell lines showed the cell viability range of 73% to 307% in different test formulation groups. Overall, the MTT data suggested that the test formulation along with DMEM groups were found safe at all the tested concentrations range up to maximum 10 µg/mL in the tested cell lines.

Cellular elasticity in H9C2 cells was determined using percentage change in young’s modulus (YM). The cells were co-treated with the test formulation and stimulated with 0.01 µg/mL to 10 µg/mL for 72 hours. The level of YM was calculated in all the groups, which were compared and presented graphically (Figure 2). The decreased values of YM were calculated on the basis of the untreated test group of DMEM and test formulation. Calcitriol data showed a significant decreased value of YM by 11.1%, 48.3%, and 56.9% at 10, 100, and 500 nM concentrations, respectively. YM in all the treated experimental groups were significantly decreased as compared with the H9C2 cells in DMEM control group, which suggest an improved cellular elasticity of H9C2 cells. Furthermore, the data of the Biofield Energy Treated/untreated test formulation/DMEM combination was compared with respect to the untreated test formulation and DMEM group. Thus, the data suggested that UT-DMEM + BT-TI group showed increase YM by 44.9% and 25.5% in the 0.01 and 0.1 µg/mL, respectively; while decreased YM by 48.8% at 1 µg/mL as compared with the UT-DMEM + UT-TI group. Moreover, 66.1% increased the value of YM was also reported in the BT-DMEM + UT-TI group at 1 µg/mL as compared with untreated test group. Besides, BT-DMEM + BT-TI group showed a increased YM value by 96.8% and 24.9% at 0.01 and 0.1 µg/mL, respectively; while YM value was decreased by 11.5% at 1 µg/mL as compared with the UT-DMEM + UT-TI group. Overall, H9C2 cells (human cardiac muscle cells) treated with Biofield Energy Treated/Blessed medium and TI showed a modulation of elasticity. Modulation in elasticity of cardiac cells (cardiomyocytes) is important for normal heart functions (contractile functions) 3233.

The C2C21 cells were evaluated for cellular elasticity, which was determined using percentage change in young’s modulus (YM) and the data was compared. The cells were co-treated with the test formulation and stimulated with 0.01 µg/mL to 10 µg/mL for 48 hours. The level of YM was calculated in all the groups, which are compared and presented graphically (Figure 3). The increased values of YM were calculated on the basis of untreated test group of DMEM and test formulation. Calcitriol data showed a significant increased the value of YM by 203.8%, 525.3%, and 733.7% at 50, 100, and 500 nM concentrations, respectively as compared with the C2C21 cells in DMEM. The data of Biofield Energy Treated/untreated test formulation/DMEM combination was compared with respect to the untreated test formulation and DMEM group. The increased values of YM were calculated on the basis of untreated test group of DMEM and test formulation. Thus, the data suggested that UT-DMEM + BT-TI group showed decreased value of YM by 103.8% and 84.2% at 0.01 and 0.1 µg/mL, respectively while increased the value of YM by 443.9% at 1 µg/mL as compared with untreated test group (UT-DMEM + UT-TI group). However, 74.1% and 869.6% increased the value of YM was also reported in the BT-DMEM + UT-TI group at 0.1 and 1 µg/mL, respectively as compared with the untreated test group. Besides, BT-DMEM + BT-TI group showed an increased YM value by 314% at 1 µg/mL as compared with the UT-DMEM + UT-TI group. Therefore, in present study increase in YM with Biofield Energy Treatment suggests an increase of differentiation of skeletal muscle cells. Muscle cells have different mechanical properties depending on the physiological processes (cell differentiation, growth, adhesion, etc.). In muscle cells, an increase in YM has been observed which correlates with the extent of differentiation 34. Overall, the present study increase(s) in YM after Biofield Energy Treatment/Blessing suggested increase of differentiation of skeletal muscle cells.

The cellular elasticity in HaCaT cells was determined using percentage change in young’s modulus (YM). The cells were co-treated with the test formulation and stimulated with 0.1 µg/mL to 10 µg/mL for 48 hours. The level of YM was calculated in all the groups, which are compared and presented graphically (Figure 4). The altered values of YM were calculated on the basis of untreated test group of DMEM and test formulation. The values of YM were calculated on the basis of the untreated DMEM and test formulation. Calcitriol data showed a significant decreased value of YM by 7.4%, 28.5%, and 52.7% at 10, 50, and 100 nM concentrations, respectively as compared with the HaCaT cells in DMEM control group. YM in all the treated experimental groups were significantly decreased as compared with the HaCaT cells in DMEM control group, which suggest an improved cellular elasticity of HaCaT cells. Furthermore, the data of Biofield Energy Treated/untreated test formulation/DMEM combination was compared with respect to the untreated test formulation and DMEM group. Thus, the data suggested that UT-DMEM + BT-TI group showed a significant reduced the value of YM by 247.7% at 1 µg/mL as compared with the untreated test group. However, 225.8% and 59.7% decreased the value of YM was also reported in the BT-DMEM + UT-TI group at 0.1 and 1 µg/mL, respectively as compared with the untreated test group. Besides, BT-DMEM + BT-TI group showed a significant decreased YM value by 25.9% and 97.9% at 0.1 and 1 µg/mL, respectively as compared with the UT-DMEM + UT-TI group. Thus, present study concluded that when cells were treated with Biofield Energy Treated/Blessed medium and test formulation, decrease in YM was observed, which indicates an increase in cellular elasticity. In the present study parameter, decrease in YM with Biofield Energy Treatment/Blessing suggests an increase in elasticity of keratinocytes. However, it was reported that modulation in elasticity of skin cells is important for maintenance of skin firmness and elastic properties 35, which was significant improved after Biofield Energy Treatment/Blessing.

The cellular elasticity in HaCaT cells was determined using percentage change in young’s modulus (YM). The cells were co-treated with the test formulation and stimulated with 0.1 µg/mL to 10 µg/mL for 48 hours. The level of YM was calculated in all the groups, which are compared and presented graphically (Figure 5). The altered values of YM were calculated on the basis of untreated test group of DMEM and test formulation. The values of YM were calculated on the basis of the untreated DMEM and test formulation. Calcitriol data showed a significant decreased value of YM by 7.4%, 28.5%, and 52.7% at 10, 50, and 100 nM concentrations, respectively as compared with the HaCaT cells in DMEM control group. YM in all the treated experimental groups were significantly decreased as compared with the HaCaT cells in DMEM control group, which suggest an improved cellular elasticity of HaCaT cells. Furthermore, the data of Biofield Energy Treated/untreated test formulation/DMEM combination was compared with respect to the untreated test formulation and DMEM group. Thus, the data suggested that UT-DMEM + BT-TI group showed a significant reduced the value of YM by 247.7% at 1 µg/mL as compared with the untreated test group. However, 225.8% and 59.7% decreased the value of YM was also reported in the BT-DMEM + UT-TI group at 0.1 and 1 µg/mL, respectively as compared with the untreated test group. Besides, BT-DMEM + BT-TI group showed a significant decreased YM value by 25.9% and 97.9% at 0.1 and 1 µg/mL, respectively as compared with the UT-DMEM + UT-TI group. Thus, present study concluded that when cells were treated with Biofield Energy Treated/Blessed medium and test formulation, decrease in YM was observed, which indicates an increase in cellular elasticity. In the present study parameter, decrease in YM with Biofield Energy Treatment/Blessing suggests an increase in elasticity of keratinocytes. However, it was reported that modulation in elasticity of skin cells is important for maintenance of skin firmness and elastic properties 36, which was significant improved after Biofield Energy Treatment/Blessing.

In this research plan, the results showed the significant improved level of cellular elasticity in H9C2, C2C12, HaCaT, and SH-SY5Y cell-lines, which helps in slowdown of the disease progression, disease-related all other symptoms/complications and also reduced the chances of disease susceptibility. This improved cellular differentiation, contractile functions, exonal extensions, and skin elasticity and firmness of the cell lines used in the study after treatment was very significant. Based on the overall data, it suggests that the Biofield Energy Healing/Blessing Therapy was found to be most effective and benefited in order to prevent and protect from the occurrence of any type of diseases. It can also be used as significant way for energy boosting in various disease states that ultimately improve the overall health and quality of life in human.