Zinc chloride, magnesium (II) gluconate hydrate, pyridoxine hydrochloride, resveratrol, and cyanocobalamin (vitamin B₁₂) were purchased from TCI, Japan. Iron (II) sulphate, copper chloride, and cholecalciferol (vitamin D₃) were purchased from Sigma-Aldrich, USA. Telomerase assay was performed using 9 µg protein employing TeloTAGGG Telomerase PCR ELISA kit purchased from Roche Applied Science, USA. SIRT1 activity was assessed using Universal SIRT Activity Assay kit procured from Abcam, USA. Fetal bovine serum (FBS), epidermal growth factor (EGF) and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco, ThermoFisher, USA. Antibiotics solution was purchased from HiMedia, India, while 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium) (MTT), direct red 80 and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma, USA. All the other chemicals used in this experiment were analytical grade procured from India.

For evaluation of anti-aging activity, two cell lines were used viz. mouse pre-adicocytes (3T3-L1) for SIRT1 activity and freshly isolated PBMCs for telomerase assay, respectively. 3T3-L1 cells were originated procured from embryo of Mus musculus, mouse. PBMCs cells were isolated from blood tissue from human, healthy volunteers. Both the two cell lines were maintained in the DMEM growth medium supplemented with 15% FBS, with added antibiotics penicillin (100 U/mL) and streptomycin (100 μg/mL). Standard cell line growth conditions were maintained such as 37C, 5% CO₂, and 95% humidity. Positive control, resveratrol was used specifically at different non-cytotoxic concentration in MTT assay 35.

The experimental test groups in anti-aging model were divided into baseline control group which included cells with DMEM, normal control group, vehicle control group (0.08% DMSO), positive control group, and the experimental tested groups at different safe concentrations. The experimental test groups included the Biofield Energy Treated and untreated test formulation with DMEM.

The test formulation was divided into two parts. One part each of the test formulation was treated with Biofield Energy by a renowned Biofield Energy Healer, Mahendra Kumar Trivedi remotely for ~3 minutes under standard laboratory conditions and coded as the Biofield Energy Treated test formulation. While, the second part did not receive any sort of treatment and coded as the untreated test formulation group (Control). Biofield Energy Healer in this study never visited the laboratory (Dabur Research Foundation, New Delhi, India), nor had any contact with the test formulation. This Biofield Energy Healing Treatment was provided through Healer s unique Energy Transmission process to the test formulation. Further, the control groups were treated by a sham healer for comparative purposes. The sham healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed conditions for experimental study 35.

The non-cytotoxic concentrations of the test samples were estimated in both the cell lines using MTT assay. 3T3-L1 cells were trypsinized, counted and then plated in 96-well plates at the density corresponding to 5 X 10³ cells/well/180 µL of growth medium, while PBMCs were isolated from human blood (healthy volunteers) using HiSep(R) density gradient centrifugation method. PBMCs were counted and plated in 96-well plates at the density corresponding to 50 X 10³ cells/well/180µL of growth medium. Both the cells were counted and plated in 96-well plates and were incubated overnight under specific growth conditions, which were allowed for cell recovery and exponential growth followed by serum stripping or starvation. The cells were subsequently treated with the Biofield Energy Treated and untreated test formulation at different concentrations followed by incubation from 24 to 72 hours in a CO₂ incubator at 37°C, 5% CO₂, and 95% humidity. Further, serum-free MTT media was added followed by incubation for 3 hours at 37°C. The supernatant were aspirated and 150 µL of DMSO was added to each well to dissolve the formazan crystals. Thereafter, at 540 nm absorbance was recorded of each well using Synergy HT micro-plate reader, BioTek, USA. The concentrations that exhibited percentage cytotoxicity of less than 30% were considered as non-cytotoxic 35.

The 3T3-L1 cells were trypsinized, counted and then plated in 6-well plates at the density corresponding to 5 X 10⁵ cells/well, which was then incubated overnight under standard growth conditions as to allow the cell recovery and exponential growth. The cells were then treated with positive control and Biofield Energy Treated and untreated test formulation and incubated in a CO₂ incubator at 37°C, 5% CO₂, and 95% humidity. After 72 hours of treatment, nuclear extracts were prepared and protein estimation was carried out for the extracts using Pierce BCA Protein Assay Kit (Thermofischer Scientific) 7. SIRT1 activity was assessed using Universal SIRT Activity Assay kit (Abcam) by manufacturer s protocol, followed by the absorbance reading at 450 nm using Synergy HT microplate reader.

The percentage increase in SIRT1 activity was calculated using Equation 1:

% increase = (((X-T)/(R)) x 100) ----------- (1)

Where, X = SIRT1 activity corresponding to positive control and test groups after 72 hours

R = SIRT1 activity corresponding to Baseline group after 72 hours

The effect of the test formulation for the estimation of telomerase activity in PBMCs cell line was estimated. PBMCs were isolated from human blood using HiSep density gradient centrifugation method. PBMCs were counted and then plated in a 6-well plates at the density corresponding to 1.5 X 10⁶cells/well. The above cells were then treated with positive control and Biofield Energy Treated and untreated test formulation and incubated in a CO₂ incubator at 37°C, 5% CO₂ and 95% humidity. After 72 hours of incubation, lysates were prepared and protein estimation was carried out for the extracts using Pierce BCA Protein Assay Kit (Thermofischer Scientific), which was used for the estimation of telomerase activity 8. Telomerase assay was performed using 9 µg protein employing TeloTAGGG Telomerase PCR ELISA kit (Roche Applied Science) as per manufacturer s protocol followed by absorbance reading at 450 nm using Synergy HT microplate reader.

The percentage increase in the telomerase activity was calculated using Equation 2:

% increase = (((X-T)/(R)) x 100) ------------ (2)

Where, X = Absorbance of cells corresponding to positive control and test groups after 72 hours

R = Absorbance of cells corresponding to baseline group after 72 hours

All the values for anti-aging activity were represented as percentage or specific concentrations of the respective parameters. For multiple group comparison, one-way analysis of variance (ANOVA) was used and all the statistically significant values were set at the level of p≤0.05.

MTT assay was used to study the non-cytotoxic concentrations of the test formulation in both the cell lines i.e., 3T3-L1 and PBMCs. The results in term of percentage are shown in Figure 1. All the results in different groups were compared with respect to the different positive controls viz. resveratrol and phytohemagglutinin (PHA) in respective cell lines. MTT assay showed that the test formulation concentrations were found to be safe and non-toxic with more than 70% cell viability. 3T3-L1 cells showed more than 125% cell viability at 100 µg/mL test formulation concentration, which was significantly increased after Biofield Energy Treatment (Figure 1A). On the other hand, PBMCs also showed significant improved cell viability with more than 70% cell viability at 100 µg/mL test formulation concentration, which was significantly increased after Biofield Energy Treatment (Figure 1B). Thus, the non-toxic and safe test formulation concentration was further used for the assessment of anti-aging activity like SIRT assay and telomerase assay.

The effect of test formulation and positive control was tested for SIRT1 activity and the results are illustrated in Figure 2. The positive control, resveratrol showed a significant increased SIRT1 activity by 108.56%, 112.18%, 85.67%, and 63.41% at 10, 15, 30, and 40 µM, respectively. However, the data was presented using the protein estimation in each group. The data showed that protein amount was 21.97, 23.17, 21.39, 22.12, and 18.14 µg at 10, 25, 50, 75, and 100 µg/mL, respectively in the untreated test formulation group. Similarly, Biofield Energy Treated Test formulation group data showed that protein amount was 30.04, 23.12, 21.76, 22.07, and 22.12 µg at 10, 25, 50, 75, and 100 µg/mL, respectively. However, the overall data suggested that on the basis of protein content, SIRT1 activity in terms of OD/min/mg did not show any significant increased percentage activity with respect to the untreated test formulation group.

Telomere (enzyme which maintain telomeric length) length and aging are having direct correlation, as they are present at the extreme ends of chromosomes, which are the best indicators of biological age 36. It was reported that the increased age would result in telomere length shortening, which leads to apoptosis, cellular senescence, or oncogenic transformation of somatic cells, and significant affects the lifespan of individuals 37. Besides, it also increases the number of diseases and it was correlated with the lifestyle factors, diet, and activities that results in affecting the length of telomere. Our experimental results with Biofield Energy Treated Test formulation was studied for telomerase activity, which showed a significant improved activity after Biofield Energy Healing Treatment. The effect of test formulation and positive control was tested for telomerase activity and the results are illustrated in Figure 3. The positive control, PHA (phytohemagglutinin, µg/mL) showed a significant increased activity by 5.28%, 16.06%, and 31.75% at 0.1, 1, and 5 µg/mL, respectively as compared with the control group. However, the Biofield Energy Treated Test formulation showed a significant improved telomerase activity by 39.25%, 6.20%, 20.86%, and 17.95% at concentrations 0.01, 1, 5, and 100 µg/mL, respectively as compared with the untreated test formulation group. Thus, the overall data suggested that telomerase activity, which was directly related with aging was significantly increased that improves the overall human health. Besides, it was studied using in vitro model that the life-span can be increased by introduction of telomerase in human cell and its improved activity would reduce the age-related diseases 38.

The level of telomerase activity is very important for the determination of “telomere length” in aging cells. The significance of telomerase reactivation causes cancer development and for immortalizing the cells. Thus, telomere shortening occur in the human body during aging 39. From the mechanistic point of view the telomere length is controlled by two ways: attrition and elongation. Attrition occurs as each cell divides. However, elongation process is partially modulated by the enzyme telomerase, which adds more repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates the cells 40. Thus, it can be concluded that the Trivedi Effect^® would be the best approach towards anti-aging activity through improved telomerase activity by extending the telomere length.