Pyridoxine hydrochloride (vitamin B₆), zinc chloride, cyanocobalamin (vitamin B₁₂), magnesium (II) gluconate, and resveratrol were purchased from TCI, Japan. Copper chloride, cholecalciferol (vitamin D₃), sodium carboxymethylcellulose (Na-CMC), and iron (II) sulfate were procured from Sigma-Aldrich, USA. D (+) galactose obtained from Amresco, LLC. Any other chemicals used in this study were analytical grade acquired from India. 

Randomly breed male Sprague Dawley (SD) rats (n=10) with body weight ranges from 238 to 420 gm were used in this study. The animals were purchased from M/s. National Institute of Biologicals, India. Animals were randomly divided into nine groups based on their body weights consist of ten animals in each group. 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.

The test formulation was separated into two parts. The first part of each ingredient was considered as control, where no Biofield Energy Treatment was provided. The second part of each component was received Biofield Energy Treatment by Mr. Mahendra Kumar Trivedi (the Trivedi Effect^®) under laboratory conditions for ~3 minutes through Mr. Trivedi s unique Energy Transmission process to the test formulation. The blessing/treatment was given to the test items/animals remotely without touching in the laboratory of Dabur Research Foundation, near New Delhi, India. Similarly, the untreated test samples were subjected to sham healer under the same laboratory conditions for ~3 minutes. The sham healer did not have knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated test samples were kept in the similar sealed condition and used as per the study plan. The Biofield Energy Treated animals were also be taken back to an experimental room for further proceedings.

Five days after acclimatization, animals were randomized and grouped based on 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 5 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 (D (+) Galactose + 0.5% CMC); G3 as reference item (resveratrol, 200 mg/kg body weight); G4 includes D (+) Galactose along with untreated test formulation; G5 as D (+) Galactose along with the Biofield Energy Treated test formulation; G6 group includes D (+) Galactose along with Biofield Energy Treatment per se to animals from day -15; G7 as D (+) Galactose along with the Biofield Energy Treated test formulation from day -15; G8 group includes D (+) Galactose along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted D (+) Galactose along with Biofield Energy Treatment per se animals plus the untreated test formulation. Dosing for group G7 and G8 was also initiated on a day -15 till the end of the experiment. However, G1 to G6 and G9 animals were dosed from day 1 until the end of an experiment. All the animals except G1 received D-Galactose, daily (500 mg/kg; i.p.) from day 1 to the end of the investigation. After completion of an experiment i.e., during 9^th week, the animals were sacrificed and a portion of the brain and kidney samples was homogenized and stored in -80°C for estimation of BDNF, SIRT-1 protein, and SIRT-1 activity in brain homogenate, and klotho protein in kidney homogenate by ELISA method.

The data were expressed as a mean ± standard error of the mean (SEM) and subjected to statistical analysis using Sigma Plot (Version 11.0). For multiple comparison one-way Analysis of Variance (ANOVA) followed by post-hoc analysis by Tukey s test. Between two group comparisons, Student s t-test was performed. The p≤0.05 (n=10) was considered as statistically significant.

The BDNF level was estimated in rat brain homogenate after administration of test formulation and the results obtained are shown in Figure 1. The normal control group (G1) showed 142.56 ± 12.78 pg/mL and it was significantly (p≤0.05) reduced by 30.58% in the aging control (G2) group (98.97 ± 11.35 pg/mL). The positive control, resveratrol (200 mg/kg) significantly (p≤0.05) increased the level of BDNF by 48.84% compared to the G2 group. Moreover, the level of BDNF was increased by 25.83%, 19.35%, 4.92%, 14.67%, and 5.43% in the Biofield Energy Treated test formulation (G5), Biofield Energy Treatment per se at day -15 (G6), Biofield Energy Treated test formulation at day -15 (G7), Biofield Treatment per se to animals plus Biofield Treated test formulation from day -15 (G8), and Biofield Treatment per se (day -15) to animals plus untreated test formulation group (G9) compared to the G2 group. High level of oxidative stress is the key initiative for the development of neurodegenerative disorders 38, leads to the reduction of BDNF expression. Reduced level of BDNF plays a vital role in the pathogenesis of neurodegenerative disorders, widely distributed neurotrophin in the central nervous system, and it also plays in synaptic plasticity and neuronal survival 39.

Sirtuin 1 (SIRT1) is a member of silent information regulator 2 in mammals responsible for age-related diseases, such as cancer, diabetes, metabolic diseases, cardiovascular disease, neurodegenerative diseases, osteoporosis and chronic obstructive pulmonary disease (COPD) 40. Aging is defined as the dysfunction of organ functions. Nowadays, sirtuins is a famous molecule responsible for slow the aging process and thus reduced the chances of age-related disorders 41. The impact of the test formulation on the expression of sirtuin 1 (SIRT1) is a member of silent information regulator 2 is shown in Figure 2. The expression of a SIRT-1 protein in the normal control group (G1) was 29.13 ± 4.31 ng/mL, and it was reduced by 29.21% in the aging control (G2) group (20.62 ± 3.21 ng/mL). The positive control group (G3) showed 3.88% increased the level of SIRT-1 compared to the G2 group. Moreover, the expression of SIRT-1 was increased by 14.63% and 4.55% in the Biofield Energy Treated test formulation (G5) and Biofield Energy Treatment per se at day -15 (G6) groups, respectively compared to the untreated test formulation group (G4).

The effect of the test formulation on SIRT-1 activity in rat brain homogenate as shown in Figure 3. The SIRT-1 activity in the normal control group was found 0.11 ± 0.03 OD/min/mg, and it was reduced by 18.18% in the aging control (G2) group (0.09 ± 0.03) OD/min/mg. SIRT-1 activity was increased by 16.8% in the positive control group as compared to the G2 group. The SIRT-1 activity was increased by 39.7%, 32.5%, 15.9%, and 136% in the Biofield Energy Treatment per se at day -15 (G6), Biofield Energy Treated test formulation at day -15 (G7), Biofield Treatment per se to animals plus Biofield Treated test formulation from day -15 (G8), and Biofield Treatment per se (day -15) to animals plus untreated test formulation group (G9) compared to the untreated test formulation (G4) group (Figure 3). Further, it was increased by 57.3% in the G9 group as compared to the disease control (G2) group. Overall, improvement of brain SIRT-1 activity which might be due to Biofield Energy Treatment. Sirtuin protein expression and their activity resembled life-span by regulating cellular metabolism and aging process 42.

The impact of the test formulation on the expression of Klotho protein in kidney homogenate is shown in Figure 4. The level of Klotho protein in the normal control group was 664.79 ± 53.09 ng/mL and it was significantly (p≤0.001) decreased by 34.15% in the aging control (G2) group (437.75 ± 15.21 ng/mL). The positive control (resveratrol) showed 11.39% increased the level of Klotho protein expression as compared to the G2 group. Further, expression of Klotho protein was increased by 16.67% and 6.83% in the Biofield Energy Treated test formulation (G5) and Biofield Energy Treatment per se at day -15 (G6) groups, respectively compared to the aging control group (G2). Others Biofield Treatment groups have altered the level of kidney Klotho expression. Klotho gene is recognized as a putative aging-suppressor gene, has a great interest and provides more useful information of the aging process. Data obtained from one experiment in mice reported that the overexpression of the Klotho gene extends the life-span, and mutations to the klotho gene which shorten the life-span 4344.

Based on the literature, it has been stated that SIRT1 can regulates the expression of BDNF in the brain. It also mentioned that increased SIRT1 level can sometimes diminished the BDNF signaling 3. However, in this experiment, authors found that 50% groups increased level of SIRT-1 can acclerate the level of BDNF and Klotho and 50% groups can suppressed the levels of Klotho only. As per literature, it has been stated that zinc/magnesium can suppressed the deacetylase activity of SIRT1 45. Thus, it could be due to the presence of minerals zinc/magnesium in the test proprietary formulation. In this research design, four groups were considered as preventive maintenance groups. These groups were G6 (Biofield Energy Treatment per se to animals at -15 days), G7 (Biofield Energy Treated test formulation from day -15), G8 (Biofield Energy Treatment per se to animals along with Biofield Treated test formulation from day -15), and G9 (Biofield Treatment per se at -15 days to animals with untreated test formulation). The results showed the significant slowdown of the disease progression, complications and also reduced the chances of disease susceptibility in these groups. Based on the overall data, it suggests that the Biofield Energy Healing Therapy has improved antiaging biomarkers that could be effective and benefited in order to prevent and protect from the occurrence of diseases in rat model that correlate with longevity, life-span, overall health, and quality of life in human.