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

Randomly breed maleSD rats with body weight around 300 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 of 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.

Each ingredient of the test formulation was divided into two parts. One part of each ingredient was considered as control, where no Biofield Energy Treatment/Blessing was provided. Another part of each ingredient was received Biofield Energy Treatment/Blessing by Mr. Mahendra Kumar Trivedi (known as the Trivedi Effect^®) under laboratory conditions for ~3 minutes. Besides, three group of animals were also received Biofield Energy Treatment under laboratory conditions for ~3 minutes. The Blessing/Treatment was given to the test items remotely in the laboratory of Dabur Research Foundation, near New Delhi, India. Similarly, the control samples were subjected to “sham” healer under the same laboratory conditions for ~3 minutes. The “sham” healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated samples were kept in the similar sealed condition and used as per the study plan. The Biofield Energy Treated animals were also taken back to the experimental room for further proceedings.

Five days after acclimatization, animals were randomized and grouped based on their body weight. The dosing for group G7 and G8 was also initiated on day -15 till the end of the experiment. However, group G1 to G6 and G9 animals were dosed from day 1 till the end of experiment. All the animals except G1 received D-galactose, daily (500 mg/kg; i.p.) from day 1 to the end of the experiment. At the end of the experimental period, i.e., during 9^th week, animals were bled and the blood and serum samples were subjected for the test such as immunoglobulins (IgA, IgE, IgG, and IgM), CD markers (CD4⁺, CD8⁺, and CD28⁺), hematology parameters, biochememistry, and organs were subjected to histopathological analysis.

In order to study the humoral immune response, IgA, IgE, IgG, and IgM were estimated using Mini Vidas, Biomeurix (French) from serum, using commercially available kits as per manufacturer’s instructions. Flow cytometry was used to evaluate the CD4⁺, CD8⁺, & CD28⁺ cells in whole blood as a measure of the cellular immune response using Guava Flow Cytometer, EasyCyte. The mean value was calculated for each group with SEM. The percent change in the Biofield Energy Treated/Blessed group was calculated compared to the vehicle treatment group.

In order to determine the effect of test formulation on blood profile, blood was collected from the retro-orbital plexus using heparinized or non-heparinized capillary tubes in all the experimental animals. Blood was placed in plain bottles for isolation of serum in order to perform the biochemical analysis. The other portion of the blood samples were subjected to estimation of various haematological parameters using Hematology analyzer (Abbott Model-CD-3700). The levels of hemoglobin (Hb), red blood cell count (RBC), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets were analyzed. In addition, magnesium, blood urea, creatinine, uric acid, calcium, phosphorus, potassium, sodium, and chloride ion concentration were also analyzed.

All the experimental animals were daily analyzed for their change in body weight and feed intake, which was calculated by weighing the daily feed supply and the left-over amount that evaluate the average daily feed intake. The average of the feed intake was computed for every three days of the experimental period. All the data were reported through the study treatment regimen.

All the animals in different test groups were analyzed for various clinical signs and symptoms in accordance with in-house protocol. Abnormal behaviour in animals was recorded with the time of onset and disappearance.

All the rats were subjected to histopathological analysis at the end of the experiment, while the organs of all the animals such as liver, kidneys, heart, spleens, lungs and uterus were excise for relative organ weight. The organ to body weight ratio percentage was identified by comparing the weight of each organ with the final body weight of individual rat. Histopathological examination of all the collected samples was placed in 10% neutral buffered formalin.

Relative organ weight was calculated using the formula mentioned below-

Relative organ weight = Absolute organ weight (g)/weight of rat on sacrifice day (g) x 100

All the animal experimental data of immunological studies were expressed as mean ± standard error of mean (SEM) followed by Student’s t-test. Statistical significance was considered at p≤0.05.

Immunoglobulin’s levels (IgM, IgG, IgA, and IgE) after treatment with the test formulation are shown in Figure 1 (A-D). IgM level of rats treated with D-galactose (G2) showed 0.47 ± 0.07 mg/mL, which was higher than the normal control (G1) group 0.37 ± 0.03 mg/mL. However, rats treated with Biofield Energy Treatment per se, reference compound, different combination of Biofield Energy Treated and untreated test formulation to the Biofield Energy Treated and untreated D-galactose induced animals, showed significant change in the level of IgM. Overall, the level of IgG was significantly increased by 2.18%, 10.70%, and 8.03% in G5, G6, and G8 groups, respectively while IgE level was increased by 7.83% and 5.79% in G5 and G8 groups, respectively after Biofield Energy Treatment as compared with the untreated test formulation. Overall, it can be concluded that the Biofield Energy Healing Treatment altered the humoral immune response with respect to the untreated test formulation.

IgM, IgG, IgA, and IgE are considered as the major immunoglobulins, which regulates the immune system 36. The test formulation used contained vitamins and minerals, which are reported to have significant immunomodulatory activity. Moreover, various studies reported that there is a correlation between immune response and activation of anti-aging gene Sirtuin-1 expression 3738. Therefore, the effects of the novel test formulation and the Trivedi Effect^®-Biofield Energy Healing may activate the anti-aging gene Sirtuin 1 that is connected to the immune system, oxidative stress and improving overall health. Besides, literature also cited that activation of heat shock gene Sirtuin-1 is due to improvement of immune system 3940. Thus, the effects of the Trivedi Effect^®-Biofield Energy Healing may possibly involve activation of the heat shock gene Sirtuin 1 that is connected to mitochondrial function, immune system, cell senescence and apoptosis. All-inclusive, the data suggested that the Biofield Energy Healing Treatment has significantly modified the immune response and altered the immunoglobulin production in various test groups, could be used as antiaging potential.

The test formulation was tested for cellular immune response, which was estimated by calculating the percentage of vital biomarkers such as CD4⁺, CD8⁺, and CD28⁺ and the results are presented in the Figure 2 (A-B). CD28⁺ level of rats treated with D-galactose (G2) was 61.20 ± 1.00%, which was higher than the control (G1) group 54.28 ± 1.49% (p<0.01). The results showed the percentage of CD8⁺ was significantly increased by 20.67% in the G8 group, while % of CD28⁺ was significantly increased by 5.44%, 11.70%, 8.32%, and 9.82% in the G5, G7, G8, and G9 groups, respectively after Biofield Energy Treatment to animals as compared with untreated test formulation. This experimental data suggested that the Biofield Energy Healing Treatment has shown a significant improved cellular immune response in the test groups as compared with the untreated test formulation, which showed its use in many inflammatory and autoimmune disorders.

Cellular immunity plays a vital role against various antigens, while CD4⁺ or T4 cells manage infection control and its spread and CD8⁺ T lymphocyte T8 cells or CD8⁺i.e. suppressor or killer cells have the capacity to kill the infected cells or cancerous cells. CD28⁺ cells are antigen specific cytotoxic T cells, which also plays a vital role in immunity to fight against numerous infections 41. T cells activation and its proliferation play a major role in strong immunity to fight against infections and its associated diseases 42. The present study concluded that Biofield Energy Healing (the Trivedi Effect^®) Treatment in test formulation showed a significant improved cellular response, which results in increased number of CD4⁺, CD8⁺, and CD28⁺ cells that would significantly improved the cellular immunity to fight against various infections.

Table 1 showed the result of change in major hematological parameters after treatment with the Biofield Energy Treated and untreated test formulation. However, all the treatment groups showed similar levels, except G5 which was marginally higher than G2. The RBC (10⁶/μL) count in the Biofield Energy Treated test formulation group (G5) was slightly increased i.e., 10.03 ± 0.23 X 10⁶/μL as compared with the disease control group. However, significant increase in the platelet count was reported in G5, G6, G7, G8, and G9 by 40.69%, 27.95%, 26.67%, 38.58%, and 28.28%, respectively compared with the disease control (G2) group. However, the data suggested the change was not statistically significant in the following hematological parameters across all the treatment groups (G3 to G9) including normal (G1) and D-galactose induced aging group (G2), viz. neutrophils, platelets, PCV, MCV, MCHC, hemoglobin, MCH, RDW-CV and RBC.

Overall, the haematological data suggested that Biofield Energy Treated test formulation showed an improved haematological profile of animals. The minerals and vitamins in the test formulation showed improved animal hematology parameters as compared with the untreated test formulation. This suggests that the improved immunomodulatory activity of the Biofield Energy Treated test formulation, which can be significantly active against various inflammatory and autoimmune diseases.

The general biochemistry after treatment with the test formulation showed statistically non-significant alterations, which were observed in the following ion panel of parameters across all the treatment groups (G3 to G9) including normal (G1) and D-galactose induced aging group (G2), magnesium, sodium, chloride, potassium, calcium and phosphorus. Besides, the level of creatinine was significantly decreased by 32.14% in the G9 group as compared with the G2 group. However, the detailed analysis of biochemical study after oral administration of Biofield Energy Treated and untreated test formulation are presented in Table 2. The study data showed vital role of Biofield Energy Healing Treatment on test formulation against many inflammatory diseases.

Lipid profile analysis after treatment with the Biofield Energy Treated test formulation as per the experimental protocol showed a marginal increase in the HDL level in the group injected with D-galactose (G2) for 8 weeks (16.63 ± 0.94 mg/dL), when compared to the normal control (G1, 14.85 ± 0.73 mg/dL). However, all the treatment groups showed similar levels as of group G2. Besides, marginal increase in LDL level was also noticed in the group injected with D-galactose (G2) for 8 weeks (31.83 ± 1.75 mg/dL), when compared to normal control (G1, 25.79 ± 1.32 mg/dL). However, all the treatment groups showed similar levels as of group G2. Similarly, data suggested that slight increase in cholesterol level in G2 group for 8 weeks (54.67 ± 2.13 mg/dL) when compared to the normal control (G1, 48.11 ± 2.41 mg/dL). However, all the treatment groups showed similar level of group G2. Besides, marginal reduction in the VLDL and triglyceride level was noticed in the group injected with D-galactose (G2) for 8 weeks when compared to normal control. However, all the treatment groups showed similar levels as of G2, except G5 which was higher than Group G2 and almost equal to group G1. All the results have been compiled in Table 3. Therefore, the Biofield Energy Treated test formulation showed altered lipid profile, which can be used against inflammatory diseases. It can be concluded that Biofield Energy Healing Treatment has the capacity to improve the lipid profile that modulates after oral administration of Biofield Energy Treated test formulation, which might suggested its importance in the immunomodulation and its associated disorders.

The test biomarkers used in hepatic and cardiac biomarkers such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and cardiac enzyme creatine kinase myocardium band (CK-MB), and others biomarkers such as, total bilirubin, albumin, and globulin showed alterations with respect to other groups, the results are summarized in Table 4. Liver toxicity is defined by the hepatic enzymes, and these are considered as the biomarkers for any infection in liver damage 43. Besides, minerals and vitamins were reported with vital importance against hepatic enzymes with protective effect 4445. Statistically non-significant increase in CK-MB level was noticed in the group injected with D-galactose (G2) for 8 weeks (244.83 ± 45.66 U/L) when compared to the normal control (G1, 166.44 ± 32.64 U/L). However, no significant change was observed in all the treatment groups (G3 to G9). Besides, no significant change was observed in other tested biochemical parameters such as TB, TP, A, G and A/G.

The test formulation was tested in animals with respect to weight parameters, feed intake, and histopathology against Biofield Energy Treated and untreated test formulation. The results of animal tested organ weight parameters are summarized in the Table 5. The study data suggested that the initial and final weight were changed as per normal physiology pattern. In addition, the relative organ weight parameters did not show any significant change in the tested organ weight throughout the experiment in liver, lungs, kidneys, brain, heart, eyes, spleens, pancreas, thymus, adrenal gland, small intestine, large intestine, testis, prostrate, epididymis, and vas deference. Relative organ weight of rats treated with the Biofield Energy Treatment per se, reference compound, different combination of the Biofield Energy Treated and untreated test formulation to the Biofield Energy and untreated to the D-galactose induced animals were estimated. Organ to body weight ratio is the valuable index for any experimental test procedure with respect to the documentation of swelling, atrophy, or hypertrophy after exposure of test samples 46. All the data concluded that no direct cellular constriction and any associated inflammation, which suggested the safe treatment strategy. Overall, the animal weight data, relative organ weight, along with feed intake data suggest no significant change with respect to the disease control group, it suggest that Biofield Energy Treated test formulation and Biofield Energy Treatment per se were found safe in all the tested animals.