There were numerous studies done on demonstrating the adverse effects of stress on health^[1,2]. According to such studies, the chronic or long-term stressors may pose health-aversive effects; among which, some are mediated through the immune mechanism of body. Although, during short-term stress events occurring in nature, the physiological systems of the body act in synchrony to each other and help in the survival of the organisms^[3]. For example, in event of the stress response, the body prepares the musculoskeletal, cardiovascular, and neuro-endocrine systems for fight or flight; similarly, the short-term stress may also helps in preparing the immune system under certain conditions, for facing the challenges such as, wounds or infection that may occur in the presence of stressor such as, predator or surgical procedure^[4,5]. It was observed in various studies that short duration stressors helps in inducing the immune cells redistribution within the body; along with the significant increase in the immune functions of certain organs such as, skin to which leukocytes traffic during the event of acute stress. However, in case of chronic stress, the studies reported the dysregulation in the immune responses of the body^[6,7], either by altering the cytokine balance from type-1 to type-2 cytokine-driven responses^[8] and / or by accelerating the immunosenescence^[9]. The other activities that contribute to this dysregulation involve suppression of immunity by reducing the numbers, trafficking, as well as the function of protective immune cells; while, increasing the regulatory / suppressor T cells^[10,11]. Such activities involve the action of antibodies (Ab), which are also known as the immunoglobulin (Ig). Immunoglobulins are the large, Y-shaped proteins, which are basically produced by the plasma cells, and used to neutralize the pathogens such as pathogenic bacteria and viruses. The antibodies found in immune system are of five kinds; in which, IgA is observed in secretions; IgE is involved in allergic reactions and it binds to mast cells; IgM helps in clearing the antigen from the bloodstream; IgG diffuses into tissue and crosses the placenta due to smaller size; and IgD that may be produced by immature B cells. Moreover, the production of such antibodies in response to the antigens is basically controlled by the humoral immune system^[12,13].

On the other hand, there is another term used when mentioning the immune response, i.e., CD. It refers to the cluster of differentiation, which is used as a protocol for the identification and investigation of cell surface molecules; and thereby providing the targets for the immune-phenotyping of the cells^[14]. CD antigens are considered as the cell-surface molecules that may be expressed on the leukocytes and other cells related to the immune system. It helps in providing a unified designation system for the cell-surface molecules and monoclonal antibodies (mAbs), recognized by them. Besides, there is a non-descriptive cluster of differentiation number e.g., CD2 or CD3, which was assigned to a group of mAbs, recognizing the same cell surface molecule, and the particular cellular differentiation pattern^[15,16]. Human CD antigens are currently numbered up to CD363, and the presence or absence of a specific antigen from the surface of particular cell population is mentioned with the help of “+” or “-” symbols, respectively. However, there is no any particular class of CD antigens, physiologically, due to their functions ranging from cell surface receptors to adhesion molecules^[17].

In this study, discussed mainly about the CD4 and CD8 cells. Among them, the CD4 is a glycoprotein, which is observed on the surface of T helper cells, macrophages, monocytes, and dendritic cells. It is well known that the CD4⁺ T helper cells (T4 cells), also known as white blood cells are considered as the essential part of the immune system. They are referred as the helper cells due to their main role in sending the signals to other types of immune cells, such as CD8 killer cells, which further helps in killing the infectious particle[18,19]. Similarly, CD8 is a trans-membrane glycoprotein that mainly acts as a co-receptor for the T cell receptor, and may also binds to a major histo-compatibility complex (MHC) molecule (specifically class I MHC protein). This affinity helps in close bonding between the T cell receptor of the cytotoxic T cell and the target cell during the antigen-specific activation. Besides aiding with cytotoxic T cell antigen interactions, the CD8 co-receptor also helps in T cell signalling. Moreover, the cytotoxic T cells with CD8 surface protein are called as CD8⁺ T cells[20-22]. The present study aimed to evaluate the effect of unpredictable chronic stress (USC) on immune system of male Sprague-Dawley rats in presence of the novel test formulation, which was treated with Biofield Energy Treatment (a Complementary and Alternative Medicine, CAM) by a renowned Biofield Energy Healer. The test formulation is the combination of vital minerals (selenium, zinc, iron, calcium, copper, and magnesium), essential vitamins (cyanocobalamin, ascorbic acid, pyridoxine HCl, alpha tocopherol, and cholecalciferol), and β-carotene, Ginseng, cannabidiol isolate (CBD).

Besides, the Biofield Energy Healing has been considered as the form of Energy therapy, which was accepted by National Center for Complementary and Alternative Medicine (NCCAM) under CAM therapies due to its beneficial effect against many diseases^[23.24]. It is based on the fact that a human has the ability to harness energy from the universal and can transmit it to any living organism(s) or nonliving object(s) around the globe. National Centre of Complementary and Integrative Health (NCCIH) accepted the Biofield Energy Healing as a CAM health care approach along with the other therapies such as yoga, therapeutic touch, Reiki, pranic healing, guided imagery, chiropractic/osteopathic manipulation, meditation, hypnotherapy, mindfulness, Ayurvedic medicine, and traditional Chinese herbs and medicines in biological systems^[25,26]. Various scientific literatures reported the impact of the Trivedi Effect^®- Consciousness Energy Healing Treatment on the physicochemical properties of metals, chemicals, ceramics and polymers^[27-29], as well as in the field of antimicrobial activity^[30-32], agricultural productivity^[33,34], biotechnology^[35,36], skin health^[37,38], herbomineral formulations^[39,40], bone health^[41,42], and cancer research^[43,44], etc. Thus, the present study was designed to scientifically evaluate the impact of the Biofield Energy Treatment (the Trivedi Effect^®) on the given novel test formulation and Biofield Energy Treatment per se to the animals for its major cellular and humoral immune biomarkers such as IgA, IgE, IgG, and IgM along with the expression of CD4⁺, CD8⁺, and CD4⁺CD25⁺ cells.

Materials and Methods

Chemicals and Reagents

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 carboxymethyl cellulose (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.

Maintenance of Animal

Randomly breed male Sprague Dawley (SD) rats with body weight ranges from 200 to 300 gm were used in this study. The animals were purchased from M / s. Vivo Bio Tech, Hyderabad, India. Animals were randomly divided into nine groups based on their body weights consist of 6 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.

Consciousness Energy Healing Strategies

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 and magnesium (II) gluconate. Each ingredient of the novel test formulation was divided into two parts. The test formulation was divided into two parts, one part of the test compound did not receive 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 Treatment (Biofield Energy Treatment) by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi under laboratory conditions for ~3 minutes. Besides, three group of animals also received Biofield Energy Healing Treatment (known as the Trivedi Effect^®) by Mr. Mahendra Kumar Trivedi under similar laboratory conditions for ~3 minutes to the test formulation were located in the research laboratory of Dabur Research Foundation, New Delhi, India. The energy transmission was done without touching the samples or animals. After that, the Biofield Energy Treated 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 energy treatment, under the same laboratory conditions. The “sham” healer did not have any knowledge about the Biofield Energy Treatment. The Biofield Energy Treated animals were also taken back to experimental room for further proceedings.

Detailed Experimental Procedure

Seven days after acclimatization, animals were randomized and grouped based on the 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 10 mL / kg in morning and evening based on body weight. The experimental groups were divided as G1 as normal control; G2 as disease control (UCS: Unpredictable Chronic Stress with 0.5% CMC); G3 as reference item (UCS along with imipramine hydrochloride, 30 mg / kg); G4 includes UCS along with the untreated test formulation; G5 as UCS along with the Biofield Energy Treated test formulation); G6 group includes UCS along with Biofield Energy Treatment per se to animals from day -15; G7 as UCS along with Biofield Energy Treated test formulation from day -15; G8 group includes UCS along with Biofield Energy Treatment per se plus Biofield Energy Treated test formulation from day -15), and G9 group denoted UCS along with Biofield Energy Treatment per se animals plus untreated test formulation. G1 and G2 animals were treated orally with 0.5% w / v CMC-Na in distilled water for 8 weeks (From day 1 to 56). Group G3 animal was treated orally with reference item, imipramine hydrochloride at a dose of 30 mg / kg body weight for 8 weeks. The freshly prepared suspensions of untreated test formulation and Biofield Energy Treated Proprietary Product was administered orally to the G4 and G5 group animals, respectively, at a dose of 1257.80 mg / kg body weight in the morning and 2012.75 mg / kg body weight in the evening, respectively for 8 weeks. G6 group was not to be dosed with the test formulation. In addition; G7 and G8 group were dosed similar to the G4 and G5 dosing regimen, but from the day of Biofield Energy Treatment (i.e. from day-15 to day 56). G9 group, Biofield Energy Treated per se animal was treated with untreated test formulation for 8 weeks. Body weight and clinical signs were taken daily throughout the experimental period. All the animals except G1 group received stress induced procedures such as stress procedures like sound stress, tilted cages and crowd stress, cold and warm water swim stress, food and water deprivation, stress due to change in the light and dark cycle were undergo seven different types of unpredictable stress procedures after scheduled dosing daily at specified interval to the end of the experiment for 8 weeks after the initiation of stress, which vary every week interval i.e. shuffling of stress type. At the end of (8 week) experimental period, all the animals were individually subjected for blood collection using retro-orbital route to the experimental purpose.

Assessment of Cellular and Humoral Immune Responses

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⁺, and CD4⁺CD25⁺ cells in whole blood as a measure of the cellular immune response using Guava Flow Cytometer, Easy Cyte. The mean value was calculated for each group with SEM. The percent change in the Biofield Energy Treated group was calculated compared to the vehicle treatment group.

Statistical Analysis

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 (n = 6) was considered as statistically significant.

Results and Discussion

Evaluation of Humoral Immune Response

Immunoglobulin’s levels (IgM, IgG, IgA, and IgE) after treatment with the test formulation in different experimental groups are shown in Figure 1 (A-D). The data showed the IgM, IgG, IgA, and IgE level in the unpredictable chronic stress (G2) was found to be 0.26 ± 0.02 mg / mL, 21.33 ± 0.66 mg / mL, 3.31 ± 0.04 mg/mL, and 0.12 ± 0.07 mg/mL respectively. Imipramine treatment (G3) increased IgM, IgG, and IgE levels by 39.50%, 6.72%, and 96.43% as compared to the G2. The untreated test formulation to the untreated rats (G4) showed increased IgM level by 60.78% as compared to the G2. Biofield Energy Treated test formulation to the untreated rats (G5) showed a significant increased IgG and IgA level by 10.82% and 2.28% respectively, as compared to the G4 group. Biofield Energy Treatment to the rats (G6) increased the IgM and IgE level by 45.12% and 631.25% respectively, as compared to the G4 group. 15 days pre-treatment of Biofield Energy Treated test formulation (G7) increased the IgM and IgE level by 87.20% and 213.54% respectively, as compared to the G4 group. 15 days pre-treatment of Biofield Energy Treated test formulation to the Biofield Energy Treated rats (G8) showed increased IgM, IgG, and IgE level by 69.82%, 3.28%, and 355.21% respectively, as compared the G4. Untreated test formulation to the Biofield Energy Treated rats (G9) showed increased IgM, IgG, and IgE level by 94.51%, 7.89%, and 247.92% respectively, as compared the G4. However, if the immunoglobulins levels were compared with respect to the disease control G2 group, the results were observed as 52.45%, 133.33% (p ≤ 0.001), 200.98% (p ≤ 0.001), 173.04% (p ≤ 0.001), and 212.75% ( p ≤ 0.001) increase in IgM in the G5, G6, G7, G8, and G9 groups, respectively. Immunoglobulins or antibodies are defined as the glycoproteins, which are synthesized by plasma cells. IgM, IgG, IgA, and IgE are considered as the major immunoglobulins, which regulates the immune system^[45,46]. Among them, IgM is the one of the very first immunoglobulin expressed during B cell development. All the immunoglobulins exhibit one or more functions with activation of the complement system and neutralization^[47]. Besides, these immunoglobulins are responsible for opsonisation (phagocytosis of bacteria), tumors killing behaviours, mast cell degranulation, activation of antigen-presenting cells (APC), for regulation of cellular and humoral immune response^[48]. Overall, it can be concluded that the Biofield Energy Healing Treatment per se and Biofield Energy Treated test formulation significantly improved the humoral immune response with respect to the untreated test formulation as well as disease control groups.

Figure 1: The effect of the test formulation on the immunoglobulin, (A) IgM, (B) IgA, (C) IgG, and (D) IgE in various test groups G1 to G9 in male Sprague Dawley rats. G: Group; G1: Normal control; G2: Disease control (UCS: Unpredictable Chronic Stress + 0.5% CMC); G3: Reference item (UCS + Imipramine hydrochloride 30 mg / kg); G4: (UCS + untreated test formulation); G5: (UCS + Biofield Energy Treated test formulation); G6: (UCS + Biofield Energy Treatment per se to animals from day -15; G7: (UCS + Biofield Energy Treated test formulation from day -15); G8: (UCS + Biofield Energy Treatment per se plus Biofield Energy Treated test formulation from day -15), and G9: (UCS + Biofield Energy Treatment per se animals plus untreated test formulation). Values are presented as mean ± SEM (n=6). ***p ≤ 0.001 vs. G2.

Measurement of Cellular Responses

The test formulation was tested for cellular immune response, which was estimated by calculating the percentage of vital biomarkers such as CD4⁺, CD8⁺, and CD4⁺CD25⁺, while the results are presented in the Figure 2 (A-C). CD4⁺ and CD8⁺ levels of rats were reported in terms of percentage mean as 43.35% and 14.17% respectively, in the G1 group. However, the level of CD4⁺ and CD8⁺ in reference control group was 40.07% and 12.37%, which was significantly increased as compared with the disease control group. Similarly, the CD4⁺ level was significantly improved by 1.77%, 27.39%, 3.16%, 18.80%, and 25.79% in the G5, G6, G7, G8, and G9 groups respectively, as compared with the untreated group, G4. However, the CD8⁺ level was also significantly improved by 18.42%, 43.93%, 27.43%, 43.02%, and 37.75% in the G5, G6, G7, G8, and G9 groups respectively, as compared with the untreated group, G4. However, the level of CD4⁺CD25⁺ was also altered in experimental group, which was not significant with respect to the disease control and untreated test samples. CD4⁺ or T4 cells manage cellular immunity against various antigens; on the other hand CD8⁺ T lymphocyte T8 cells or CD8⁺i.e. suppressor or killer cells manage infection control and have the capacity to kill the infected cells or cancerous cells^[49]. Thus, T cell activation are very crucial in achieving a significant immune response against pathogens, which helps in maintaining the proper cellular functioning for survival^[50,51]. Overall, it can be concluded that Biofield Energy Healing (the Trivedi Effect^®) based test formulation showed a significant improved cellular response, which results in increased number of CD4⁺ and CD8⁺ cells that would significantly improved the cellular immunity to fight against various infections against many inflammatory and autoimmune disorders.

Figure 2: The effect of the test formulation on the cellular biomarkers in blood sample of male SD rats on various groups (G1 – G9). (A) CD4₊, (B) CD8₊, and (C) CD4₊CD25₊. G: Group; G1: Normal control; G2: Disease control (UCS: Unpredictable Chronic Stress + 0.5% CMC); G3: Reference item (UCS + Imipramine hydrochloride 30 mg / kg); G4: (UCS + untreated test formulation); G5: (UCS + Biofield Energy Treated test formulation); G6: (UCS + Biofield Energy Treatment per se to animals from day -15; G7: (UCS + Biofield Energy Treated test formulation from day -15); G8: (UCS + Biofield Energy Treatment per se plus Biofield Energy Treated test formulation from day -15), and G9: (UCS + Biofield Energy Treatment per se animals plus untreated test formulation). Values are presented as mean ± SEM (n=6).

In this research plan, 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, stress disease related all other symptoms /complications and also reduced the chances of disease susceptibility in these groups. Specifically, group G6 (preventive Biofield Energy Treatment group per se at -15 days) showed the best results as a prophylactic / preventive treatment group compared to the other groups. Based on the overall data, it suggests that the Biofield Energy Healing Therapy was found to be most effective and benefited in order to prevent and protect from the occurrence of any type of diseases in rat model. It indicated that this therapy can act as a preventive maintenance therapy to prevent the occurrence of the disease, slow down the disease progression and disease related complications of the existing aliments that will ultimately improve the overall health and quality of life in human.

Conclusion

Unpredictable chronic stress animal model was used experimental for the estimation of cellular (CD4⁺, CD8⁺, and CD4⁺CD25⁺) and humoral immune (IgA, IgE, IgG, and IgM) in whole blood after treatment with the Consciousness Energy Healing Treatment (the Trivedi Effect^®) based novel test formulation. The results were reported in percentage mean values, which were reported as significant improved the level of IgM and IgE level by 133.33% (p ≤ 0.001) and 631.25%, respectively in the Biofield Energy Treatment per se to the rats (G6) group as compared to the disease control group (G2). Similarly, 15-days pre-treatment of the Biofield Energy Treated test formulation (G7) group was reported with an increased IgM and IgE level by 200.98% (p ≤ 0.001) and 213.54%, respectively as compared to the G2 group. 15-days pre-treatment of the Biofield Energy Treated test formulation to the Biofield Energy Treated rats (G8) group showed an increased IgM and IgE level by 173.04% (p ≤ 0.001) and 355.21%, respectively as compared the G2. However, the untreated test formulation to the Biofield Energy Treated rats (G9) also showed an increased the level of IgM and IgE by 212.75% (p ≤ 0.001) and 247.92%, respectively as compared the G2. The cellular immune data found with improved level of CD4⁺ in the G6, G8, and G9 groups by 27.39%, 18.80%, and 25.79%, respectively as compared with the G4. CD8⁺ level were significantly increased by 18.42%, 43.93%, 27.43%, 43.02%, and 37.75% in the G5 (Biofield Energy Treated test formulation), G6, G7, G8, and G9 groups respectively, as compared with the untreated group, G4. Thus, Biofield Energy Healing Treatment (the Trivedi Effect^®) per se showed outstanding results with high efficacy in the preventive maintenance group, G6 as compared to the other preventive maintenance groups (G7, G8, and G9) in rat model study. It also helped to slow down the disease progression and disease related complications of the overall animal’s health. These data suggested that Biofield Energy Treatment per se and / or Biofield Energy Treated Test formulation in combination would be the best treatment strategies in order to prevent and protect from the occurrence of any type of diseases. Therefore, the Biofield Energy Treatment might act as a preventive maintenance therapy in order to maintain good health, or full restoration of health or improve the overall health and quality of life in human. This therapy might also reduce the severity of any type of acute / chronic disease (auto-immune related and inflammatory disorders) progression rate and can be used in both before and after the manifestation of most of the immunity related disorders in healthy, unhealthy, and ill peoples such as human body immune responses, enhance resistance towards diseases, Cancer, Hashimoto’s thyroiditis, allergies, lethargic conditions, energy booster action, improve exercise capacity in heart related disorders, Crohn’s disease, autoimmune diseases, and its various immune deficiency diseases. Overall, the data suggested the Biofield Energy Treated test formulation and Biofield Energy Treatment per se in showed significant action on cellular and humoral immune response with respect to biomarkers, as a Complementary and Alternative Medicine (CAM). This test formulation also can be used against Addison Disease, Multiple Sclerosis, Myasthenia Gravis, Rheumatoid Arthritis, Crohn’s Disease, Vitiligo, and Alopecia Areata, as well as various inflammatory disorders such as Ulcerative Colitis, Dermatitis, Hepatitis, Diverticulitis, Mental Disorders, Parkinson’s and Other Movement Disorders, Stroke and in the improvement of overall health and quality of life.

Acknowledgements

The authors are grateful to Dabur Research Foundation, Trivedi Science, Trivedi Global, Inc., and Trivedi Master Wellness for the assistance and support during the work.

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