In vitro Assessment of Biofield Energy Treated DMEM on Thermogenesis Using Myoblasts Cell Line (C2C12) - Juniper Publishers

 Cell Science & Molecular Biology - Juniper Publishers

Abstract

Mitochondrial dysfunction lead to various serious disorders, which are considered as one of the important components related with the aging, such as type-2 diabetes and Alzheimer’s disease. The aim of the present study was to examine the effect of Consciousness Energy Healing based DMEM medium on murine myoblasts (C2C12) cells to evaluate the mitochondrial mass content using 10-N-nonyl acridine orange (NAO) dye assay. The test item (DMEM medium) was divided into two parts, one part received Consciousness Energy Healing Treatment by a renowned Biofield Energy Healer, Dahryn Trivedi and was labeled as the Biofield Energy Treated DMEM group, while the other part did not receive any kind of Treatment and denoted as the untreated DMEM group. The level of mitochondrial mass content was assessed using 10-N-nonyl acridine orange (NAO) dye method. Cell viability of the test items using MTT assay showed 72.32% and 125.32% viable cells in the untreated DMEM and Biofield Energy Treated DMEM groups, respectively suggested a safe and nontoxic profile of the test items. Besides, the mitochondrial mass content in terms of Fluorescence Unit (FU) was significantly (p≤0.05) increased by 81.78% in the Biofield Energy Treated DMEM group compared to the untreated DMEM group. Overall, the experimental data suggested that the Consciousness Energy Healing Based DMEM showed a significant improvement of mitochondrial mass content and results in better thermogenesis with respect to naive DMEM. Thus, an increased level of NAO dye accumulation in muscle cells indicated increased mitochondrial mass content and hence, better thermogenesis. In the present study, results demonstrated that an increased mitochondrial mass content in the cells when treated with The Trivedi Effect®. This indicates that the test sample has the potential to improve thermogenesis, which can be used against various metabolic diseases, such as insulin resistance, type-2 diabetes, and cardiovascular diseases, etc.

Keywords: Biofield energy; The Trivedi Effect®; Thermogenesis; Mitochondrial biogenesis; Metabolic disorders; Murine myoblast cell; DMEM

Abbreviations: CAM: Complementary and Alternative Medicine, NCCAM: National Center for Complementary and Alternative Medicine; DMEM: Dulbecco’s Modified Eagle’s Medium; FBS: Fetal Bovine Serum

Introduction

Mitochondria (also known as power generator of the cell) produce most of the vital energy required for the cellular function through oxidative phosphorylation involved in electron transport and ATP synthesis. They produce ATP through the process of cellular respiration mainly aerobic respiration, which requires oxygen. Number and amount of mitochondria in a cell be governed by the energy requirement of the cell [1]. For example, the muscle cells have found comparatively more number of mitochondria since, they need to produce energy to move the body. On the other hand, red blood cells carry oxygen to other cells, do not need to produce energy as compared with the muscle cells. Mitochondria are the powerhouse in the cell, which produce energy from basic components. They undergo fusion, fission, transport, and degradation, all of the process is vital to maintain a healthy mitochondrial population [1]. However, the mitochondrial biogenesis process is involved an increased and controlled mitochondrial mass with number that helped to produce greater production of ATP as a response to greater energy expenditure [2]. Physiologic, metabolic, and pathologic changes along with morphological and functional adaptability are the vital factor to regulate the process of mitochondrial biogenesis. In addition, proteins and transcription factors, upstream regulatory proteins and secondary mechanisms are also involved in the biogenesis process, which also stabilizes the new mitochondrial DNA [3].

Mitochondrial biogenesis regulates and control various therapeutic interference in wide number of diseases such as metabolic syndrome, neurodegenerative disorders, sarcopenia, cardiac pathophysiology and physiological processes like aging [4]. Nonyl-acridine orange (NAO) is a non-fluorescent dye that converts into fluorescent dye in the presence of oxidative species [5]. NAO assay is one of the gold standard assays to detect the mitochondrial mass alteration, which is a metachromatic dye that binds to cardiolipin, an inner mitochondrial membrane lipid, regardless of the energetic state of the cell. Therefore, mitochondrial mass of the cells could be estimated by studying accumulation of the fluorescent dye in the mitochondria. Furthermore, an alternative therapies such as nuclear gene was reported to regulate total mitochondrial mass in response to mitochondrial dysfunction [6]. In order to improve the mitochondrial mass content via thermogenesis process, some alternative treatment approach without any associated side-effect is needed.

Biofield Energy Healing is a categorized as one of the Complementary and Alternative Medicine (CAM) accepted worldwide for the various treatment. Biofield Energy Therapy was accepted by National Center for Complementary and Alternative Medicine (NCCAM). Biofield Energy Healing is one of the emerging frontier in medicine, which has been increased in order to promote wellness by uncovering the root cause of diseases with universal solutions. CAM therapies have shown various significant clinical benefits. Over the past few decades, many energy healing practices have been reported a significant outcomes in various clinical and non-clinical fields. The effects of the CAM therapies have great potential, which include external qigong, Johrei, Reiki, therapeutic touch, yoga, Qi Gong, polarity therapy, Tai Chi, pranic healing, deep breathing, chiropractic/osteopathic manipulation, guided imagery, meditation, massage, homeopathy, hypnotherapy, progressive relaxation, acupressure, acupuncture, special diets, relaxation techniques, Rolfing structural integration, healing touch, movement therapy, pilates, mindfulness, Ayurvedic medicine, traditional Chinese herbs and medicines in biological systems both In vitro and in vivo [7]. Every living organisms possess some kind of unique energy known as Biofield Energy, which is infinite, para-dimensional and electromagnetic field surrounding the human body. Biofield (Putative Energy Fields) based Energy Healing Therapies have been reported to have significant outcomes against various disease conditions. Biofield Energy Healing Treatment (The Trivedi Effect®) contain a putative bioenergy, which is channeled by a renowned practitioner from a distance. Biofield Energy Healing as a CAM showed a significant results in biological studies [8]. However, the National Center for Complementary and Alternative Medicine (NCCAM), well-defined Biofield Therapies in the subcategory of Energy Therapies [9]. The Trivedi Effect®- Consciousness Energy Healing Treatment has been reported with significant revolution in the physicochemical properties of metals, chemicals, ceramics and polymers [10- 12], improved agricultural crop yield, productivity, and quality [13,14], transformed antimicrobial characteristics [15-17], biotechnology [18,19], improved bioavailability [20-22], skin health [23, 24], nutraceuticals [25,26], cancer research [27,28], bone health [29-31], human health and wellness. On the basis of outstanding benefits of Biofield Energy Treatment, the present study was aimed to evaluate the impact of the Biofield Energy Treatment (The Trivedi Effect®) on DMEM as test sample to alter the mitochondrial mass content on thermogenesis using NAO dye staining using standard in vitro assay in murine myoblasts (C2C12) cells.

Material and Methods

Chemicals and reagents

Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Life Technology, USA. Antibiotics solution (penicillin-streptomycin) were procured from HiMedia, India, and ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma, USA. All the other chemicals used in this experiment were analytical grade procured from India.

Cell culture

C2C12 (murine myoblasts) was used as a test system in the present study. The C2C12 cell line was maintained in DMEM growth medium for routine culture supplemented with 10% FBS. Growth conditions were maintained at 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. Before initiation of the experiment, cells were incubated in DMEM+2% horse serum (HS) for 3 days to allow the cells to differentiate into myotubes.

Experimental design

The experimental groups consisted of group 1 (G-I) with untreated DMEM and group 2 (G-II) included the Biofield Energy Treated DMEM.

Consciousness energy healing treatment strategies

The test item, DMEM was divided into two parts. One part of the test item was treated with the Biofield Energy by a renowned Biofield Energy Healer, Dahryn Trivedi remotely for ~5 minutes and coded as the Biofield Energy Treated DMEM, while the second part did not receive any sort of treatment and denoted as the untreated DMEM group. Biofield Energy Healer was located in the USA, while the test items were located in the research laboratory of Dabur Research Foundation, New Delhi, India. This Biofield Energy Treatment was administered through Healer’s unique Energy Transmission process to the test sample under laboratory conditions. Dahryn Trivedi in this study never visited the laboratory in person, nor had any contact with the test item (DMEM). Further, the untreated DMEM group was treated with 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.

Assessment of cell viability using MTT assay

The cell viability was performed by MTT assay in C2C12 cell line. The cells were counted and plated in 96-well plates at the density corresponding to 10 X 10³ cells/well/180μL of cell growth medium (DMEM+2% HS). The above cells were incubated overnight under growth conditions and allowed the cell recovery and exponential growth, which were subjected to serum stripping or starvation. The cells were treated with the Untreated and Biofield Energy Treated test item. The cells in the above plate(s) were incubated for 24 to 72 hours in a CO₂ incubator at 37°C, 5% CO₂, and 95% humidity. Following incubation, the plates were taken out and 20μL of 5 mg/mL of MTT solution were added to all the wells followed by additional incubation for 3 hours at 37°C. The supernatant was aspirated and 150μL of DMSO was added to each well to dissolve formazan crystals. The absorbance of each well was read at 540nm using Synergy HT microplate reader, Bio Tek, USA [29]. The percentage cytotoxicity at each tested concentrations of the test items were calculated using the following equation (1):

%cytotoxicity = (1− X / R)*100...........(1)

Where, X = Absorbance of the Biofield Treated cells; R = Absorbance of untreated cells

The percentage cell viability corresponding to each treatment was obtained using the following equation (2):

% Cell Viability = (100- % Cytotoxicity).........(2)

The concentrations exhibiting ≥70% cell viability was considered as non-cytotoxic.

Assessment of mitochondrial content

For the assessment of mitochondrial mass, the cells were counted using an hemocytometer and plated at 4500 cells/well in dark walled 96-well plates in DMEM supplemented with 2% HS. The cells were incubated overnight under standard growth conditions to allow the cell recovery and exponential growth, which were treated by the test items in different groups followed by incubation with the test items for 72 hours. After incubation with the test items, mitochondrial content was determined by 10-N-nonyl acridine orange (NAO) dye. 50nM dye was added to each well and the cells were incubated for 30 minutes at 37°C and 5%CO2. After 30 minutes of incubation, media was discarded and cells were washed with phosphate buffer saline (PBS). 150μL of PBS was added to each well and fluorescence was read at 485/20 excitation, 528/20 emission filter using synergy HT microplate reader. The percentage increase in mitochondrial content was calculated using following equation

Where, FU denotes Fluorescence unit

Statistical analysis

All the values were represented as Mean ± SEM (standard error of mean) of three independent experiments. The statistical analysis was performed using Sigma Plot statistical software (v11.0). For two groups comparison student’s t-test was used. Statistically significant values were set at the level of p≤0.05.

Results and Discussion

Cell viability using MTT assay

Cell viability data of the untreated and Biofield Energy Treated DMEM groups in C2C12 cells using MTT assay is shown in Figure 1. The percentage of cell viability in the untreated DMEM group was 72.32%, while it was 125.32% in the Biofield Energy Treated DMEM group (Figure 1). Overall, data suggest that all the test samples were found safe against the tested C2C12 cells, which were used for the estimation of mitochondrial mass content, which indicate extend of thermogenesis.

Extend of mitochondrial mass content in C2C12 cells

Mitochondrial mass content or biogenesis showed a significant effects against various metabolic diseases. The increased cell capacity to control and maintain the cell metabolism, signal transduction, and regulation of mitochondrial ROS production [2]. Alteration or decrease in the mitochondrial biogenesis are related with the mitochondrial dysfunction and mitochondrial oxidative stress, which leads to various diseases [32]. Mitochondrial mass content results in the improved production of ATP as a response to greater energy expenditure [33]. Various factors such as physical exercise, nutritional factors, etc. reported to have an improved mitochondrial mass content, which results in greater glucose uptake by muscles, along with an increased metabolic enzymes level for glycolysis, oxidative phosphorylation and ultimately a greater mitochondrial metabolic capacity [34]. Aging process leads to decrease level of mitochondrial mass content and results in various diseases such as enhanced aging, insulin resistance, type-2 diabetes, cardiovascular diseases, obesity, etc. [35]. The experiment was conducted to study the influence of Biofield Energy Healing Treatment on mitochondrial content in C2C12 cells using NAO dye assay. The results of mitochondrial mass content in terms of increase number of fluorescence unit (FU) among different groups in C2C12 cells using NAO dye assay are presented in Figure 2. The untreated DMEM group showed 272.3 ± 12.14 FU. Besides, the Biofield Energy Treated DMEM group showed 81.78% increase the level of mitochondrial mass in terms of FU, compared to the untreated DMEM group (Figure 2).

Thus, the data suggested that the Consciousness Energy Blessed DMEM showed a significant improvement of thermogenesis, which results in mitochondrial mass content. This phenomenon can be significantly used against various metabolic diseases, such as insulin resistance, type 2 diabetes, and cardiovascular diseases. Overall, the Biofield Energy Healing Treatment (The Trivedi Effect®) has the significant capacity to improve the overall Quality of life with an improved thermogenesis and mitochondrial content.

Conclusion

MTT data showed a significant cell viability with ranges from 72.32% to 125.32% in different test item groups, which indicated that the test items were safe and non-toxic in nature. Correspondingly, The Trivedi Effect® showed a significant change in mitochondrial mass among different groups. The Biofield Energy Treated DMEM group demonstrated significant increase in the mitochondrial mass by 81.78% as compared to the untreated DMEM group. Thus, Biofield Energy Healing based DMEM can be significantly used to improve the energy level, endurance, body energy, which can be utilized against many diseases such aging, diabetes, cancer, depression, hypertension, cardiovascular disease, aging, and physical strength. The Biofield Energy Treated (The Trivedi Effect®) DMEM were found to have a significant impact on thermogenesis, which might significantly improve the mitochondrial content in muscle cells. Therefore, the Consciousness Energy Healing based DMEM might be a suitable alternative media for cell growth. It can be useful for the management of various disorders such as Lupus, Systemic Lupus Erythematosus, Fibromyalgia, Addison Disease, Hashimoto Thyroiditis, Celiac Disease (gluten-sensitive enteropathy), Multiple Sclerosis, Dermatomyositis, Graves’ Disease, Myasthenia Gravis, Pernicious Anemia, Aplastic Anemia, Scleroderma, Psoriasis, Rheumatoid Arthritis, Reactive Arthritis, Diabetes, Sjogren Syndrome, Crohn’s Disease, Vasculitis, Vitiligo, Chronic Fatigue Syndrome and Alopecia Areata, as well as inflammatory disorders such as Irritable Bowel Syndrome (IBS), Asthma, Ulcerative Colitis, Alzheimer’s Disease, Parkinson’s Disease, Atherosclerosis, Dermatitis, Hepatitis, and Diverticulitis. Further, the Biofield Energy Healing Treatment can also be used in the prevention of immune-mediated tissue damage in cases of organ transplants (for example heart transplants, kidney transplants and liver transplants), for anti-aging, stress prevention and management, and in the improvement of overall health and Quality of Life.

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Oxidative Stress and Inflammatory Biomarkers Analysis of Biofield Treated Proprietary Test Formulation in Heart Tissues in Cecal Slurry, LPS and E. Coli-induced Systemic Inflammatory Response Syndrome (SIRS) in Sprague Dawley Rats - Juniper Publishers

 Annals of Reviews and Research - Juniper Publishers

Abstract

The study was aimed to evaluate the antioxidant and anti-inflammatory biomarkers in heart tissues after treatment with the Biofield Energy Treated Proprietary Test Formulation and Biofield Energy Treatment per se to the animals on Cecal Slurry, LPS and E. coli-induced systemic inflammatory response syndrome (SIRS) model in Sprague Dawley rats. In this experiment, different antioxidants biomarkers such as myeloperoxidase (MPO), superoxide dismutase (SOD), lipid peroxidase (LPO) and proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein-2 (MIP-2), and matrix metallopeptidase 9 (MMP-9) were analysed using ELISA assay in heart homogenate. A proprietary test formulation was formulated including minerals (magnesium, zinc, calcium, selenium, and iron), vitamins (ascorbic acid, pyridoxine HCl, vitamin E, cyanocobalamin, and cholecalciferol), Panax ginseng extract, β-carotene, and cannabidiol isolate. The constituents of the test formulation were divided into two parts; one section was defined as the untreated test formulation, while the other portion of the test formulation and the animals received Biofield Energy Healing Treatment remotely for about 3 minutes by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi.

The level of MPO was reduced by 12.07% in the G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15) group as compared to the untreated test formulation (G4) group. Moreover, the level of SOD was significantly (p≤0.05) increased by 19.03%, 17.26%, and 11.81% in the G6, G7, and G9 groups, respectively as compared to the G4 group. The level of TNF-α was significantly decreased by 25.97%, 40.28% (p≤0.01), 24.86%, 36.54% (p≤0.01), and 34.30% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the disease control (G2) group. Moreover, the level of IL-6 was significantly (p≤0.001) decreased by 23.5%, 31.0%, 26.3%, and 39.8% in the G5, G6, G8, and G9 groups, respectively as compared to the G2 group. Additionally, the level of MIP-2 was reduced by 26.7% and 19.5% in the G6 and G8 groups, respectively as compared to the G4 group.

Besides, the level of MMP-9 was significantly (p≤0.001) reduced by 15.1%, 21.5%, and 34% in the G6, G8, and G9 groups, respectively as compared to the G4 group. Altogether, the data imply the antioxidant and anti-inflammatory potential of the Biofield Energy Treated test formulation and Biofield Energy Treatment per se along with preventive measure on the animal with respect to various inflammatory conditions that might be beneficial various types of systemic inflammatory disorders specially sepsis, trauma, septic shock or any types of cardiac injuries. Therefore, the results showed the significant slowdown the inflammation-related disease progression and its complications/symptoms in the preventive Biofield Energy Treatment group per se and/or Biofield Energy Treated Test formulation groups (viz. G6, G7, G8, and G9) comparatively with the disease control group.

Keywords: Biofield Treatment; Inflammatory cytokines; The Trivedi Effect®; ELISA; SIRS; Antioxidant; Heart biomarker

Abbreviations: SIRS: Systemic inflammatory response syndrome; MPO: Myeloperoxidase; SOD: superoxide dismutase; LPO: Lipid peroxidase; CAD: Coronary artery disease; LDL: Low-density lipoprotein; CAM: Complementary and Alternative Medicine; NCCAM: National Center for Complementary/Alternative Medicine; NCCIH: National Centre of Complementary and Integrative Health; SD: Sprague Dawley; LPS: Lipopolysaccharide; SEM: Standard error of mean; RA: Rheumatoid arthritis; AD: Addison disease

Introduction

Cardiovascular diseases are very common cause of health burden worldwide [1]. Heart disease is the leading cause of death for all age’s population in the United States. In 2010, coronary artery disease (CAD) accounted for one in six deaths in the United States [2]. However, in 2020, one person dies every 36 seconds that’s one in every four deaths in the United States from cardiovascular disease [3,4]. Oxygen free radicals promote low-density lipoprotein (LDL) peroxidation, and increase the number of foam cells, that causes vascular endothelial cell injury, and induce expression of proinflammatory cytokines [5]. Cytokines (TNF-α, TGF-β) and interleukins (IL-1, IL-4, IL-6, IL-8, and IL-18) are responsible for the development of various inflammatory pathologies of various vital systems such as cardiac, brain, renal, lymphatic, etc. [6]. MIP-2 is produced by a variety of cells in response to infection or injury. It is regulated by multiple factors like by signalling through Toll-like receptor 2 (TLR2), TLR3, and TLR4 in response to diverse pathogens [7]. Superoxide dismutases (SODs) an antioxidant enzyme and also acts as a good therapeutic agent against reactive oxygen species-mediated diseases [8]. Thus, in order to study the change in heart cytokines in presence of Cecal Slurry, LPS and E. coli-induced systemic inflammatory response syndrome model in Sprague Dawley rats, a novel test formulation was designed with the combination of vital minerals (selenium, zinc, iron, calcium, and magnesium), essential vitamins (cyanocobalamin, ascorbic acid, pyridoxine HCl, vitamin E, and cholecalciferol), and nutraceuticals (β-carotene, Ginseng, cannabidiol isolate (CBD)). All the minerals and vitamins used in the test formulation have significant functional role to provide vital physiological responses [9,10]. Besides, cannabidiol itself has wide range of pharmacological profile and has been reported to role in different disorders [11,12], while ginseng extract is regarded as the one of the best immune booster for overall immunity [13]. The present study was aimed to evaluate the antioxidant and anti-inflammatory potential of the Biofield Energy Treated Proprietary Test Formulation and Biofield Energy Treatment per se to the animals on Cecal Slurry, LPS and E. coli-induced systemic inflammatory response syndrome model in Sprague Dawley rats.

Biofield Energy Healing Treatment has been reported with significant effects against various disorders and defined as one of the best Complementary and Alternative Medicine (CAM) treatment approach [14-16]. National Center for Complementary/Alternative Medicine (NCCAM) recommended CAM with several clinical benefits as compared with the conventional treatment approach [17]. National Centre of Complementary and Integrative Health (NCCIH) accepted Biofield Energy Healing as a CAM health care approach in addition to other therapies such as deep breathing, natural products, Tai Chi, yoga, therapeutic touch, Johrei, Reiki, pranic healing, chiropractic/osteopathic manipulation, guided imagery, meditation, massage, homeopathy, hypnotherapy, special diets, relaxation techniques, movement therapy, mindfulness, Ayurvedic medicine, traditional Chinese herbs and medicines in biological systems [18,19]. The Trivedi Effect®-Consciousness Energy Healing Treatment was scientifically reported on various disciplines such as in the materials science [20,21], agriculture science [22], antiaging [23], gut health [24], nutraceuticals [25], pharmaceuticals [26], overall human health and wellness. In this study, the authors want to evaluate the effect of the Biofield Energy Treatment (the Trivedi Effect®) on the given novel test formulation and Biofield Energy Treatment per se to the animals on heart biomarkers in presence of Cecal Slurry, LPS and E. coli-induced systemic inflammatory response syndrome model in in Sprague Dawley rats using standard ELISA assay.

Material and Methods

Chemicals and Reagents

Pyridoxine hydrochloride (vitamin B6), zinc chloride, magnesium (II) gluconate, and β-carotene (retinol, provit A) were purchased from TCI, Japan. Cyanocobalamin (vitamin B12), calcium chloride, vitamin E (Alpha-Tocopherol), cholecalciferol (vitamin D3), iron (II) sulfate, and carboxymethyl cellulose sodium were procured from Sigma-Aldrich, USA. Ascorbic acid (vitamin C) and sodium selenate were obtained from Alfa Aesar, India. Panax ginseng extract and cannabidiol isolate were obtained from Panacea Phytoextracts, India and Standard Hemp Company, USA, respectively. Dexamethasone was obtained from Clear synth, India. For the estimation of heart antioxidant and inflammatory biomarker panels, such as myeloperoxidase (MPO), superoxide dismutase (SOD), lipid peroxidation (LPO), tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein-2 (MIP-2), and matrix metallopeptidase 9 (MMP-9) were procured from CUSABIO, USA using specific ELISA kits.

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 10-12 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

Each ingredient of the novel 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. The Blessing (prayer)/Treatment was given to the test items/animals (present in the laboratory of Dabur Research Foundation, near New Delhi, India), remotely from USA for about 3 minutes via online web-conferencing platform. 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 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.

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 (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Dosing for groups G7 and G8 were started on Day -15 and continued till end of the experiment. However, Group G1 to G5 and G9 animals were dosed with respective formulations from Day 1 and continued till the end of the experiment. Group G6 animals received Biofield Energy Treatment on Day-15 and were not dosed throughout the experimental period. At the end of the experimental period (8 weeks treatment), the animals were sacrifice and heart were collected, homogenised, and the supernatant subjected for estimation of antioxidants (MPO, SOD, and LPO) and cytokines (TNF alpha, IL-6, MIP-2, and MMP-9).

Induction of Systemic Inflammatory Response Syndrome (SIRS) Model

A combination model of sepsis was developed in SD rats by administering Cecal slurry (from donor animals, intraperitoneally, at the dose of 400 mg/kg) in combination with LPS (at the dose of 100 µg/animal) and E. coli [Escherichia coli; 0.2 mL (2M CFU)/animal]). The animals were monitored for various parameters for up to 56 days after disease (SIRS) induction. Ten Donor (~20 weeks old) rats were anesthetized. A midline laparotomy was performed on them and the cecum was extruded. A 0.5 cm incision was made on the anti-mesenteric surface of the cecum, and the cecum was squeezed to expel the feces. The feces from different donor animals was collected and weighed. Immediately after collection, the feces were pooled, diluted 1:3 with 5% dextrose solution and filtered to get a homogeneous suspension. Bacterial viability in the cecal slurry was analyzed. Cecal slurry prepared from donor rats was injected intraperitoneally into experimental rats (G2 to G9) at the dose of 400 mg/kg within 2 hours of preparation. After 3 hours, lipopolysaccharide (LPS) at the dose of 100 µg/animal, and gram-negative viable bacteria such as E. coli [0.2 mL (2M CFU)/animal] were injected, intraperitoneally (G2 to G9).

Preparation of Sample for the Estimation of Antioxidant and Cytokines

With the continued treatment to the respective groups of 8th week of the experimental period, all the animals were sacrificed, heart were collected, homogenized and subjected for the estimation of antioxidants and cytokines. The tissue from all the groups was stored at -20°C for further estimation. Alternatively, aliquot all the samples and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles, which may alter the level of cytokines during final calculations.

Estimation of Antioxidants and Cytokine Levels

The heart from all the groups was subjected for the estimation of level of antioxidants such as MPO (CSB-E08722r), SOD (706002), and LPO (700870) and cytokines such as TNF-α (CSB-E11987r), IL-6 (CSB-E04640r), MIP-2 (CSB-E07419r), and MMP-9 (CSB-E08008r). All the biomarker panel was estimation using ELISA method as per manufacturer’s recommended standard procedure. This was a quantitative method and the principle was based on the binding of antigen and antibody in sandwich manner assay.

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 was considered as statistically significant.

Results and Discussion

Assessment of Antioxidants in Heart Homogenate

Estimation of Myeloperoxidase (MPO): Myeloperoxidase (MPO) was estimated in the presence of the test formulation and the data are graphically shown in Figure 1. The data suggested that the disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) + 0.5% CMC) group (G2) showed value of MPO as 2.41 ± 0.0.28 ng/mL, which was increased by 0.45% as compared with the normal control (G1, 2.40 ± 0.1 ng/mL). However, positive control (Dexamethasone) treatment (G3) showed the level of MPO in heart i.e., 2.80 ± 0.14 ng/mL.

The level of MPO in heart tissues was decreased by 9.98% and 12.07% in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation) and G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15) groups, respectively as compared to the untreated test formulation (G4) group. High expression of MPO level in circulation are associated with inflammation and increased oxidative stress that leads to cardiovascular disease (CVDs) like coronary artery disease, congestive heart failure, arterial hypertension, pulmonary arterial hypertension, myocardial ischemia, stroke, cardiac arrhythmia and venous thrombosis [27]. Multiple lines of evidence suggested that MPO may play a role in atherogenesis in humans. However, MPO has little role as atheroprotective in the murine atherosclerosis model [28]. MPO plays an important role in the host defense against different types of bacteria and viruses. MPO is also an important enzyme in the inflammatory process, and inflammation is a key component in the development and progression of atherosclerotic and other forms of cardiovascular disease [29]. Overall, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Healing Treatment per se reduced the level of MPO in the heart tissues, which could be helpful for the management of oxidative stress and inflammatory conditions related to cardiovascular disorders.

Figure 1 The effect of the test formulation on the level of heart myeloperoxidase (MPO) in Sprague Dawley rats. G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Values are presented as mean ± SEM (n=6-9).

Estimation of Superoxide Dismutase (SOD): The effect of the test formulation and Biofield Energy Treatment per se was assessed by estimating the level of heart superoxide dismutase (SOD), and the results are graphically presented in the Figure 2. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) + 0.5% CMC) group (G2) showed value of SOD as 3.79 ± 0.13 U/mL, which was decreased by 3.2% as compared to the normal control group i.e., 4.10 ± 0.18 U/mL. However, positive control (Dexamethasone) treatment (G3) showed the level of SOD in heart i.e., 4.49 ± 0.22 U/mL, which was increased by 13.3% as compared to G2.

The level of SOD was increased significantly by 1.27%, 19.03% (p≤0.05), 17.26% (p≤0.05), 6.15%, and 11.81% (p≤0.05) in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15), G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively with reference to disease control group (G2). Further, the level of SOD was significantly increased by 3.8%, 22.01% (p≤0.05), 20.19% (p≤0.05), 8.80%, and 14.6% in the G5, G6, G7, G8, and G9 groups, respectively with reference to untreated test formulation group (G4). Studies in the heart suggest that extra-cellular SOD is important for preventing oxidative injury after myocardial infarction and may contribute to cardiac remodeling [30]. SOD is one of the main intracellular antioxidant defence mechanisms is associated with cardiac and vascular defects leads to hypertension and atherosclerosis. It is also protecting thermogenesis [31]. Therefore, in this experiment the Biofield Energy Treated test formulation significantly increased the level of heart SOD, which could be beneficial inflammation and oxidative damage.

Figure 2: The level of superoxide dismutase (SOD) measured in heart tissue in Sprague Dawley rats after administration with Biofield Treated test formulation and Biofield Treatment per se. G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli + Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se + Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Values are presented as mean ± SEM (n=6-9). *p≤0.05 vs. G2 and #p≤0.05 vs. G4.

Estimation of Lipid Peroxidation (LPO): The level of lipid peroxidation (LPO) end product in terms of malondialdehyde (MDA) was detected in all the experimental groups and the data are presented in Figure 3. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) and positive control (Dexamethasone) treatment (G3) groups showed value of MDA as 4.20 ± 0.48 µM and 4.33 ± 0.37 µM, respectively.

The level of MDA was decreased by 5.6%, 2.9%, and 18% in the G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively with reference to disease control group (G2). Additionally, the level of MDA was significantly reduced by 4.1%, 11.1%, 8.5%, and 22.8% (p≤0.05) in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation), G7, G8, and G9 groups, respectively as compared to the untreated test formulation group (G4). Oxidative stress and inflammation are two major mechanisms leading to atherosclerosis. Under oxidative stress, phospholipids and cholesterol esters can readily oxidized through a free radical-induced lipid peroxidation (LPO) process to form a complex mixture of oxidation products. These oxidized lipids are responsible for inflammatory responses in atherosclerosis by interacting with immune cells (macrophages) and endothelial cells [32]. The LPO products are highly reactive and causes selective alterations in cell signaling, protein and DNA damage, and cytotoxicity [33]. In this experiment, the Biofield Energy Treated preventive groups significantly reduced the level of LPO in heart tissues, which could be beneficial inflammation and oxidative damage in heart.

Figure 3: The level of heart lipid peroxidation (LPO) in Sprague Dawley rats after dosed with the Biofield Treated test formulation and Biofield Energy Healing per se. G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli + Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Values are presented as mean ± SEM (n=6-9). *p≤0.05 vs. G4.

Assessment of Cytokines in Heart Homogenate

Estimation of Tumour Necrosis Factor Alpha (TNF-α): The expression of heart tumour necrosis factor alpha (TNF-α) in Sprague Dawley rats after administration of Biofield Treated test formulation and exposure of Biofield Treatment to the animals per se, and the results are shown in Figure 4. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of TNF-α as 204.12 ± 46.49 pg/mL, which was significantly (p≤0.01) increased by 399% as compared with the normal control (G1, 40.91 ± 3.85 pg/mL).

Further, the positive control (Dexamethasone) treatment (G3) showed significant (p≤0.01) decreased TNF-α level by 66% i.e., 69.31 ± 8.52 pg/mL as compared to the G2 group. TNF-α level was decreased significantly by 25.97%, 40.28% (p≤0.01), 24.86%, 36.54% (p≤0.01), and 34.30% in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15), G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 (Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se + Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se animals + untreated test formulation) groups, respectively as compared to the disease control group (G2). Further, the expression of TNF-α was reduced by 16.2%, 10.9%, and 7.8% in the G6, G8, and G9 groups, respectively as compared to the untreated test formulation group (G4). Pro-inflammatory cytokines are consistently increased in congestive heart failure. In the cardiovascular system, TNF-α activate signal transduction pathways may causes vascular dysfunction, development, and progression of atherosclerosis, and thus ultimately leads to myocardial infarction and heart failure [34]. Another, study reported that TNFα is responsible for the progression of heart failure as a mediator of myocardial dysfunction and adverse remodeling, that leads to elevated levels of circulating TNFα in heart failure patients as compared with the control [35]. Moreover, TNF modulates both cardiac contractility and peripheral resistance, the two most important haemodynamic determinants of cardiac function [36]. Therefore, here the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of TNF-α, which could be beneficial in the cardiovascular disorders.

Figure 4: The expression of heart tumour necrosis factor alpha (TNF-α) in Sprague Dawley rats after administration of Biofield Treated test formulation and exposure of Biofield Treatment to the animals per se. G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli + Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Values are presented as mean ± SEM (n=6-9). ##p≤0.01 vs. G1 and **p≤0.01 vs. G2.

Estimation of Interleukin-6 (IL-6)

The expression of heart interleukin-6 (IL-6) in Sprague Dawley rats after administration of Biofield Treated test formulation and exposure of Biofield Treatment to the animals per se, and the results are graphically shown in Figure 5. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of IL-6 as 19.05 ± 2.29 pg/mL, which was significantly (p≤0.001) increased by 98.7% as compared with the normal control (G1, 9.59 ± 0.44 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed the level of IL-6 i.e., 10.46 ± 0.71 pg/mL, which was decreased by 45.1% as compared to the G2 group. The level of IL-6 was significantly decreased by 23.5% (p≤0.001), 31.0% (p≤0.001), 19.5%, 26.3% (p≤0.001), and 39.8% (p≤0.001) in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15), G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2).

Further, the expression of IL-6 was decreased by 3.2%, 12.7%, 6.7%, and 23.9% in G5, G6, G8, and G9 groups, correspondingly with reference to untreated test formulation (G4) group. Based on the one of the studies from myocardial infarction which shows that IL-6 signaling plays a causal role in cardiovascular disease [37]. The patients with high titre of circulating inflammatory biomarkers get more susceptible to cardiovascular events. It is more common in patients with high IL-6, associated with an increased incidence of myocardial infarction and mortality among patients with acute coronary syndromes [38]. There is an extensive body of the literature that supports that an increased level of inflammatory cytokine like IL-6 is associated with acute ischemic conditions and predictor of coronary artery disease [39]. Overall, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of IL-6, which could be reduce the risks of inflammatory diseases specially in the heart.

Figure 5: The expression of heart interleukin-6 (IL-6) in Sprague Dawley rats after administration of Biofield Treated test formulation and exposure of Biofield Treatment to the animals per se. G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli + Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Values are presented as mean ± SEM (n=6-9). ###p≤0.001 vs. G1 and ***p≤0.001 vs. G2.

Estimation of Macrophage Inflammatory Protein-2 (MIP-2): The expression of macrophage inflammatory protein-2 (MIP-2) in heart tissue after administration of the Biofield Treated/Blessed proprietary test formulation and Biofield Energy Healing Treatment per se to the animals was estimated, and the results are graphically shown in Figure 6. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of MIP-2 as 1734.78 ± 237.57 pg/mL, which was decreased by 51.8% as compared with the normal control (G1, 3598.50 ± 395.77 pg/mL).

Further, the positive control (Dexamethasone) treatment (G3) showed increased heart MIP-2 level by 40.6% i.e., 2438.50 ± 255.71 pg/mL as compared to the G2 group. The level of MIP-2 was decreased by 26.7% and 19.5% in the G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15) and G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15) groups, respectively as compared to the untreated test formulation group (G4). The MIP-2 is a murine counterpart of IL-8. MIP-2 is a naturally occurring inflammatory cytokine biomarker in myocardium and its expression is increased during myocarditis. Study reported that plasma MIP-2 levels are significantly elevated in mice on days 7 and 14 of post-infection with encephalomyocarditis (EMC) virus [40]. Taken together, our data suggest that the Biofield Energy Treated test formulation and Biofield Energy Treatment per se reduced the level of MIP-2 in heart tissues, which could prevent the cardiovascular-inflammation.

Figure 6: The expression of heart macrophage inflammatory protein-2 (MIP-2) in Sprague Dawley rats after treatment with Biofield Treated test formulation and Biofield Energy treatment per se to the animals. G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli + Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Values are presented as mean ± SEM (n=6-9).

Estimation of Matrix Metallopeptidase-9 (MMP-9): The expression of matrix metallopeptidase-9 (MMP-9) in heart tissue after administration of the Biofield Treated/Blessed proprietary test formulation and Biofield Energy Healing Treatment per se to the animals was estimated, and the results are graphically presented in Figure 7. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of MMP-9 as 155.85 ± 12.62 pg/mL, which was increased by 13.8% as compared with the normal control (G1, 136.96 ± 4.68 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) group decreased MMP-9 level by 8.1% i.e., 143.28 ± 7.66 pg/mL as compared to the G2 group.

The level of MMP-9 was decreased by 6.4%, 9%, 2%, 15.8%, and 29.3% in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2).

Besides, the level of MMP-9 was significantly reduced by 12.6%, 15.1% (p≤0.001), 8.6%, 21.5% (p≤0.001), and 34% (p≤0.001) in the G5, G6, G7, G8, and G9 groups, respectively with reference to untreated test formulation (G4) group. MMP-9 is one of the most widely investigated MMPs. MMP-9 expression has increases during cardiovascular disorders like hypertension, atherosclerosis, and myocardial infarction. MMP-9 degrades extracellular matrix proteins and activates cytokines and chemokines to regulate pathological remodeling processes that involve inflammation and fibrosis in cardiovascular disease [41]. According to one of the extensive research work done by Swedish researchers, they found the high level of MMP-9 in coronary artery disease (coronary artery ectasia) patients and a predictor of increased mortality in that patients [42]. In this study, the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of MMP-9, which could be beneficial to combat inflammatory disease conditions in the cardiovascular patients.

Figure 7: The effect of the test formulation on the level of heart macrophage inflammatory protein-2 (MIP-2) in Sprague Dawley rats. G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli + Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se + Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli + Biofield Energy Treatment per se animals + untreated test formulation. Values are presented as mean ± SEM (n=6-9). ***p≤0.001 vs. G4. Experiment includes four preventive maintenance groups (G6, G7, G8 and G9). The findings showed the significant slowdown of inflammation-related symptoms and also reduced the chances of disease susceptibility. All-inclusive, it indicate that the Trivedi Effect® was found to be most effective and benefited to protect different kinds of diseases and also improve the overall health and quality of life.

Conclusion

Based on the study outcome it was found that the level of MPO was decreased by 12% in the G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15) group as compared to the untreated test formulation (G4) group. Expression of SOD was significantly (p≤0.05) increased by 19.03%, 17.26%, and 11.81% in the G6, G7, and G9 groups, respectively as compared to the G4 group. Moreover, the level of TNF-α was significantly reduced by 25.97%, 40.28% (p≤0.01), 24.86%, 36.54% (p≤0.01), and 34.30% in the G6, G7, G8, and G9 groups, respectively with reference to disease control (G2) group. Additionally, IL-6 was significantly (p≤0.001) decreased by 23.5%, 31.0%, 26.3%, and 39.8% in the G5, G6, G8, and G9 groups, respectively as compared to the G2 group. Further, MIP-2 was decreased by 26.7% and 19.5% in the G6 and G8 groups, respectively as compared to the G4 group. Besides, the level of MMP-9 was significantly (p≤0.001) reduced by 15.1%, 21.5%, and 34% in the G6, G8, and G9 groups, respectively as compared to the G4 group.

Altogether, the Biofield Energy Treated test formulation and Biofield Energy Healing Treatment (the Trivedi Effect®) per se showed significant results with respect to different inflammatory biomarkers (cytokines) in the preventive maintenance group, G6 as well as other preventive maintenance groups (G7, G8, and G9) in Cecal Slurry, LPS and E. coli-induced systemic inflammatory response syndrome model rat model study. It also helped to slowdown the inflammatory disease progression and disease-related complications. The study data showed that Biofield Energy Treated Test formulation and Biofield Energy Treatment per se would be one of the best treatment strategies to prevent the manifestation of diseases. Thus, the Biofield Energy Treatment might act as a preventive maintenance therapy to maintain and improve the overall health and quality of life and simultaneously reduce the severity of acute/chronic diseases. The test formulation can also be used against rheumatoid arthritis (RA), fibromyalgia, aplastic anaemia, Addison disease (AD), multiple sclerosis, myasthenia gravis, psoriasis, Crohn’s disease, ulcerative colitis, dermatitis, hepatitis, Parkinson’s, stroke, etc.

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Liquid Chromatography-Mass Spectrometry Based Isotopic Abundance Ratio Analysis of the Consciousness Energy Healing Treated L-Cysteine - Juniper Publishers

 Juniper Online Journal Material Science - Juniper Publishers

Abstract

L-cysteine is a semi-essential sulfur-containing amino acid found in nails, skin, hair, etc. in the body. This study was performed to investigate the impact of the Trivedi Effect® on the structural properties and the isotopic abundance ratio of L-cysteine using LC-MS analytical techniques. L-cysteine sample was divided into control and treated parts. The treated part only received the Trivedi Effect®-Consciousness Energy Healing Treatment remotely by a renowned Biofield Energy Healer, Dahryn Trivedi. The LC-MS spectra of both the control and treated samples at retention time (Rt) 1.96 minutes exhibited the mass of the molecular ion peak adduct with hydrogen ion at 122 along with low molecular fragmented mass peaks at m/z 105, 102, 87, 76, and 59 for C₃H₅O₂S⁺, C₃H₂O₂S^•+, C₃H₅NO₂²⁺ or C₃H₅NS^•+, C₂H₆NO₂⁺, and C₂H₃O₂⁺, respectively were also observed. The peak area of the treated sample (1960679.58) was significantly increased by 8.02% compared to the control sample (1815060.18). The isotopic abundance ratios of P_M+1/P_M (²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O or ³³S/³²S) and P_M+2/P_M (³⁴S/³²S) in the treated L-cysteine was significantly increased by 41.86% and 32.39%, respectively compared with the control sample. Hence, the ¹³C, ²H, ¹⁵N, ¹⁷O, ³³S, and ³⁴S contributions from C₃H₈NO₂S⁺ to m/z 123 and 124 in the treated L-cysteine were significantly increased compared to the control sample. The changes in peak area and isotopic abundance ratios might be the cause of changes in nuclei, possibly through the interference of neutrino particles via the Trivedi Effect®-Consciousness Energy Healing Treatment. The increased isotopic abundance ratio of the treated L-cysteine may increase the intra-atomic bond strength, increase its stability, and shelf-life. The novel Biofield Energy Treated L-cysteine might have increased the stability, solubility, bioavailability, and shelf-life compared to the control sample. 

The new form of treated L-cysteine would be a better and more stable precursor in the food, cosmetics, pharmaceuticals, personal-care products, additives to cigarettes (act as an expectorant), preventative or antidote for some of the negative effects of alcohol, acetaminophen overdose, clinically used ranging from baldness to psoriasis, excellent for the treatment of asthmatics by enabling them to stop theophylline and other medications, enhances the effect of topically applied silver, tin and zinc salts for preventing dental cavities. In the near future, this Biofield Energy Treated L-cysteine may play a better role in the treatment of diabetes, psychosis, cancer, and seizures.

Keywords: Biofield Energy; Consciousness Energy Healing Treatment; L-cysteine; The Trivedi Effect®; LC-MS

Introduction

Cysteine is a semi-essential sulfur-containing amino acid found in nails, skin, hair, etc. in the body. It contains a thiol group and available as a chiral molecule with dextrorotation (D) and levorotation (L) forms [1]. Cysteine is a non-essential amino acid but may be essential for new-borns, the elderly, and individuals with specific metabolic disease or malabsorption syndromes. The cysteine plenty available in egg, meat, milk, garlic, onions, red peppers, oats, broccoli, wheat germ, brussels sprout, sprouted lentils, etc. Industrially it is also prepared from animal feathers, hair, and even from chemical synthesis [1-3].

Due to its high reactivity of the sulfhydryl group of cysteine (nucleophilic in nature) has numerous biological functions, i.e., it acts, as a precursor to the antioxidant glutathione and iron-sulfur clusters, metal cofactors in enzymes, detoxification, metabolic functions, protein synthesis, collagen production, translation of messenger RNA molecules to produce polypeptides, etc. [1-6]. It is also a precursor in the food, cosmetics, pharmaceuticals, personal-care industries, additives to cigarettes (as an expectorant), preventative or antidote for some of the harmful effects of alcohol (i.e., liver damage and hangover), acetaminophen overdose, production of more wool from sheep, clinically used ranging from baldness to psoriasis, used for the treatment of asthma, enhances the effect of topically applied silver, tin and zinc salts for preventing dental cavities [1,6-9]. Many research work claiming that, in the near future, cysteine may play an important role in the treatment of diabetes, psychosis, cancer, and seizures [10]. The stability of L-cysteine is an issue in the neutral or slightly alkaline aqueous solutions, which is oxidized to cystine by air, and on decomposition, it emits very toxic fumes of sulphur oxides and nitrogen oxides [6].

The physicochemical properties of L-cysteine pay a very important role in the food, cosmetic, pharmaceutical, nutraceutical, and other industries. The Trivedi Effect®- Consciousness Energy Healing Treatment has the astonishing abilities to transform the characteristic properties of both living and non-living object(s) [11-15]. The Trivedi Effect® is a natural and only scientifically proven phenomenon in which an expert can harness this inherently intelligent energy from the “Universal Energy Field” and transmit it anywhere on the planet via the possible mediation of neutrinos [16]. An energy field generated around the body due to the continuous movement of the charged particles in the body known as “Biofield”. The object(s) received the “Energy Therapy” respond to a useful way is known as the Biofield Energy Healing Treatment. There are several Biofield based Energy Therapies that are used nowadays against various disease conditions [17-19]. Biofield Energy Healing therapy has been recognized worldwide as a Complementary and Alternative Medicine (CAM) health care approach by the National Center of Complementary and Integrative Health (NCCIH) with other therapies, medicines and practices such as Ayurvedic medicine, yoga, meditation, homeopathy, traditional Chinese herbs and medicines, naturopathy, chiropractic/osteopathic manipulation, Qi Gong, Tai Chi, aromatherapy, acupressure, acupuncture, healing touch, hypnotherapy, Reiki, cranial-sacral therapy, etc. [20]. These CAM therapies have been adopted by most of the U.S.A. population with several advantages [21]. Similarly, the Trivedi Effect®- Consciousness Energy Healing Treatment also been reported with significant impact on the properties of polymers, ceramics, metals, organic compounds, cancer cell line, microbes, improved skin health, bone health, improved agricultural crop yield, productivity, and quality, and altered the isotopic abundance ratio, improved bioavailability of pharmaceutical/ nutraceutical compounds [22- 37].

The analysis of the natural stable isotope has the importance of many applications to understand the isotope effects resulting from the alterations of the isotopic composition [38-40]. Gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) analytical techniques are the widely used analytical techniques for the analysis of isotope ratio with sufficient precision [39]. The Trivedi Effect®-Consciousness Energy Healing Treatment could be an economical approach to alter the isotopic abundance of L-cysteine with improved physicochemical properties for the food, cosmetic, pharmaceutical/ nutraceutical, and other industries. Thus, this study was designed and evaluated the LC-MS based structural characterization and the isotopic abundance ratios in the Trivedi Effect® - Consciousness Energy Healing Treated L-cysteine compared to the control sample.

Materials and Methods

Chemicals and Reagents

The test sample L-cysteine (>98%, titration method) was purchased from Alfa Aesar, India. Other chemicals like methanol, acetonitrile, and ammonium acetate were purchased from Merck, India.

Consciousness Energy Healing Treatment Strategies

The test sample L-cysteine powder was divided into two parts. One part of the L-cysteine powder sample did not receive the Biofield Energy Treatment called the control sample. However, the other part of L-cysteine was received the Trivedi Effect®- Consciousness Energy Healing Treatment remotely under standard laboratory conditions for 3 minutes by the renowned Biofield Energy Healer, Dahryn Trivedi, USA, known as the Biofield Energy Treated L-cysteine. Further, the control sample was treated with a “sham” healer, who did not have any knowledge about the Biofield Energy Treatment. After that, both the Biofield Energy Treated and untreated L-cysteine samples were kept in sealed conditions and characterized using LC-MS analytical techniques.

Characterization

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis and Calculation of Isotopic Abundance Ratio

The liquid chromatography-mass spectrometric analysis of the L-cysteine was carried out with the help of LC-MS ThermoFisher Scientific, USA, equipped with an ion trap detector connected with a triple-stage quadrupole mass spectrometer. The column used here was a reversed phase Thermo Scientific Synchronis C18 (250mm × 4.6mm × 5micron), maintained at 25˚C. The diluent used for the sample preparation was methanol. The L-cysteine solution injection volume was 20μL and the analyte was eluted using acetonitrile (92%) + 0.1% ammonium acetate (8%) pumped at a constant flow rate of 0.8mL/min. Chromatographic separation was achieved using gradient condition and the total run time was 10 min. Peaks were monitored at 210 nm using the PDA detector. Mass spectrometric analysis was performed under ESI +ve ion mode. The total ion chromatogram, peak area% and mass spectrum of the individual peak which was appeared in LC along with the full scan were recorded.

The natural abundance of each isotope (C, H, N, O, and S) can be predicted from the comparison of the height of the isotope peak with respect to the base peak. The values of the natural isotopic abundance of the common elements are obtained from the literature [40-43]. The LC-MS based isotopic abundance ratios (P_M+1/P_M and P_M+2/P_M) for the control and Biofield Energy Treated L-cysteine (C₃H₈NO₂S⁺) were calculated.

Percentage (%) change in isotopic abundance ratio = [(IAR_Treated–IAR_Control)/ IAR_Control)] × 100

Where IAR_Treated = isotopic abundance ratio in the treated sample and IARControl = isotopic abundance ratio in the control sample.

Results and Discussion

Liquid Chromatography-Mass Spectrometry (LC-MS)

The LC-SM of the L-cysteine showed a single major peak at retention time (Rt) of 1.96 minutes in both the chromatograms (Figure 1). The peak area of the Biofield Energy Treated sample (1960679.58) was significantly increased by 8.02% compared to the control sample (1815060.18). This indicated that the solubility of the Biofield Energy Treated L-cysteine might have increased compared to the control sample. The finding was supported by the published literature data [12].

The mass spectra of both the samples of the L-cysteine are shown in Figure 2. The mass spectra of both the samples at Rt of 1.96 minutes exhibited the presence of the molecular ion of L-cysteine (Figure 2) at m/z 122 (calcd for C₃H₈NO₂S⁺, 122.03). Along with the molecular ion peak, low molecular fragmented mass peaks at m/z 105, 102, 87, 76, and 59 for C₃H₅O₂S⁺, C₃H₂O₂S^•+, C₃H₅NO₂²⁺ or C₃H₅NS^•+, C₂H₆NO₂⁺, and C₂H₃O²⁺ were observed in case of both the samples (Figures 2 & 3). The experimental data were well supported by the published literature [44].

Isotopic Abundance Ratio Analysis

The L-cysteine samples showed the mass of a molecular ion at m/z 122 (calcd for C₃H₈NO₂S⁺, 122.03) with 100% relative abundance in the spectra. The theoretical calculation of isotopic peak PM+1 for the protonated L-cysteine presented as below:

P (¹³C) = [(3 x 1.1%) × 100% (the actual size of the M+ peak)] / 100% = 3.3%

P (²H) = [(8 x 0.015%) × 100%] / 100%= 0.12%

P (¹⁵N) = [(1 x 0.4%) × 100%] / 100% = 0.4%

P (¹⁷O) = [(2 x 0.04%) × 100%] / 100% = 0.08%

P (³³S) = [(1 x 0.08%) × 100%] / 100% = 0.08%

P_M+1 i.e. ¹³C, ²H, ¹⁵N, ¹⁷O, and ³³S contributions from C₃H₈NO₂S⁺ to m/z 123 = 3.98%

Similarly, the theoretical calculation of PM+2 for L-cysteine was presented as below:

P (³⁴S) = [(1 × 4.21%) × 100%] / 100% = 4.21%

P_M+2, i.e. ³⁴S contributions from C₃H₈NO₂S⁺ to m/z 124 = 4.21%

The calculated isotopic abundance of P_M+1 (3.98%) and P_M+2 (4.21%) values was very close to the experimental values 4.3% and 4.6% (Table 1). From the above calculation, it has been found that ¹³C, ¹⁵N, and 34S have the major contribution to m/z 123 and 124.

The isotopic abundance ratio analysis P_M, P_M+1, and P_M+2 for L-cysteine near m/z 122, 123, and 124, respectively of both the samples were obtained from the observed relative peak intensities of [M⁺], [(M+1)⁺], and [(M+2)⁺] peaks, respectively in the mass spectra (Table 1). The isotopic abundance ratio of P_M+1/P_M (²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O or ³³S/³²S) and P_M+2/P_M (³⁴S/³²S) in Consciousness Energy Healing Treated L-cysteine was significantly increased by 41.86% and 32.39% compared to the control sample (Table 1). Thus, the ¹³C, ²H, ¹⁵N, ¹⁷O, ³³S, and ³⁴S contributions from C₃H₈NO₂S+ to m/z 123 and 124 in the Biofield Energy Treated sample was significantly increased compared to the control sample.

The isotopic abundance ratios of P_M+1/P_M (²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O or ³³S/³²S) and P_M+2/P_M (³⁴S/³²S) in the Biofield Energy Treated L-cysteine were significantly increased compared to the control sample. The changes in isotopic abundance could be due to the possible interference of neutrino particles via the Trivedi Effect®-Consciousness Energy Healing Treatment [16]. The altered isotopic composition in the molecular level of the treated L-cysteine might have altered the neutron to proton ratio in the nucleus. A neutrino is an elementary particle that interacts through the weak subatomic force and gravity. The neutrinos have the ability to interact with protons and neutrons in the nucleus, which indicated a close relationship between neutrino and the isotope formation [39,40]. The isotopic abundance ratios ²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O or ³³S/³²S or ³⁴S/³²S would influence the atomic bond vibration of treated L-cysteine [45]. The increased isotopic abundance ratio of the treated L-cysteine may increase the intra-atomic bond strength, increase its stability, and shelf-life. The novel Biofield Energy Treated L-cysteine might have increased the stability, solubility, bioavailability, and shelf-life compared to the control sample. The novel Biofield Energy Treated L-cysteine would be more important to the food, cosmetic, pharmaceutical/ nutraceutical, and other industries compared to the control sample.

Conclusion

The Trivedi Effect®-Consciousness Energy Healing Treatment showed a significant impact on the chromatographic peak area and isotopic abundance ratio of L-cysteine. The LC-MS spectra of both the control and Biofield Energy Treated samples at Rt 1.96 minutes exhibited the mass of the molecular ion peak adduct with hydrogen ion at 122 along with low molecular fragmented mass peaks were also observed. The peak area of the Biofield Energy Treated sample was significantly increased by 8.02% compared to the control sample. The isotopic abundance ratios of P_M+1/P_M (²H/¹H or ¹³C/¹²C or ¹⁵N/¹⁴N or ¹⁷O/¹⁶O or ³³S/³²S) and P_M+2/P_M (³⁴S/³²S) in the Biofield Energy Treated L-cysteine was significantly increased by 41.86% and 32.39%, respectively compared with the control sample. Hence, the ¹³C, ²H, ¹⁵N, ¹⁷O, ³³S, and ³⁴S contributions from C₃H₈NO₂S⁺ to m/z 123 and 124 in the Biofield Energy Treated L-cysteine was significantly increased compared to the control sample. The changes in peak area and isotopic abundance ratios might be the cause of changes in nuclei possibly through the interference of neutrino particles via the Trivedi Effect®-Consciousness Energy Healing Treatment. The increased isotopic abundance ratio of the Biofield Energy Treated L-cysteine may increase the intra-atomic bond strength, increase its stability, and shelf-life. The novel Biofield Energy Treated L-cysteine might have increased the stability, solubility, bioavailability, and shelf-life compared to the control sample. The new form of Biofield Energy Treated L-cysteine would be a better and more stable precursor in the food, cosmetics, pharmaceuticals, personal-care products, additives to cigarettes (act as an expectorant), preventative or antidote for some of the negative effects of alcohol, acetaminophen overdose, clinically used ranging from baldness to psoriasis, excellent for the treatment of asthmatics by enabling them to stop theophylline and other medications, enhances the effect of topically applied silver, tin and zinc salts for preventing dental cavities. In the near future, this Biofield Energy Treated L-cysteine may play a better role in the treatment of diabetes, psychosis, cancer, and seizures.

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