American Journal of Health Research 2016; 4(6): 179-188 http://www.sciencepublishinggroup.com/j/ajhr doi: 10.11648/j.ajhr.20160406.15 ISSN: 2330-8788 (Print); ISSN: 2330-8796 (Online) Effect of Biofield Energy Healing Treatment ® (The Trivedi Effect ) Based Herbomineral Formulation on Pro-Inflammatory Cytokines Expression in Murine Dendritic and Splenocyte Cells Mahendra Kumar Trivedi1, Alice Branton1, Dahryn Trivedi1, Gopal Nayak1, Cathryn Dawn Nykvist1, Celine Lavelle1, Daniel Paul Przybylski1, Dianne Heather Vincent1, Dorothy Felger1, Douglas Jay Konersman1, Elizabeth Ann Feeney1, Jay Anthony Prague1, Joanne Lydia Starodub1, Karan Rasdan1, Karen Mie Strassman1, Leonid Soboleff1, Maire Anne Mayne1, Mary M. Keesee1, Padmanabha Narayana Pillai1, Pamela Clarkson Ansley1, Ronald David Schmitz1, Sharyn Marie Sodomora1, Sambhu Charan Mondal2, Snehasis Jana2, * 1Trivedi Global, Inc., Henderson, Nevada, USA 2Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, Madhya Pradesh, India Email address: [email protected] (S. Jana) *Corresponding author To cite this article: Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Cathryn Dawn Nykvist, Celine Lavelle, Daniel Paul Przybylski, Dianne Heather Vincent, Dorothy Felger, Douglas Jay Konersman, Elizabeth Ann Feeney, Jay Anthony Prague, Joanne Lydia Starodub, Karan Rasdan, Karen Mie Strassman, Leonid Soboleff, Maire Anne Mayne, Mary M. Keesee, Padmanabha Narayana Pillai, Pamela Clarkson Ansley, Ronald David Schmitz, Sharyn Marie Sodomora, Sambhu Charan Mondal, Snehasis Jana. Effect of Biofield Energy Healing Treatment (The Trivedi Effect®) Based Herbomineral Formulation on Pro-Inflammatory Cytokines Expression in Murine Dendritic and Splenocyte Cells. American Journal of Health Research. Vol. 4, No. 6, 2016, pp. 179-188. doi: 10.11648/j.ajhr.20160406.15 Received: November 15, 2016; Accepted: November 26, 2016; Published: December 8, 2016 Abstract: The utilization and demand of self-medication with herbomineral-based formulations have increased day-by-day across the globe over the last decade. A new proprietary herbomineral formulation was prepared with the mixture of minerals (zinc, magnesium, and selenium) and the herbal root extract of ashwagandha. The current study was undertaken to evaluate the Biofield Energy Healing (The Trivedi Effect®) on the test herbomineral formulation using murine dendritic cells (DCs) and splenocytes in vitro. The formulation was divided into two parts, one part was control without any Biofield Energy Treatment, while the other part was defined as the Biofield Energy Treated sample, which received the Biofield Energy Healing Treatment remotely from eighteen renowned Biofield Energy Healers. The effect of the Biofield Energy Treated formulation in murine cells was monitored with an estimation of pro-inflammatory cytokines levels such as tumor necrosis factor (TNF-α), macrophage inflammatory protein-1α (MIP-1α) and interleukin (IL-1β) in cell culture supernatants along with estimations of non-cytotoxic concentrations of the test formulation by MTT assay. The Biofield Treated formulation showed 114.2%, 122.6%, 141.2%, 127.8%, and 114.1% cell viability at concentrations 1.05, 5.2, 10.5, 25.6, and 51.2 µg/mL, respectively in DCs. Similarly, the Biofield Energy Treated and untreated formulations showed more than 100% cell viability in mice splenocytes at 5 µg/mL. The level of TNF-α in DCs was significantly (p≤0.05) inhibited by 19.21% in the Biofield Treated formulation at concentration 5.2 µg/mL as compared to the untreated test formulation. The level of MIP-1α in LPS induced mice splenocyte cells was reduced by 15.35% in the Biofield Energy Treated formulation at 0.0105 µg/mL as compared to the untreated formulation. Similarly, the level of IL-1β in LPS induced mice splenocyte cells was significantly (p≤0.05) reduced by 31.59% in the Biofield Treated formulation at 1.05 µg/mL as compared to the untreated formulation. Altogether, the results suggest that The Trivedi Effect® (Biofield Energy Healing Treatment) showed significant down-regulation of the tested pro- inflammatory cytokines expression and potentiated the immunosuppressive effect of the treated formulation to modulate the immune system. These data also suggest that the Biofield Treated test formulation can be used for autoimmune and 180 Mahendra Kumar Trivedi et al.: Effect of Biofield Energy Healing Treatment (The Trivedi Effect®) Based Herbomineral Formulation on Pro-Inflammatory Cytokines Expression in Murine Dendritic and Splenocyte Cells inflammatory diseases, stress management and anti-aging by improving overall health. Keywords: Biofield Energy Healing Treatment, Biofield Energy Healers, The Trivedi Effect®, Immunomodulation, Dendritic Cells, Splenocytes, Cytokines trace elements such as zinc, magnesium, selenium, copper 1. Introduction and manganese etc. have strong immunomodulatory effects [10]. Different cytokines are involved in host responses with Since ancient times, natural products have played primary respect to the inflammation and immune activation process roles in Complementary and Alternative Medicine (CAM) that lead to various diseases [11]. Therefore, anti- and have been used worldwide for the treatment and inflammatory phytomedicines are used as an adjunct therapy prevention of various diseases or ailments. Moreover, the use for the management of chronic inflammatory disorders [12- of alternative and complementary medicine has increased in 14]. circumstances where conventional medicine and the Dendritic cells (DCs) play an important role in the immune allopathic model of health care is ineffective, i.e. cases of system and possess the ability to stimulate naive T cells. advanced diseases like cancer or with ailments/problems that Murine DCs are characterized by CD11c marker expression, are not limited to only the brain and the body. Many which is a common marker among plasmacytoid and myeloid medicinal plants and minerals have immunomodulatory DCs [15]. Thymus derived lymphocyte helper cells 1 (TH1) properties. The ashwagandha (Withania somnifera) plant is are the primary sources for interleukin-1β (IL-1β), tumor one important herbal product that is widely used in necrosis factor-α (TNF-α), IL-2, interferon-ϒ (IFN-ϒ), complementary and alternative medicine for various types of lymphotoxins and other factors which characterize the cell- diseases. There has been increased interest in the use of mediated immune responses [16]. The different types of natural substances, especially pertaining to plants, derived immune cells such as DCs, macrophages and spleen play an herbal extracts and various essential minerals, which are important role in stopping acute/chronic inflammation and collectively known as nutraceuticals [1]. The herbal extracts retrieving a steady state strategy through the secretion of report various pharmacological benefits such as immuno-modulating cytokines [17]. These immune cells immunostimulation, stress reduction, appetite stimulation, have been reported to be useful as cellular models for in vitro growth promotion and antimicrobial properties. These effects studies. Therefore, murine bone marrow derived dendritic were found to be caused by the presence of active cells (BMDCs) and splenocytes were used to assess the effect constituents such as steroids, flavonoids pigments, alkaloids, of the test formulation on in vitro cell cultures. In recent phenolics, and terpenoids [2]. Based on the literature, a new years, Biofield Energy Healing Treatment has been accepted proprietary herbomineral formulation was prepared that as an alternative and complementary method of health care combined the herbal root extract of ashwagandha with three and has been shown to have impacts on various properties of minerals viz. zinc, magnesium, and selenium. Ashwagandha living organisms in a cost-effective manner [18]. The use of root is a traditional Indian herb (also known as Indian alternative and integrative medicine approaches have Ginseng) commonly used as a constituent in herbal tonic increased in the facilitation of wellness and because of their preparation. It is considered an 'adaptogen', (a term used to minimal side-effects for cancer patients and people suffering describe herbs that improve physical energy, athletic ability from chronic diseases. Recent studies report that the uses of and vitality, as well as increase sexual capacity, fertility and energy medicine provide the highest benefit to cancer immunity to cold infections) [3]. Herbal preparations that patients as compared to other complementary and alternative contain ashwagandha root extract have shown anti- forms of medicine [19]. inflammatory, anti-arthritic, antibiotic, anti-tumor, and Human Biofield Energy has subtle energy that has the immunomodulatory effects with withanolides [4, 5]. capacity to work in an effective manner [20]. Reports show Ashwagandha root extract has been shown to modulate the that CAM therapies have been practiced worldwide with immune response via cytokines expression level [6]. reports of clinical benefits in different health disease profiles Mazumder et al. (2012) reported that zinc, along with [21]. Biofield Energy comes under the category of energy Glycyrrhiza glabra, had increased the leukocyte count and medicine, which is well defined and accepted by National phagocytic index and potentiated immunomodulatory effects Center for Complementary and Integrative Health (NCCIH), [7]. Sodium selenate greatly improved the process of a subdivision of the National Institutes of Health (NIH). This lymphocytes proliferation to the increased level of IL-2. The energy can be harnessed and transmitted by individuals into literature reports that sodium selenate increased the living and non-living things via the process of Biofield phagocytic index and down-regulated the expression levels Energy Healing. Biofield Energy Healing Treatment (The of pro-inflammatory cytokines viz. IL-1, IL-6, and TNF-α Trivedi Effect®) have been known to transform the structural, [8]. Selenium produces antioxidant properties by scavenging physical, and thermal properties of various metals, chemicals, free-radicals. Selenium also plays an important role in ceramics and polymers in materials science [22-25], several immunological and metabolic processes of improved the overall productivity, yield and quality of crops phagocyted bacteria during intracellular digestion [9]. Many American Journal of Health Research 2016; 4(6): 179-188 181 [26-29], altered characteristics and features of microbes [30- Ghaziabad, India. The treated sample was subjected to 33], improved excellent outcomes of various nutraceutical Biofield Energy Healing (also known as The Trivedi compounds in the fields of nutraceuticals [34-37], and Effect®), which was remotely administered for a period of 5 improved the growth and anatomical characteristics of minutes by a group of eighteen Biofield Energy Healers, various medicinal plants [38, 39]. Based on the literature eleven of which were remotely located in the U.S.A., four in related to Biofield Energy Treatment, the authors of this Canada, one in the UK, one in Russia and one in Ireland. study sought to investigate the effects of Biofield Energy Similarly, the control sample was subjected to “sham” (The Trivedi Effect®) on the test herbomineral formulation, healers under the same laboratory conditions for 5 minutes. which might modulate the key pro-inflammatory cytokines The sham healers did not have any knowledge about the expression in in vitro murine DCs and splenocytes models. Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed 2. Materials and Methods conditions and used for the in vitro study on DCs and splenocytes for cytokines estimation. 2.1. Chemicals and Reagents 2.4. Experimental Animal 3-(4, 5-diamethyl-2-thiazolyl) 2, 5 diphenyl-2H- C57BL/6 male mice (8 weeks old) were purchased from tetrazolium) (MTT), Lipopolysaccharide (LPS), L-glutamine, M/S Vivo Bio Tech Ltd., Hyderabad, India and acclimatized RPMI-1640, penicillin, HEPES, streptomycin, 2- for one week prior to the experiment. The mice were mercaptoethanol were purchased from Sigma Chemical maintained under controlled conditions with a temperature of Company in St. Louis, MO, a subsidiary of Sigma-Aldrich 22 ± 3°C, humidity of 30–70% and a 12-hour light/12-hour Corporation. ELISA (enzyme-link immunosorbent assay) dark cycle and rodent laboratory diet and drinking tap water assay kits for all cytokines such as TNF-α, macrophage were provided ad libitum. All the procedures were followed inflammatory protein-1α (MIP-1α), and IL-1β were in strict accordance with the Guide for the Care and Use of purchased from R&D systems, U.S.A. Fetal bovine serum Laboratory Animals published by the US National Institutes (FBS) was procured from GIBCO, U.S.A. Ashwagandha root of Health. Approval was obtained from the Institutional extract powder was procured from Sanat Products Ltd., India. Animal Ethics Committee prior to carrying out the animal Zinc chloride and magnesium (II) gluconate hydrate were experiments. The various concentrations of the test procured from Tokyo Chemical Industry Co., Ltd. (TCI, formulation were used from 1.05 to 51.2 µg/mL for BMDCs Japan). Sodium selenate was procured from Alfa Aesar, and 0.0000105 to 1.05 µg/mL for LPS stimulated splenocytes U.S.A. Rapamycin, NaHCO , and EDTA were procured from 3 culture. The respective vehicle controls (DMSO) were kept Sigma. All other chemicals used in this experiment were of in the assay. analytical grade and procured from local vendors. 2.5. Mouse Bone Marrow-Derived Dendritic Cells (DCs) 2.2. Test Formulation and Reference Standard Cultures The test formulation contained the combination of four C57BL/6 male mice were euthanized by CO asphyxiation. ingredients: zinc chloride, magnesium gluconate, sodium 2 Subsequent experimental steps were conducted in a laminar selenate, and ashwagandha root powder extract. Different air flow. BMDCs were induced from bone marrow (BM) concentrations of the test formulation were used for the study cells using the modified method of Inaba et al. (1992) [40]. i.e. concentration range of 1.05 to 1052.5 µg/mL for bone Briefly, a single cell suspension was prepared from BM marrow DCs and 0.0000105 to 10.5 µg/mL with LPS obtained from the femur. After removing all muscle tissues stimulated splenocyte culture for cell viability assay. LPS from the femur using gauze, the bones were placed in a 90 was used as an immunostimulant and as an inducing agent at mm dish with 70% alcohol for 1 minute, washed twice with 0.5 µg/mL and rapamycin (1 and 10 nM) was used as the PBS, and then transferred into a fresh dish containing RPMI reference standard (positive control) in splenocyte culture. 1640 medium. Both ends of the bones were cut with scissors TNF-α estimated in DCs, which was treated with 0.1% in the dish, and then the bone marrow (BM) was flushed out DMSO, served as the vehicle control group. MIP-1α and IL- using 2 mL of RPMI 1640 medium with the help of a syringe 1β were estimated in splenocyte cells, which includes attached with a 25-gauge needle. To remove small pieces of splenocyte cells treated with LPS (0.5 µg/mL) along with bone and debris, the tissue was suspended and passed 0.005% DMSO, and was defined as the vehicle control through a nylon mesh and red blood cells were lysed with group. ammonium chloride. After washing, lymphocytes and other 2.3. Biofield Energy Treatment Strategies cells were killed with a cocktail of monoclonal antibodies (mAbs) and rabbit complement for 60 minutes at 37°C. After The herbomineral formulation was divided into two parts. that, whole BM cells (2 × 106 cells/mL) were cultured in One part was considered the control sample and the other RPMI 1640 medium in 90 mm Petri dishes (Sigma Aldrich, part was defined as the treated sample. Both samples were St. Louis, MO) at 37°C, 5% CO , supplemented with 10% 2 kept under standard laboratory conditions at the research FBS, 2 mM L-glutamine, 100 U/mL penicillin G, 100 mg/mL laboratory Dabur Research Foundation near New Delhi in 182 Mahendra Kumar Trivedi et al.: Effect of Biofield Energy Healing Treatment (The Trivedi Effect®) Based Herbomineral Formulation on Pro-Inflammatory Cytokines Expression in Murine Dendritic and Splenocyte Cells streptomycin, 20 ng/mL recombinant mouse GM-CSF (R & The concentrations that resulted in >95% viability were D systems Inc., U.S.A.). The cells were incubated for 24 selected for subsequent cytokine estimation. hours. The petri-plates were then gently swirled and the 2.8. Cytokines Assays Using ELISA medium containing non-adherent cells, and which was then removed and replaced with the nutrient medium as described The effects of the Biofield Energy Treated and untreated above. The supplemented medium was replaced every three test samples on the production of TNF-α, MIP-1α and IL-1β days. On day 6, non-adherent and loosely adherent DCs were were measured by ELISA method using culture supernatants collected, washed, and seeded in culture plates for further collected from the treated cells. Briefly, ELISA plates were assays. Cell counts were performed using a hemocytometer coated overnight and kept at 4◦C with coating buffer and cell viability was determined with the help of trypan-blue containing capture antibody at the recommended dye exclusion technique, results showing ≥95% of viable concentration. After washing with PBS-T (PBS with 0.05% cells. The cells were cultured in 96-well tissue culture plates Tween 20), the plates were blocked with assay diluent for at with 5 x 103 cells per well. They were incubated at 37°C in a least 2 hours at room temperature. 100 µL of culture humidified atmosphere with 5% CO for the indicated period. 2 supernatants from different experimental samples and standards were incubated overnight at 4◦C and, after three 2.6. Mouse Splenocyte Cultures washes, biotinylated anti-mouse cytokine (TNF-α, MIP-1α C57BL/6 male mice were sacrificed and the spleens were and IL-1β) antibodies at the recommended concentrations removed and ground by passing them through a sterile plastic were incubated for 1 hour at room temperature and the plate strainer under aseptic conditions. After that, the cells were was incubated for 45 minutes at room temperature with centrifuged twice at 1000 g for 5 minutes. Erythrocytes were gentle shaking. Plates were again washed 3 times and then lysed by lysis buffer (0.15 M NH Cl, 0.01 M NaHCO , and 100 µL of horseradish per-oxidase (HRP)-streptavidin 4 3 0.1 mM EDTA, pH 7.4) and then the cell pellets were washed conjugate solution was added and the plate was incubated for twice with RPMI-1640 medium. Then, the cells were 45 minutes at room temperature with gentle shaking in a resuspended in a complete RPMI-1640 medium (RPMI 1640 shaker. Next, the plate wells were washed 3 times and 100 medium plus 10% FBS, 2 mM glutamine, streptomycin and µL of 3,3,5,5'-tetramethylbenzidine (TMB) was added to the 100 IU/mL of penicillin and 15 mM HEPES, and 50 mM 2- wells, followed by 30 minutes of incubation at room mercaptoethanol). Cell counts were performed with the help temperature in the dark. Then 50 µL of 0.2 mol/L sulphuric of hemocytometer and cell viability was determined using acid was added to each well to stop the reaction. After that, trypan-blue dye exclusion technique, the results showing the plates were read for absorbance at 450 nm using a Biotek ≥95% of viable cells. The cells were cultured in 96-well reader (SIAFRT/Synergy HT multimode reader). Standards tissue culture plates with 0.2 x 106 cells per well. They were were run in parallel to the samples. Concentrations were incubated at 37°C in a humidified atmosphere containing 5% determined and the experiment was done in triplicates. CO for the specified period. 2 2.9. TNF-α Level in Mouse Dendritic Cells (DCs) 2.7. Cytotoxicity by MTT Assay DCs were seeded in a 24-well plate at a density of 0.5 x The number of viable cells was estimated based on the 106 cells per well with 1 mL of culture medium and co- conversion of MTT to formazan dye using mitochondrial incubated for 24 hours with different non-cytotoxic enzyme. Both cells were cultured overnight in 96-well plates, concentrations ranging from 25 to 500 µg/mL of the Biofield at a density of 5 x 103 cells per well for BMDCs and 0.2 x Treated and untreated test samples. DCs treated with 0.1% 106 cells per well for splenocytes. After the Biofield Energy DMSO was included as the vehicle control. After 24 hours of Treatment on the test formulation with the desired incubation incubation, culture supernatants were collected and assayed. period, the medium was removed. 20 µL of 5 mg/mL MTT The supernatants were analyzed for the estimation of TNF-α was then added to each well and incubated for 3 hours further using ELISA as per the manufacturer’s instructions [41]. at 37oC in a humidified 5% CO atmosphere. The cells were Concentrations were determined in two wells in each sample 2 centrifuged and supernatants were removed. The cell pellet in and this experiment was conducted in triplicates. each well was resuspended in 150 µL of DMSO to dissolve 2.10. MIP-1α and IL-1β in Splenocytes formazan crystals. The optical density of each well was read at 540 nm using Biotek Reader (SIAFRT/Synergy HT For estimation of MIP-1α and IL-1β in LPS (0.5 µg/mL) multimode reader). The effect of the test formulation on cell induced splenocytes (2 x 106 cells/well in 24-well culture viability of DCs and splenocyte cells was determined as per plates) were treated with the Biofield Treated and untreated equation 1: test formulations at selected non-toxic concentrations (0.00005 to 5 µg/mL) in triplicates. Splenocytes treated with % Cell viability=(cid:13)100−% Cytotoxicity(cid:20) (1) LPS along with 0.005% DMSOs were included as control Where; % Cytotoxicity = {(O.D. of Control cells – O.D. of cells. Untreated cells were included as a negative control. cells treated with the test formulation)/ OD of Control Rapamycin was included as a positive control. After 48 hours cells}*100. of incubation, the supernatants were analyzed for the secreted American Journal of Health Research 2016; 4(6): 179-188 183 levels of MIP-1α and IL-1β using ELISA as per the the test formulation on the viability of DCs and splenocytes manufacturer’s instructions [42, 43]. are shown in Figure 1 and Figure 2, respectively. The cell viability in the untreated test group showed 136.30%, 2.11. Statistical Analysis 141.90%, and 113.00% at concentrations of 5.2, 25.6, and 51.2 µg/mL, respectively in DCs. Further, increasing the The experiment was performed in triplicates and the data dose of the untreated sample resulted in reduced cell viability are presented as mean ± standard error of mean (S.E.M.). up to 26% at 210.5 µg/mL. So, the dose ranges from 1.05 to Comparisons of the means of control and test groups were 51.2 µg/mL were selected for the cytokine (TNF-α) subjected to Student’s t-test for two group comparison. estimation. Similarly, the Biofield Energy Treated Statistical significance was considered at p≤0.05. formulation showed 114.2%, 122.6%, 141.2%, 127.8%, and 114.1% cell viability at concentrations of 1.05, 5.2, 10.5, 3. Results 25.6, and 51.2 µg/mL, respectively in DCs. The increased cell viability with respect to the vehicle control might be due 3.1. In Vitro Immune Cells Viability Assay by MTT Assay to proliferation in the cell culture. Further, increasing the The effects of the Biofield Energy Treated and untreated dose of the Biofield Treated formulation resulted in test formulations on the proliferation of mouse BMDCs and decreased cell viability up to 25% at concentration 210.5 splenocyte cells were examined after 24 hours and 48 hours, µg/mL. respectively using MTT cell viability assay. The effects of Figure 1. MTT Assay in mouse bone marrow dendritic cells (DCs) after 24 hours of treatment with various concentrations of the test formulation in the absence of LPS. The absorbance of the MTT formazan was determined at 540 nm in an ELISA reader. Cell viability was defined as the absorbance ratio (expressed as a percentage) of the Biofield Energy Treated cells relative to the untreated vehicle cells. LPS induced splenocytes were treated with the test 74.2% and 81.6% cell viability at 1 and 10 nM, respectively. formulation at the concentration ranges from 0.0000105 to Both the Biofield Energy Treated and untreated test 1.05 µg/mL for 48 hours. The result of percent cell viability formulations showed more than 100% cell viability at 5 is presented in Figure 2. The cytokines analysis was also µg/mL concentration. Therefore, the dose range from conducted using the same concentration range. The cell 0.0000105 to 1.05 µg/mL of the test formulation was selected viability of the LPS group was 122.8%, while rapamycin (the for the cytokines (MIP-1α and IL-1β) estimation. reference standard of the immunosuppressive agent) showed Figure 2. MTT Assay in splenocyte cells after 48 hours of treatment with various concentrations of the test sample in the presence of 0.5 µg/mL of LPS. The absorbance of the MTT formazan was determined at 540 nm in an ELISA reader. Cell viability was defined as the absorbance ratio (expressed as a percentage) of the Biofield Treated cells relative to the untreated vehicle cells. 184 Mahendra Kumar Trivedi et al.: Effect of Biofield Energy Healing Treatment (The Trivedi Effect®) Based Herbomineral Formulation on Pro-Inflammatory Cytokines Expression in Murine Dendritic and Splenocyte Cells 3.2. Effect of the Test Formulation on the Expression of 3.3. Modulation of TNF-α Expression in Mouse Bone Pro-inflammatory Cytokines TNF- α, MIP-1α and Marrow Dendritic Cells (BMDCs) IL-1β in Immune Cells DCs were treated with the test formulation at non-cytotoxic Lymphocyte proliferation and the activation of natural concentrations for 24 hours. The effect of the formulation on killer (NK) cells are cytokine dependent [44]. The type of TNF-α secretion in DCs is represented in Figure 3. Both the cytokine up/down regulation affects the course of immune untreated and Biofield Energy Treated test formulation groups response and the whole network of immune regulation. The demonstrated decreased levels of TNF-α secretion at different pro-inflammatory cytokines TNF- α, MIP-1α and IL-1β play concentrations i.e. 1.05, 5.2, 10.5, 25.6, and 51.2 µg/mL as a pivotal role in inflammation, immune modulation, and compared to the vehicle control group. The maximum down- lymphocyte activation. Various herbomineral formulations regulation effect was observed at 25.6 µg/mL of the Biofield have the potential to modulate the expression and activation Treated and untreated formulations with respect to the vehicle of cytokines. Therefore, we examined the effect of the test control group. The Biofield Energy Treated and untreated formulation on the levels of TNF-α, MIP-1α and IL-1β formulations showed 59.3 and 73.4 pg/mL of TNF-α levels at expression at two different immune cells: dendritic and 5.2 µg/mL as compared to the control group. Therefore, the spleen cells. The production of the cytokines by these level of TNF-α in the Biofield Treated group was decreased by immune cells was tested in the culture supernatants using the 19.21% as compared to the untreated group, which was commercial ELISA kits. statistically significant (p≤0.05) (Figure 3). Figure 3. Dose-dependent inhibition of TNF-α by the test formulation. For each concentration treatment, the level of TNF-α release was measured after 24 hours of treatment. The values are represent as means ± SEM of independent ELISA experiment, *p≤0.05 as compared to the untreated test formulation. 3.4. Modulation of MIP-1α and IL-1β Expression in Mouse formulation on each cytokine production was observed after Splenocytes 48 hours of incubation at various concentrations of the Biofield Energy Treated and untreated test formulations. Splenocytes were cultured in RPMI-FBS (10%) with LPS Rapamycin was used as the positive control. and the levels of MIP-1α and IL-1β cytokines were measured by ELISA in the culture supernatants. The effect of the test Figure 4. Dose-dependent inhibition of LPS mediated production of MIP-1α by test formulation. For each concentration treatment, the level of MIP-1α release was measured after 48 hours of treatment. The values are represented as mean ± SEM of independent ELISA experiments. American Journal of Health Research 2016; 4(6): 179-188 185 in splenocytes. Both the untreated and the Biofield Treated 3.4.1. MIP-1α Expression test formulations demonstrated inhibition of IL-1β as The effect of the test formulation on MIP-1α secretion in compared to the LPS stimulated cells (vehicle control). The splenocytes is shown in Figure 4. This figure demonstrates Biofield Energy Treated formulation exhibited better the comparative expression profile of MIP-1α secretion in inhibition of IL-1β secretion than the untreated formulation splenocytes of the Biofield Treated and untreated groups. group at most of the tested concentrations. The level of IL-1β Both the untreated and Biofield Treated formulations was observed as 329.5 pg/mL and 349.9 pg/mL in the demonstrated inhibition of MIP-1α expression as compared Biofield Treated and untreated groups, respectively at to the LPS stimulated cells (vehicle control). However, the 0.00105 µg/mL. There was 5.83% reduction of IL-1β in the Biofield Energy Treated formulation group enhanced the Biofield Treated group as compared to the untreated group. down-regulation of MIP-1α. At concentration 0.105 µg/mL, Moreover, at concentration 0.0105 µg/mL, the level of IL-1β the level of MIP-1α was observed as 1915.5 and 2144.1 was observed as 332.3 pg/mL in the case of the Biofield pg/mL in the Biofield Treated and untreated formulation Energy Treated group, whereas 378.4 pg/mL was found in groups, respectively. Moreover, there was 10.66% reduction the untreated group. There was 12.18% reduction of IL-1β in of MIP-1α in the Biofield Treated group as compared to the the Biofield Treated group as compared to the untreated untreated group. The level of MIP-1α was observed as group. Additionally, at concentration 0.105 µg/mL, level of 2087.8 pg/mL and 2466.4 pg/mL in the Biofield Treated and IL-1β was unchanged between the Biofield Treated and untreated test formulation groups, respectively at 0.0105 untreated test formulation groups. However, at concentration µg/mL. There was 15.35% reduction of MIP-1α in the 1.05 µg/mL the level of IL-1β was found at 272.4 pg/mL and Biofield Treated group as compared to the untreated group. 398.2 pg/mL in the Biofield Treated and untreated group, respectively. There was statistically significant (p≤0.05) 3.4.2. IL-1β Expression reduction of IL-1β by 31.59% in the Biofield Treated group Figure 5 demonstrates the comparative effect of the Biofield as compared to the untreated group. Energy Treated and untreated formulation on IL-1β secretion Figure 5. Dose-dependent inhibition of LPS mediated production of IL-1β by test formulation. For each concentration treatment, the level of IL-1β release was measured after 48 hours of treatment. The values are represented as mean ± SEM of independent ELISA experiment, *p≤0.05 as compared to the untreated test formulation. dose-dependent response. The mechanism of action may 4. Discussion reflect the ability of zinc to either induce or inhibit the activation of NF-κB. The major involvement of zinc in the The use of herbomineral products to maintain or improve immune system [47] includes an ability to influence the overall health has gradually increased across the globe over the production and signaling of numerous inflammatory cytokines last couple of years. Herbal extracts have also been shown to in a variety of cell types [48, 49]. Magnesium sulphate reduced modulate immune responses during inflammation. A new cytokine production in intrapartum women and term and proprietary herbomineral formulation was developed, preterm neonates, demonstrating effectiveness in those at risk consisting of four ingredients viz. ashwagandha, zinc, for inflammation associated with adverse perinatal outcomes, magnesium and selenium. Ashwagandha was shown to inhibit whereby magnesium plays a critical regulatory role in NF-κB TNF-α induced NF-κB activation in human myelomonoblastic activation, cytokine production, and disease pathogenesis [50]. leukemia cells in a study conducted by Singh and Aggrawal Recently, Chen Y. et al. (2009), reported that the (1995) [45, 46]. Zinc deficiency influences the generation of supplementation of MC3T3-E1 with methylseleninic acid cytokines, including IL-1β, IL-2, IL-6, and TNF-α, and in (MSA) (0.5 mM to 4 mM) reduced the activation of NF-kB response to zinc supplementation, plasma cytokines exhibit a 186 Mahendra Kumar Trivedi et al.: Effect of Biofield Energy Healing Treatment (The Trivedi Effect®) Based Herbomineral Formulation on Pro-Inflammatory Cytokines Expression in Murine Dendritic and Splenocyte Cells leading to a decrease in IL-6, MCP-1, COX-2 and iNOS in supernatants of all tested concentrations. Importantly, these response to MDA-MB-231 conditioned medium [51]. The effects were not attributed to a decrease in cellular viability, authors of this study developed a product which showed indicating that this formulation was not adversely affecting these immunostimulating effects that could contribute to the cultured murine DCs and splenocytes. It is worthwhile to note maintenance of the immune system, which may prevent the that the anti-inflammatory effects of the test formulation were progression of acute or chronic diseases. Inflammation is the observed in both DCs and mouse splenocytes with the first response of the immune system to infection or irritation. suppression of pro-inflammatory cytokines (TNF-α, MIP-1α and It is caused by cytokines such as TNF-α, IL-1 and IL-6, and IL-1β) production. Several cytokines are deeply associated with by eicosanoid such as PGE2. Thus, inhibitors of these most inflammatory diseases. In particular, TNF-α and IL-1β are cytokines have been considered as a candidate for anti- prominent contributors to chronic inflammatory disorders. Our inflammatory drugs. The immune cells, especially the bone data suggests that the test formulation modulates DC and marrow cells such as dendritic and splenocyte cells, are splenocytes function. The Biofield Energy Treated formulation always used to screen the potential immunomodulatory effect showed significant inhibition of pro-inflammatory cytokines of a substance/product. This is because the thymus is the expression like TNF-α, MIP-1α and IL-1β as compared to the major primary lymphoid organ for T cell development, while untreated formulation. Therefore, The Trivedi Effect® may act bone marrow is for B and NK cells. These immune cells then via the specific inhibition of nuclear factor-kappa B (NF-κB), a migrate to the spleen, which is the secondary lymphoid transcription factor involved in the activation of many tissue, and respond to antigens in there. Therefore, the inflammatory mediator genes. regulation of thymus and spleen cell proliferation, which is closely related to maintaining immune homeostasis, can be 5. Conclusions considered as an important marker for immune response control. Lymphocytes/monocytes/macrophages are key Overall, results suggest that the Biofield Energy Treated mediators of inflammation and are widely distributed in the formulation exerts better and significant inhibition of pro- body. Therefore, mouse bone marrow derived DCs and inflammatory cytokines (TNF-α, MIP-1α and IL-1β) splenocytes, which represent appropriate model systems to expression levels as compared to the untreated group in both study the immune responses, were utilized to investigate the immune cells viz. of DCs and splenocytes. The level of TNF-α anti-inflammatory effects of the newly developed proprietary was significantly suppressed (p≤0.01) by 19.21% in DCs in the herbomineral formulation. Biofield Treated test group at 5.2 µg/mL as compared to the Based on the literature, it has been shown that human untreated test formulation group. The level of MIP-1α in LPS emotions such as loneliness are directly correlated with the induced splenocyte cells was reduced by 15.35% in the down-regulation of genes bearing anti-inflammatory Biofield Energy Treated test formulation at 0.0105 µg/mL as response elements and the up-regulation of genes bearing compared to the untreated test formulation group. Moreover, response elements for pro-inflammatory NF-kappaB/Rel the level of IL-1β in LPS induced splenocyte cells was transcription factors [52]. In this study, the cytotoxic effect of significantly (p≤0.05) reduced by 31.59% in the Biofield the Biofield Energy Treated formulation was screened by Treated test group at the concentration of 1.05 µg/mL as assessing the metabolic activity of immune cells through compared to the untreated group. In conclusion, the Biofield MTT assay. MTT assay was previously proven to show the Energy Treated test formulation could be used as an effective similar result in cell proliferation as the bromodeoxyuridine complementary and alternative treatment and/or method of (BrdU) assay, because its accurate correlation between the prevention for various types of autoimmune and inflammatory metabolic activity and cell growth was proven [53]. The anti- diseases, stress management and anti-aging as an effective inflammatory effects of both the Biofield Energy Treated and anti-inflammatory and immunomodulatory product without untreated test samples were evaluated here in two different any adverse effects. The Biofield Energy Treated test immune cells from the mice. The metabolic activity is formulation can be a better complementary and alternative evaluated by measuring the activity of a mitochondrial medicine to prevent the immune-mediated tissue damage in enzyme succinate dehydrogenase using the MTT test. This cases of organ transplants (for example kidney transplants, test is widely used in the in vitro evaluation of the toxicity of liver transplants and heart transplants), various autoimmune any test item. Both DCs and splenocytes (immune cells) were disorders such as Lupus, Addison Disease, Celiac Disease exposed to various concentrations (1.05 to 51.2 µg/mL for (gluten-sensitive enteropathy), Dermatomyositis, Graves’ DCs and 0.0000105 to 1.05 µg/mL for splenocytes) of the Disease, Hashimoto Thyroiditis, Multiple Sclerosis (MS), test formulation for 24 hours and 48 hours, respectively. Myasthenia Gravis, Pernicious Anemia, Aplastic Anemia, There was a significant reduction in the DCs viability at Reactive Arthritis, Rheumatoid Arthritis, Sjogren Syndrome, concentration >51.2 µg/mL and splenocyte cells showed Systemic Lupus Erythematosus, Type 1 Diabetes, Alopecia cytotoxic at 10.5 µg/mL of the test formulation. Areata, Crohn’s Disease, Fibromyalgia, Vitiligo, Psoriasis, The Biofield Treated test formulation demonstrated the Scleroderma, Chronic Fatigue Syndrome and Vasculitis, as greatest potential for modulating the inflammatory response of well as inflammatory disorders such as Asthma, Ulcerative DC and splenocyte cells in vitro assay. There was a significant Colitis, Alzheimer’s Disease, Atherosclerosis, Dermatitis, reduction of TNF-α, MIP-1α and IL-1β levels in the Diverticulitis, Hepatitis, Irritable Bowel Syndrome, and American Journal of Health Research 2016; 4(6): 179-188 187 Parkinson’s Disease etc. along with autoimmune and [10] Bahi A, Necib Y (2014) Anti-inflammatory and inflammatory diseases, stress management and anti-aging to immunomodulatory activities of argan oil (Argania Spinosa. L) and sodium selenite after exposure to mercuric chloride in improve overall health and quality of life. rats. Int J Pharm Sci Rev Res 29: 211-215. 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